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TABLE OF CONTENTS

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KEYNOTE….…………………………………...………………………………..……………………………..…….2

SPECIAL REPORT BY DGRC……………….…………………………………………………………….….….4

PLENARY TALKS (DAY 1) …………………………………………….…………………….…….….………...5

PLENARY TALKS (DAY 2)….……………………………………………………………….….….….………...7

PLENARY TALKS (DAY 3)…………………………………………………………….….………….………...15

PLENARY TALKS (DAY 4)…………………………………………………………….….………….………...17

PLATFORM TALKS (DAY 1).………………………………………………….….…………………….…….19

PLATFORM TALKS (DAY 2).………………………………………………….….……………………………23

PLATFORM TALKS (DAY 3).…………………………………………….….………………………….…….32

PLATFORM TALKS (DAY 4).…………………………………………………….….………………….…….41

POSTER: STEM CELLS, CELL DIVISION AND GROWTH CONTROL…………….……………..45

POSTER: CELL DEATH……………………………………….…….….……………………………..…………70

POSTER: NEURAL PHYSIOLOGY AND BEHAVIOR………………………………….….……………80

POSTER: NEUROGENETICS AND NEURAL DEVELOPMENT…………………………….…….110

POSTER: IMMUNITY AND PATHOGENESIS………………………………….….……...………….150

POSTER: PHYSIOLOGY AND METABOLISM…………………………….….……..………………..155

POSTER: REGULATION OF GENE EXPRESSION………………………….….……….…….……..164

POSTER: CHROMATIN AND EPIGENETICS ……………………………………….……..…….……177

POSTER: PATTERN FORMATION………………………………….….……………………….…………182

POSTER: GAMETOGENESIS AND ORGANOGENESIS…………………………………..……….195

POSTER: CELL BIOLOGY AND SIGNAL TRANSDUCTION…………………………..….….……204

POSTER: TECHNIQUES AND FUNCTIONAL GENOMICS…………………………….….………236

POSTER: EVOLUTION AND QUANTITATIVE GENETICS……………………………….….…….240

POSTER: OTHERS…………………………….….…………….……….….…………………..……….…….247

SPEAKER AND AUTHOR INDEX…………………………………………………………………………..254

KEYNOTE

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K1 Dendrite Morphogenesis: Sensory Stimulation Dependent Plasticity and Functional Implications Yuh-Nung Jan Howard Hughes Medical Institute, Department of Physiology, University of California, 1550 4th Street, San Francisco 94158, USA.

KEYNOTE

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K2 Physiological and Regenerative Homeostasis in Drosophila Nobert Perrimon Department of Genetics and Howard Hughes Medical Institute, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA

The central objective of developmental biology is to understand how organisms grow to adulthood. In the past 30 years, studies using genetically tractable model systems have led to a detailed understanding of the genetic mechanisms involved in the control of developmental events, as illustrated by our intimate knowledge of patterning and morphogenesis. The next big questions are how complex phenotypes arise in the context of the whole organism and how the programs regulating their development and function are influenced by genetic background and environment. For example, little is understood about how the simultaneous growth and differentiation of different tissues are coordinated and how the development of different cell types and tissues is integrated within an organ. Furthermore, many of the mechanisms by which growth factor–triggered signaling events intersect with cell metabolism, which is regulated by nutrients and hormones, remain to be identified.

We are using Drosophila as a model system to characterize the responses of specific cells to extracellular signals. Our previous work focused on the characterization of the signaling pathways that orchestrate embryonic patterning and morphogenesis. More recently, however, as we now have a rather good knowledge of these processes, we have become interested in studying (1) the mechanisms involved in the control of cell and tissue growth, and especially the roles of the insulin pathway in these processes, and (2) how signaling mechanisms are used in the context of homeostasis. Homeostasis, from the Greek words for same and steady, refers to ways in which the body acts to maintain a stable internal environment, despite perturbations. We are interested in two kinds of homeostatic regulation: (1) physiological homeostasis, which encompasses the mechanisms by which differentiated tissues, such as muscles, grow and maintain their mass during the aging process; and (2) tissue/regenerative homeostasis, which addresses the maintenance of tissue integrity by stem cell systems, as in the gut, which exhibits slow regeneration under normal conditions but accelerated regeneration when injured.

DGRC

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D1 Role, Capability, and Prospects of DGRC Masa-Toshi Yamamoto

Drosophila Genetic Resource Center, Kyoto Institute of Technology Saga-Ippongi-cho, Ukyo-ku, Kyoto 616-8354, JAPAN

Drosophila Genetic Resource Center (DGRC), Kyoto Institute of Technology was establised in 1999 as a Ministry ordinance research institute to maintain, develop and provide the living resources of Drosophila. The major aim is to collect and maintain useful and important genetic resources, and provide them to the researchers who are devoted to the basic biology, life sciences, bio-technology and related fields of biological sciences in order to promote the progress. The National Bio-Resource Project (NBRP) which started in 2002 supports our mission. DGRC plays the principal role as the core institute of NBRP "Drosophila". As sub-institutes, National Institute of Genetics (RNAi strains), Ehime University (wild-type strains of other species) and Kyorin University (mutants of other species) participate in NBRP "Drosophila". We at present maintain more than 36,000 strains and provide the resources to the fly community all over the world. We not only distribute the resources but also accept donations of useful fly stocks from researchers and develop new stocks.

PLENARY TALKS (DAY 1)

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P1 Regulation of Gastric Emptying in Drosophila Duc N.T. Nguyen1, Jing Ren2, Tian Xu1,2

1) Howard Hughes Medical Institute, Department of Genetics, Yale University School of Medicine, New Haven, CT 06536, USA 2) Institute of Developmental Biology and Molecular Medicine, School of Life Science, Fudan University, Shanghai 200433, China

Gastric emptying, a process for food transit from the stomach to the gut, is important for efficient digestion and absorption. To better understand this process, we used Drosophila to first examine the physiology associated with gastric emptying. During ingestion, food is directed by inward peristalsis of the crop duct and stored in the crop, which displays rhythmic rather than continuous contractions. Gastric emptying is accomplished by outward peristalsis coupled with constriction of the crop duct sphincter. In a genetic screen for gastric emptying defective mutants, we identified bajie, which catalyzes a product from nucleic acids digestion to actively trigger the physiological responses for inducing gastric emptying. The identification of the gastric emptying inducer molecule provides a tool for studying the interactions between gastrointestinal compartments and a potential interventional means for nutritional and metabolic disorders.


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P2 Development of Blood Cells in the Fly Julian A. Martinez-Agosto, Bama Charan Mondal, Tina Mukherjee, Lolitika Mandal, Cory J. Evans, Sergey A. Sinenko, Utpal Banerjee Department of Molecular, Cell, and Developmental Biology, Department of Biological Chemistry, Molecular Biology Institute, University of California Los Angeles, Los Angeles, CA 90095, USA

Hematopoiesis in Drosophila takes place in two phases, where the earliest phase initiates in the embryonic head mesoderm and the first blood cells get specified. Hematopoietic precursors are again specified later in embryogenesis, in a specialized organ called the lymph gland. The lymph gland comprises of two primary lobes and several secondary lobes. The primary lobe harbors immature hematopietic stem like progenitors in an inner core region, the Medullary zone (MZ) that are maintained by Wingless signaling within the MZ and by a Hedgehog derived signal emanating from the hematopoietic niche, termed the posterior signaling center (PSC). These pluripotent progenitors can differentiate into three types of terminally mature hemocytes largely akin to the vertebrate myeloid lineage, the plasmatocytes, crystal cells and lamellocytes that populate the outermost layer of the lymph gland, the Cortical zone (CZ). Our recent work has identified that the maintenance of stemness/quiescence of the progenitors in the MZ is achieved by a combined action of Hedgehog derived niche signal and a CZ derived backward signal generated by Pvf1/Pvr mediated signaling. This we term the Equilibrium signal. The ligand Pvf1 expressed in the PSC, transported via exocytosis to the CZ activates Pvr in the CZ where it is expressed at high levels. This Pvf1/Pvr interaction via activation of STAT generates adenosine derived growth factor-A (Adgf-A), a secreted factor from the differentiated hemocytes that functions to maintain low levels of extracellular adenosine in the MZ and inhibits progenitor proliferation and differentiation. Adenosine signaling via the adenosine receptor (AdoR) in the MZ activates cAMP-dependent PKA function that promotes proliferation and differentiation of the progenitors by opposing the Hh derived niche signal at the level of Ci activation within the MZ progenitors. Therefore this leads to a model where a niche derived forward signal and a CZ derived equilibrium signal represent two opposing forces in the maintenance of stem like progenitors by regulating the levels of extracellular adenosine via Adgf-A. A certain threshold level of adenosine is required for proliferation and Adgf-A lowers this threshold causing quiescence in the entire MZ population. In this context, the lymph gland can function as an effective stress sensor as adenosine levels are often upregulated in various stress conditions. Our work has demonstrated that mitochondrial stress, causes rapid proliferation and differentiation of the progenitors. This is because of an in increase in the levels of ROS. A certain low level of ROS in these progenitors is used as a developmental signal and as a stress sensor. Similarly, Sima/Hif-alpha mediated activation of full-length Notch signaling via the endosomal pathway is important for hemocyte survival both during development, and during hypoxic stress. Such dual role played by various signaling pathways in the Drosophila hematopoietic system provide an interesting paradigm for the formation and expansion of myeloid cell types from their stem-like precursors both during development and in stress.


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P3 Localization of Intermediate-Term Memories within the Drosophila Mushroom Body Ann-Shyn Chiang Brain Research Center, National Tsing Hua University, Hsinchu 30013, Taiwan

Pavlovian olfactory learning in Drosophila produces two genetically distinct forms of

intermediate-term memories: anesthesia-sensitive memory (ASM), which requires the amnesiac gene and anesthesia-resistant memory (ARM), which requires the radish gene. Here we show that two modulatory neurons, the anterior paired lateral APL neurons and the dorsal paired medial DPM neurons, form heterotypic gap junctions within the mushroom body (MB), a learning and memory center in the Drosophila brain. Using RNAi-mediated knockdowns of inx7 and inx6 in the APL and DPM neurons respectively, we found that flies showed normal olfactory associative learning and intact anesthesia-resistant memory (ARM) but failed to form anesthesia-sensitive memory (ASM). Our results reveal that the heterotypic gap junctions between the APL and DPM neurons are an essential part of the MB circuitry for ASM formation. On the other hand, we also found that ARM is specifically enhanced or inhibited in flies with elevated or reduced serotonin (5HT) levels, respectively. The requirement for 5HT was additive with the memory defect of the amnesiac mutation but was occluded by the radish mutation. This suggests that 5HT and RADISH protein act on the same pathway for ARM formation. Three supporting lines of evidence indicate that ARM formation requires 5HT released from only two DPM neurons onto the MBs, the olfactory learning and memory center in Drosophila. (i) DPM neurons were 5HT-antibody immunopositive. (ii) Temporal inhibition of 5HT synthesis or release from DPM neurons, but not other serotonergic neurons, impaired ARM formation. (iii) Knocking down the expression of d5HT1A serotonin receptors in a/b MB neurons, which are innervated by DPM neurons, inhibited ARM formation. Thus, in addition to the AMNESIAC peptide required for ASM formation, the two DPM neurons also release 5HT acting onto MB neurons for ARM formation. Together, we propose that direct APL-DPM electrical communications and their multiple products consolidate olfactory associative short-term memory into intermediate-term ASM and ARM in the Drosophila MB neurons.


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P4 Detection and Processing of Color Information in Drosophila

Claude Desplan Center for Developmental Genetics, Department of Biology, New York University, 100 Washington Square East, New York, NY 10003, USA

The Drosophila compound eye is made of 800 unit eyes (ommatidia) that each contains eight photoreceptors: Six (R1-R6) are involved in motion detection while two (R7 and R8) are required for color vision. Ommatidia belong to two subsets distributed stochastically throughout the retina in a 30:70 ratio: p-type ommatidia contain UV-sensitive Rh3 in photoreceptors R7 and Blue-Rh5 in R8 while y-type express another UV-Rh4 in R7 and green-Rh6 in R8. Comparison between the outputs of R7 and R8, and between p and y ommatidia allows flies to discriminate between colors.

I will describe the cascade of genes that specify the different subsets of photoreceptors through a series of fate restrictions and how this cascade is modified to define the various regions of the retina in Drosophila. In particular, spineless is expressed in a stochastic manner in R7 cells that express Rh4 (yR7). It allows the specification of the whole retina by specifying the y choice in R7 and allowing R7 to instruct R8 of its choice.

Processing of color information occurs in the medulla part of the optic lobe. It is formed by ~40,000 neurons surrounding a neuropil where photoreceptors and medulla neurons interconnect. Associated with each set of R7/R8 projections, there are ~800 ‘columns’, the functional units in the medulla. We are addressing how medulla cells process color information coming from R7 (sensitive to UV) and R8 (sensitive to blue or green) and send it to higher processing centers in the lobula complex and central brain to mediate color behavior.

We are silencing subsets of medulla neurons using specific Gal4 lines and testing the consequence for color discrimination. We have adapted to color vision the flight simulator originally designed by the Dickinson/Frye labs. In an operant paradigm, the fly is trained to associate color with a reward or punishment before being tested in the absence of the reward.


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P5 A Novel Gene of Circadian Rhythm in Drosophila Joonho Choe Department of Biological Sciences, KAIST, Daejeon 305-701, Korea.

In Drosophila, the Clock/cycle dimer activates the transcription of period, timeless, vrille, Par domain protein 1, and clockwork orange genes, which in turn feed back to inhibit CLK-activated transcription or regulate Clock transcription. These components are also modified posttranslationally to alter core clock timing. Regulation at multiple levels is thought to impose temporal delays in feedback allowing sustained oscillations on a circadian time scale. To discover novel clock components, we performed a genome-wide behavioural screen. Using the KAIST-GenExel Drosophila library, we identified ~4000 EP lines containing P elements bearing the Upstream Activating Sequence (UAS) for the yeast GAL4 transcription factor inserted near transcription start sites. One EP line identified by a long-period rhythm was the G10872 line that contains an insertion 893 bp upstream of the CG4857 transcription start site. Sequence analyses of the predicted amino acid sequence for CG4857 did not reveal any apparent functional domains or obvious vertebrate homologues but do reveal conservation with genes from different Drosophila species and other insects. We termed this novel gene twenty-four (tyf). TYF function is especially important in pacemaker neurons. Both tyf and per expression in the PDF+ LNv is sufficient to strongly rescue behavioural rhythms in tyf mutants. Moreover, tyf effects on PER are, by far, most evident in these pacemaker neurons. The brain pacemaker neurons are also among the few clock cells, which demonstrate robust free-running molecular rhythms in constant dark. Thus, TYF-mediated translational control may be a specialization of networked pacemakers in the brain crucial for sustaining free-running rhythmicity. Our data also strongly support the model that TYF acts at the level of translational control.


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P6 Neural Stem Cell Plasticity Changes with Time and is Associated with Chromatin Changes in vivo Angela Giangrande IGBMC, ILLKIRCH, FRANCE

One of the most challenging issues in neurobiology is to understand how is cell diversity generated in the nervous system. Neurons and glia, the major cell types in this tissue, share a common precursor population called neural stem cells or NSCs. Transcription factors play an important role in cell-fate decision and, more in general, in cell plasticity, however, the glial versus neuronal decision in the vertebrate Central Nervous System (CNS), involves a rather complex gene network, making it difficult to assess the role and mode of action of such determinants in vivo.

In the simple Drosophila model, we have identified the glial precursors and the glial-promoting activity, which provides us with a unique opportunity to dissect the regulatory pathways controlling gliogenesis. The glial cell deficient/glial cell missing (glide/gcm) and glide2 genes are necessary to induce the glial fate in vivo, with glide/gcm playing the major role in the process. We have recently found that Glide/Gcm fully and efficiently converts NSCs towards the glial fate when overexpressed. Glide/Ggcm acts in a dosage-dependent and autonomous manner by repressing the NSC program and inducing the glial one, both in terms of gene expression and proliferation profiles. Plasticity changes with time and does not require cell division, because Glide/Gcm is able to convert mutant NSCs that cannot divide but not wild-type NSCs at late developmental stages. Moreover, the postmitotic NSC offspring loses plasticity, as neurons cannot be converted into glia upon Glide/Gcm overexpression. Finally, the Glide/Gcm pathway controls a chromatin mark that characterizes glial cells. The endogenous as well as the ectopic glia produced upon fate conversion express low levels of histone 3 lysin 9 acetylation (H3K9ac) and low levels of the Drosophila CREB binding protein (dCBP) Histone Acetyl-Transferase (HAT), which targets the H3K9 residue. This mark is necessary for gliogenesis, as overexpressing dCBP specifically downregulates the expression of glial genes whereas a HAT inactive dCBP does not. Interestingly, glial cells of the vertebrate nervous system also need low histone acetylation levels.

Thus, a single transcription factor efficiently induces the stable and complete conversion of NSCs into glial cells in vivo. NSC plasticity relies on an internal clock that is independent of cell division and the induction of glial cells involves a specific chromatin mark that is conserved in evolution.


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P7 Interaction between Circadian Clocks and Metabolism Kanyan Xu, Justin Diangelo, Michael Hughes, John Hogenesch, Amita Sehgal

University of Pennsylvania School of Medicine, Philadelphia, PA

Circadian rhythms of behavior and physiology are regulated by a synchronized system of central and peripheral clocks. We showed previously that neuronal and metabolic clocks have opposing effects on metabolic activity. Clocks in neurons deplete nutrient stores while clock in metabolic tissues promote the storage of nutrients. To identify genes controlled by clocks in metabolic tissues, we conducted a microarray analysis for genes expressed cyclically in the fat body. We find that the clock in the Drosophila fat body drives rhythmic expression of genes involved in many different processes- lipid and carbohydrate metabolism, detoxification, the immune response and steroid hormone regulation. While the clock in the fat body controls expression of most of these genes, some continue to cycle when the fat body clock is disrupted indicating they are under the influence of exogenous factors. Food is clearly an important stimulus as feeding paradigms that limit food availability to a six-hour interval each day drive rhythmic expression of circadian genes in the fat body. Restricting food to a time of day when consumption is typically low confers an altered phase of expression on the fat body clock. However, the brain clock is unaffected by food, and thus food consumption at the wrong time of day desynchronizes internal clocks.We have examined the effects of such desynchrony on fitness of the animal.


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P8 Tissue Damage and Intestinal Stem Cell Response in the Drosophila Midgut Tony Ip1, Alla Amcheslavsky1, Madhurima Chatterjee1, Eun-Young Yun2, Fang-Fang Ren3, Jin Jiang3 1) Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA 2) Department of Agricultural Biology, National Academy of Agricultural Science, Rural Development Administration, Suwon 441-100, Korea 3) Department of Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Stem cell division is essential for tissue integrity during growth, aging, and pathogenic assaults. Intestinal stem cells (ISCs) in adult Drosophila midgut have recently been identified and shown to replenish the various cell types within the tissue. By feeding pathogenic bacteria and tissue damaging chemicals to flies, we show that Drosophila ISCs can increase the rate of division in response to tissue damage. Intrinsic factors such as the Tuberous Sclerosis Complex and the Myc oncoprotein are used to regulate ISC growth and division, in a way different from the developing imaginal discs. We also demonstrate that loss of Hpo signaling in either midgut precursor cells or epithelial cells stimulates ISC proliferation, through increased production of Upds and EGFR ligands. The Hpo pathway mediator Yorkie (Yki) is also required in precursor cells for injury-induced ISC proliferation in response to tissue-damaging agent DSS. Meanwhile, the insulin receptor signaling pathway is required within ISC to regulate division, in response to endocrines from other organs such as the brain. Adult gastrointestinal tract encounters numerous stimulations, and impaired tissue regeneration may lead to inflammatory diseases and cancer. The uncovering of genetic requirements for intestinal stem cell biology should provide important insights into gastrointestinal tissue regeneration and diseases.


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P9 Nuage: A Potential Site for Processing of Germline piRNA Toshie Kai Temasek Life Sciences Laboratory and Department of Biological Sciences, 1 Research Link, The National University of Singapore, Singapore 117604

The most important cell lineage for the survival of sexually reproducing metazoans is the germline, due to its unique role in gamete production and species continuity. The fidelity of genomic information in the germline has to be tightly regulated for accurate transmission to the next generation. In many animal germline cells, Piwi-interacting RNAs (piRNAs) are reported to silence the expression of one class of mobile genetic elements, retroelements, whose transposition may afflict the genome with mutational burden. We and others have proposed that nuage, a well-conserved perinuclear organelle found in germline cells, may be the sites where germline piRNAs are amplified in a feed-forward loop, called ping-pong cycle. I will introduce our recent findings supporting this model. Proteins required for biogenesis of piRNAs, including the Piwi-subfamily proteins, Aubergine and Argonoute3, localize to perinuclear nuage region as a large complex to produce germline piRNAs. We have reported some tudor domain proteins which contribute this process. They exhibit hierarchal genetic interaction which may reflect the sequential process of ping-pong cycle.

Here I describe a conserved component of germline piRNA pathway, kumo. kumo mutants exhibits some common phenotypes shared with other nuage/piRNA pathway mutants, including insufficient germline piRNA production, displacement of other nuage components from perinuclear region, and the polarity defect during oogenesis. Kumo localizes not only to perinuclear nuage, but also to the nucleus. kumo mutant germarium shows higher expression of HP1, which were more frequently juxtaposed with centromeric/pericentromeric harbouring piRNA loci. Consistently, the occupancy of HP1 to the germline piRNA loci but not to somatic piRNA is increased in kumo mutant ovary. We also observed Kumo physically interacts with HP1. Our results suggest that in kumo mutant ovary, germline piRNA loci are transcriptionally repressed and therefore the piRNA precursors are reduced. We propose that kumo functions to sequester HP1 from the germline piRNA loci to facilitate transcription of the piRNA precursors.


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P10 Linking Transcription Factor Occupancy and Chromatin State to Gene Expression during Embryonic Development Eileen E. Furlong EMBL, Heidelberg, Germany One of the central challenges in biology is to understand how the genome is utilized to give rise to diverse cell types. Embryonic development occurs through the progressive restriction of cell fates, from pluripotent fields of cells to complex organs and tissues. This process requires a directed progression through interlinked regulatory states, each defined by the total set of active transcription factors. At each stage of development, the combined inputs of signalling and transcriptional networks regulate the expression of specific sets of genes that drive the transition to the next, often more specialized, state. Understanding how the underlying cis-regulatory networks produce spatial and temporal gene expression is therefore an essential step towards deciphering metazoan development and ultimately evolutionary change. While genetic studies have identified a growing number of essential transcription factors (TFs) required for cell fate specification, little is known about the mechanisms by which these regulators function. Conversely, recent global approaches assaying TF binding enable the location and even combinatorial occupancy of CRMs to be experimentally measured as specific stages of development, at a genome-wide scale. A current major challenge is to interpret these TF binding data in terms of their resulting spatio-temporal cis-regulatory activity. Our work attempts to bridge this gap, by integrating genetic, genomic and computational approaches to understand how transcriptional networks drive cell fate selection, using mesoderm specification in Drosophila as a well-defined model system. I will present our current status on an on-going effort to build a predictive cis-regulatory network defining multiple stages of development.


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P11 Dpp, Gradients and Growth Konrad Basler Institute of Molecular Life Sciences, Winterthurerstr. 190, 8057 Zurich, Switzerland.

Decapentaplegic (Dpp) acts as a secreted morphogen in the Drosophila wing disc, and spreads through the target tissue in order to form a long range concentration gradient. Despite extensive studies, the mechanism by which the Dpp gradient is formed remains controversial. Two opposing mechanisms have been proposed: receptor-mediated transcytosis (RMT) and restricted extracellular diffusion (RED). In these scenarios the receptor for Dpp plays different roles. In the RMT model it is essential for endocytosis, re-secretion and thus transport of Dpp, whereas in the RED model it merely modulates Dpp distribution by binding it at the cell surface for internalization and subsequent degradation. We analyzed the effect of receptor mutant clones on the Dpp profile in quantitative mathematical models representing transport by either RMT or RED. Using new genetic tools, we experimentally monitored the actual Dpp gradient in wing discs containing receptor gain-of-function and loss-of-function clones. Gain-of-function clones reveal that Dpp binds in vivo strongly to the type I receptor Thick veins, but not to the type II receptor Punt. Results with the loss-of-function clones then refute the RMT model for Dpp gradient formation, while supporting the RED model in which the majority of Dpp is not bound to Thick veins. Our results show that receptor-mediated transcytosis cannot account for Dpp gradient formation, and support restricted extracellular diffusion as the main mechanism for Dpp dispersal. The properties of this mechanism, in which only a minority of Dpp is receptor-bound, may facilitate long-range distribution.


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P12 Linking Global Tissue Asymmetry to Cell Polarity Toshiyuki Harumoto1,2, Tetsuya J. Kobayashi3,4, Yuko Shimada1, Hiroki R. Ueda5, Tadashi Uemura1,2

1) Graduate School of Biostudies, Kyoto University 2) CREST, JST 3) Institute of Industrial Science, the University of Tokyo 4) PRESTO, JST 5) Center for Developmental Biology, RIKEN Cells sense global axes of the tissue to which they belong and manifest polarity for specialized functions. One such example is planar cell polarity (PCP) and underlying mechanisms of PCP have been best studied in the Drosophila wing, where epidermal cells are oriented along the proximal-distal (P-D) axis. The pertinent molecular players have been classified into at least the 2 following categories: The first group includes atypical cadherins Dachsous (Ds) and Fat (Ft) that are thought to contribute to the tissue patterning information across the axis. Second, members of the “core group,” including Frizzled (Fz) and Flamingo (Fmi), assemble into asymmetric complexes that straddle the proximodistal junctions between adjacent cells. Unsolved questions include how the above 2 categories of regulators are functionally related to each other and why Fz is relocalized at distal cell borders in the first place. We previously proposed that cellular mechanisms underlying this relocalization include polarized transport of Fz-containing vesicles along P-D-oriented non-centrosomal microtubules (Shimada et al., 2006). We have been analyzing dynamics of the microtubules (MTs) and movements of the vesicles to elucidate 2 critical questions: First, how do the MTs become aligned along the P-D axis? Second, why are the Fz vesicles transported distally? Our quantitative in vivo imaging has shown that Ds and Ft control alignment and asymmetry of the MT growth, and it has also revealed statistical properties of the vesicle movements, which will give us insight into the asymmetric relocalization of the core group (Harumoto et al., 2010 and in prep.) .


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P13 Gene Expression and Protein Evolution in Drosophila Hiroshi Akashi Division of Evolutionary Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540 Japan

Rates of protein evolution are strongly associated with gene expression patterns in yeast, Drosophila and mammals. Broadly/highly expressed genes evolve at slower rates than tissue-specific/lowly expressed genes across taxa. Protein-specific functional constraints were thought to be the main factor underlying rates of protein evolution, but these patterns suggest that global forces may be important for understanding proteome evolution. In particular, biosynthetic constraints (selection for accurate and efficient synthesis and folding) could have a strong impact on the size, composition, and evolutionary rates of proteins. We attempt to distinguish among potential causes of relationships between expression patterns and protein evolution using available transcript abundance data from 14 tissues/developmental stages in Drosophila melanogaster and genome sequences of closely related species. In these data, tissue-specific proteins evolve roughly 3-fold faster than ubiquitously expressed proteins. However, expression intensity and breadth are highly correlated. When we control for expression breadth, we find that evolutionary rates decline for abundant proteins as predicted under biosynthetic constraints. Interestingly, among tissue-specific genes, Drosophila and mammals show similar patterns; brain and nervous tissue genes evolve slowly and male reproductive genes evolve at high rates.


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P14 Phenotypes of New Genes: Evolution of Developmental Genetic Programs Manyuan Long Department of Ecology and Evolution, The University of Chicago, 1101 E 57th Street, Chicago, IL 60637, USA

New genes have been observed to frequently originate through various mutational mechanisms in the Drosophila genomes (Long et al, 2003, Nature Rev Genet; Long, 2007, Nature; Yang et al, 2008, PLoS Genet). Adaptive evolution was observed to be a major evolutionary force acting on the early origination of a new gene, suggesting significant phenotypic effects new genes contributed to the evolution of Drosophila species (Kaessmann et al, 2009, Nature Rev Genet; Emerson et al, 2008, Science). Furthermore, most new genes generated from retroposition were identified to have testis-expressed functions, implicating their phenotypic effects on the male fertility (e.g. Vibranovski et al, 2009a, Genome Res; 2009b, PLoS Genet). Computational and experimental analyses of a number of Drosophila genomes identified ~1000 Drosophila-specific new genes which originated in recent 45 million years (Zhang et al, 2010, Genome Res). To detect the fitness phenotypes of these new genes, we have conduced genetic analyses ranging from knockout, knockdown to mutational analyses (Dai et al, 2008, Proc Natl Acad Sci USA; Chen et al, 2010, Science; Chen, Yang and Long, unpublished data). Unexpectedly, we found that a high proportion of the new genes were lethal and/or male sterile when silenced, countering a conventional conclusion that only conserved ancient genes were essential. Further inspection of these essential new genes indicated that they if silenced terminated development stages from embryotic stage to eclosion, mostly in pupal stages or control adult organ development, e.g. the development of wings, notum and testis. Because the new genes are species- or linage-specific, thus, the identified developmental effects reveal the existence of specific-specific or lineage-specific developmental genetic programs of which these new genes are components. These findings from the examination of young protein-coding genes showed that the developmental genetic programs in Drosophila were rapidly evolving, contrary to the widely believed conservation of development with the conserved developmental genes and the non-variable tool-kits for evolution of development.

PLATFORM TALKS (DAY 1)

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T1 Regulations of Drosophila Larval Brain Neural Stem Cell Self-Renewal

Kai Chen Chang1,2, Gisela Garcia-Alvarez2, Gregory Somers3, Rita Sousa-Nunes4, Fabrizio Rossi5, Cayetano Gonzalez5, William Chia1, Hongyan Wang2 1) Temasek life Sciences Laboratory, Singapore 2) Duke-NUS Graduate Medical School Singapore, Singapore 3) La Trobe University, Australia 4) National Institute for Medical Research, UK 5) IRB-Barcelona, Spain

How a cell decides to self-renew or differentiate is a critical issue in stem cell and cancer biology. Atypical protein kinase (aPKC) promotes self-renewal of Drosophila larval brain neural stem cells, neuroblasts. However, it is unclear how aPKC cortical polarity and protein levels are regulated. Here we have identified a novel zinc-finger protein, Zif, which regulates the expression and asymmetric localization of aPKC. aPKC displays ectopic cortical localization with upregulated protein levels in dividing zif mutant neuroblasts, leading to neuroblast overproliferation. We show that Zif is a transcription factor that directly represses aPKC transcription. We further show that Zif is directly phosphorylated by aPKC both in vitro and in vivo. Phosphorylation of Zif by aPKC excludes it from the nucleus, leading to Zif inactivation in neuroblasts. Thus, reciprocal repression between Zif and aPKC act as a critical regulatory mechanism for establishing cell polarity and controlling neuroblast self-renewal.


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T2 Non-Autonomous and Context-Dependent Control of Apoptosis by Deregulated Hedgehog Signaling Andreas Bergmann, Tian Ding, Yun Fan, Audrey E. Christiansen The University of Texas M. D. Anderson Cancer Center, Department of Biochemistry and Molecular Biology, Houston, TX 77030, USA

Hedgehog (Hh) signaling is an important signaling pathway for development and homeostasis. Deregulated, i.e. increased Hh signaling can give rise to disease including cancer. In Drosophila and mammals, deregulated Hh signaling causes excessive cell proliferation leading to overgrowth and tumor phenotypes. However, here we show in eye imaginal discs that deregulated Hh signaling caused by loss of the negative regulators cos2, ptc and pka also promotes cell survival by increasing apoptosis resistance. Surprisingly, it is not the cells with deregulated Hh activity that have increased apoptosis resistance. Instead, these mutant cells promote apoptosis resistance of neighboring wild-type cells. This non-autonomous effect is mediated through Hh-induced accumulation of the protein levels of Drosophila Inhibitor of Apoptosis-1 (DIAP-1) in neighboring wild-type cells, conferring apoptosis resistance. This activity is context-dependent and occurs only in and anterior to the morphogenetic furrow in the eye disc. The posterior part of the eye disc is inert to deregulated Hh activity with respect to DIAP-1. We also observe non-autonomous and context-dependent up-regulation of DIAP-1 by deregulated Hh activity in wing imaginal discs. In summary, we demonstrate that deregulated Hh signaling not only promotes proliferation, but also cell survival of adjacent tissue. Potentially, in humans a similar non-autonomous effect on apoptosis by deregulated Hh signaling may be needed to generate a supportive micro-environment for tumor growth.


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T3 Genetic Dissection of Tumor Growth and Progression through Cell-Cell Communications Tatsushi Igaki, Mai Nakamura, Yoshitaka Sato, Aya Betsumiya, Shizue Ohsawa Department of Cell Biology, G-COE, Kobe University Graduate School of Medicine, Japan

Tumor progression is regulated by both intra- and inter-cellular cooperations of oncogenic alterations. However, the underlying mechanism of how each oncogenic mutation cooperates with other mutations through cell-cell communications to progress toward malignancy remains elusive. To systematically analyze such mechanisms, we performed a genetic screen in Drosophila for identifying genes that drive tumor growth and progression through cell-cell interactions. Clones of cells bearing oncogenic alteration of Ras (RasV12) leads to the development of benign tumors in imaginal epithelia. We introduced additional mutations in RasV12-tumors and screened for mutations that cause non-cell autonomous overgrowth of surrounding wild-type tissue (which we call nag mutations). We identified a series of nag mutations that cause not only overgrowth in their neighboring wild-type tissue but also malignancy in their neighboring RasV12-induced tumors to exhibit metastatic behaviors. The molecular mechanisms by which nag mutations trigger non-cell autonomous overgrowth and the inter-clonal oncogenic cooperation will be presented.


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T4 Spatial Fluctuation of Notch Activity Coordinates Maintenance and Differentiation of Optic Lobe Neuroepithelia Mo Weng1, Jill M. Haenfler2, Cheng-Yu Lee1, 2, 3, 4 1) Department of Cell and Developmental Biology, 2) Program in Cellular and Molecular Biology, 3) Division of Molecular Medicine and Genetics, Department of Internal Medicine, 4) Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109 USA

Regulation of the switch from stem cell expansion to stem cell differentiation coordinates an increase in cell number and tissue morphogenesis during normal development. Differentiation of neuroepithelial cells into neuroblasts in the Drosophila larval optic lobe provides an in vivo model system for investigating the regulation of the switch from symmetric to asymmetric stem cell divisions. Neuroepithelia lacking Notch function or defective in Notch activation prematurely differentiate into neuroblasts while constitutive activation of Notch signaling prevents neuroepithelia differentiation. Notch maintains the identity of undifferentiated neuroepithelial cells through anterior open (aop). While aop is necessary and sufficient for the maintenance of neuroepithelia, over-expression of aop suppresses precocious differentiation of Notch mutant neuroepithelia. In differentiating neuroepithelial cells, up-regulation of Notch coincides with transient cell cycle arrest but precedes the onset of pointedP1 (pntP1) expression, and functions to prevent precocious transition into immature neuroblasts by raising the threshold of the response to PntP1. Down-regulation of Notch through cis-inhibition by Delta and a high level of PntP1 trigger the differentiating neuroepithelial cells immediately preceding immature neuroblasts to undergo the transition. Thus, a dynamic fluctuation in Notch signaling ensures precise spatial-control of expansionary symmetric and differentiative asymmetric stem cell divisions during optic lobe neurogenesis.


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T5 Reconstruction of Neural Circuits in the Drosophila Brain through Lineage Analysis Hung-Hsiang Yu1,2, Tzumin Lee2 1) Institute of Cellular and Organismic Biology, Academia Sinica, Taiwan 2) Janelia Farm Research Campus, Howard Hughes Medical Institute, USA

A mature brain is composed by complex neural circuits, and reconstructing neural circuits with all involved neurons is a challenging task. In the Drosophila central brain, roughly 20,000 neurons are derived from about 120 neural precursors (defined as 120 neural lineages), and the types of neurons produced from a common precursor are in an invariant order. To comprehensively identify neurons, we sought to identify all neural lineages for picturing overall neuronal connections and break down each neural lineage into individual neurons. We have, so far, identified 103 neural lineages, and observed a stereotypy of each morphological pattern of neural lineages. However, there are several non-canonical patterns within the stereotyped neural lineages, indicating a non-classical neurogenesis pattern. We then started to reconstruct the olfactory neural circuit by identifying all neurons innervating the antennal lobe (AL), the primary olfactory center in the Drosophila brain. Neurons from five lineages compose the AL, including one local interneuron (LN) lineage, three projection neuron (PN) lineages (adPN, vPN and lvPN) and a lineage with both PNs and LNs (lALN). Most adPNs and PNs of the lALN lineage have uniglomerular dendritic projections in the AL, while many of vPNs and lvPNs have multiglomerular dendritic projections in the AL. Interestingly, different types of vPNs and lvPNs posses distinct multiglomerular dendritic patterns, suggesting that distinct olfactory information could be pre-integrated within vPNs and lvPNs in the AL. Applying our strategy on reconstructing the olfactory neural circuit to other neural circuits should promise the future construction of a complete cellular and developmental brain map.


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T6 Cell-Surface Molecules Specify Synaptic-Layer Targeting in the Drosophila Visual System Satoko Hakeda-Suzuki1*, Sandra Berger-Mueller1*, Klaudiusz Mann1, Tadao Usui2, Shin-ya Horiuchi2, Tadashi Uemura2,3, Takashi Suzuki1

1) Max-Planck Institute of Neurobiology, Martinsried, Germany 2) Graduate School of Biostudies, Kyoto University, Japan 3) Japan Science and Technology Agency, CREST, Japan

Information-processing in the brain is critically dependent on precise synaptic communication between specific neurons. Synaptic connections are often organized in layered structures, which contain synapses between neurons that have similar functions. For instance, in the Drosophila visual system, two types of photoreceptor cells (R7 and R8) which detect different wavelengths form synapses in distinct layers in the medulla (M6 and M3, respectively). Although several axon guidance and cell adhesion molecules have been shown to be required for the layer targeting of photoreceptor axons, the underlying molecular mechanisms are still unclear.

We found that Golden Goal (Gogo) and Flamingo (Fmi), two cell-surface proteins involved in photoreceptor targeting, functionally interact in R8s. We show that Gogo localization at the photoreceptor growth cone is largely dependent on Fmi. Our genetic studies indicated that Gogo promotes R8 adhesion to the temporary layer M1, whereas Gogo and Fmi collaborate to mediate axon targeting to the final layer M3. Moreover, Structure-function analysis suggests that Gogo and Fmi interact with intracellular components through the Gogo cytoplasmic domain and that the Gogo signaling is regulated by the phosphorylation of a conserved tyrosine in the cytoplasmic domain. Fmi is also required in a subset of target cells for R8 axon targeting, suggesting Fmi homophilic interaction between photoreceptor axons and target cells. Altogether, these results indicate that Gogo acts as a functional partner of Fmi for R8 axon targeting and that the dynamic regulation of Gogo-Fmi collaboration specifies the synaptic-layer selection of photoreceptors. We propose that synaptic-layer specificity is achieved by combinatorial codes of cell-surface molecules.


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T7 neuronal synaptobrevin Functions in Neuronal Maintenance Independent of Its Role in Neurotransmitter Release Adam Haberman1, Daniel Epstein1, W. Ryan Williamson1, Ian A. Meinertzhagen2, P. Robin Hiesinger1 1) UT Southwestern Medical Center, Dallas, USA 2) Dalhousie University, Halifax, Canada

The neuron-specific SNARE neuronal synaptobrevin (n-syb) has a well-characterized role in synaptic vesicle fusion. Here we report the discovery that loss of n-syb leads to adult-onset degeneration because of an intracellular degradation defect that is independent of n-syb’s role in synaptic transmission. Adult n-syb mutant photoreceptor neurons degenerate in a light-dependent manner and exhibit increased sensitivity to human Alzheimer-related disease proteins tau and Abeta. Electron microscopy of n-syb photoreceptor synapses reveals large accumulations of undegraded endocytic and autophagic organelles. In order to map the intracellular membrane fusion reaction blocked by loss of n-syb, we assayed the trafficking of the degradative enzyme Cathepsin L, which is activated by strongly acidic pH in degradative compartments. Loss of n-syb leads to dramatic accumulation of inactive Cathepsin, indicating a failure of ‘degradation machinery’ vesicles to fuse with endosomes and autophagosomes. This phenotype is very similar to loss of the vesicle ATPase protein V100, another neuron-specific protein required for neurotransmission and neuronal maintenance. We have recently shown that V100 functions in both endosomal fusion as well as acidification. Strikingly, we find that over-expressing either the wild type or an acidification-defective variant of V100 partially rescues n-syb-dependent degeneration, but not neurotransmission. Our analyses reveal that n-Syb and V100 function in the same novel neuron-specific degradation pathway. We propose that this pathway increases neuronal degradative capacity and is required for neuronal maintenance.


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T8 Decreased Ribose-5-Phosphate Isomerase Expression in Neurons Enhances Oxidative Stress Resistance, Increases Lifespan, and Attenuates Polyglutamine Toxicity in Drosophila Ching-Tzu Wang1, Yi-Yun Wang1, Ming-Hao Huang1, Tzu-Kang Sang1, Yi-Chun Chen1, Si-Chih Cho1, Chiou-Hwa Yuh2, Chao-Yung Wang3, Theodore J. Brummel4, Horng-Dar Wang1 1) Institute of Biotechnology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, R.O.C. 2) Division of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan, R.O.C. 3) Second Section of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital at Linkou, Chang Gung University College of Medicine, Taoyuan, Taiwan, R.O.C. 4) Department of Biology, Long Island University, Brookville, NY 11548, U.S.A.

Aging and age-related diseases can be viewed as the result of the lifelong accumulation of stress insults. The identification of mutant strains and genes which are responsive to stress and can alter longevity profiles provides new therapeutic targets for age-related diseases. Here we reported that a Drosophila strain EP2456, with reduced expression of ribose-5-phosphate isomerase (rpi), exhibits increased resistance to oxidative stress and enhanced lifespan. In addition, the strain also displays higher levels of NADPH. The knockdown of rpi in neurons by double-stranded RNA interference recapitulated the lifespan extension and oxidative stress resistance in Drosophila. This manipulation was also found to ameliorate the effects of genetic manipulations aimed at creating a model for studying Huntington’s disease by overexpression of polyglutamine in the eye, suggesting that modulating rpi levels could serve as a treatment for normal aging as well as for polyglutamine neurotoxicity.


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T9 Drosophila Short Neuropeptide F (sNPF) Signaling Regulates Lifespan, Body Growth, and Carbohydrate Metabolism through the Insulin Signaling Kweon Yu Aging Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon, Korea

Drosophila short neuropeptide F (sNPF) is processed and expressed in the nervous systems of various developmental stages. Inhibition of sNPF in the nervous system shows increased lifespan, small body size, and increased blood carbohydrate level compared to the Wild-type control flies. These are typical phenotypes of mutants in the insulin signaling, which suggest that sNPF may regulate insulin signaling. Therefore, we investigated whether sNPF regulates insulin signaling in the insulin producing cells (IPCs) and insulin target tissues.

After immunostaining with the anti-serum against sNPF receptor (sNPFR1) in the fly brain, we found that sNPFR1 is localized in the plasma membrane of IPCs. When sNPF was inhibited in the neurons, the expression level of the Drosophila insulin-like peptides (Dilps) gene was decreased compared to that of the Wild-type control. Likewise, sNPFR1 suppression in IPCs reduced Dilps expression. These regulations of Dilp expression by sNPF and sNPFR1 in IPCs are mediated by ERK. In the fat body, which is the homologous tissue of mammalian liver and adipocytes, sNPF mutants inhibited the activation of Akt, induced the nuclear localization of FOXO, and increased the expression of the translational inhibitor 4E-BP. It indicates that sNPF mutants inhibited the insulin receptor signaling in the fat body. These IPCs and fat body data demonstrate that sNPF and sNPFR1 regulate lifespan, body size, and glucose metabolism through the insulin signaling.


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T10 Reactive Oxygen Species Induced by Ecc15 Oral Infection Triggers Systemic Innate Immune Response via IMD Signaling Pathway in Drosophila Shih-Cheng Wu1,3, Shih-Sheng Jiang2, Rong-Long Pan3, Jyh-Lyh Juang1 1) Division of Molecular and Genomic Medicine, NHRI, Taiwan 2) National Institute of Cancer Research, NHRI, Taiwan 3) Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Taiwan

How natural infection by oral route induces innate immune response in Drosophila is largely unknown. Here we show that the oral infection of larvae with Ecc15 induces systemic expressions of antimicrobial peptide (AMP) genes and this immune response is suppressed by a ROS scavenger or by depletion of Duox. The overexpression of duox in larval intestinal epithelial cells or H2O2 or paraquat feeding to larvae also induce AMP genes expression, suggesting that ROS play a critical role in regulating natural infection-induced innate immune response. Our biochemistry and genetic data show that Relish is the key transcription factor initiating systemic AMP response to ROS. It is thus of interest to investigate how Drosomycin (Drs) is regulated via Imd, but not Dif/Dorsal-dependent Toll signaling pathway. We found that the phosphorylation level of JNK is suppressed by Ecc15 in larval fat body with natural infection. Since AP1 is a key downstream effector of JNK signaling, we overexpressed AP1 in fat body and found that the Ecc15-induced Drs is diminished, suggesting that JNK activity which suppresses Drs expression is negatively regulated during Ecc15 natural infection. The same finding is also observed in S2 cells in vitro assays. Together, we propose a model in which the excess ROS level induced by intestinal Ecc15 triggers Imd signaling pathway in fat body to activate systemic innate immune response; a phenomenon that is commonly called `cytokine storm' in mammalian system.


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T11 The Cytoophidium: Another Organelle for Energy Metabolism? Ghows Azzam, Gabriel Aughey, Kangni Chen, Zillah Deussen, Ji-Long Liu* Medical Research Council Functional Genomics Unit, University of Oxford

Compartmentation is essential for the localization of biological processes within a cell. We recently identified a novel intracellular filamentary structure containing CTP synthase, termed the cytoophidium, in Drosophila cells. More recently, CTP synthase-containing filaments have been reported in bacteria and yeast, suggesting that the cytoophidium is well-conserved from prokaryotes to eukaryotes. Because of the central role of CTP in energy metabolism, the biosynthesis of nucleic acids, phospholipids, and sialic acid, CTP synthase has been an attractive target for the development of anti-tumour, antiviral and antiprotozoal agents. Discoveries from our lab, and from others’, on the intracellular compartmentation of CTP synthase in prokaryotes and eukaryotes are timely and promise much not only for a better understanding of fundamental cell biology but also for the study of associated diseases. An emerging question is whether the cytoophidium is similar to the mitochondrion and serves as one of the special subcellular domains for energy metabolism.


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T12 Repression of Early Zygotic Transcription in Drosophila Primordial Germ Cells Akira Nakamura, Kazuki Matsuda, Kazuko Hanyu-Nakamura Laboratory for Germline Development, RIKEN CDB, Kobe, Japan

In many animal species, germ cell formation depends on maternal factors in the germ plasm. One of key activities in the germ plasm is to repress mRNA transcription in newly formed germ cells, a mechanism that is though to ensure germ cell specification by blocking somatic developmental programs. In Drosophila embryos, polar granule component (pgc), encodes a small 71-aa protein that is essential for repressing zygotic mRNA transcription in newly formed germ (pole) cells. Pole cells lacking Pgc die through apoptosis during embryonic stages 10-14. We found that pole cell death in pgc– embryos was, at least in part, due to precocious loss of Nanos (Nos) protein, an evolutionally conserved factor essential for germ cell maintenance. We also found that the loss of Nos protein in pgc– embryos is a consequence of the precocious degradation of maternally supplied nos mRNA in stage-5 pole cells.

It has long been known that maternal mRNAs including nos have to be degraded in somatic cells during the maternal-to-zygotic transition (MZT). MicroRNA (miRNA) is a zygotically expressed factor that promotes maternal RNA degradation in the soma during the MZT. We found that many miRNAs were ectopically expressed in pgc– pole cells during stages 4-5. Luciferase assays using S2 cells suggested that these miRNAs repressed the expression of reporter genes possessing the nos 3’ UTR. Given that the germ plasm contains maternal factors sufficient for germ cell formation, Pgc-mediated transcriptional repression is essential to protect maternal messages in the germ plasm from their precocious degradation during the MZT.


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T13 Atrophin, a Transcriptional Cofactor, Positively Influences the Activity of Notch in Multiple Developmental Pathways in Drosophila Hongxing Gui1, Hui Wang2, Bryan W. Heck1, Xin Tong1, Michael P. Matise2, Chih-Cheng Tsai1 1) Department of Physiology and Biophysics 2) Department of Cell Biology and Neuroscience, UMDNJ-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA

The Atrophin-family proteins, including Drosophila Atrophin (Atro) and its vertebrate homolog, RE-dipeptide repeat protein (RERE), have been characterized as transcriptional cofactors involved in diverse developmental processes, including embryonic segmentation, somite bilateral symmetry, neurogenesis, and planar polarity. Mutations of human Rere and the related gene atrophin-1 have been implicated in diseases, such as neuroblastoma and the neurodegenerative DRPLA, respectively. Thus far, information about which specific transcriptional regulatory pathways mediate the Atrophin proteins’ biological and pathological properties remains limited. We have shown that Atro is involved in retinal development in Drosophila: reduced expression of Atro causes small eyes. Building on this initial observation, we investigated how Atro controls retinal growth and found that it does so by participating in the Notch pathway. Not only does Atro interact with Notch genetically to promote retinal cell proliferation, Atro also assists Notch to induce the expression of its target genes, such as E(spl)mβ and four-jointed, in the eye disc. We further demonstrate a similar role for Atro in regulating Notch activity in additional developmental pathways in Drosophila, as well as in the developing chick neural tube. We conclude that Atrophin proteins represent a previously unidentified class of positive regulators of the Notch pathway. In my presentation, I will discuss the mechanisms by which Atrophin proteins influence the Notch pathway in both Drosophila and vertebrate tissues.


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T14 A Role for Vasa in Regulating Mitotic Chromosome Condensation in Drosophila Jun Wei Pek, Toshie Kai

Department of Biological Sciences and Temasek Life Sciences Laboratory, 1 Research Link, The National University of Singapore, Singapore 117604

Vasa (Vas) is a conserved DEAD-box RNA helicase expressed in germline cells that localizes to a characteristic perinuclear structure called nuage. Previous studies have shown that Vas has diverse functions, with roles in regulating mRNA translation, germline differentiation, poleplasm assembly and piwi-interacting RNA (piRNA)-mediated transposon silencing. Although vas has also been implicated in the regulation of germline proliferation in Drosophila and mice, little is known about whether Vas plays a role during the mitotic cell cycle. Here, we report a translation-independent function of vas in regulating mitotic chromosome condensation in the Drosophila germline. During mitosis, Vas facilitates robust chromosomal localization of the condensin I components, Barren (Barr) and CAP-D2. Vas specifically associates with Barr and CAP-D2, but not with CAP-D3 (a condensin II component). The mitotic function of Vas is mediated by the formation of peri-chromosomal Vas bodies during mitosis, which requires the piRNA pathway components, aubergine and spindle-E. Our results suggest that Vas functions during mitosis and may link the piRNA pathway to mitotic chromosome condensation in Drosophila.


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T15 Functions of a Helix-Loop-Helix Transcription Factor, Extramacrochaetae, in Development of Left-Right Asymmetry in the Drosophila Embryonic Hindgut Ryo Hatori, Kiichiro Taniguchi, Naotaka Nakazawa, Reo Maeda, Kenji Matsuno Department of Biological Science and Technology, Tokyo University of Science

Left-right asymmetrical morphogenesis is a critical aspect of many animal organogenesis. To understand the mechanisms involved in left-right asymmetrical morphogenesis, we are using the embryonic hindgut of Drosophila. In our genome wide genetic screen, we previously found that DE-Cadherin (DE-Cad) and Myosin31DF (Myo31DF) mutants show randomized and reverse laterality of the hindgut, respectively. More recently, we identified extramacrochaetae (emc), in the same screen. emc encodes a negative helix-loop-helix transcription factor involved in a variety of biological processes such as cell-fate determination. Our epistatic analysis implies that Emc functions upstream of Myo31DF and DE-Cadherin. In wildtype embryos, DE-Cadherin tended to be localized in a planar left-right asymmetrical manner at cell boundaries. However, in emc mutants, there was no left-right bias in the planar localization of DE-Cadherin.

Detailed observation revealed that the shape of epithelial cells of the hindgut were chiral with respect to the anterior-posterior axis before the left-right asymmetrical morphogenesis. This new type of cell chirality was named, planar cell chirality (PCC). PCC tended to be slanted to the left in wild-type flies, but emc mutants showed no left-right bias in PCC. Moreover, our in silico simulation suggested that PCC is sufficient for the left-right asymmetric morphogenesis of the hindgut. In summary, Emc controls the left-right asymmetrical localization of DE-Cad, which in turn introduces PCC that drives the left-right asymmetrical morphogenesis of the hindgut.


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T16 Directional Transport of Dpp/BMP Draws Diversified Wing Vein Patterns in Insects Osamu Shimmi1, Shinya Matsuda1, Naotoshi Yoshiyama2, 3, Jaana Künnapuu1, Stuart J. Newfeld4, Masatsugu Hatakeyama2

1) Institute of Biotechnology, University of Helsinki, Finland 2) Division of Insect Sciences, National Institute of Agrobiological Sciences, Japan 3) Graduate School of Science and Technology, Shinshu University, Japan 4) School of Life Sciences, Arizona State University, USA

A key issue in biology is to uncover the mechanisms employed by conserved signaling

pathways to generate morphological diversity. The Bone Morphogenetic Proteins (BMPs) are a family of evolutionarily conserved, secreted growth factors involved in a myriad of diversified events. In Drosophila, BMP type-ligand Decapentaplegic (Dpp) is required for wing vein development during pupal stage. However, how diffusible Dpp specifies complex and diversified insect wing vein patterns remains unknown. Here we show that spatial distribution of Dpp is tightly regulated to reflect the distinct wing vein patterns in Dipteran Drosophila melanogaster and Hymenopteran Athalia rosae (sawfly). In Drosophila, Dpp is directionally transported from longitudinal veins (LVs) to posterior crossvein (PCV) by BMP binding proteins, Short gastrulation (Sog) and Crossveinless (Cv). In contrast, majority of Dpp is actively retained in LVs through BMP type I receptor Thickveins (Tkv) and positive feedback mechanisms. In sawfly, we found that Dpp signal recapitulates complex fore- and hind- wing venations throughout the wing vein development. Furthermore, we provide the evidence that Dpp transport mechanism is critical for producing the distinct fore- and hind-wing vein patterns by inducing crossveins at different positions. These observations suggest that variations in the directional transport of Dpp lead to local modifications in its spatial distribution and subsequently to diverse wing vein patterns.


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T17 Mechanical Tension Guides Cell Sorting at Compartment Boundaries in Drosophila Daiki Umetsu1, Katharina Landsberg1, Reza Farhadifar2, Jonas Ranft2, Thomas Widmann1, Thomas Bittig2, Amani Said1, Benoit Aigouy1, Suzanne Eaton1, Frank Jülicher2, Christian Dahmann1. 1) Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 2) Max Planck Institute for the Physics of Complex Systems, Dresden, Germany

The formation and maintenance of straight and sharp boundaries separating neighboring groups of cells with different identities is central to building body plans. Lineage restriction is a major strategy to form boundaries by preventing two groups of cells from mixing. These boundaries, called compartment boundaries, often serve as landmarks of tissue development by positioning and stabilizing organizing centers, and therefore are important for morphogenesis. Here, we show by live imaging of the developing Drosophila abdomen that cell proliferation causes prominent cell rearrangements, yet the anteroposterior compartment boundary remains straight despite of cell proliferation. Thus mechanisms are required that counteract cell rearrangements caused by cell division. By analyzing the response to laser ablation of cell bonds in the vicinity of the anteroposterior compartment boundary in developing Drosophila wings, we found that mechanical tension is increased on cell bonds along this compartment boundary as compared to the remaining tissue. Cell bond tension is decreased in the presence of Y-27632, an inhibitor of Rho-kinase whose main effector is Myosin II. Simulations using a vertex model showed that an increased cell bond tension suffices to guide cell rearrangements after cell division to maintain compartment boundaries. These results provide a physical mechanism in which the local increase in Myosin II-dependent cell bond tension directs cell sorting at compartment boundaries.


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T18 Border-Cell Migration Requires Integration of Spatial and Temporal Signals by the BTB Protein Abrupt Anna C.-C. Jang1,3, Yu-Chiuan Chang1, Jianwu Bai2, Denise Montell1

1) Department of Biological Chemistry, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA. 2) Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02175, USA. 3) Institute of Biotechnology, National Cheng Kung University, Taiwan

During development, elaborate patterns of cell differentiation and movement must occur in the correct locations and at the proper times. Developmental timing has been studied less than spatial pattern formation, and the mechanisms integrating the two are poorly understood. Border-cell migration in the Drosophila ovary occurs specifically at stage 9. Timing of the migration is regulated by the steroid hormone ecdysone, whereas spatial patterning of the migratory population requires localized activity of the JAK–STAT pathway. Ecdysone signalling is patterned spatially as well as temporally, although the mechanisms are not well understood. In stage 9 egg chambers, ecdysone signalling is highest in anterior follicle cells including the border cells. We identify the gene abrupt as a repressor of ecdysone signalling and border-cell migration. Abrupt protein is normally lost from border-cell nuclei during stage 9, in response to JAK–STAT activity. This contributes to the spatial pattern of the ecdysone response. Abrupt attenuates ecdysone signalling by means of a direct interaction with the basic helix–loop–helix (bHLH) domain of the P160 ecdysone receptor coactivator Taiman (Tai). Taken together, these findings provide a molecular mechanism by which spatial and temporal cues are integrated.


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T19 Differential Positioning of Adherens Junctions Initiates Epithelial Folding during Drosophila Gastrulation Yu-Chiun Wang, Eric Wieschaus Department of Molecular Biology and Howard Hughes Medical Institute, Princeton University, Princeton, New Jersey, USA

During tissue morphogenesis, simple epithelial sheets undergo folding to form complex structures. While it is well known that spatially restricted cell shape change introduces geometric heterogeneities to initiate epithelial folding, it has not been determined whether modification of epithelial polarity is involved in this process. Epithelial cells are polarized along the apical-basal axis by mutually antagonistic activities of the polarity proteins Par-3 and Par-1, which position adherens junctions in the subapical regions of the cells. Using live embryo imaging, we show that during Drosophila gastrulation adherens junctions shift basally in the cells that initiate the formation of two dorsal epithelial folds, while maintain subapical positioning in the neighboring cells. Abrogation of Drosophila Par-3 homolog Bazooka or Par-1, which causes uniformly apical or lateral positioning of junctions, respectively, results in disruption of dorsal fold initiation. While Bazooka levels are similar across the dorsal epithelium, Par-1 levels decrease selectively in the dorsal fold initiating cells. Overexpression of Bazooka causes ectopic basal shift of junctions and formation of ectopic dorsal folds, suggesting that increased Bazooka/Par-1 ratio enables basal shift of junctions and initiation of epithelial folding. We propose a model whereby initiation of dorsal folds requires spatially restricted modification of epithelial polarity. Differential positioning of adherens junctions mediated by modulation of Bazooka/Par-1 ratio likely represents a general mechanism that organizes epithelial folding.


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T20 Coordination of Cell Death and Selective Adhesion Interlocks Dorsal and Ventral Wing Edges for Zig-zag Pattern of Drosophila Wing Margin Hairs Masahiko Takemura1, 2, Takashi Adachi-Yamada1, 2, 3 1) Department of Biology, Graduate School of Science, Kobe University, Japan 2) Institute for Biomolecular Science, Gakushuin University, Japan 3) Department of Life Science, Faculty of Science, Gakushuin University, Japan

Animal tissues and organs are comprised of several types of cells, which are often arranged in a well-ordered manner. The posterior part of the Drosophila wing margin is covered with a double row of long hairs, which are equally and alternately derived from the dorsal and ventral sides of the wing, exhibiting a zig-zag pattern. How this geometrically regular pattern is formed has not yet been fully understood. In this study, we show that the zig-zag patterning of the wing margin hairs requires surrounding wing margin cells to rearrange during metamorphosis. This cell rearrangement is induced by selective cell death of a subset of the wing margin cells. After the cell death period, the remaining wing margin cells can be classified into two types. One is alternately aligned with hair cells along the dorsoventral boundary. The other is located on the dorsal and ventral edge sides of hair cells, which forms corrugated wing margin edges on both sides to interlock them for zigzag hair patterning.

We further show that the corrugated topology of these two types of wing margin cells is generated by a combination of distinct sets of heterophilic binding with four NEPH1/nephrin family proteins. Homophilic binding of E-cadherin is also required to attach the corrugated dorsoventral edges. Taken together, these results demonstrate that sequential coordination of cell death and epithelial architecture with selective adhesion generates the geometrically ordered zig-zag hair alignment.


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T21 Dsnx3 Influences Wingless Secretion by Recycling Wntless from Endosomes to the Trans-Golgi Network Peng Zhang1, Yihui Wu1, Tanya Belenkaya2, Xinhua Lin1,2＊ 1) State Key Laboratory of Biomembrane and Membrane Biotechnology, and Key Laboratory of Stem Cell and developmental Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China 2) Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, 45229, USA

Recent studies have identified Wntless (Wls) and Retromer complex as key components involved in Wingless (Wg) secretion, but the mechanism(s) remain elusive. As Snx molecules are essential for anchoring Retromer on membrane, we hypothesize that specific Snx(s) might be required for Wg secretion by regulating Retromer activity. To address this question, we generated Drosophila mutants for 8 Snx members, and Dsnx3 was found to be involved in regulating Wg secretion. In wing discs, Wg secretion was defective in Dsnx3 mutant clones. In Dsnx3 dsRNA treated Drosophila S2 cells, Wg levels in culture medium were also markedly reduced. To investigate whether the reduction of Wg secretion is due to Wls degradation, we examined Wls levels in Dsnx3 mutant clones and found that Wls levels were striking reduced. Furthermore, overexpression of Wls can rescue the Wg secretion defect observed in Dsnx3 mutant cells. In vitro experiments showed that Dsnx3 can interact with Drosophila Wls and its mammalian homologue, human Wls. These data indicated Dsnx3 influenced Wg secretion by regulating Wls activity. Additionally, Dnsx3 can interact with Drosophila vps35 in S2 cells, and co-localize with human vps35 in early endosomes through the PtdIns3P binding motif of PX-domain in Hela cells. Together, these data argue that Dsnx3 functions as a cargo-specific accessory component of Retromer complex, which is required for endocytic recycling of Wls.


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T22 The Initiation and Maintenance of Segregation of Eye and Antennal Fates Cheng Wei Wang1, 2, Y. Henry Sun1,2

1) Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan, Republic of China 2) Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan, Republic of China

In Drosophila melanogaster, the eye and antennae originate from a cluster of 20~30 cells

set aside during embryonic development. These cells uniformly express ey gene. The eye or antennal identity of these cells is not determined until mid or late second instar with the restricted expression of Cut, a homeodomain transcriptional repressor in the antennal field and ey in the eye field. The mechanisms responsible for subdividing this epithelium into distinct eye and antennal fields are poorly understood. In this study, we showed that ey is redundantly repressed by Cut and another homeodomain transcription factor, Hth, which has been demonstrated to suppress eye development. On the other hand, the restriction of Cut to antennal field is established by repression by another homeodomain transcription factor, SO, which is a direct downstream target of Ey in eye field. These results demonstrate that the segregation of antenna and eye fates are maintained by the mutual antagonisms of the eye and antennal genes. Furthermore, we demonstrate that the initial segregation of the antennal and eye fates is dependent on EGFR signaling which is required and sufficient for Cut expression in the antennal field. Our results provided the molecular mechanisms for the initiation and maintenance of antennal and eye fate segregation.


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T23 The Genetic Architecture of Coordinately Evolving Male Wing Pigmentation and Courtship Behavior in Drosophila elegans and D. gunungcola

Shu-Dan Yeh1 2, John R. True1 1) Department of Ecology and Evolution, Stony Brook University, USA 2) Current address: Department of Ecology and Evolutionary Biology, University of California, Irvine, USA

Many adaptive phenotypes consist of complexes of simpler traits that act in combination, including morphologies and the behaviors that utilize those morphologies. Genetic correlations between components of such combinatorial traits, in the form of pleiotropic or tightly linked genes, could in principle promote the evolution and maintenance of these traits but this idea is largely untested. In the Oriental Drosophila melanogaster species group, male wing pigmentation shows phylogenetic correlations with male courtship behavior; species with male-specific apical wing melanin spots also exhibit male visual wing displays whereas species lacking these spots generally lack the displays. We investigated the quantitative genetic basis of divergence in male wing spots and displays between D. elegans, which possesses both traits, and its sibling species D. gunungcola, which lacks them. We found that divergence in both wing spot size and male courtship score is determined by at least five quantitative trait loci (QTL). On the autosomes, QTL locations for pigmentation and behavior were generally separate. But on the X chromosome two clusters of QTL were found affecting both wing pigmentation and courtship behavior. The co-occurrence of X-linked QTL for male pigmentation and behavior is consistent with the coordinate evolution of these traits and motivates fine scale mapping studies to elucidate the nature of the contributing genetic factors in these intervals.


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T24 Genetics of Hybrid Inviability and Sterility between D. melanogaster and D. simulans Kyoichi Sawamura Graduate School of Life and Environmental Sciences, University of Tsukuba, Japan

One hundred years ago Quakenbush (1910) observed unisexual broods in “D. melanogaster”. But later it turned out that “D. melanogaster” includes two species, D. melanogaster and D. simulans (Sturtevant, 1919). In the cross between D. melanogaster females and D. simulans males, only sterile female hybrids appear; male hybrids die at the larval stage. On the other hand in the reciprocal cross, sterile male hybrids appear; most female hybrids die at the embryonic stage. Several genes for hybrid inviability and sterility have recently been characterized at the molecular level. Here I summarize the history of the research.

The hybrid inviability and sterility genes isolated to date are as follows. (1) D. melanogaster Hybrid male rescue (Hmr) that codes a DNA-binding protein causes hybrid inviability (Barbash et al., 2003). (2) D. simulans Lethal hybrid rescue (Lhr) that codes a heterochromatin protein (HP3) causes hybrid inviability (Brideau et al., 2006; Prigent et al., 2009). (3) D. simulans Nucleoporin 96 (Nup96) causes inviability when made hemizygous in the hybrid (Presgraves et al. 2003). (4) D. simulans Nucleoporin 160 (Nup160) causes inviability and female sterility when introgressed into D. melanogaster (Tang & Presgraves, 2009; Sawamura et al., 2010). (5) D. melanogaster zygotic hybrid rescue (zhr) consisting of heterochromatic 359 bp repetitive sequences causes inviability of hybrid from D. simulans mothers (Sawamura et al., 1993; Ferree & Barbash, 2009); (6) JYalpha, a gene located on different chromosomes between D. melanogaster and D. simulans, causes male sterility of introgression homozygotes (Masly et al., 2006).

Characterization of the genes has been leading to a generalization: genomic conflict (e.g., arms races between heterochromatin and the binding proteins) might be the major source of reproductive isolation.


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T25 Does Positive Selection Drive Transcription Factor Binding Site turnover? A Test with Drosophila cis-Regulatory Modules Bin He

University of Chicago

Transcription factor binding site(s) (TFBS) gain and loss, or turnover, is a well-documented feature of cis-regulatory module (CRM) evolution, yet little attention has been paid to the evolutionary force(s) driving this turnover process. The predominant view, motivated by its widespread occurrence, emphasizes the importance of compensatory mutation and genetic drift. Positive selection, in contrast, although it has been invoked in specific instances of adaptive gene expression evolution, has not been considered as a general alternative to neutral compensatory evolution. In this study we evaluate the two hypotheses by analyzing patterns of single nucleotide polymorphism in the TFBS of well-characterized CRM in two closely related Drosophila species, D. melangoaster and D.simulans. A novel feature of the analysis is classification of TFBS mutations according to the direction of their predicted effect on binding affinity, which allows gains and losses to be evaluated independently along the two phylogenetic lineages. The observed patterns of polymorphism and divergence are not compatible with neutral evolution for either class of mutations. Instead, multiple lines of evidence are consistent with contributions of positive selection to TFBS gain and loss. This result challenges the prevailing view that turnover of TFBS is a neutral process. As an alternative, we propose a model to reconcile the finding of selection driving TFBS turnover with constrained CRM function over long evolutionary time.


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T26 Molecular Evolution of desaturase Gene Family in Drosophila Shun-Chern Tsaur1, Kuang-Hsi Chu2, Wei-Chin Ho2, Wan-Ju Shen3, Chau-Ti Ting2,4, Shu Fang3 1) Department of Life Sciences & Institute of Genome Sciences, National Yang-Ming University, Taiwan, ROC 2) Institute of Zoology, National Taiwan University, Taiwan, ROC 3) Biodiversity Research Center, Academia Sinica, Taiwan, ROC 4) Department of Life Science & Institute of Ecology and Evolutionary Biology, National Taiwan University, Taiwan, ROC

Fatty acid desaturases play important roles in the lipid metabolism and the biosynthesis of pheromones in insects. Our previous work in Drosophila showed that functional divergence driven by positive selection in the desaturase genes is responsible for the sex pheromonal diversity contributing to species recognition. We also found that recurrent duplication events of a desaturase gene, desatF, took place in several Drosophila lineages. This multiple duplication of desatF further contributes to the pheromonal diene complexity in several species. While gene duplication events are relatively frequent, gene degeneration is not scarce. These degenerated copies provide a good model to understand the pseudogenization process. Among nine members of D. melanogaster species subgroup, six show no desatF gene expression with various levels of degeneration in the coding region. The fact that no shared degenerated alleles were observed in our population samples suggests that these pseudogenization events might occur independently. In addition, high indel and nucleotide polymorphisms have been accumulated in both D. mauritiana and D. yakuba lineages whereas a reduction of polymorphism was found in their sibling species. Based on population genetics analyses, this reduction in multiple species might result from recent selective sweeps. This further implies that pseudogenes like desatF could evolve non-neutrally and at very different rates between sibling species. In conclusion, the rapid independent turnover of desaturase genes is one of the driving forces in pheromonal diversity and speciation.

POSTER: STEM CELLS, CELL DIVISION AND GROWTH CONTROL

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1 The Apical-Basal Cell Polarity Determinant Crumbs Regulates Hippo Signalling in Drosophila Chiao-Lin Chen1,2, Kathleen Gajewski3, Fisun Hamaratoglu4, Wouter Bossuyt1, Leticia Sansores-Garcia1, Chunyao Tao1, Georg Halder1,2,4

1) Dept. of Biochemistry and Molecular Biology, 2) Program in Genes & Development, 3) Department of Systems Biology, Univ. of Texas M. D. Anderson Cancer Center, USA 4) Program in Developmental Biology, Baylor College of Medicine, USA

Proper establishment and maintenance of apical-basal polarity is essential for normal development. Defects in apical-basal cell polarity are often associated with cancer in vertebrates. In Drosophila, abnormal expression of apical-basal determinants, such as overexpression of Crumbs (Crb), can lead to loss of cell polarity and proliferation control, which are two hallmarks of cancer. However, the pathways through which apical-basal polarity determinants affect growth are poorly understood. Here, we investigated the mechanism by which the apical determinant Crb affects growth. We found that both Crb gain and loss of function caused overproliferation in imaginal discs and defects in Hippo signaling, a key signaling pathway that controls tissue growth in Drosophila and mammals. Manipulation of Crb levels caused the up-regulation of Hippo target genes, genetically interacted with known Hippo pathway components, and required Yorkie, a transcriptional coactivator that acts downstream in the Hippo pathway, for target gene induction and overgrowth. Interestingly, Crb regulates growth and cell polarity through different motifs in its intracellular domain. The Hippo pathway component Expanded, an apically localized adaptor protein, is mislocalized in both crb mutant cells and Crb overexpressing tissues, whereas the other Hippo pathway components are unaffected. Taken together, our data show that Crb regulates growth through Hippo signaling, and thus identify Crb as a novel upstream input into the Hippo pathway.


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2 Insulin Signals Control the Competence of the Drosophila Female Germline Stem Cell Niche to Respond to Notch Ligands Hwei-Jan Hsu1,2,5, Daniela Drummond-Barbosa1,2,3,4

1) Department of Cell and Developmental Biology, Vanderbilt University Medical Center, 465 21st Avenue South, Nashville, TN 37232, USA. 2) Departments of Biochemistry and Molecular Biology 3) Departments of Environmental Health Sciences, 4) Division of Reproductive Biology, Johns Hopkins Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205, USA 5) Present address: Institute of Cellular and Organismic Biology, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei 115, Taiwan

Stem cells reside in niches, a population of specialized cells, that provide both physical contact and diffusible factors to control stem cell self-renewal and proliferation; however, little is known about niche regulation itself. We previously showed that insulin signals control niche size and germline stem cell (GSC)-niche interaction in Drosophila females. Insulin signals modulate Notch signaling to maintain cap cells in the niche, and control cap cell-GSC attachment, likely via E-cadherin. Here, we further dissect the molecular mechanisms underlying these processes, and reveal that Notch ligands produced within the niche stimulate Notch in cap cells, and insulin signals act via phosphoinositide 3-kinase and FOXO to control the competence of cap cells to respond to Notch ligands to regulate niche size. Insulin signals, however, control cap cell-GSC attachment independently of Notch. These results are potentially relevant to many systems in which Notch signaling modulates stem cells, and demonstrate that complex interactions between local and systemic signals are required for proper stem cell niche function.


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3 Datou Functions Downstream of Lethal Giant Larvae and is Required for Mislocalization of Cell Fate Determinants in lgl Mutants Lihui Goh1,2, Shuping Lin1, Xiaohang Yang1

1) Institute of Molecular and Cell Biology, Agency for Science Technology and Research, Singapore 2) National University of Singapore Graduate School for Integrative Sciences and Engineering

Asymmetric cell division (ACD) of stem cells produces two daughter cells, one remains the stem cell and the other becomes committed or differentiated cell. This process is highly controlled; failure of which would result in cell over-proliferation or development defects. ACD is well studied in the model organism Drosophila neuroblast and sensory organ precursor cells. It is known that proteins in the apical complex such as Bazooka, Partitioning Defective 6, and atypical Protein Kinase C direct cell fate determinants such as Numb and Miranda (Mira), to localize as a basal crescent and inherited by only one of the daughter cells late in mitosis. However, detailed mechanism of this exclusive segregation of cell fate determinants is largely unknown.

Using a UAS-GAL4 system to drive expression of RNAi constructs, we identified novel genes that are involved in ACD of the larval neuroblasts and sensory organ precursor cells. datou was first identified in an RNAi knockdown screen, which showed bristle missing phenotype.

Datou is specifically expressed in the larval brain neuroblasts. datou mutants display a weak phenotype of mislocalized Mira and Numb crescent in a small percentage of dividing neuroblast. However, deletion of datou rescues the lethal giant larvae (lgl) phenotype. In double mutations of datou and lgl, mislocalization of both Mira and Numb in larval brain neuroblasts is largely restored. In addition, the brain size of datou, lgl double mutant is also considerably smaller than that of lgl mutant alone.

Hence, we hypothesize datou to be a protein which function downstream of the tumor suppressor gene lgl. In the absence of Lgl, Datou disturbs asymmetric localization of Mira and Numb in the larval brain nuroblasts, resulting in defective asymmetric division, which causes cell over-proliferations in lgl mutants. More studies on datou may shed light in uncovering its role in tumor progression and ACD.


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4 Knock out Jy001 and Jy002 by Homologous Recombination Di Wu, YingYing Sung, Xiu Zhou, Xiaohang Yang

College of Life Sciences, Zhejiang University

Asymmetric neural stem cell division plays a critical role in neurogenesis during animal development. In Drosophila, neural stem cells divide and generate one stem cell and one ganglion mother cell. Two groups of proteins are asymmetrically localized oppositely in dividing cells. Baz, Par6, aPKC, Insc, Pins, Gai and Loco are apically enriched and Mira, Brat, Pros, Pon and Numb are basally localized in the mitotic cells. We have screened a collection of RNAi lines and identified several candidate genes which were required for Mira basal localization. Knock-down of JY001 and JY002 causes cytoplasmic localization of Mira. Unfortunately P-elements inserted in or near these two genes are not available. Transgenic flies carrying constructs for homologous recombination knockout of these two genes have been generated. All genetic crosses have been done .We are in the final stage of identifying the knockout lines by PCR method. More data will be presented in the poster.


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5 A Novel Mutant Effecting Drosophila Neuroblast (NB) Asymmetric Divisions Huanping An1, Yingying Song2, Xiaohang Yang1 1) The School of Life Sciences, Zhejiang university, China 310058 2) Institute of Molecular and Cell Biology, ASTAR, Singapore 138673

Drosophila neuroblast (NB) asymmetric divisions generate tow daughters of unequal size and non-identical fate. A complex of apically localized molecules mediates basal localization of cell fate determinants and apical-basal orientation of the mitotic spindle.

To further understand the mechanism of it. Our lab has used RNAi method to screen novel regulators of NB asymmetric division; we isolated a mutant which was named Exes. The homozygous mutant died during the embryonic stage. Our preliminary MARCM analysis in the 3rd instar larval brain indicated abnormal phenotype. We had rough mapped it on a small region of L3 chromosome. Now we are fine mapping it and try to find the gene.


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6 Investigation of the Hippo Signaling Pathway Function on Drosophila Polar Cells Differentiation Hsi-Ju Chen, and Jenn-Yah Yu

Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taiwan

During development, the balance between cell division and differentiation is important. A new signaling pathway revealed in Drosophila melanogaster, the Hippo signaling pathway, plays an important role in development process. However, the interactions among the Hippo pathway and other signaling pathways remain unknown.

We test whether the Hippo pathway participates in polar cell formation during Drosophila oogenesis. We generated follicle cell clones mutant for hpo, wts, or yki, and observed that yki mutant clones form ectopic polar cells. In addition, polar cells formation may be attenuated in hpo mutant clones based on the expression of polar cell markers Fas III and A101. We also noticed that wts mutation results in egg chambers fusion, which may be caused by loss of polar cells. Our results suggest that the Hippo pathway plays important roles in regulating polar cell formation. Previous reports showed that Notch is required for polar cells formation. To test if the Hippo pathway affects the Notch signaling pathway during polar cell formation, we generated the yki mutant clones in a reduced Notch background. Compared with the Fas III expression levels in the normal polar cells at the terminus of egg chambers, most yki mutant clones express less Fas III. This suggests the Hippo pathway acts upstream to the Notch signaling pathway.

Since cell proliferation and differentiation require accurate regulation, this work provides an insight into the function of the Hippo pathway and helps our understanding of how the Hippo pathway cooperates with other signaling pathways to direct homeostasis during development.


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7 The Drosophila Retinoblastoma Protein, Rbf1, Controls Germline Stem Cell Differentiation through Non-Cell-Autonomous Regulation of BMP Signalling Gregory W. Somers1, Leonie. M. Quinn2, Gary. R. Hime2

1) Genetics, La Trobe University, Melbourne, Australia 2) Anatomy and Cell Biology, University of Melbourne, Melbourne, Australia

Stem cells are essential for tissue growth and maintenance throughout the lifetime of an organism. Conserved molecular signals from the local tissue environment are necessary for regulating stem cell divisions. Our research interests involve understanding the complex molecular signals that govern the balance between stem cell self-renewal versus differentiation during development. Using the male germline stem cell model system of Drosophila melanogaster, a genetic screen was conducted to identify new molecules that regulate stem cell biology. This screen identified the Drosophila Retinoblastoma gene, Rbf1, to be essential for differentiation of the germline. Although many research groups investigating the function of the mammalian pRb family have also reported a disruption to cellular differentiation, the mechanism of action is uncertain due to complications associated with functional redundancy. Rbf1 was identified to be both necessary and sufficient within the somatic stem cell lineage to regulate differentiation of both the somatic and germline stem cell populations. This function was independent of its role in regulating cell cycle progression. Rbf1 was identified to be essential for regulating the Bone Morphogenetic Protein (BMP) signalling pathway, recently shown to promote stem cell properties in the Drosophila testis. In addition Rbf1 was identified to be important for regulating chromatin modifications during stem cell differentiation. We conclude that Rbf1 plays an essential role with respect to regulating germline stem cell development, and may be a primary downstream target of niche signals to regulate self-renewing signals.


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8 Brat Establishes the Neural Progenitor Cell Potential through PointedP1 and Apc2 Hideyuki Komori, Cheng-Yu Lee Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan, 48109 USA

Stem cells generate progenitor cells with restricted potential to amplify their output in

generating progeny while safeguarding their genomic integrity, but how the progenitor cell potential is regulated remains unknown. Type II neuroblasts in Drosophila larval brains repetitively generate immature intermediate neural progenitors (INPs) that acquire the restricted potential. The tumor suppressor gene brain tumor (brat) plays a crucial role in establishing INP potential because brat mutant brains show massive ectopic type II neuroblasts at the expense of INPs. We discovered that Brat establishes the INP potential via novel pointedP1 (pntP1) and adenomatous polyposis coli 2 (apc2) dependent mechanisms. Brat promotes nuclear localization of PntP1 in immature INPs where it promotes timely differentiation via a RTK-independent mechanism. Furthermore, Brat maintains Apc2 expression and antagonizes Armadillo-mediated Wnt target gene expression in type II neuroblast lineages. Apc2 is dispensable in differentiation to INPs although restoration of Apc2 expression suppresses ectopic neuroblasts phenotype in brat mutant brains. Thus, Brat establishes the restricted potential in INPs by promoting differentiation via PntP1 and suppressing de-differentiation through Apc2.


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9 The Roles for Helix-Loop-Helix Proteins in Cellular Growth and Apoptosis Lan-Hsin Wang, Nicholas E. Baker Department of Genetics, Albert Einstein College of Medicine, NY, USA

Class I and Class V helix-loop-helix (bHLH) transcription factors regulate cell fate commitment and differentiation in various cell lineages. In humans, dysfunction of the Class I proteins (E proteins) and Class V proteins (Id proteins) are associated with cell cycle arrest and many human cancers, respectively. However, little is known about the underlying pathological mechanisms due to the complexity of the protein family. Fortunately, these growth affects can be investigated easier in Drosophila, which has only one Class I protein (Daughterless, Da) and one Class V protein (Extramacrochaetae, Emc). To completely understand the mechanisms by which Class I and V bHLHs regulates tumorigenesis, I established a Da overexpressing fly model, which mimics the deficiency of Emc. I found that Da has roles in regulating cell proliferation through disrupting G1/S progression and stimulating apoptosis. Overexpression of Da during eye development can inhibit eye growth. Interestingly, the only Drosophila CDK inhibitor, Dacapo, has no effect on the growth inhibition caused by overexpressing Da. This pointed to an unidentified connection between Da and proliferation/growth in Drosophila. To identify novel components involved in this process, I further carried out a genetic screen using DrosDel and Exelixis deficiency collection. 33 genomic regions were identified from this screen, 25 showed suppressed and 8 showed enhanced effects on Da dependent growth inhibition. I will present results from the ongoing study.


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10 Elimination of Oncogenic Neighbors by JNK-mediated Engulfment in Drosophila Shizue Ohsawa1, Kaoru Sugimura2, Kyoko Takino1, Tian Xu3, Atsushi Miyawaki2, Tatsushi Igaki1

1) Department of Cell Biology, G-COE, Kobe University Graduate School of Medicine, Japan 2) Laboratory for Cell Function and Dynamics, Brain Science Institute, RIKEN, Japan. 3) Howard Hughes Medical Institute, Department of Genetics, Yale University School of Medicine, Boyer Center for Molecular Medicine, USA.

Most cancers arise from a single cell of origin in an epithelial sheet. Therefore, a newly emerged oncogenic cell in the epithelial population has to confront anti-tumor selective pressures in the host tissue. However, the mechanisms by which surrounding normal tissue exerts anti-tumor effects against oncogenically transformed cells are poorly understood. In Drosophila imaginal epithelia, clones of cells mutant for evolutionally conserved tumor suppressor genes such as scribble (scrib) and discs large (dlg) are eliminated from the tissue. This elimination of oncogenic cells requires the presence of surrounding wild-type tissue, suggesting that normal imaginal tissue possesses a tumor-suppressor effect that eliminates oncogenic cells from epithelium. We found that normal imaginal cells activated non-apoptotic JNK signaling in response to the emergence of oncogenic cells. Furthermore, this non-apoptotic JNK activation promoted elimination of oncogenic neighbors by engulfment through activation of intracellular cytoskeletal rearrangement. Our data suggest that JNK-mediated cell engulfment could be evolutionarily conserved tumor suppression mechanism that eliminates pre-malignant cells from epithelia. The underlying mechanisms will be presented.


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11 Maintenance of Undifferentiated State of Stem Cell Precursors in the Drosophila Ovary Shinya Matsuoka1, 2, Miho Asaoka1, 2, Yasushi Hiromi1, 2

1) National Institute of Genetics, Japan 2) SOKENDAI, Japan

Many types of sexually reproducing animal possess germline stem cells (GSCs). GSCs are undifferentiated cells and have an ability to produce not only gamete but GSCs themselves after every cell division. This self-renewal ability allows GSCs to produce gamete unlimitedly to maximize success in fertilization. Drosophila is one of such organisms equipped with GSC systems.

During larval stage, Drosophila ovary is filled with stem cell precursors, primordial germ cells (PGCs), which are kept in an undifferentiated state. At pupal stage, some of PGCs start to differentiate to eggs, but the rest of PGCs are selected as GSCs and remain in an undifferentiated state. A previous study has shown that it is a crucial step to prevent PGC differentiation to establish adequate number of GSCs. However, how PGC differentiation is suppressed is largely unknown.

Here we show that novel gene gone early is involved in prevention of PGC differentiation. Gone early is a membrane-bound protein with an unknown extracellular domain. When gone early is over-expressed in PGCs in which it is endogenously expressed, PGC differentiation is excessively blocked. In contrast, in gone early mutant, the increased number of differentiating germ cells is observed and this phenotype can be rescued by expressing gone early. Although the number of differentiating germ cells in gone early mutant is greater than in wildtype, the number of PGCs does not decline. This is due to dedifferentiation of differentiating germ cells into PGCs in order to ensure certain number of PGCs. Even though PGC number does not decline in gone early mutant, the number of GSCs in favorable background is lower than in wildtype, supporting the notion that preventing PGC differentiation is prerequisite to establish adequate number of GSCs.


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12 Analysis of dMyc-Mediated Competitive Interactions during Proliferation and Cell Death Nanami Senoo-Matsuda1, 2, Mai Kanai1, 2, Naoya Fukawa1, Nobuhito Goda1, 2

1) Department of Life Science and Medical Bio-Science, School of Advanced Science and Engineering, Waseda University, Tokyo, Japan 2) CREST, JST, Japan

Our studies have revealed that developing wing cells in Drosophila melanogaster that differ in expression levels of Drosophila c-Myc (dMyc) can compete, leading to the apoptosis of the cells with less dMyc (“losers”) and over-representation of cells with more dMyc (“winners”) in the wing (de la Cova et al., 2004). This phenomenon, called cell competition, seems to play a crucial role in the control of organ size. We have developed two assays to identify the molecular mechanisms underlying cell competition. First, a cell-culture based cell competition assay has determined that competition is mediated by diffusible factors produced by winner and loser cells that allow recognition and determine the response to each other's presence (Senoo-Matsuda & Johnston, 2007). Second, proteomics and transcriptomic approaches with competition induced in the tissue culture models and living animals have identified a handful of factors. We postulate that cell competition is a broadly used and evolutionarily conserved process of tissue homeostasis of metabolism that may be exploited by cancer cells during tumorigenesis in humans.


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13 Tousled-Like Kinase Involves in Stress-Activated Protein Pathway (p38) Inducing Antephase in Drosophila Chih Hsin Ou-Yang, Hsiao-Hsi Chou and Gwo-Jen Liaw

Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan

Function of Tousled-Like Kinases (TLKs) in cell-cycle progression has been well studied.

To explore function of Tlk in cell division, Drosophila tlk gene expressed in eye imaginal disc resulted in a small-eye phenotype without defects on ommaditial morphology, suggesting that progression of cell cycle was delayed. To reveal what phase of cell cycle was affected, the second mitotic wave (SMW) was used as an assay system that cells behind the morphogenetic furrow synchronously divide once and the cell division completes within 10 rows of neuronal clusters. Results showed that cells entering metaphase was delayed, delayed antephase, supported by delayed degradation of both Cyclins A and B. Genetic interaction experiments were carried out to test whether tlk functions in the two pathways whose activities prolong antephase. Preliminary results indicated that Tlk acts upstream Dp38. Therefore, western blotting was used to demonstrate whether tlk overexpression increases Dp38 phosphorylation. To solve Mr. Chou’s problems of irreproducible results, I tested several factors, such as ageing time, mixing antibody and usage of inhibitors for phosphatase or proteases. Results showed ageing for 30 min and usage of phosphatase inhibitor greatly improved quality of western results. Low and high levels of tlk expression increase level of phosopho-Dp38 by 1.35~1.43-fold. To show that files heterozygous to tlk 14 failed to cope with stresses like flies homozygous for Dp38 did, the flies were treated with various stresses, such as heat, starvation and feeding H2O2. Lethality was further decreased by 12% when one copy of tlk was removed from fly heterozygous for Dp38, consistent with results from the epistatic analysis.


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14 Gene-Hunting in Asymmetry Field Lihui Goh1, Ying Ying Sung1, Zhenxing Huang1, Shuping Lin1, Xiaohang Yang2

1) Institute of Molecular and Cell biology, Proteos, 61 Biopolis Drive Singapore 138673 2) College of Life Sciences, Zhejiang University, 388 Yuhangtang Road, Hangzhou, China

In the Drosophila central nervous system, Baz, Par6, aPKC, Insc, Pins and Gi, as well as

Loco, are concentrated on the apical cortex of mitotic neural stem cells, forming a functional apical complex; while cell fate determinants such as Pros, Brat and Numb, together with their respective adaptor proteins Mira and Pon, are enriched on the opposite side (basal) of the cell cortex. The apical protein complex controls basal localization of cell fate determinants, coordinates spindle orientation and regulates spindle asymmetry and displacement late in mitosis. In order to understand the detailed molecular mechanism, we carried out several screens and identified a couple of dozens of novel genes that play roles in asymmetric divisions. Currently we are focusing on three genes: datou, startled and p93. Mira localization is disrupted in the larval brain neuroblasts in mutants of these genes. Removal of datou in lgl mutant background rescues lgl tumor phenotype. Startled appears to regulate aPKC activity. Understanding the functions of these genes will provide us new clues to elucidate the mechanism of asymmetric division.


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15 Expanded Regulation in the SWH Pathway Ashesha Sinha1,2, Claire Milton1,2, Kieran Harvey1,2 1) Peter MacCallum Cancer Centre, St Andrew's Place, East Melbourne VIC 3002 2) Department of Pathology, University of Melbourne, Parkville, VIC, Australia, 3010

The Salvador-Warts-Hippo (SWH) pathway is a newly identified growth regulatory pathway, which was first discovered in genetic mosaic screens in Drosophila. Since the pathway’s inception in 2003, much information on the mechanism of the core members and downstream transcriptional regulation by Yorkie (Yki) has been elucidated. However, upstream regulation of the SWH pathway is less well understood. Towards this end, the current study is investigating the regulation of one of the upstream members, Expanded (Ex) which is also a transcriptional target of the pathway. In earlier studies, Ex protein localization and stability was shown to be regulated by two upstream SWH pathway proteins; the Fat receptor and Crumbs (Crb). We investigated whether Fat and Crb act in the same pathway to regulate Ex and results so far suggest that they act independent to each other. We further explored how Fat might be regulating Ex. Translational regulation by a known regulator of Ex, microRNA (miR) -278 (or miRvana) was investigated and miRs more generally, using an ex miR sensor tool which showed that miRs act independently of Fat to regulate Ex levels. Therefore, further experiments to look at the effect of Fat on Ex protein stability have been devised. These studies will provide further insight into the mechanism by which upstream SWH pathway proteins modulate tissue growth.


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16 Growth of Eye-Antenna Disc Compartments is Differently Regulated by Hedgehog, Decapentaplegic and Wingless Jong-Hoon Won, Orkhon Tsogtbaatar, Wonsuk Son, Kyung-Ok Cho* Cellular and Developmental Biology Lab. Department of Biological Sciences, KAIST, Daejeon, Korea

The eye-antenna imaginal disc (ead) develops into part of complex adult head structures like eye, head cuticle and antenna by programmed cell proliferation and pattern formation. While other imaginal discs are derived from a single embryonic segment, ead is composed of seven embryonic segments that fuse together to form a sac of epithelial cells during the late embryogenesis. Actual retina tissue containing photoreceptor clusters is generated from the posterior end of the ead at the early third instar stage, but it is not clear whether the retina is originated from a single or multiple embryonic segments. Therefore, studying the early development of ead is essential for understanding the origin of retina tissue.

We have previously shown that Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg) are essential morphogens for the formation of ead during the first instar larval stage. Interestingly, the ventral domain of ead is added onto the preexisting dorsal domain in the first instar. The growth of cells in the dorsal domain is critically dependent on Hh, while that of the ventral domain is not. Cell death occurring in the dorsal domain in the absence of Hh can be rescued by CycE but not by p35 expression, suggesting that the primary effect of Hh is on cell proliferation. Unlike Hh, Dpp was important for both retina growth in the posterior region and the head part in the anterior region of ead. Wg appeared to be essential for the birth of the ventral domain. Our data suggest that these morphogens affect the growth of various embryonic segments differently. Consistent with this idea, there were only a few founder cells for the entire ead that may represent different embryonic segments.


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17 Multiple Signals Produced from a Muscular Niche Regulate the Maintenance of Adult Drosophila Intestinal Stem Cells Chen-Hui Wang, Na Xu, Fu Yang, Guonan Lin, Rongwen Xi National institute of Biological Sciences, Beijing, 100206, China

Adult Drosophila intestinal stem cells (ISCs) are multipotent stem cells scattered along a thin layer of basement membrane surrounded by visceral muscle cells. Our previous work has demonstrated the canonical Wnt signaling and Jak/Stat signaling are required for ISC division and long-term maintenance. Specifically, the wnt ligand wingless and the major Jak/Stat signal ligand upd are exclusively expressed in the muscle cells in healthy intestines, suggesting the muscle cells may act as the ISC niche microenviroment. Recently, we find that large patches of ISC clones mutant for all the three upds (upd, upd2, upd3) can maintain normal epithelial homeostasis and retain normal Jak/Stat activity, further supporting that upd produced from the muscle cells is crucial for Jak/Stat activity and intestinal homeostasis. Here we find that EGFR/Ras signaling pathway is also required for ISC proliferation and maintenance. Interestingly, one of EGFR/Ras pathway ligands vein is specifically expressed in the muscular niche. Knockdown of vein expression level in the muscular niche significantly reduces ISCs number as well as their proliferation activity. Furthermore, two additional EGFR ligands (spitz and Krn) ,expressed in the progenitor cells, are also required for ISC division and maintenance. Taken together, our studies suggest that ISCs are maintained by multiple signaling pathways and reinforce the idea that the viceral musle cells act as an important constitute of the ISC niche.


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18 Notch Activates Dally to Define Drosophila Female GSC Niche Chan Wu1,2, Zheng Guo1,2, Zhaohui Wang1 1) Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China 2) Graduate school, Chinese Academy of Sciences, China

Germline stem cells (GSCs) are maintained by the somatic niche cells. In Drosophila ovary, cap cells form the major part of GSC niche and provide signals that are essential for GSCs identity. It has been reported that Notch signaling is responsible for cap cells formation and consequently for GSC maintenance. However, it is still unclear how Notch signaling endows the cap cells the abilities to sustain the GSCs. Previously, we discovered that glypican Dally is specifically expressed in cap cells to maintain GSCs fate and short-range BMP signaling. Here, we found that Notch signaling could activate Dally expression, and thus enabled cap cells to function as GSC niche. Expanding Notch activation in more somatic cells led to the formation of more Dally positive cap cells, which supported more GSCs; whereas this GSC over-proliferation could be suppressed by Dally mutant. In our clonal analysis, we showed that cap cells with compromised Notch signaling failed to express Dally and consequently resulted in GSC loss. Now we are trying to rescue Notch mutant clone phenotype by restoring Dally expression. Our data suggest that Notch activates Dally expression in cap cells to define GSC niche and to maintain the GSC identity.


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19 Control of Neural Lineage Topology by a Notch-Mediated Genetic Switch in Neural Progenitor Division Mode Ryan MacDonald, Carina Ulvklo, Caroline Bivik, Magnus Baumgardt, Daniel Karlsson, Erika Lundin, Stefan Thor Department of Clinical and Experimental Medicine, Linkoping University, SE-581 85, Linkoping, SWEDEN

During nervous system development, neural progenitor cells divide asymmetrically, renewing themselves and budding off daughter cells with more restricted potential. Daughter cells can either differentiate directly, or divide to expand a certain branch of a lineage tree. However, how the mitotic potential of any given daughter cell is controlled during neural lineage progression is not well understood.

In the Drosophila embryo, neural progenitor cells, neuroblasts, generate the CNS by series of asymmetric cell divisions. Daughter cells from such divisions, ganglion mother cells (GMCs), typically divide once to generate two neurons or glia. However, in certain lineages, such as thoracic neuroblast 5-6, there is an intriguing switch in division mode, such that the four last-born daughter cells never divide, instead differentiating directly into the Apterous (Ap) neurons. The prospero gene controls cell cycle exit of GMCs, and in pros mutants there is an expansion of the early NB5-6 lineage, as a result of extra GMC divisions. However, the directly-generated late-born Ap neurons are not affected, and thus the lineage topology switch is not controlled by pros.

In a genetic screen scoring for affects upon NB5-6 lineage progression, we have identified two loci that fail to execute the lineage topology switch. Here, Ap cells undergo one ectopic round of division before differentiating, resulting in a doubling of the number of Ap neurons. These two loci map to kuzbanian and neuralized–two components of the Notch signaling pathway. We find that the Notch pathway is not critical for the specification of Ap neurons, but acts exclusively to control the switch in daughter cell division potential. The reason why Ap neurons only divide ectopically one round in Notch mutants is that each Ap neuron is converted into a GMC, and GMCs are prevented from dividing by pros. Strikingly, in pros, Notch double mutants, Ap cells now divide multiple times. To our knowledge, this is the first identified mechanism for a genetic switch in neural lineage topology.


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20 Dissection of Functional Modifications on Drosophila Wee Changqing Li, Hanqing Chen, Renjie Jiao State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China

The tyrosine kinase Wee1 is a crucial negative regulator of mitotic entry by suppressing cyclin B/Cdk1 activity. It phosphorylates Cdk1 at the inhibitory Tyr15 residue until the cell is ready to divide. Mutations in Wee1 may cause either apoptosis or tumorgenesis depending on how the mutation affects its activity and the cellular contexts. Wee1 is regulated at multiple levels, such as phosphorylation, protein-protein association and proteasome-mediated degradation. Phosphorylation is the most important regulation, which occurs mainly in the N-terminal non-catalytic region of Wee1, both in vertebrates and yeasts. Several phosphorylation sites have been identified, for example, we have reported that Cyclin A/Cdk2 complex associates with “RXL” motif and mediates phosphorylation of T239 in human somatic Wee1 for inhibition of Wee1 activity. In addition, phosophrylation by Cyclin A/Cdk2 complex promotes Wee1 re-localization from the nucleus to the cytoplasm. Drosophila Wee1 （Wee） protein shows high similarity to both embryonic and somatic human Wee1. Wee plays important roles during development and is involved in the checkpoint pathway induced by DNA damage. However, how Wee is post-translationally regulated is far from being understood. We are employing genetic tools and biochemical methods to characterize the phosphorylation and/or other modification sites of Wee in an aim to understand the molecular mechanisms of how Wee is regulated.


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21 Non-Cell-Autonomous Control of the Orientation of Stem Cell Polarity and Divisions Shigeki Yoshiura, Nao Ohta, Fumio Matsuzaki RIKEN Center for Developmental Biology, Japan

During the formation and maintenance of tissues, the directional division of stem cells is a major mechanism for the establishment of tissue polarity. Drosophila embryonic neural stem cells, neuroblasts, asymmetrically divide perpendicular to the overlying epithelial layer, budding off daughter ganglion mother cells and their descendant neurons on the opposite side, thereby determining the initial orientation of neural tissue growth. The formation of neuroblast polarity is known to be regulated by Par-complex in a cell-autonomous manner. On the other hand, non-cell-autonomous mechanisms, dependent on a cue from the epithelial cells, are believed to regulate the relative orientation of neuroblast polarity, although the mechanisms underlying this process remain unknown. To identify this signaling from the epithelium, we have performed deficiency screens for genes encoding transmenbrane proteins, and found that one mutation compromises the orientation of neuroblast polarity without affecting its formation, resulting in defective neural tissue growth. We will discuss about the mechanism of this cell-extrinsic orientation of neuroblast polarity, and as the Par-complex polarizes various types of cells, dictating asymmetric divisions, our finding may help to understand the universal mechanism for the control of tissue polarity, by orienting polarized stem cells and their divisions.


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22 Isolation of QF Enhancer Trap Lines Expressed in Adult Intestinal Stem Cell System in Drosophila Koji Takeda, Takashi Okumura, Kiichiro Taniguchi and Takashi Adachi-Yamada

Department of Life Science, Faculty of Science, Gakushuin University, Japan.

Gal4/UAS system, a binary expression system composed of a DNA-binding transcription factor and its target gene, has commonly been used for transgene expression in Drosophila. Recently, a new similar system QF/QUAS has been developed. Combining this new system with the traditional GAL4/UAS system allows us to manipulate transgenes expression independently.

In this study, we focused on a newly isolated QF enhance trap line named “QF003”, which showed QF expression in all of the intestinal stem cells (ISCs), enteroblasts (EBs, immature enterocytes), and 90% of the enteroendocrine (ee) cells in the adult posterior midgut. The homeostasis of the Drosophila midgut is maintained by multipotent ISCs, each of which gives rise to a pair of a new ISC and an EB/ee through a single round of cell division. The ee cells were known to be classified into two subgroups (T- and A-types) by the differential expression of hormones such as Tachykinin (T) and Allatostatin (A), which showed a paired distribution among enterocytes. Further analysis of QF003 revealed that it is commonly expressed in both the T- and A-types ee cells. On the other hand, the 10% remainder of the ee cells lacking QF003 expression belonged to neither T- nor A- types (nonTA-type), and did not exist as a pair with other ee cell. A DNA sequencing analysis of QF003 revealed that the QF-containing P-element was inserted at the 5’ flanking site of the causal candidate gene (CG32243 or CG11357) on the third chromosome.

Using this QF line as a new marker of ee cells subgroups, together with antibody staining of Tachykinin and Allatostatin markers, we are currently working to clarify the underlying mechanisms of ee-cell subgroups differentiation.


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23 Cytokinesis-Deficient Binucleation in Drosophila Accessory Gland for Providing Plasticity of Organ Size Kiichiro Taniguchi1, Akihiko Kokuryo1,2, Takao Imano1,2, Rumi Sakata1, Ryunosuke Minami3, Hideki Nakagoshi3, Takashi Adachi-Yamada1,2 1) Department of Life Science, Faculty of Science, Gakushuin University, JAPAN 2) Department of Biology, Graduate School of Science, Kobe University, JAPAN 3) Department of Biology, Faculty of Science, Okayama University, JAPAN

As cytokinesis theoretically follows karyokinesis during the M phase in a cell cycle, most eukaryotic cells contain only a single nucleus. Nonetheless, cytokinesis does not occur in particular kinds of cells, such as hepatocytes or myocytes, which results in cells containing two nuclei. However, mechanisms for skipping cytokinesis and the significance of binucleation are largely unknown. The Drosophila male accessory gland, a reproductive organ, shows binucleation in all epithelial cells.

Here, we examined the mechanisms and roles for the binucleation in the accessory gland. The binucleation occurs by synchronous M-phase entry after the standard cell proliferation. Both a central spindle and a contractile ring did not form during binucleation. The elevated activity of Rho signaling induced the contractile ring formation and the following cytokinesis. This result suggests that insufficient formation of central spindle, which restricts the activation of Rho signaling, leads to the cytokinesis skipping in binucleation. In the accessory gland, the apical area of binucleated cells became more enlarged than that in endoreplicated mononucleate cells. On the other hand, the apical area of binucleated cells became more shrunken after mating than that of divided mononucleate cells. These results suggest that binucleation is an effective strategy as it provides a plasticity in the organ size. This size plasticity of the accessory gland should lead to a effective reproductive success.


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24 A Drosophila GATA Transcription Factor Gene, GATAe, is Required for Maintaining Adult Intestinal Stem Cells Takashi Okumura, Takashi Adachi-Yamada

Department of Life Science, Faculty of Science, Gakushuin University, Japan Adult stem cells having a prolonged self-renewal ability and multipotency are essential for

maintaining tissue homeostasis throughout an organism’s life. Recently, a population of adult intestinal stem cells (ISCs) was identified in the Drosophila adult midgut and studied as a new model system of adult stem cells. The Drosophila ISCs produce enteroblasts (EBs) which directly differentiate into two types of mature cells, enterocytes (ECs) and enteroendocrine cells (ees), without cell division in weekly turnovers.

GATAe is a Drosophila member of the C4 zinc finger-containing transcription factors. Its orthologs in both protostomia and deuterostomia are known to be involved in heart and endoderm differentiation. In this study, we found that the RNAi-mediated depletion of GATAe caused a decrease in the frequency of the ISC/EB pairs marked by escargot (esg) expression. In addition, whereas the normal ISCs and EBs showed a pyramidal morphology and were located in an epithelial niche adjacent to the basement membrane and visceral musculature surrounding the midgut epithelium, ISCs and EBs treated with GATAe RNAi resulted in not only their spherical morphology and detachment from the niche but also cell death detected by TUNEL staining, suggesting that GATAe was required for survival of the ISCs and EBs. Since GATA transcription factors have been reported to be involved in differentiation of various tissues both in invertebrates and vertebrates, we are also currently studying whether GATAe is involved in differentiation of ECs and ees and will discuss this possibility.


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25 Downregulation of Notch Mediates the Seamless Transition of Individual Drosophila Neuroepithelial Progenitors into Optic Medullar Neuroblasts during Prolonged G1 Minako Orihara-Ono1, Masako Toriya1, Keiko Nakao2*, Hideyuki Okano1* 1) Department of Physiology, Faculty of Medicine, Keio University, Japan 2) Department of Physiology, Faculty of Medicine, Saitama Medical University, Japan

During development of the Drosophila optic medullar primordium, symmetrically dividing neuroepithelial cells (NEs) first expanding themselves and asymmetrically dividing neuroblasts (NBs) arise subsequently. However, It is still unclear that what mechanisms specify the NEs to change into NBs. Here, we performed detailed analyses demonstrating that individual NEs converted into NBs. We could show that this transition occurred during an elongated G1 phase. During this G1 phase, each columnar NE changed morphological features and gene expression dynamically. Once the NE-to-NB transition was complete, the former NE changes its cell-dividing behavior, commencing asymmetric division. Moreover, we found that Notch signaling was activated just before the transition, which accompanies the Su(H)-dependent downstream gene activation and was rapidly downregulated. Furthermore, the clonal loss of Notch wildcopy in the NE region near the medical edge causes ectopic accumulation of Delta, leading to the precocious onset of transition. Taken together, activation of Notch signaling during a finite window coordinates the proper timing of the transition of NEs to NBs.

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26 The Effects of the Gamma-Secretase Complex Components on Cell Survival in Drosophila Wing Development Joel Brown1, Emilie Cooper1,2, Hui-Min Chung1

1) Dept. Biology, Univ. West Florida, Pensacola, FL, USA 2) Center for Disease Control and Prevention, GA, USA

γ-Secretase is an important protease complex for regulating processing of several type I

transmembrane proteins like the Amyloid Precursor Protein (APP) and Notch proteins. Interrupting its function has substantial effects on signaling transduction and cell health during animal development. The essential components of γ-secretase include: Presenilin (Psn), Aph-1, Nicastrin (Nct), and Presenilin enhancer 2 (Pen-2). During assembly of the protease, Aph-1, Nct and Pen-2 stabilize the newly synthesized Psn holoprotein to facilitate generation of the active form of Psn, which is a Psn-NTF/Psn-CTF heterodimer produced through an endoproteolytic cleavage of the Psn holoprotein. We found that in Drosophila, in addition to its requirement for γ-secretase activity, Aph-1 is needed to promote cell survival in the wing imaginal disc; aph-1 mutant cells are lost either through cell death or because of a defect in cell proliferation. This function of Aph-1 is independent of its role in regulating γ-secretase activity, but possibly involves downregulating the activity of the uncleaved Psn holoprotein, raising the possibility that Psn might have multiple activities, depending on the statuses of the other three components of the γ-secretase complex. Using various modified Psn forms that do not require endoproteolysis or have a large deletion of the cytosolic loop, we would like to learn whether removal of Nct or Pen-2 affects cell survival as Aph-1 does. The finding would impact our understanding on the mechanism of Psn functioning inside and outside of the γ-secretase activity in regulating cell health and cell proliferation.

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27 Novel Functions of APP in FoxO Mediated Apoptosis in Drosophila Xing-Jun Wang1, Qiang-Zhi Wang1, Lei Xue1 1) Shanghai Key Laboratory for Signaling and Diseases, School of Life Science and Technology, Tongji University, Shanghai, China

The amyloid precursor protein (APP) is a widely expressed transmembrane protein that is cleaved to generate Aβ peptides in the central nervous system and plays a significant role in the pathogenesis of Alzheimer’s disease. Despite many studies focusing on the Alzheimer’s disease, the physiological functions of APP remain poorly understood. While a commonly accepted model argues that Aβ peptides are the main cause of onset and early pathogensis of Alzheimer’s disease, recent studies have challenged this hypothesis and proposed a direct role of APP in this neurodegenerative disease. Here we demonstrate, for the first time, a physiological function of APP in regulating the FoxO mediated apoptosis in Drosophila.

First, ectopic expression of APP induces apoptosis in various tissues including the nervous system and non-neuronal tissues of the Drosophila, which mimics JNK activation. Second, APP expression in 3rd instar wing discs and eye discs induces endogenous puc expression, a read-out of the JNK signaling. Third, ectopic APP expression in the developing thorax and wing produces small scutella and loss of vein phenotype, respectively. Both phenotypes could be suppressed by loss of FoxO, a transcription factor of JNK pathway. Finally, deletion of the APP intracellular domain or downregulation of psn, a Drosophila γ-secretase responsible for releasing of APP intracellular domain, failed to induce JNK activation, indicating that AICD is required for APP- induced FoxO- mediated apoptosis.

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28 Expression of Mutant Human p53 Enhances the Formation of Tumor Clones wts/wts. Kirsanov K.I, Lesovaya E.A, Sidorov R.A, Belitsky G.A, Yakubovskaya M.G Blokhin Cancer Research Center RAMS, Moscow, Russia

Structural and functional homology of human p53 and Drosophila Dmp53 is under intensive investigation. Drosophila Dmp53 controls damage-induced apoptosis and possibly affects cell-circle progression, while human p53 signal pathways are much more complex. Inactivating mutations of p53 is known to increase probability of mammal cell malignant transformation.

Previously we have shown that either mutation of Drosophila p53 or its inhibition by RNA-interference increased frequency of spontaneous and carcinogen induced clone formation in Somatic Mutation And Recombination Test with heterozygotes wts/+ .

To study p53 and Dmp53 functional homology we have obtained Drosophila melanogaster transgenic strain bearing mutant human p53 gene (p53Y234C) under control of UAS promoter and demonstrated expression and hyperexpression of the transgene by RT-PCR. Tumor clone frequencies in heterozygotes wtsP4/+(III), diheterozygotes wtsP4 + /+ P{UAS-p53Y234C}(III) and triheterozygotes act-Gal4(II); wtsP4 + /+ P{UAS-p53Y234C}(III) were compared. After oxoplatin treatment the clone frequency in wtsP4 + /+ P{UAS-p53Y234C} flies was three times as much as in wtsP4/+ heterozygotes, moreover in the case of transgene hyperexpression this index had 4,5-fold increase. For spontaneous mutagenesis we registered statistically significant increase of tumor frequency in the case of mutant p53 hyperexpression only.

Thus, our data show the functional homology between human p53 and Drosophila Dmp53 ability to control the level of spontaneous as well as carcinogen induced somatic mutations and recombination.

POSTER: CELL DEATH

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29 The Function of Ecdysone Signal in the Optic Lobe Cell Death in Drosophila Yusuke Hara1,2, Yu Togane1,2, Hiromi Akagawa1,2, Tatsuya Sudo1, Ayano Ishitsuka1, Masashi Iwamura1, Rie Ayukawa1, Keiichiro Hirai1, Kengo Beppu1, Takashi Takahashi1, Kikuo Iwabuchi2, Hidenobu Tsujimura1 1) Developmental Biology, Tokyo University of Agriculture and Technology 2) Applied Entomology, Tokyo University of Agriculture and Technology

Development of the central nervous system accompanies many cell deaths. Defining the mechanism and the role of the cell death is important to understand the basis of neural development. We have been studying the cell death in the developing optic lobe, the visual center in Drosophila. The adult optic lobe develops de novo during metamorphosis. Enormous cells die along with the development and the number of dying cells peaks at 24hours after puparium formation. This indicates factors controlling the timing of the cell death. Here, we provide evidences showing ecdysone is one of them. Disruption of ecdysone signal transduction involving βFTZ-F1, a competence factor for ecdysone at an early stage of metamorphosis, reduced the number of dying cells in the optic lobe. The number was also reduced in ecdysone receptor-B1(EcR-B1) null mutant. These results suggest ecdysone promotes the optic lobe cell death.

Then we analyzed tissue-specific requirement of EcR for the cell death with RNAi. Ectopic expression of EcR RNAi in neurons resulted in the reduction of the number of dying cells, whereas the expression in glia resulted in the increase of the number. These results suggest ecdysone signal functions differently in neurons and glia and the optic lobe cell death may be regulated via proper combination of these functions.

We also checked the organization of the optic lobe in EcR-B1 null-mutant and the RNAi expressed animals and found marked abnormalities in the organization of their optic lobe. These results indicate cell death may be involved in the organization.

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30 Gradients of a Ubiquitin E3 Ligase Inhibitor and a Caspase Inhibitor Determine Differentiation or Death in Spermatids Yosef Kaplan, Liron Gibbs-Bar, Yossi Kalifa, Yael Feinstein-Rotkopf, Eli Arama* Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel

Caspases are executioners of apoptosis, but also participate in a variety of vital cellular processes. Here, we identified Soti, an inhibitor of the Cullin-3–based E3 ubiquitin ligase complex required for caspase activation during Drosophila spermatid terminal differentiation (individualization). We further provide genetic and biochemical evidence that the giant inhibitor of apoptosis-like protein dBruce is a target for the Cullin-3–based complex, and that Soti competes with dBruce for binding to Klhl10, the E3 substrate recruitment subunit. We then demonstrate that Soti is expressed in a subcellular gradient within spermatids, and in turn promotes proper formation of a similar dBruce gradient. Consequently, caspase activation occurs in an inverse graded fashion, such that the regions of the developing spermatid that are the last to individualize experience the lowest levels of activated caspases. These findings elucidate how the spatial regulation of caspase activation can permit caspase-dependent differentiation while preventing full-blown apoptosis.

This work has been recently published in Kaplan et al (2010) Gradients of a Ubiquitin E3 Ligase Inhibitor and a Caspase Inhibitor Determine Differentiation or Death in Spermatids. Dev. Cell 19, 160-173.

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31 Epigenetic Regulation of Stress-Induced Apoptosis Lei Zhou, Can Zhang, Nianwei Lin, Michael Novo Department of Molecular Genetics and Microbiology & UF Shands Cancer Center.University of Florida, Gainesville, FL

IAP-antagonists such as reaper and hid play pivotal role in mediating cell death during development and in response to cytotoxic stimuli. A 33kb genomic region upstream of reaper, the irradiation responsive enhancer region (IRER), is required for mediating the induction of both reaper and hid following irradiation. Interestingly, IRER is subject to epigenetic regulation. While it is open in undifferentiated cells during early embryogenesis, chromatins in IRER become enriched for H3K27Me3 and H3K9Me3 and form facultative heterochromatin in late embryogenesis. This epigenetic modification of IRER blocks the irradiation-responsiveness of both reaper and hid and renders the cells resistant to irradiation. Several Polycomb group (PcG) proteins are required for this epigenetic regulation of IRER (Zhang et al. Dev. Cell 2008).

Using an ubi-DsRed reporter knocked into IRER, we found that about 2% cells in

Drosophila larvae are DsRed positive, i.e. have an open IRER. These cells have higher levels of reaper expression and are much more sensitive to stress-induced cell death. In addition, mosaic clone analysis showed that cells deficient for IRER developed hyperplasia. Mechanistic analysis indicated that cells without IRER or with silenced IRER are much more resistant to cell competition-induced cell death than cells with open IRER. Overall, our data indicated that epigenetic regulation of pro-apoptotic genes plays an important role in controlling cellular sensitivity to developmental and environmental stresses.

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32 Controlled Cell Death of Secondary Cells in Accessory Gland of Drosophila Rumi Sakata1, Akihiko Kokuryo1, Kiichiro Taniguchi1, Ryunosuke Minami2, Hideki Nakagoshi2, Takashi Adachi-Yamada1

1) Department of Life Science, Faculty of Science, Gakushuin University, Japan 2) Graduate School of Natural Science and Technology, Okayama University, Japan

The accessory gland (AG) of Drosophila is a male-specific internal reproductive organ that

produces more than 80 kinds of peptides [Accessory gland proteins (Acps)] that affect female reproductive characters. All AG epithelial cells have two nuclei. There are two kinds of cells; one is the paved and polygonal “main cell,” and another is the scattered and round “secondary cell”. There are about 1000 main cells in the whole of the AG and about 40 secondary cells around its tip. Both types of cells secrete Acps which are transported with sperm into the female reproductive organs.

We found that, prior to binucleation without cytokinesis, the number of secondary cells decreases from 80 to 40 by cell death during the pupal stage. Consistently, around 30 hours after puparium formation, Caspase-3 was sporadically activated around the AG tip. The mechanism to induce this apoptosis was investigated by focusing on the proteins that work around the tip of the AG. At this developmental stage, we also detected the activities of two kinds of transmembrane proteins, Notch and Dachsous. There is a significant spatiotemporal overlap in the activation of these two proteins, and the activity of each protein is expected to regulate that of the other. The survival abilities of the secondary cell precursors are also seemed to be affected by both proteins. For example, forced expression of p35, a baculoviral anti-apoptotic protein, in the Notch-active cells resulted in an increase of the secondary cell number. We will further analyze the detailed roles of these proteins in activation of Caspase-3 in the regulation of secondary cell apoptosis.

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33 Excessive Dpp Signaling Induces Cardial Apoptosis through dTAK1 and dJNK during Late Embryogenesis of Drosophila Sheng-An Yang1.2, Ming-Tsan Su1 1) Department of Life Science, National Taiwan Normal University, TAIWAN 2) Institute of Cellular and Organismic Biology, Academia Sinica, TAIWAN

Previous study has suggested a cardiogenic function for the dorsal open group gene raw. Nevertheless, its role in the developing heart of Drosophila has not been fully elaborated. Here, we show that raw mutation produced an over-specification of cardial cells at stage 14, but these overproduced cells were mostly eliminated in late mutant embryos due to apoptosis. Aberrant decapentaplegic (dpp) signaling was responsible for the cardial phenotype found in raw mutants, because expression of dpp or constitutively activated thickven (tkvCA), the type I receptor of Dpp, induced a raw-like phenotype. Additionally, we show that dpp induced non-autonomous apoptosis-expression of a dominant negative form of Drosophila TAK1 (dTAK1DN) was able to suppress cell death in raw mutants or embryos overexpressing dpp. Importantly, we demonstrated that dpp can induce its own expression through dTAK1, which also leads to the hyperactivation of Drosophila JNK (DJNK). The hyperactivated DJNK was attributed to be the cause of Dpp/DTAK1-induced apoptosis because overexpression of a dominant negative DJNK, basket (bskDN), suppressed cell death induced by Dpp or DTAK1. Moreover, targeted overexpression of P35 or a dominant negative P53 (P53DN) blocked Dpp/DTAK1-induced apoptosis, and rescued heart cells under the raw mutation background. Our study has thus delineated the pro-apoptotic nature of ectopic Dpp during late stage cardiogenesis of Drosophila.

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34 Roles of the Kelch Repeat Protein KLHDC10 in Oxidative Stress-Induced ASK1 Activation Yusuke Sekine, Ryo Hatanaka, Takeshi Watanabe, Kohsuke Takeda, Hidenori Ichijo

Cell Signaling, Grad. Sch. Pharmaceut. Sci., Univ. of Tokyo, Japan

Apoptosis signal-regulating kinase 1 (ASK1), a member of the MAP3K family, constitutes the JNK and p38 MAPK cascades and plays pivotal roles in a wide variety of stress response including apoptosis and inflammation. Although ASK1 is activated by various stresses, signaling components that serve as activators of ASK1 have not been fully understood. From a genetic screen using Drosophila, we identified Slim as an upstream gene of the Drosophila ASK1 (DASK1)-p38 pathway. Overexpression of Slim activated DASK1 in S2 cells, and Kelch repeat domain containing 10 (KLHDC10), the mammalian counterpart of Slim, also activated ASK1 in HEK293 cells, suggesting that Slim and KLHDC10 serve as evolutionarily conserved activators of ASK1. Both Slim and KLHDC10 are Kelch repeat-containing proteins, but their molecular functions have been unknown. KLHDC10 was recently reported to be one of the substrate recognition subunits of the Cullin2-RING ubiquitin ligase (CRL2) complex. Our pull-down screen to search for KLHDC10-binding molecules revealed that KLHDC10 interacted not only with the CRL2 complex but also with Protein phosphatase 5 (PP5) that had been shown to negatively regulate ASK1 in response to oxidative stress. We also found that KLHDC10 suppressed PP5 activity in vitro and was required for oxidative stress-induced activation of the ASK1-p38 pathway in Neuro2a cells. These results suggest that KLHDC10 contributes to oxidative stress-induced ASK1 activation by suppressing PP5 activity.

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35 Mitochondrial Dysfunction and Oxidative Stress Contribute to the Pathogenesis of SCA12. Yu-Chun Wang, Chi-Mei Lee, Li-Chu Tung, Hsiu-Mei Hsieh-Li, Guey-Jen Lee-Chen, Ming-Tsan Su* Department of Life Science, National Taiwan Normal University, Taipei 11677, Taiwan, Republic of China

Spinal cerebellar ataxia type 12 (SCA12) has been attributed to the elevated expression of ppp2r2b. To better elucidate the pathomechanism of the neuronal disorder and to search for a pharmacological treatment, Drosophila models of SCA12 were generated by overexpression of a human ppp2r2b and its Drosophila homolog, tws. Ectopic expression of ppp2r2b or tws caused various pathological features, including neurodegeneration, apoptosis and shortened lifespan. More detailed analysis revealed that elevated ppp2r2b and tws induced fission of mitochondria accompanied by increases in cytosolic reactive oxygen species (ROS), cytochrome c (Cyt c) and caspase 3 activity. TEM revealed that fragmented mitochondria with disrupted cristae were engulfed by autophagosomes in photoreceptor neurons of flies overexpressing tws. Additionally, transgenic flies were more susceptible to oxidative injury induced by paraquat. By contrast, ectopic dSod2 expression and antioxidant treatment reduced ROS and caspase 3 activity, and extended the lifespan of the SCA12 fly model. In summary, our study demonstrates that oxidative stress induced by mitochondrial dysfunction plays a causal role in SCA12, and reduction of ROS is a potential therapeutic intervention for this neuropathy.

POSTER: NEURAL PHYSIOLOGY AND BEHAVIOR

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36 desaturase1 Plays an Essential Role in the Discrimination of Courtship Targets. Tetsuya Nojima1,2, Francois Bousquet1, Benjamin Houot1, Daisuke Yamamoto2, Jean-Francois Ferveur1 1) University of Burgundy, France 2) Graduate School of Life Sciences, Tohoku University, Japan

The desaturase1 (desat1) gene acts to produce sexually dimorphic composition of unsaturated cuticular hydrocarbons (CHs), which function as sex pheromones. desat1 mutants lack conspicuous sexual dimorphism in the composition of CHs, and mutant males are vigorously courted by wild-type males. Our previous studies have demonstrated that desat1 acts not only in the production but also in the perception of sex pheromones, as desat1 mutant males cannot discriminate between wild-type males and females as courtship targets. To elucidate which cells are required for the desat1-dependent sexual discrimination, we established transgenic fly strains, each of which carries different putative transcription regulatory region of the desat1 gene and the Gal4 coding sequence (desat1-Gal4). Here, we show that transgenic males could not discriminate between wild-type males and females, when UAS-desat1 RNAi was driven by one of the desat1-Gal4s, tentatively named RDio-Gal4. The expression of RDio-Gal4 was detected in subsets of neurons. We carried out the MARCM experiment for the rigorous identification of the RDio-Gal4-expressing neurons. This experiment revealed that RDio-Gal4 drives expression in several classes of olfactory receptor neurons, which include those innervating DA1 glomerulus in the antennal lobe. DA1 is significantly larger in males than in females, and involved in the processing of cis-vaccenyl acetate, a sex pheromone that inhibits male courtship. We also found the RDio-Gal4 expression in some interneuronal clusters in the brain, which are involved in male courtship behavior. These results will provide insights into how desaturation of hydrocarbons in the neurons contributes to sexual behavior


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37 RNAi Screening of nAChR Subunits that Mediated Nicotine-Induced Locomotor Activity Increase in Drosophila Jing Ren1, Jing-Han Sun2, Yun-Peng Zhang2, Ke Zhang1, Yan Li2, Aike Guo1,2 1) Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. 2) State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.

Affecting one third of the global adult population, tobacco use can lead to nicotine

addiction along with serious health problems, both physically and psychologically. Recent studies revealed that nicotine functions through certain nAChR subunits in mammalian nervous system. We established a locomotor behavior assay in Drosophila and characterized a stable increase of locomotor activity upon chronic nicotine treatment. By RNAi screening, we characterized two Drosophila nAChR subunits, nAChR-gfa (α7-like) and α96Ab (α2-like), responsible for this nicotine effect. In addition, we identify a RNA binding protein as a key target downstream of nAChR that mediated the nicotine-induced activity change. Further study on the regulatory signaling will advance our understanding on the molecular mechanisms of nicotine addiction.


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38 Sexually Dimorphic Projection Patterns of Taste Neurons on the Forelegs in Drosophila Ken-ichi Kimura1, Akira Urushizaki1, Chiaki Sato1, Rie Matsuda1, Daisuke Yamamoto2 1) Lab. Biol., Sapporo Campus, Hokkaido Univ. of Education, Sapporo 068-8642, Japan 2) Graduate School of Life Sci., Touhoku Univ., Sendai 980-8578, Japan

In Drosophila melanogaster, sex pheromone plays an important role in courtship behavior. Cuticular pheromone, which is received by the taste neurons on the male forelegs, has an important role to decide the initiation of the courtship behavior. Males have more chemosensory bristles on the tarsus of the foreleg than females. Sex differences in the projection pattern of the taste neurons are present. GFP expression driven by a poxn-Gal4 showed that the axon of the taste neurons pass through the midline in males, but not in females. Two sex determination genes, fruitless (fru) and doublesex (dsx), express in some taste neurons which showed the sexually dimorphic projections. A fru-Gal4 line labels two taste neurons out of four ones attaching with each chemosensory bristle. Applying MARCM method using the fru-Gal4, we identified the CNS projection pattern of each neuron on the tarsal segment. Results showed that the neurons with some specific bristles in males have male-typical projection. Furthermore, the projection area of fru and/or dsx expressing taste neurons was distinct from that of neurons involved in sweet or bitter taste. These results suggest that taste information possibly for pheromone is encoded by labeled line in the fru and/or dsx expressing taste neurons.


83

39 A Behavioural Odour-Similarity 'Space' in Larval Drosophila Chen Yi-chun1,2, Mishra Dushyant1,2, Schmitt Linda1, Schmuker Michael3,4, Gerber Bertram1,2 1) Department of Neurobiology and Genetics, Biozentrum, Universität Würzburg, Würzburg, Germany 2) Department of Genetics, Institute of Biology, Universität Leipzig, Leipzig, Germany 3) Neuroinformatics and Theoretical Neuroscience, Institute for Biology, Freie Universität Berlin, Berlin, Germany 4) Bernstein Center for Computational Neuroscience Berlin, Berlin, Germany

To provide a behaviour-based estimate of odour similarity in larval Drosophila, we use

four recognition-type experiments: (i) We train larvae to associate an odour with food, and then test whether they would regard another odour as the same as the trained one. (ii) We train larvae to associate an odour with food, and test whether they prefer the trained odour against a novel, non-trained one. (iii) We train larvae differentially to associate one odour with food, but not the other one, and test whether they prefer the rewarded against the non-rewarded odour. (iv) In an experiment like (iii), we test the larvae after a 30min-break. This yields a combined, task-independent estimate of perceived difference between odour-pairs. Comparing these perceived differences to published measures of physico-chemical difference reveals a weak correlation. A notable exception are 3-octanol and benzaldehyde, which are distinct in published accounts of chemical similarity, and in terms of their published sensory representation, but nevertheless are consistently regarded as the most similar of the ten odour pairs employed. It thus appears as if at least some aspects of olfactory perception are 'computed' in post-receptor circuits on the basis of sensory signals, rather than being immediately given by them.


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40 Functional Analysis of ALS2/Alsin in a Drosophila Familial Amyotrophic Lateral Sclerosis Model Tsutsumi Kanako1, Nobuhito Goda1, 2 and Nanami Senoo-Matsuda1,2

1) Department of Life Science and Medical Bio-Science, School of Advanced Science and Engineering, Waseda University, Tokyo, Japan 2) CREST, JST, Japan

Although many neurodegenerative diseases are induced by different responsible genes, these diseases might share common pathophysiological mechanism such as neurodegeneration, neuronal cell death, aggregation of abnormal protein, and abnormal energy metabolism.

Amyotrophic lateral sclerosis (ALS) is a progressive neuromuscular degenerative disease. It is characterized by progressive degeneration of motor neurons in the brain (upper neurons) and spinal cord (lower motor neurons). Recently several responsible genes of ALS have been identified and generated model animals for each disease, however the pathogenic mechanism is still unknown.

In this study, we have planned to analyze for physiological and pathological functions of ALS2/Alsin, the one of the responsible factors of autosomal juvenile onset recessive familial ALS. We established ALS model flies by neuronal tissue-specific knock down of Drosophila ALS2 (dals2).

We found shorter life spans and lower locomotor activities in pan-neuronal dals2 knockdown flies. Over-expression of dALS2 suppressed neuronal cell death and degeneration induced by reaper (rpr), pathogenic Machado-Joseph Disease (MJD) glutamine (Q) 78/MJD-Q78 protein, and Alzheimer disease-linked mutant of human tau. These results suggest that dALS2 acts as a neuronal anti-apoptotic and anti-degenerative factor.

We would also discuss our recent data that we have been performing genetic analysis between dALS and the downstream signaling.


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41 Modulation of Sphingolipids, Cholesterol and Saturated Fatty Acids Interact with the Neurodegeneration in the Drosophila blue cheese Mutant George Boo, Kathleen Amy Osborne, Joan Sim and Rachel Susan Kraut School of Biological Sciences, Nanyang Technological University, Singapore 637551

The accumulation of insoluble ubiquitinated protein aggregates characterizes many neurodegenerative diseases such as Alzheimer’s disease. Some of these diseases are associated with perturbation of sphingolipids and cholesterol homeostasis. To elucidate the association between aberrant metabolism of lipids and age-related neurodegeneration, we study the Drosophila neurodegenerative blue cheese (bchs) mutant. bchs is associated with lysosomal trafficking and autophagosomal degradation of aggregated proteins. In this study, pharmacological interventions as well as augmented cholesterol and fatty acid diets were used to assay possible interactions between lipid metabolism and the autophagic pathway which might bring about the bchs neurodegenerative phenotype. Cholesterol levels in wandering bchs third instars larvae, whose phenotypes that have either been exacerbated or ameliorated by different treatments, were enzymatically analyzed and quantified. Drugs associated with induction of autophagy such as Rapamycin, Metformin and Resveratrol rescued bchs neurodegenerative phenotype. Whereas drugs associated with suppression of autophagy such as Myriocin and Fumonisin B1 exacerbated bchs neurodegeneration. Cholesterol feeding led to excessive sterol accumulation in larvae and exacerbated the bchs neurodegenerative phenotype. Moreover, sphingolipids may be involved in this process; sphingolipid metabolic inhibitors such as Myriocin and Fumonisin B1 which lower ceramide levels in bchs mutants resulted in further exacerbation of the degenerative phenotype.


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42 Direct Expression of a Schizophrenia Susceptibility Gene, DISC1, Impairs Sleep Homeostasis, Associative Learning and Axonal Branching in Drosophila Kojiro Takayama1, Daisuke Tanaka1, Mai Ando1, Tetsuya Ando1, Ken Honjo1, Masami Shimoda2, Atsushi Kamiya3, Norio Ishida4, Minoru Saitoe5, Akira Sawa3, Katsuo Furukubo-Tokunaga1 1) Life and Environ. Sci., Univ. Tsukuba, Japan 2) Natl. Inst. Agrobiol. Sci., Tsukuba, Japan 3) Psychiatry Neurosci., Johns Hopkins Univ., Baltimore, USA 4) Advanced Inst. Industrial Sci. Tech., Tsukuba, Japan 5) Tokyo Met. Inst. Neurosci., Tokyo, Japan

Schizophrenia is a debilitating mental illness that affects 1% of the population worldwide.

Increasing evidences suggest that schizophrenia is a neurodevelopmental disorder with psychosis as a late symptom. Although its molecular etiology remains enigmatic, recent advances in psychiatric genetics identify a large number of risk factor genes that extend stress susceptibility by epistatic interactions. Disrupted-in-Schizophrenia-1 (DISC1), originally identified at the breakpoint of a chromosome (1;11)(q42.1; q14.3) translocation in a Scottish family with frequent mental disorders, is a promising susceptibility gene for both schizophrenia and affective disorders. We have introduced the human DISC1 gene in fruit flies to analyze its neuronal functions in vivo. Whereas DISC1 causes no gross anatomical abnormalities, overexpression of DISC1 in the mushroom body alters sleep-arousal state and impairs olfactory associative learning. Moreover, DISC1 regulates terminal axonal branching of mushroom body gamma neurons. A series of deletion constructs uncovered multiple functional domains involved in cytoplasmic and nuclear functions that might be important for the pathophysiology of this protein. Representing the first example of schizophrenia modeling in flies, our study demonstrates the potency of the Drosophila system for the analysis of the underlying genetic mechanisms of human mental diseases.


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43 Dysfunction of TBP Dependent Transcription may Contribute to the Pathogenesis of Tauopathy Wang, Hsiang-Yu, Su, Ming-Tsan

Department of Life Science, National Taiwan Normal University, Taipei, Taiwan.

Tauopathies are a group of neurodegenerative diseases which is characterized by abnormal deposition of the microtubule-associated protein tau. Previous studies found that the TATA box binding protein (TBP) is sequestered by tau-mediated neurofibrillary tangles (NFTs) in the brain of postmoterm AD patients, suggesting that down-regulation of TBP may play a role in tauopathy. Using Drosophila notum bristle as an assay system for tau induced toxicity, we found that notural bristles of the transgenic flies overexpressing tau can be modulated by the expression level of the endogenous TBP. Moreover, both transactivation and DNA binding ability of TBP were affected by tau. Our findings strongly suggested that ectopic tau can lead to dysfunction of TBP. Interestingly, loss of TBP function in flies also caused various age-dependent neurodegeneration, including formation of vacuole in brain, motor dysfunction and premature death. Since TBP controls virtually the transcription of all genes, to further demonstrate that transcription dysfunction is a causative factor of tauopathy, we treated tansgenic fly model with various HDAC inhibitors, including SAHA. We found that HDACi can alleviate effectively the toxicity of tau.


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44 Antennal Lobe VP Glomeruli are Responsible for Drosophila Hygro-Perception Li-An Chu*, Jason Sih-Yu Lai*, Chen-Wei Chu, Shiuan-Tze Wu, Ann-Shyn Chiang Institute of Biotechnology and Department of Life Science, National Tsing Hua University, Hsinchu, 30013, Taiwan, ROC

Ablation of the arista or the antenna third segment eliminates the humidity choices ability in Drosophila. However, the hygrosensory neurons at the antenna were not clearly identified. In this study, we identified two types of arista neurons are sent to the antennal lobe VP2 and VP3 glomeruli respectively. At the antenna third segment, we considered the sacculus neurons, which not involved in olfaction are hygrosensory neurons too.

We use a new technology called “Brainbow” to identify each sacculus neurons terminates at either VP1 or VP3 glomerulus. Blockage neural transmission of both arista-VP2 and sacculus-VP1,3 neurons show significant defect in behavior preferences to dry and moist air choices but not arista-VP3 neurons. Functional images show both arista and sacculus neurons are response to humidity changes. The findings provide direct evidences that antennal lobe VP glomeruli are responsible for hygro-perception. These studies reconstruct the first level of the hygrosensory system in the Drosophila brain.


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45 Degeneration of Optic Lamina Caused by Defective Endocytic Function in Glia Cells Yuan-Ming Lee1,2, Y. Henry Sun1,2. 1) Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 2) Departmentof Life Science and Institute of Genome Science, National Yang-Ming University, Taipei, Taiwan.

The visual system is composed of neurons and glial cells. In the optic lamina and medulla, there are also several distinct groups of glial cells. These are known to play a role in photoreceptor axonal projection and maintain the physiological function of the lamina monopolar neurons. We are interested in the function of glias in the adult visual system. By blocking the endocytic pathway by expressing the temperature-sensitive dominant-negative dynamin (shits1) in glia, the degenerative vacuoles formed in the optic lamina. Our analyses also suggest that the vacuoles are formed cell-autonomously by the epithelial and marginal glia in the optic lamina in MARCM experiment. The visual synaptic transmission was abolished in phototaxis and in ERG assays. The vacuolized lamina was rescued by the coexpression of dTOR but not anti-apoptotic p35. Therefore the endocytic function may be required for the glia to survive by inhibit caspase-independent or autophagy. Molecular definition of the autophagy features in degeneration neuropile is still studying.


90

46 Automated Real-Time Drosophila Behavioral Training and Analysis System Ming-Chin Wu1*, Li-An Chu3*, An-Kuo Hong3, Yen-Yin Lin4,5, Tsung-Ho Liu1, Chien-Chung Fu1,2,4, Ann-Shyn Chiang3,4 1) Department of Power Mechanical Engineering, National Tsing Hua University, Taiwan 2) Institute of Nanotechnology and Microsystems Engineering, National Tsing Hua University, Taiwan 3) Institute of Biotechnology, National Tsing Hua University, Taiwan 4) Brain Research Center, National Tsing Hua University, Taiwan 5) Department of Electrical Engineering, National Tsing Hua University, Taiwan

Here we develop an automated behavioral training system for Drosophila, the training system use method base on machine vision to track flies, extract wings pose and combine external stimulator—blue laser, through the method and external tool, let the training system not only accurately track flies trace and calculate wings angles but also according to researcher’s requirement to train assigned fly.

Courtship conditioning is the first social memory behavior in Drosophila. Using the behavioral training system, we can real-time analysis flies movement and feedback controlled the laser to train the assigned fly during courtship or only when fly’s wing extension. Punishments only showed with wing-extension make little difference of courtship index (CI), but with whole courtship process make a great decrease of CI.

The behavioral training system not only analyze the courtship index but also could analyze many others data, ex., longest courtship length, average courtship length, bout number of wing extension, and total time of wing extension. In the future training method can accord researcher’s requirement to add more different training method. We expect that our behavioral training system, which help scientist to explore more behavior knowledge in Drosophila.


91

47 Dissection of Visual Orientation in Drosophila Yanqiong Zhou, Zhefeng Gong, Li Liu

State Key Lab of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, 15 Da Tun Road, Chao Yang District, Beijing, 100101

Visual motor control is very important for insect to survive in nature. It begins with the visual perception and ends with motor control. But the intervening processes related to analyze, evaluate and code the visual input for motor control are especially critical. However, the neural mechanism of this procedure remains poorly understood.

We used a modified Buridan’s paradigm to explore the basic visual parameters involved in visual motor control in Drosophila. We found that the same pattern presented at different height had significantly different attractiveness to flies. But altering the visual angle of a strip in certain range didn’t change its attractiveness. And the contrast of a visual stimulus also affected the performance index of orientation preference. Wild type flies showed visual orientation toward edges between the blue and the green circumstance. At the same time wild type flies showed a slight preference for blue area as monitored by the performance index of regional preference, which imply that the color channel also participate in motor control. All our results indicate that different visual parameters affect different parts of motor control. We will use genetic tools to further analyze its underlying neural mechanism.


92

48 Screen for Genes Related with Drosophila Larval Photo-Behavior Liu Jiangqu Institute of Biophysics, Chinese Academy of Sciences

Photo-behavior is one of Drosophila larval innate behaviors. Wild type feeding Drosophila larvae tend to select to stay in the dark part when they face a dark-light choice. Drosophila larvae spend most of their time feeding by digging into food which can help keep them warm and wet. Negative photo-behavior is one of the important factors that help to keep larvae in food. However, opposite to feeding larvae, wandering larvae have positive photo-behavior which may take part in the process of pupation. This phenomenon implies that there should be some molecular basis underlying this transition.

I carry out a screen aiming at finding molecules functioning in Drosophila larval photo-behavior. More than 500 NP stocks are screened in dark/light selection paradigm. Early 3rd instars are used in experiments. The results are divided into 3 groups: movement disability (37), normal photo-behavior (360) and abnormal photo-behavior (115). These results show that there exists a balance between light and dark choice in larvae, and one (NP5) of these 5 stocks which have abnormal photo-behavior is picked out. P-element of NP5 inserts in an intron of kuafu. Qpcr results show that NP5 larvae have a decreased transcriptional level compared with control. Western blot assays also infer that this insertion reduces kuafu expression level. Consequently another 3 stocks (ln1, ln2, ln3) that have P-element insertion in kuafu are also tested. They all show abnormal photo-behavior. Qpcr and western blot experiments of these three stocks also imply that there exists a relationship between expression levels of kuafu and photo-behavior. I also generate RNAi and overexpression lines of kuafu. Primary rescue results imply that overexpression of kuafu can rescue abnormality.

In conclusion, I find a new gene kuafu which takes part in photo-behavior of Drosophila larvae.


93

49 The Identificaiton of PKG-related Genes in Drosophila Qionglin Peng, YiJin Wang, Haiyun Gong, Zhefeng Gong, Li Liu State Key Lab of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, 15 Da Tun Road, Chao Yang District, Beijing, 100101, China

cGMP-dependent protein kinase (PKG) is a critical signaling molecule with varied functions on behaviour regulation and synaptic plasticity in animals. Previous studies have found that PKG is required for visual learning and memory in the central complex body in Drosophila. However, the molecular basis of cGMP-PKG signaling in associative learning and memory is still unknown. Here we have set up a developmental and behavioral model for screening the PKG- related genes in Drosophila. Using this model, we have found some candidate genes, which may also involved in visual learning and memory via cGMP pathway. These results would provide us further clues for molecular mechanism of PKG functions.


94

50 Physiological Functions and Adaptive Roles of Drosophila Rhodopsin 7 Maki Maeda1,2, Keita Tsujimoto1,2, Osamu Nishimura1, Minako Izutsu1,2, Kiyokazu Agata2, Naoyuki Fuse1 1) Laboratory for Biodiversity, Global COE Program, Kyoto University, Japan 2) Laboratory for Molecular Developmental Biology, Kyoto University, Japan

In order to study adaptive evolution, a unique Drosophila strain, named the “Dark-fly”, has been kept under constant dark conditions for 56 years (1,400 generations). The whole genome sequence of the Dark-fly was determined (data of Izutsu et al.). We detected 40 genes carrying nonsense mutations in the Dark-fly genome and one of them is a light-receptor, Rhodopsin 7 (Rh7), gene. This mutation leads to truncation of the C-terminal 21 amino acids of Rh7 protein (483 amino acids for the wild-type protein). Since Rh7 is the only rhodopsin whose function is unknown and is highly conserved among the Drosophila genus, we examined the function of Rh7 in this study. Immuno-staining experiments using an enhancer-trap line suggested that Rh7 is not expressed in the compound eye, but in antennal JO-AB neurons involved in sound-sensing. RT-PCR experiments showed that Rh7 mRNA is indeed expressed in adult antennae. The deficient mutant with deletion around the Rh7 gene was defective for auditory-based behavior. Transposon-inserted lines in the Rh7 gene locus also showed defects in sound-sensing. These results suggest the involvement of Rh7 in the auditory signaling pathway and the integration of senses for light and sound. Interestingly, the Dark-fly seems to be more sensitive to sound than the wild-type flies. The causal relationship between the C-terminal truncation of Rh7 and the sensitive behavior of the Dark-fly will be elucidated in the future.


95

51 Network Analysis of Neural Computation in Drosophila Antennal Lobe Nan-Yow Chen1,2, Meng-Fu Maxwell Shih3, Jheng-Jian Wu3,6, Li-An Chu3, Tung-Hung Chueh4, Pei-Ling Liu4, Meng-Ying Chou4, Hao-Chiang Shao5, Henry Horng-Shing Lu4, Yung-Chang Chen5, Chi-Tin Shih6,7, Ting-Kuo Lee2, Ann-Shyn Chiang3,* 1) National Center for High-Performance Computing, Hsinchu, Taiwan 2) Institute of Physics, Academia Sinica, Nankang, Taipei, 115, Taiwan 3) Department of Life Science, National Tsing Hua University, Hsinchu, 30013, Taiwan 4) Institute of Statistics, National Chiao Tung University, Hsinchu 30010, Taiwan 5) Engineering and System Science group, Department of Electrical Engineering National Tsing Hua University, Hsinchu 30013, Taiwan 6) Department of Physics, Tunghai University, Taichung 40704, Taiwan 7) Physics Division, National Center for Theoretical Sciences, Hsinchu 30013, Taiwan

A lot of models have been proposed to simulate various powerful features of neural computation. The virtual network under these models could give us inspiration but not reality. Here we demonstrate a distinct way to investigate neural computation, from real hardwiring of neuron connection. Taking computation in the Drosophila olfactory system as example, we first extract stereotyped circuit of antennal lobe local neurons (LNs) from numerous individual-neuron images, and then analyze this circuit in terms of network via statistical tools. Functional silencing of genetically selected LNs changes the odor preference of flies as our network-based prediction, validating the connectivity of the network as well as the feasibility of our paradigm to decipher neural computation.


96

52 Heterotypic Gap Junctions between Two Mushroom Body Modulatory Neurons are Necessary for Drosophila Memory Formation Chia-Lin Wu1,7, Meng-Fu Maxwell Shih2,7, Jason Sih-Yu Lai2, Hsun-Ti Yang2, Glenn C. Turner4, Linyi Chen3, Ann-Shyn Chiang1,2,5,6,* 1) Brain Research Center, 2)Institute of Biotechnology, 3) Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 30013, Taiwan. 4) Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724, USA. 5) Genomics Research Center, Academia Sinica, Nankang, Taipei 11529, Taiwan. 6) Kavli Institute for Brain and Mind, University of California at San Diego, La Jolla, CA 92093-0526, USA. 7) These authors contributed equally to this work.

Gap junctions play an important role in nervous system function. By serving as connections that enable small molecules under 1kD in size, they play an important role in the regulation of neuronal metabolism and homeostasis. Gap junctions also electrically couple neurons, synchronizing the activity of neurons connected with such so-called electrical synapses. Although recent studies have linked gap junctions to memory formation, it remains unclear how neuronal gap junctions contribute to this process. Gap junctions are formed from hexameric hemichannels that link two cells across the extracellular space. These hexamers are composed of subunits from the connexin and pannexin gene families in chordates and the innexin (inx) gene family in invertebrates. Here we show that two modulatory neurons, the anterior paired lateral APL neurons and the dorsal paired medial DPM neurons, form heterotypic gap junctions within the mushroom body (MB), a learning and memory center in the Drosophila brain. Using RNAi-mediated knockdowns of inx7 and inx6 in the APL and DPM neurons respectively, we found that flies showed normal olfactory associative learning and intact anesthesia-resistant memory (ARM) but failed to form anesthesia-sensitive memory (ASM). Our results reveal that the heterotypic gap junctions between the APL and DPM neurons are an essential part of the MB circuitry for memory formation, potentially constituting a recurrent neural network to stabilize ASM.


97

53 Anatomical Characterization of Medulla Tangential Neurons in the Adult Drosophila Optic Lobe An-Lun Chin1, Ann-Shyn Chiang1,2* 1) Brain Research Center, 2) Institute of Biotechnology, National Tsing Hua University, Hsinchu 30013, Taiwan.

The second dipteran optic neuropil, the medulla, processes the combination of chromatic and achromatic visual signals by two crossing systems: a class of various retinotopically columnar neurons between the medulla and the upstream (the lamina) or downstream (the lobula complex) optic neuropil, and a class of medulla tangential neurons (Mts) that connect the whole or partial of specific medulla strata with the central brain or with the contralateral optic lobe. The morphology and function of dipteran Mts are seldomly discussed, restricting our knowledge about the rule of the medulla in the dipteran visual system.

We screened 329 Mts from FlyCircuit, an image database of adult Drosophila brain wiring maps. They were categorized into 5 groups by their innervating patterns in the central brain: 1) Mtis that only innervate in the medulla tangentially as interneurons; 2) sMt-VMPs that converge signals from different visual-fields in the medulla to the posterior ventralmedial protocerebrum (VMP); 3) AL7SMts that share the signature of their partial innervation in the accessory medulla (aMe), the proximal layer of the lobula (L7 layer), and the lateral edge of superpeduncular protocerebrum (SPP); 4) other unilateral Mts that project to various regions of central brain from distinct medulla tangential elements; 5) cMts that project deurites centrifugally from central brain to different forms of medulla tangential elements. Anatomical study of Mts explored the relationships between the medulla and the whole brain system, providing a novel perspective of Drosophila visual outputs from the optic lobe.


98

54 Semi-Automated Reconstruction of Synaptic Circuits in the Fly’s Medulla Shinya Takemura, Shiv Vitaladevuni, Lou Scheffer, Aljoscha Nern, Zhiyuan Lu, Ian Meinertzhagen, Dmitri Chklovskii Janelia Farm Research Campus, HHMI, Ashburn, VA, USA

Recent serial-section EM (ssEM) studies in the fly’s visual system reveal the synaptic contacts made by input terminals in the medulla, the second optic neuropile. Even so, the synaptic contributions made by most other neurons in the medulla remain unclear. The principle obstacle is that manual tracing of neuron profiles is time-consuming. For example it took a couple of month to trace out a single medulla cell using wholly manual methods. The chief reason is that medulla neurons have lots of tiny neurites that approximate both the size of a synaptic vesicle and the thickness of an ultrathin section (<40 nm) and that render this neuropile densely complex. We are therefore establishing more efficient ways to reconstruct neurons in 3D semi-automatically as precisely as by manual reconstruction. Here as a test case we apply a semi-automated EM reconstruction pipeline developed at Janelia Farm to the medulla column.

A single medulla column has been reconstructed within a 10 x 10 x 50 µm3 volume. We have reconstructed 45 cells in this column, including 10 input terminals and 35 columnar neurons having an axon parallel to the column axis. Most are transmedulla (Tm) cells that project to the lobula, and medulla intrinsic (Mi) neurons that connect distal with proximal medulla strata. We identified 6 Tm-, 3 Mi- and 1 Dm neurons unambiguously, but final identification of other cells must await reconstruction of their more widely-extending neurites. Identification will be informed when genetic reporters have provided the total library of cell types and their distribution across the column array. Final identification will require close comparisons between ssEM and confocal reconstructions of each individual cell type.


99

55 SCA17 is Resulted from Gain of Toxicity Function of Mutant TBP and Loss Hsu, Tun-Chieh, Su, Ming-Tsan

Department of Life Science, National Taiwan Normal University, Taipei, Taiwan.

TATA box binding protein (TBP) has been implicated in many polygluatmine (polyQ) induced neuropathies as it is sequestered and inactivated in polyQ containing inclusions. Unlike most polyQ mediated neurodegenerations, spinocerebellar ataxia 17 (SCA17) is resulted from the abnormally expanded polyQ tract of TBP itself. Since transcription dysfunction has been demonstrated to be the contributing factor of many neurodegenerations, and TBP is the most critical transcription factor for all three RNA polymerases. To investigate if deactivation of TBP presents a common pathogenic mechanism of polyQ mediated neurodegenerations, including SCA17. Herein, Drosophila models for human SCA17 were first generated by target overexpression of pathogenic TBPs. Overexpression of TBP with 109 polyglutamine residues (TBP-109Q), forms protein aggregations. Electrophoretic mobility shift assay revealed that normal TBP-36Q, binds TATA box stronger than mutant TBP-109Q. Moreover, mutant TBP can interfere with the transactivation ability of normal TBP. Phenotypic analysis also revealed that down-regulation of dTBP function leads to SCA17 like phenotype in animals. We are currently investigating whether the pathological phenotypes of TBP-109Q are enhanced under dTBP mutant background.


100

56 Expression of dTRPA1 is Related to Extension of Drosophila Life Span Young Eun Lee, Donggi Paik, Joong-Jean Park Department of Physiology, College of Medicine, Korea University 126-1 Anam-Dong 5 Ga, Seongbuk-Gu, Seoul 136-705 Korea

Temperature is one of the environmental factors that induce changes of physiological responses and behaviors in many species. Fruit flies that are raised at lower or higher temperature than room temperature show longer or shorter life span, respectively. It is considered that the metabolic rates of the flies at different temperatures should be affected by the different temperatures to alter eventually longevity. However, the mechanisms of action of temperature on life span remain unclear. We hypothesized that life spans should be under control of thermo-sensing processes. Flies express several transient receptor potential (TRP) ion channels that transducer senses of sound, temperature, pain, and environmental irritants. Particularly, dTRPA1 is activated in the anterior neurons at the temperature range of 25 to 29°C. We performed the life span analysis of the fl ies that were isogenized to different wild-type flies and have a deficit function of dTRPA1 at 25 and 28°C. We found that repressing dTRPA1 expression consistently extended fly life span in diverse genetic backgrounds at all temperatures tested. Our results suggest that temperature perception should influence the longevity of Drosophila. We are now searching for possible causes of this effect and links with other known longevity pathways.

[This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2010-0015742)]


101

57 Functional Interactions between Olfactory Receptor Neurons Housed in the Same Sensillum Chih-Ying Su, John R. Carlson MCDB Department, Yale University

A unique anatomical feature of insect olfaction is that olfactory receptor neurons (ORNs) are compartmentalized in sensory hairs (sensilla). In Drosophila, most sensilla contain two ORNs, which are stereotypically grouped based on the specific olfactory receptor that each ORN expresses. The individual ORNs housed in a single sensillum often respond to different odorants. However, odor mixtures may either activate or inhibit one or both of the grouped ORNs. Here, we used single-unit electrophysiology to determine the functional significance of ORN compartmentalization. We observed that activation of one ORN influences the activity of another ORN in the same sensillum when multiple odorants are present. We observed this ORN crosstalk in multiple different sensillar types, including olfactory sensilla in the malaria mosquito Anopheles gambiae. In addition, we could recapitulate this ORN crosstalk when one ORN is directly activated by blue light by means of optogenetics. Next, using a combination of pharmacological and genetic approaches, we investigated the mechanism underlying this ORN crosstalk. Our results suggest functional implications of stereotyped ORN compartmentalization that have not been previously explored and that may bring new insights into the principles of odor coding in the periphery.


102

58 Dual-Labeling Method for Electron Microscopy to Characterize Synaptic Connectivity Using Genetically Encoded Fluorescent Reporters in Drosophila Nobuaki Tanaka1,2,3, Mark Stopfer3

1) Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Japan 2) Career Path Promotion Unit for Young Life Scientists, Kyoto University, Japan 3) National Institutes of Health, USA

Light and electron microscopy (LM and EM) both offer important advantages for

characterizing neuronal circuitry in intact brains: LM can reveal the general patterns neurons trace between brain areas, and EM can confirm synaptic connections between identified neurons within a small area. In a few species, genetic labeling with fluorescent proteins has been used with LM to visualize many kinds of neurons and to analyze their morphologies and projection patterns. However, combining these large-scale patterns with the fine detail available in EM analysis has been a technical challenge.

To analyze the synaptic connectivity of neurons expressing fluorescent markers with EM, we developed a dual-labeling method for use with pre-embedded brains. In Drosophila expressing genetic labels and also injected with markers we visualized synaptic connections among two populations of neurons in the AL, one of which has been shown to mediate a specific function, odor evoked neural oscillation.


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59 Context Dependent Odor-guided Behavioral Responses in Drosophila Megumi Mochizuki, Aki Ejima CPLS, Kyoto University, Japan

Chemosensory cues provide an animal with important environmental information for its survival and reproduction. Using olfactory cues, a fruit fly, Drosophila melanogaster, is navigated to an "appropriate" location (Fuyama 1975) and evaluates a potential mating partner (Gailey et al. 1986). Recent physiological and anatomical studies on the olfactory system shed lights on how the olfactory information is processed in the animal brain, however, most of the behavioral and physiological experiments were done with monomolecular odorants and little is known about how the information of a natural odor, a cocktail of multiple molecules is perceived by an animal and leads to the corresponding behavioral outputs (Su et al. 2009 review).

Previously we reported that the presence of food enhanced pheromone-responsive courtship inhibition, suggesting that the odor information was modified by the environmental context (Griffith & Ejima 2009). Turner and Ray (2009) showed that some food odorants inhibited CO2-responsive avoidance behavior, allowing "more appropriate" decision-making, e.g. staying on a ripe fruit which emitted high CO2. It was also reported that copulation experience changed females' preference for acetic acid (Joseph et al. 2009), indicating that the odor-guided behavioral responses were under the control of various contextual factors.

In order to investigate how the behavioral decision is made in the complex environment, we performed odor-guided trap assay under various experimental contexts and found that a fly shows different preference for acetic acid according to the odor background, reproductive and hunger status of the animals.


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60 Memory Formation by Dopamine Neurons Yoshinori Aso, Igor Siwanowicz, Chang Liu, Hiromu Tanimoto* Max-Planck-Institut für Neurobiologie, Am Klopferspitz 18, D-82152 Martinsried, Germany

A paired presentation of an odour and electric shock induces aversive odour memory in Drosophila melanogaster. Electric shock reinforcement is mediated by dopaminergic neurons, and it converges with the odour signal in the mushroom body (MB). Dopamine is synthesized in ~280 neurons that form distinct cell clusters and is involved in a variety of brain functions. Each dopaminergic cluster contains multiple types of dopaminergic neurons with different projections and physiological characteristics. Functional understanding of the circuit for aversive reinforcement and memory, therefore, requires cellular identification. Here we identified different types of dopaminergic neurons essential for signalling punishment of the electric shock. Since these neurons terminate in the restricted part of the mushroom body, the synapses undergoing associative plasticity are localized along the axonal trajectory of the Kenyon cells.


105

61 Genetic Dissection of the Sleep-Feeding Conflict Alex C. Keene Department of Biology, New York University

Sleep is affected by a number of environmental factors including light, heat, stress, social interaction and nutrition. Recent studies using Drosophila have identified genes and neurons regulating sleep, but the involvement of environmental factors remains relatively unexplored. In mammals there are strong interactions between the neural systems controlling sleep and feeding: sleep deprivation increases appetite and weight gain, while starvation suppresses sleep.

Our findings demonstrate that Drosophila potently suppress sleep during starvation and this occurs independently of light cues. It was previously shown that the mushroom bodies are required for sleep, but we find that they are dispensable for this sleep-feeding interaction. We find that disrupting function of the circadian genes Clock (Clk) and cycle (cyc) increases sleep-suppression during starvation. Silencing Clk/cyc expressing neurons phenocopies the hyper-responsiveness of Clk and cyc mutants. While disrupting Clk function in all circadian neurons hypersensitizes sleep to starvation, there is no effect of this manipulation in pacemaker neurons, suggesting that a subpopulation of non-PDF expressing Clk/cyc cells promote sleep during starvation. Thus we have uncovered an additional role for these clock genes in modulating an interaction between two homeostatically regulated behaviors.

We have also initiated a large-scale RNAi-based screen by pan-neuronally targeting neurally expressed genes. To date we have screened over 1,400 for sleep during sated and starved states. We are currently characterizing lines that result in flies that are hypersensitive to food deprivation or flies that fail to suppress sleep in response to food deprivation. We hope that these experiments will provide a novel approach investigating interactions between innate behaviors and further our understanding of the neural mechanisms underlying hierarchical control of homeostatically regulated behaviors.


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62 Two Pairs of Neurons Control Drosophila Larval Light Preference Zhefeng Gong1, Jiangqu Liu1, Chao Guo1, Yanqiong Zhou1, Yan Teng2, Li Liu1

1) State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, P.R. China 2) Protein Science Core Facility Center, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, P.R. China

Appropriate preferences for light or dark conditions can be crucial for an animal’s survival.

Innate light preferences are not static in some animals, including the fruitfly Drosophila melanogaster, which prefers darkness in the feeding larval stage but prefers light in adulthood.

To elucidate the neural circuit underlying light preference, we examined the neurons involved in larval phototactic behavior by disrupting neuronal functions. After screening a batch of Gal4 lines and comparing the expression patterns of these Gal4 lines, we found that blocking two pairs of isomorphic neurons (NP394 neurons) in the central brain was able to reverte the larval light preference from photophobic to photophilic. These neurons were found to in close proximity to pdf-expressing lateral neurons, which are innervated by larval photoreceptors. Furthermore, we proved that they might form synaptic contact with the help of GRASP (GFP reconstitution across synaptic partners) technique. In addition, the NP394 neurons were responsive to light stimulation in functional calcium imaging assay. When the pdf neurons were ablated, the calcium response in NP394 neurons upon light stimulation was stronger and much faster, suggesting that pdf neurons deliver inhibitory signals to NP394 neurons. The behavioral implication of such inhibitory effect is not clear so far.

Our results revealed a neural mechanism that could enable the adjustment of animals’ response strategies to environmental stimuli according to biological needs.


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63 Novel Cytochrome P450, cyp6a17, is Required for Temperature Preference Behavior in Drosophila. Jongkyun Kang1, Jaeseob Kim2, Kwang-Wook Choi1

1) Department of Biological Sciences, Graduate School of Nanoscience and Technology, KAIST, Korea 2) Aprogen Inc, Korea

Perception of temperature is an important brain function for organisms to survive.

Evidence suggests that temperature preference behavior (TPB) in Drosophila melanogaster, one of poikilothermal animals, is regulated by cAMP-dependent protein kinase (PKA) signaling in mushroom bodies of the brain. However, downstream targets for the PKA signaling in this behavior have not been identified.

From a genome-wide search for genes regulated by PKA activity in the mushroom bodies, we identified the cyp6a17 Cytochrome P450 gene as a new target for PKA. Our detailed analysis of mutants by genetic, molecular and behavioral assays shows that cyp6a17 is essential for temperature preference behavior. cyp6a17 expression is enriched in mushroom bodies of the adult brain. Tissue-specific knockdown and rescue experiments demonstrate that cyp6a17 is required in the mushroom bodies for normal temperature preference behavior.

This is the first study to show the expression of a cytochrome P450 gene in the mushroom bodies and its regulation by PKA. Most significantly, cyp6a17 is the first cytochrome P450 gene identified as a key factor for temperature preference behavior. Taken together, this study provides novel insights into the mechanism how PKA signaling regulates the temperature preference behavior.


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64 Propagation of Hygrosensory Information in the Drosophila Brain

Jason Sih-Yu Lai1*, Li-An Chu1*, Shyn-Jie Lou1, Chen-Wei Chu1, Christopher Masser2, Salil Bidaye2, Barry J. Dickson2, Ann-Shyn Chiang1 1) Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan. 2) Institute of Molecular Pathology (IMP) Dr. Bohr-gasse 7, A-1030 Vienna, Austria

Many animals are able to sense humidity, and the hygrosensory systems in different species are critical in multiple purposes, such as food searching, egg laying, and mating. In Drosophila, the arista is necessary for hygrosensation. Ablation of the arista disrupts the hygrosensing behavior1, and these aristal sensilla terminate at the antennal lobe VP2 and VP3 glomeruli in the antennal lobe2. To understand better the circuitry bases of the hygrosensory system, we searched for downstream neurons of the antennal lobe VP regions, which include VP1, VP2 and VP3 gloemrulus. We identified two types of projection neurons extended from the VP regions which involve hygrosensing, and exhibit different calcium responses during humidity changes. Genetically silencing only the type I VP projection neurons were sufficient to eliminate hygrosensing behavior, and artificially activated these neurons were sufficient to mimic dry air behavioral response, whereas the type II VP projection neurons are not necessary for hygrosensing behavior. The type I VP projection neurons terminate at multiple regions: the mushroom body calyx, superpeduncular protocerebrum, ventral lateral protocerebrum and posterior caudo-ventralateral protocerebrum. Since we verified that the mushroom body neurons are not involved in hygrosensing behavior, the other terminal regions might play important roles in humidity preferences. Our finding demonstrates the humidity processing regions in the brain, and suggests that the antennal lobe VP regions may function as the first level processing center.


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65 Effect of Natural Products and Synthetic Compounds on Aβ-induced Neurodegeneration in Drosophila melanogaster: A Research Proposal Vikneswaran Murugaiyah

School of Pharmaceutical Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia

Aggregation of the amyloid β (Aβ) peptides and tau proteins are pathological hallmarks in

Alzheimer’s disease (AD). Thus, there is a great interest in search for disease modifying agents that could reduce or prevent Aβ peptides-induced neurodegeneration. There are many animal models for studies of AD such as nematodes, fly, rodents and non-human primates. Rodents and primates serve as ideal animal models but due to ethical and animal welfare issues, their use for screening potential therapeutic agents is very limited. In contrast, the use of in vitro test system has a limitation of inability to characterize the behavioral changes. Based on these shortcomings, Drosophila melanogaster is proposed as an animal model for the purpose of discovery of AD modifying agents from natural products (derived from Malaysian medicinal plants) or synthetic compounds. Although anatomically different from human, fundamental molecular pathways in Aβ related neurodegenerations are highly conserved in Drosophila. Several transgenic flies are available to study human Aβ peptide-induced amyloid formation and neurodegeneration. The expression of Aβ amyloid in Drosophila results in reduced longevity, locomotor deficits and impaired memory. This model organism will be employed to study the effect of candidate compounds at various points of pathogenic cascade: (i) Aβ amyloid generation (targeting enzymes involved in formation of Aβ such as β- and γ-secretases or enzymes involved in degradation of Aβ such as neprilysin) and (ii) Aβ aggregation. Behavioral aspects to study memory in the fly will be carried out using olfactory associative learning assay, whereas the effect on Aβ-induced neurodegeneration will be evaluated by climbing assay and survival assay.

POSTER: NEUROGENETICS AND NEURAL DEVELOPMENT

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66 Mechanism of Paraquat Toxicity in a Drosophila Parkinsonism Model Sarat Chandra Yenisetti1,2, Juan Antonio Navarro2, Jose Antonio Botella2, Stephan Schneuwly2

1) Department of Zoology, Nagaland University, Lumami, Mokok Chung, Nagaland, India. 2) Institute of Zoology, University of Regensburg, Regensburg, Germany.

Parkinsonism is a complex syndrome resulting from a number of different pathogeneses that give rise to various degrees of motor deficits, akinesia, rigidity, tremors and postural disturbance along with other neurological deficits. The pathological hallmark of Parkinson’s disease (PD) is the selective loss of dopaminergic neurons in the substantia nigra. Recently, pesticide exposure has been found to be a risk factor for sporadic PD. The herbicide paraquat has been shown to cause PD related pathological traits under chronic exposure in animal models; however, the mechanism of its toxicity to dopaminergic neurons has not been yet deciphered.

Here we study the mechanism of paraquat toxicity using Drosophila. It has been assumed that paraquat, like the structurally similar MPP+, is transported into dopamine neurons by the dopamine transporter (DAT). Recently this hypothesis has been challenged and the issue remains controversial. In our study, decreasing the dopamine transporter (DAT) results in protection against paraquat neurotoxicity. We also find that overexpression of DAT in non-dopaminergic neurons makes them susceptible to the herbicide. Dopamine (DA) has been found to mediate toxicity of α-synuclein and rotenone in cell culture and in vivo models. Unexpectedly, we observe that the reduction of total DA levels does not confer protection against paraquat. Moreover, elevating the level of cytosolic DA by impairing its transport into vesicles effectively protects DA neurons against paraquat toxicity. These apparently contradictory results could be explained by a compensatory regulation of DAT function affecting the transport of paraquat.

Our findings are the first to demonstrate in vivo the role of DAT in the mechanism of paraquat toxicity. This knowledge will facilitate the identification of target molecules in treating the Parkinson’s disease and will be helpful in testing potential neuroprotective therapeutics.


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67 show Regulates Mira Localization and Spindle Orientation in Drosophila Neural Stem Cells Xiu Zhou1, Lihui Goh2, Jin Yang1, Xiaohang Yang1. 1) College of Life Sciences, Zhejiang University 2) Institute of Molecular and Cell Biology, ASTAR, Singapore 138673

Asymmetric cell division generates two daughters with non-identical cell fate. In Drosophila, neural stem cells, neuroblasts (NBs), divide and produce one large NB and one small ganglion mother cell (GMC). The mechanism of asymmetric division has been best-studied in the fruit fly. Two groups proteins are involved in asymmetric division. Baz，Par6 , aPKC , Insc , Pins and Gαi , as well as Loco, are apically enriched; while cell fate determinants such as Mira, Brat, Pros, Numb and Pon concentrate on the basal cortex of the mitotic cells. To identify novel genes involved in asymmetric division, we have screened a collection of RNAi lines with Mira as a maker. show is one of the candidate genes isolated from the screen. When show expression was inhibited, Mira localization and spindle orientation were both affected. We are generating show mutants by remobilization of P-element inserted near the show gene. The phenotypic analysis data will be presented in the post.


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68 RNAi Based Screen to Identify Genetic Modifiers of Amyotrophic Lateral Sclerosis 8 Protein, VAPB. Senthilkumar D1, Hemant Verma1, Girish Ratnaparkhi1, Ryu Ueda2, Anuradha Ratnaparkhi3. 1) Indian Institute of Science Education and Research, Pune, India. 2) National Institute of Genetics, Mishima, Japan. 3) Agharkar Research Institute, Pune, India.

Amyotrophic Lateral Sclerosis (ALS, Lou Gehrig’s disease) is a progressive neurodegenerative disorder characterized by the death of motor neurons. Most cases of ALS occur sporadically, with 5% of the cases familial, with known genetic causes. In many of the familial cases, the disease is inherited due to a dominant mutation. One such dominant missense mutation, P56S, was identified in vesicle trafficking protein VAMP (Vesicle Associated Membrane Protein) Associated Protein B (VAPB/VAP-33) (Nishimura et. al, 2004, Am. J. Hum. Genet. 75:822). VAPB is an integral trans-membrane protein of endoplasmic reticulum implicated in a variety of functions such as lipid transport, Ephrin signaling and unfolded protein response. The mutant VAPB behaves in a dominant negative manner and sequesters the wild type protein into cytoplasmic inclusions. Using a Drosophila model for ALS (Ratnaparkhi et. al, 2008, PLOS One 3:2334) we are undertaking an RNAi based genetic screen to identify genetic modifiers of VAPB function. Preliminary results from this screen will be discussed.


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69 Sadari is Involved in Glial Cell Differentiation during Drosophila Embryogenesis Kumbok Ryu, Sang-Hak Jeon*

Department of Biology Education, Seoul National University, Korea

Glial cells perform critical roles in nervous system development. They play a variety of important functions such as nourishment, homeostasis, and insulation of neurons. Although many studies of glia are constantly generated, only a few genes have been characterized.

Here, we confirm the function and role of sadari which is formally called CG11902. Transcription of sadari detected from stage 11 and decrease stage 15. Our immunostaining result using anti-Repo antibody indicated that sadari is expressed in some glial cells. We also made anti-Sadari antibody using amino acids sequence from 71 to 210. The expression pattern of Sadari antibody is same with antisense RNA probe. Sadari is expressed in the nucleus. Minos elemnet is inserted into sadari’s fourth exon. It brings about embryonic sub-lethality, partial loss and abnormal localization of glial cells. These results suggest that sadari is involved in glial cell development.


114

70 A Potential Role of Dendro-Dendritic Interaction in Remodeling into an Adult-type Arbor Ritsuko Suyama, Daisuke Satoh, Tadashi Uemura Graduate School of Biostudies, Kyoto University

During the postembryonic phase, the nervous system is reorganized at multiple structural levels. This reorganization of initial neural circuits is critical to support a wide range of animal behaviours under a variety of environmental contexts. In Drosophila, conversions from the larva-to-adult behaviors rely on, at least in part, remodeling of the circuits, which is accomplished by pruning of axons or dendrites and concomitant or subsequent growth of these processes in spatially distinct patterns.

We previously identified dendritic arborization (da) neurons in the adult abdomen and traced origins of individual cells back to the larval stage; and this has provided the basis of investigating cellular and molecular mechanisms controlling the dendritic remodeling. We then focused on the ventral neuron v’ada and its dorsal neighbors ldaA/ldaA-like in each hemisegment and observed re-growth of their dendrites during pupal stages by time-lapse analysis. At 20-30 hr after puparium formation (APF), a number of filopodia underwent extension and retraction from the cell body of v’ada, which then generated thick primary dendrites by 40 hr APF. At 45-50 hr APF, dorsal dendrites of v’ada encountered ventral ones of ldaA/ldaA-like; and the branches of v’ada started growing on those of ldaA/ldaA-like. This dendro-dendritic interaction seemed to promote the growth of v’ada, as suggested by the fact that size and complexity of v’ada dendritic arbors are dominated over those of ldaA/ldaA-like at around 70 hr APF onwards. To address this hypothetical role of the interaction, we are attempting to impair arborization of ldaA/ldaA-like selectively or ablate ldaA/ldaA-like and examine each effect on the formation of the adult-type dendritic arbor of v’ada.


115

71 In Situ Fluorescence Correlation Spectroscopy Measurement of Membrane Diffusion and Fluidity in the Central Nervous System of Drosophila Embryos Teo Lin Shin1, Rachel Kraut2, Matt Oswald3, Sean Sweeney3, Thorsten Wohland1 1) Department of Chemistry, National University of Singapore, Singapore 2) Department of Biological Sciences, Nanyang Technological University, Singapore 3) Department of Biology, University of York, UK.

We studied the biophysical properties of living neuronal membranes in the central nervous system of Drosophila melanogaster using fluorescence correlation spectroscopy (FCS). To our knowledge, this is the first time FCS has been used to examine membrane properties in vivo in Drosophila. In order to establish the method, we first investigated fluorescent proteins expressed using a specific motor neuron driver in the embryonic central nervous system (CNS). We examined GFP-tagged markers with distinct subcellular localizations—either membrane bound or cytoplasmic—in order to determine whether expected differences in diffusion properties could be detected that correlate with subcellular location. In particular we are interested in discovering whether interference with sphingolipid metabolism affects neuronal membrane fluidity, and is reflected in the diffusion properties of membrane markers. To test this, we use the raft marker, Flotillin-2-GFP and compare it to other markers that are not expected to be raft localized. Preliminary results show that over expression of ceramidase and external treatment to disrupt the cytoskeleton change the diffusion properties of Flotillin-2 in the plasma membrane. This approach promises to shed light on the biophysical properties of cellular membranes in fly models of human degenerative diseases such as Alzheimer’s and lipid storage diseases.


116

72 Gustatory Receptor Expression in Abdominal Internal Organs of Drosophila Jeong Ho Park, Jae Young Kwon Department of Biological Sciences, Sungkyunkwan University, Korea

Previous work on the Drosophila gustatory receptor gene (Gr) family has mainly focused on expression and functions in external organs, such as the legs, the pharyngeal organs, as well as the labellum. We hypothesized that the Gr genes may have unidentified roles in the internal organs of the adult fly. As a first step towards identifying potential new roles, we are identifying and characterizing the expression patterns of all Gr genes in the internal organs. We have used Gr-GAL4 transgenes to examine the expression of Gr genes in the Drosophila abdomen, including the intestine and reproductive organs. 30 of the 68 Gr genes were observed to express in the abdomen, and Gr-GAL4 transgene-expressing neuronal processes in the intestine, reproductive organs, and abdominal wall appear to project to the abdominal ganglion, a part of the central nervous system. In the intestine, Grs showed specific patterns of expression in the enterocytes, enteroendocrine cells, or intestinal stem cells, suggesting that the Grs have various roles in the intestine such as detecting nutrients or harmful substances to contribute to feeding, homeostasis, or defense mechanisms. Expression in reproductive organs or abdominal neurons was observed for certain Grs, suggesting atypical functions in addition to the conventional taste sensing functions.


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73 Identification of a Novel Suppressor of Crumbs and Its Role in Growth Regulation Eunbyul Yeom, Kwang-Wook Choi Department of Biological Sciences, Graduate School of Nanoscience and Technology, KAIST, Daejeon, Korea

Crumbs (Crb) is a transmembrane protein which regulates the apical-basal polarity in Drosophila. Recent studies have shown that Crb is also involved in growth control by regulating the Hippo signaling pathway. To gain further insights into the intracellular function of Crb, we screened a set of RNAi lines for genetic modifiers of the eye phenotype caused by overexpression of the Crb intracellular domain (Crbintra). RNAi knockdown of one of these modifiers labeled as Su(Crb) strongly suppressed the Crbintra phenotype. The C-terminal half of this gene is highly conserved in metazoan species, but no specific function has been identified yet. To investigate the biological function of Su(Crb), we generated loss-of-function mutant lines by imprecise excision of an adjacent P-element insert. Two mutant alleles Su(Crb)∆52 and Su(Crb)∆182, are likely to be null, as they are deficient in almost entire coding sequence. Partial reduction of Su(Crb) gene function in Su(Crb)/+ heterozygotes also suppressed the Crbintra eye phenotypes. These data suggest that the Crb eye phenotype is sensitive to the level or activity of Su(Crb) and that the genetic interaction between Crb and Su(Crb) is specific. Su(Crb) mutants are semi-lethal, as they die during different stages while adult escapers have smaller body sizes. Dying mutant animals show severe developmental defects. Taken together, our study suggests that Su(Crb) is a novel component that functions in the Crb pathway necessary for growth regulation.


118

74 Role of Drosophila kinesin-II and Armadillo in Cell Polarity and Wingless Signaling Thuong Linh Vuong1, Bibhash Mukhopadhyay2, Kwang-Wook Choi1

1) Department of Biological Sciences, KAIST, Graduate School of Nanoscience and Technology, Daejeon, Korea 2) Program in Developmental Biology, Baylor College of Medicine, Houston, Texas

The Drosophila Kinesin II motor subunit encoded by Klp64D is involved in the axonal transportation of choline acetyltransferase and the formation of chordotonal sensory cilia (Sarpal et al., 2003). Our previous study revealed an additional role for Klp64D in the localization of adherens junction proteins, Armadillo (Arm) and Bazooka (Baz), during photoreceptor morphogenesis in the eye. However, it has been unknown whether these AJ proteins are direct cargos for the Klp64 motor. Here, we show that Arm and Baz bind to the cargo domain of Klp64D and form a protein complex. This suggests that Klp64D is directly involved in transporting Arm and Baz to the AJ. To study the function of this interaction between Klp64D and Arm, we tested whether Klp64D is required for Wingless (Wg)-Arm signaling in the wing imaginal disc that is critical for wing outgrowth. We show that clonal loss of Klp64 in wing discs causes a reduced Cut expression regulated by Wg at the dorsoventral border. Further, klp64 mutant clones result in the loss of wing margin bristles and notching of the adult wing, indicating that Klp64D is essential for wing development. Our data suggest that kinesin-II directly transports Arm and Baz to AJ for cell polarity and is required for Wingless signaling by kinesin-dependent targeting of Arm.


119

75 Phosphorylation Alters Tau Distribution and Elongates Life Span in Drosophila Po-An Yeh1*, Ching-Jin Chang2,3, Pang-Hsien Tu1, Harry Iain Wilson4, Ju-Yi Chien2,3, Chiou-Yang Tang4 and Ming-Tsan Su5 1) Institute of Biomedical Sciences, Academia Sinica, Nankang, Taipei, Taiwan 2) Institute of Biological Chemistry, Academia Sinica, Nankang, Taipei, Taiwan 3) Graduate Institute of Biochemical Science, College of Life Science, National Taiwan University, Taipei, Taiwan 4) Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan 5) Department of Life Science, National Taiwan Normal University, Taipei, Taiwan

The microtubule-associated tau protein has long been considered an axon-specific protein. Although many articles describe the subcellular localization of tau as regulated by post-modification in cultured cells, the intracellular regulation of its distribution in living animals has yet to be elucidated. In the present study, we demonstrate that phosphorylation alters tau polarity in Drosophila melanogaster. Interestingly, it was observed that expression of phosphorylation-incompetent tau impaired neurite growth more severely than either hyperphosphorylated or pseudophosphorylated tau. We also found that inducible expression of hyper- or pseudo-phosphorylated tau in adult flies strikingly prolonged their lifespan. This study offers an alternative tauopathic model by demonstrating that hyperphosphorylated tau has a beneficial effect on the nervous system. This is also corroborated by common effects seen in a variety of organisms in response to various stresses. We hope that this important animal model leads to a paradigm shift in thinking about hyperphosphorylated tau, which plays a protective role in nervous systems rather than the toxic role that many have historically been given to it.


120

76 Coordination of Sensory Neuron Class-Specification and Topography M Rezaul Karim, Adrian W. Moore Disease Mechanism Research Core, RIKEN Brain Science Institute, Tokyo, Japan

Sensory neurons of the same class relay accurate representations of the external world to the central nervous system (CNS) via topographic mapping. Where this mapping is spatial, position-specific information must be integrated into the basic neuron class-specification program. We have developed the nociceptive multidendritic (MD) neuron lineages of the Drosophila larva as a new model in which to investigate the relationship between genetic programs that impart positional information and those that control sensory modality. We find that MD neural induction occurs through the highly localized activity of the three intersecting classic epithelial-morphogenetic pathways. In addition, prepatterning transcription factors act independently of the neural induction program to drive local convergent neuron class specification programs and link neuron position to the topographic control of axonal projections to the CNS. By manipulating these genetic processes we modulate the larval escape response to painful stimuli.


121

77 Mapping the Genetic Networks that Control Dendritic Arbor Shape Caroline Delandre1, Saori Akimoto1, Fuying Gao2, Daniel H. Geschwind2, Adrian W. Moore1

1) Brain Science Institute, RIKEN, Japan 2) University of California, Los Angeles, USA

The dendritic arbor is the chief site of signal input into a neuron and its shape varies significantly among different neuron classes. The computational properties of each neuron class arise from its specific collection and distribution of receptors and ion channels, which are in turn delineated by class-specific dendritic arbor shape. How is the basic neuro-developmental program altered to give rise to neurons with different dendritic arbor shapes?

The Drosophila larval peripheral nervous system contains a group of sensory neurons called dendritic arborization (da) neurons, classified depending on dendritic branching complexity. Several transcription factors control the morphology of da neurons: Abrupt promotes a simplified shape by inhibiting both dendritic growth and branching, whereas Cut and Knot act on different aspects of the cytoskeleton to drive branching complexity. By manipulating the expression of these transcription factors, we obtained dendritic arbors with varying degrees of branching complexity. We then investigated how the transcriptome differs among samples of wild-type da neurons and those overexpressing Abrupt, Cut, or Knot. We used the Weighted Gene Coexpression Network Analysis (WGCNA) to identify gene interactions required for dendritic development. This analysis reveals highly connected genes (hubs), which we predict to have central roles in arborization. Our future studies will validate these candidates.


122

78 Spinster is Required for Neural Degradation during Drosophila Embryonic Development Ki Seok Park1, Sang Hee Kim2, Sang Hak Jeon1*

1) Department of Biology Education, Seoul National University, Korea. 2) Department of Chemistry, KonKuk University, Korea

Programmed cell death (PCD) is a tightly regulated process that serves to eliminate unnecessary cell types during development. This PCD occurs actively during the development of the central nervous system (CNS). spinster (spin) was known as a gene encoding a lysosomal membrane protein and required for cell death in forming nervous system or neuro-muscular junction synapse during post-embryogenesis. However, the role of spin during embryogenesis has not been investigated yet. In this study, we first described the spin expression at embryonic stages in detail and examined its possible roles during embryogenesis. The double staining of spin and repo showed that spin is expressed in surface glia including subperineural glia, channel glia, and exit glia, but not expressed in neuron. In addition, spin was expressed in aminoserosa and boundary cells around dorsal closure during late embryonic development. Mutation in spin caused the increased number of surface glia. Over- or ectopic expression of spin led to progressive degeneration of exit glia and peripheral nerve. Ectopic expression of spin in the CNS also caused the degeneration of fasciclin axons. Taken together, these results indicate that spin functions in regulating embryonic neurogenesis via regulation of neural degradation.


123

79 Probing the Fly Brain with FINGR: Enhancer-trap Flippase-Mediated Gal80 Repression of Gal4 for Mosaic Analysis Taylor Fore1, Xinyun Peng1, Audrey Ojwang1, Margaret Warner1, Rudolf Bohm1,2, William Welch1, Lindsey Goodnight1, Hong Bao1, Bing Zhang1 1) Department of Zoology, University of Oklahoma, Norman, OK; 2) Department of Biology, Brandeis University, Waltham, MA

Drosophila biologists are increasingly interested in restricting Gal4 expression patterns. For neurobiologists, this drive for achieving ‘less is more’ is based in part on the assumption that most behaviors are governed by a small set of neurons. Furthermore, it is a common knowledge among fly people that rarely a Gal4 can be said to be ‘tissue-specific’. To this end, we recently developed the FINGR (enhancer-trap Flippase-induced intersectional Gal80/Gal4 Repression) method consisting of the traditional Gal4/UAS, a collection of newly created ET-FLPx2 (with two copies of FLP) lines, and two complementary Gal80-converting tools (tubP>stop>Gal80, ‘flip in’) and tubP>Gal80>, ‘flip out’). These tools will help better achieve ‘tissue-specificity’ of Gal4 expression as one concerns only the overlap of FLP and Gal4 expression. Coupled with UAS-GFP and another UAS-effector (such as UAS-polyQ), the FINGR method allows one to not only map the morphology of neural circuits or any other tissues, but to manipulate cell activities and examine the consequences on morphology or behavior. Finally, ET-FLPx2 lines can be used with MARCM and any other FRT-based methods to generate reproducible clones. Reproducibility of mosaics is particularly critical for both behavioral and morphological studies. We are characterizing the ET-FLP lines and developing a public database to highlight the expression pattern of each line. The FINGR method and reagents should be valuable to all fly researchers interested in mosaic analysis.

Supported by NSF grant IOS1025556 and internal funds from OU.


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80 Neuroligin 2 is Required for Synapse Development and Function at the Drosophila Neuromuscular Junction Mingkuan Sun1,2 , Guanglin Xing1,2 , Liudi Yuan1,3 , Guangming Gan1,2 , David Knight4 , Sheila Irene With4 , Cui He1,2 ,Junhai Han1,2 , Xiankun Zeng2 , Ming Fang1,2 , Gabrielle L. Boulianne4

and Wei Xie1,2

1) The Key Laboratory of Developmental Genes and Human Disease, Ministry of Education, Institute of Life Science, Southeast University, Nanjing 210009, China 2) Departments of Medical Genetics and Developmental Biology and 3) Biochemistry and Molecular Biology, Medical School of Southeast University, Nanjing 210009, China 4) Program in Developmental and Stem Cell Biology, The Hospital for Sick Children and Department Molecular Genetics, University of Toronto, Toronto, Ontario, Canada, M5G 1L7

Neuroligins belong to a highly conserved family of cell adhesion molecules that have been implicated in synapse formation and function. However, the precise in vivo roles of Neuroligins remain unclear. In the present study, we have analyzed the function of Drosophila neuroligin 2 (dnl2) in synaptic development and function. We show that dnl2 is strongly expressed in the embryonic and larval CNS and at the larval neuromuscular junction (NMJ). dnl2 null mutants are viable but display numerous structural defects at the NMJ, including reduced axonal branching and fewer synaptic boutons. dnl2 mutants also show an increase in the number of active zones per bouton but a decrease in the thickness of the subsynaptic reticulum and length of postsynaptic densities. dnl2 mutants also exhibit a decrease in the total glutamate receptor density and a shift in the subunit composition of glutamate receptors in favor of GluRIIA complexes. In addition to the observed defects in synaptic morphology, we also find that dnl2 mutants show increased transmitter release and altered kinetics of stimulus-evoked transmitter release. Importantly, the defects in presynaptic structure, receptor density, and synaptic transmission can be rescued by postsynaptic expression of dnl2. Finally, we show that dnl2 colocalizes and binds to Drosophila neurexin (dnrx) in vivo. However, whereas homozygous mutants for either dnl2 or dnrx are viable, double mutants are lethal and display more severe defects in synaptic morphology. Altogether, our data show that, although dnl2 is not absolutely required for synaptogenesis, it is required postsynaptically for synapse maturation and function


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81 Explore Carpet Glia Function in Drosophila Eye Disc Hong Lee1,2, Y. Henry Sun1,2

1) Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 2) Departmentof Life Science and Institute of Genome Science, National Yang-Ming University, Taipei, Taiwan

During Drosophila visual system development, photoreceptor cells (R-cells) start to differentiate in the eye disc during mid to late larval stage, and project their axon through the optic stalk into the optic lobe of brain. For proper R cells axon targeting, the retinal basal glia (RBG) cells is required. In the absence of RBGs, R cells axon can not enter the optic stalk and target to the brain.

RBGs start to migrate into the eye disc in early 3rd instar larvae when morphology furrow start to sweep over the eye disc. In the leading edge of carpet glia membrane, RBGs can reach and wrap onto R cells axon. These previous studies suggest that for Drosophila normal visual wh development, RBG migration timing should coordinate with photoreceptor differentiation and precocious migration of RBGs will cause abnormal axon projection. Based on cell morphology and position, RBGs can be classified into 6 types (Silies et al.2007). One type, carpet glia, has extended membrane processes across one half of the eye disc. Ablation of carpet glia will cause RBGs precocious migration, suggesting that carpet glia functions in restricting RBGs migration not to go beyond the extent of carpet glia membrane.

We are interesting in carpet glia’s function and its special membrane morphology. We got two carpet glia specific Gal4 line：C135-Gal4 and Np2776-Gal4. Using these Gal4 drive dominant negative form Rho GTPase family protein to block carpet glia membrane extension, we found arrest carpet membrane extension doesn’t affect RBGs migration, suggest the RBGs precocious migration phenotype cause by carpet ablation maybe cause by lost some inhibition signal secret from carpet. Although carpet glia’s membrane doesn’t function in RBGs migration, but its may probably function in regulate RBGs differentiation. Previous model shows surface glia differentiate into wrapping glia were control by FGF signal switch, carpet glia’s membrane may function as a junction to separate two different FGF signal pyramus and thisbe secret from RBGs and R-cells.


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82 Expanded Polyglutamine Domain is a Nuclear Export Signal and Mediates Nuclear Transport of Disease Proteins.

Edwin Chan1, Ho Tsoi1, Priscilla Chan1, Ronald Wu1, Eric Wong2, Tat-Cheung Cheng1, Hoi-Yeung Li2, Kwok-Fai. Lau1, Norbert Perrimon3, Pang-Chui. Shaw1.

1) School of Life Sciences, The Chinese University of Hong Kong, Shatin, Hong Kong 2) School of Biological Sciences, Nanyang Technological University, Singapore 3) Department of Genetics, Harvard Medical School, USA.

Polyglutamine (polyQ) diseases are caused by CAG trinucleotide repeat expansion in the coding region of disease genes. The cell nucleus is an important site of pathology in polyQ diseases. In this study, we showed that exportin-1 (Xpo1) regulates the nucleocytoplasmic distribution of expanded polyQ protein. We found that expanded polyQ protein, but not its unexpanded form, possesses nuclear export activity and interacts with Xpo1. Genetic manipulation of Xpo1expression levels in transgenic Drosophila models of polyQ disease confirmed the specific nuclear export role of Xpo1 on expanded polyQ protein. Upon Xpo1knockdown, the expanded polyQ protein was retained in the nucleus. Taken together, we first showed that Xpo1 is a nuclear export receptor for expanded polyQ domain, and our findings establish a direct link between protein nuclear export and the progressive nature of polyQ neurodegeneration.


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83 Meigo, a Putative UDP-Sugar Transporter, Regulates Neuronal Targeting of Synaptic Partners in the Drosophila Antennal Lobe Sayaka Sekine1, Liqun Luo4, Masayuki Miura1,3, Takahiro Chihara1,2,4 1) Dept. Genetics, Grad. Sch. Pharm. Scis, Univ. Tokyo 2) PRESTO, JST 3) CREST, JST 4) HHMI and Dept. Biol. Stanford Univ. USA

The wiring of functional neural network results from accurate synaptic matching enabled by “targeting” of numerous axons and dendrites. During the development of Drosophila olfactory system, the axon of primary neuron (olfactory receptor neuron; ORN) and the dendrites of secondary neuron (projection neuron; PN) target one of ~50 glomeruli in the antennal lobe (AL), resulting in one-to-one specific connections of proper synaptic partners.

To elucidate the mechanism of dendritic targeting of PN, we performed genetic mosaic system (MARCM)-based screen and isolated a mutant, meigo. The dendritic targeting of PN homozygous for meigo (meigo-/- PN) shifted to the medial side of the AL, however the axonal trajectory and branching were largely normal. In contrast, meigo-/- ORNs exhibited targeting defect in axon, which was similar to those seen in meigo-/- PN dendrites. The responsible gene (meigo) encodes a putative UDP-sugar transporter, suggesting that Meigo contributes to glycosylation of cell surface proteins, which is required for recognition of positional information along M-L axis of the AL. To identify the cell surface protein, we tested candidate molecules that have been previously linked to axon guidance or targeting. Together with rescue experiments, our results indicate that Meigo is cell-autonomously required in synaptic partners for neuronal targeting along the M-L axis in the AL, probably by regulating the glycosylation of cell surface proteins.


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84 Systematic Identification of the Molecules that Regulate Central Synapse Formation Atsushi Sugie, Sara Berman, Takashi Suzuki Max Planck Institute of Neurobiology

During the development of the brain, synapses are formed precisely at the loci between nerve cells in order to establish a functional neural circuit. It is a fundamental question as to how the sites of synapses are determined after axon pathfinding and targeting have occurred. In this study, we used the Drosophila visual system as a model to elucidate the mechanism for synaptic loci determination of central synapses. The visual system comprises the compound eye and four optic neuronal ganglia known as the lamina, the medulla, the lobula and the lobula plate. The compound eye consists of an array of 800 ommatidia. Each ommatidium has eight photoreceptor neurons (R1-R8 cells). The R8 axons project through the lamina and terminate at the specific layer in the second ganglion, the medulla. The R8 axons form synapses at stereotypic sites in the medulla. We focused on the R8 neuron to study the process of central synapse formation.

First, we verified whether synapses can be observed in the visual system. In order to visualize presynaptic sites in R cells, a fragment of Bruchpilot fused to mCherry (Brp::mCherry) (gift from S. Sigrist) was expressed in R8 cells by an Rh6-Gal4 driver. Brp was clearly localized at the presynaptic sites in the medulla. Next, to visualize the synapses with a Gal4 independent system, we have generated a new set of transgenic lines with a LexA/LexAop system carrying the Rh6 driver and the Brp::mCherry marker. This is the first realistic tool to monitor central synaptic sites with confocal microscopy and in a Gal4/UAS-independent manner. Using the combination of this method and the Gal4/UAS-system, we are performing large scale overexpression (and knock-down) experiments either in the R cells or target neurons. In this presentation, we will discuss the results of the screen.


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85 Anatomical Characterization of Thermosensory AC Neurons in the Adult Drosophila Brain Hsiang-Wen Shih1, Ann-Shyn Chiang1,2,3,4* 1) Institute of Biotechnology 2) Brain Research Center, National Tsing Hua University, Hsinchu 30013, Taiwan 3) Genomics Research Center, Academia Sinica, Nankang, Taipei 11529, Taiwan 4) Kavli Institute for Brain and Mind, University of California at San Diego, La Jolla, CA 92093-0526, USA.

Temperature preference is vital for the survival of all animals. A small set of warm-activated anterior cell (AC) neurons acting as an internal thermosensor in the Drosophila brain is critical for optimal temperature selection (Hamada et al., 2008). Here, we analyze the circuit components of the AC neurons by characterization of its spatial distribution, dendrite-axon polarity, and the putative type of neurontransmitter released. Our results show that the AC neurons are serotoninergic, do not have any dendrites, and send axons bilaterally to the superior dorsofrontal protocerebrum (SDFP). Searching the FlyCircuit database for neurons with serotonin receptor and dendrites in the SDFP, we found a dorsal-anterior-lateral (DAL) neuron as a candidate postsynaptic partner of the AC neurons. In conclusion, by morphological analysis of the AC neurons, we show a general strategy for predicting brain circuits orchestrating thermosensory behaviors.


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86 Activity-Dependent Retrograde Laminin A Signaling Suppresses Synaptic Growth Pei-I Tsai1,2, Manyu Wang1,2, Hsiu-Hua Kao1, Yu-Jing Lin1, Ying-Ju Cheng1, Ruey-Hwa Chen2,3, Cheng-Ting Chien1,2 1) Institute of Molecular Biology, Academia Sinica, Taipei 115, Taiwan 2) Institute of Molecular Medicine, National Taiwan University, Taipei 106, Taiwan 3) Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan

Retrograde signals derived from postsynaptic cells are elevated by synaptic activity to potentiate presynaptic properties. Activity-induced upregulation of retrograde signals have been well recognized. However, it is not known whether activity regulated retrograde signals depotentiate synaptic properties. Here we report that synaptic activity at Drosophila neuromuscular junctions (NMJs) reduced synaptic cleft-localized retrograde Laminin A (LanA) to potentiate synaptic growth. LanA activated the presynaptic integrin pathway, consisting of bn integrin and focal adhesion kinase Fak56. The LanA level at NMJs was modulated by larval crawling, synaptic excitability, postsynaptic response and anterograde Wnt/Wingless signaling, all of which modulate NMJ growth. Our data indicate that retrograde LanA acts as “negative” regulator to suppress synaptic growth, and synaptic activity promotes synaptic growth by downregulating retrograde LanA signaling.


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87 Chinmo Governs Neuronal Temporal Identity in Lineage-Specific Manners Chih-Fei Kao1, Hung-Hsiang Yu2, Yisheng He1, Jui-Chun Kao2, Tzumin Lee1,2

1) Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA 01605, USA 2) Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, VA 20147, USA

Distinct neurons are made sequentially by a multipotent neural progenitor. Chinmo, a BTB-zinc finger nuclear protein, has been shown to specify multiple temporal fates of the mushroom body (MB) lineages in a dosage-dependent manner. To determine if temporal gradients of Chinmo govern neuronal temporal identity in diverse lineages, we examined the roles of Chinmo in a lineage that makes many more neuron types in a shorter period of time, the anterodorsal projection neuron (adPN) lineage. Loss-of-function twin-spot MARCM analysis allows readily detection of temporal cell fate transformation in such a rapidly changing lineage. In the earliest chinmo mutant clones, a total of 8 adPN types born within two separate developmental windows are missing. The first window contains 3 embryonic neuron types (DM4, DL5, VM3a), while the second one includes 2 embryonic- (VM3b, DL4) and 3 larval-born adPN types (DL1, DA3, DC2). The unaffected DM3 adPN separates the two windows. Analyses of single-cell clones revealed that Chinmo acts to suppress the adoption of next Chinmo-independent temporal fate (DM3 and D fate, respectively) by neurons born within either window. Notably, even when loss of Chinmo elicited partial fate transformation, mutant neurons consistently acquired features reminiscent of the Chinmo-null default fate. Moreover, ectopic Chinmo did not promote early temporal fates in later-born adPNs. These findings argue against the gradient-dependent mechanism of temporal cell fate specification. Instead, Chinmo may underlie some hierarchical temporal patterning in the fast-tempo neuronal lineages by suppressing a later temporal fate in early siblings.


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88 Functions of COP9 Signalosome in Drosophila External Sensory Organ Development Yi-Chun Huang1,4, June-Tai Wu2, Cheng-Ting Chien1, and Haiwei Pi3 1) Institute of Molecular Biology, Academia Sinica, Taiwan 2) Institute of Molecular Medicine, National Taiwan University, Taiwan 3) Department of Biomedical Sciences, Chang Gung University, Taiwan 4) Graduate Institute of Life Sciences, National Defense Medical Center, Taiwan

The Drosophila external sensory (ES) organs, or called sensory bristles, are mechanosensory organs distributed on the surface of thorax, abdomen, legs and wing margin. For years, it has been used as a genetically tractable system to study gene regulation and function in neurogenesis.

Each ES organ consists of four different daughter cells (neurons, sheath cells, hair cell and socket cell) that are generated through asymmetric division of a SOP. After the first asymmetric division, the SOP cell produces two secondary precursors, IIa and IIb. IIa and IIb cells asymmetrically divide again to finally give rise to hair cell/socket cell and neuron/sheath cell, respectively.

To study the roles of COP9 signalosome in ES organ development, we analyzed the ES organ phenotypes in csn4 and csn5 loss-of-function mutant clones. We found COP9 is required for patterning of sensory organs alone the margin of Drosophila wings. In wild-type fly, non-innervated sensory bristles are distributed along the posterior margin. In csn4 or csn5 mutant clones, the non-innervated bristles are transformed into bristles innervated with single neuron in a proneuronal protein-independent manner. Furthermore, we found that in csn4 or csn5 mutant clones, the Broad-complex Brd-Z1 is upregulated during pupa stage. Since the Broad-complex is repressed by ecdysone receptor EcR in the absence of ecdysone and is required for Senseless expression and accumulation, we speculate that COP9 signalosome regulates neuron development through ecdysone signaling pathway.

In addition, two major phenotypes are also observed for the ES organs on the thorax: (1) the progression of ES organ development is strongly delayed, or arrested. (2) ES organ with two hair cells and a fused socket cell are observed, suggesting a possible cell fate transformation of IIb to IIa cells.


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89 The Seven-Pass Transmembrane Cadherin Flamingo Controls Dendritic Self-avoidance via Its Binding to a LIM Domain Protein Espinas in Drosophila Sensory Neurons Daisuke Matsubara, Shin-ya Horiuchi, Kohei Shimono, Tadashi Uemura and Tadao Usui Graduate School of Biostudies, Kyoto University

Drosophila Flamingo/Starry night (Fmi/Stan) is a founding member of the evolutionarily conserved family of seven-pass transmembrane cadherins and it was originally discovered in studies on epithelial planar cell polarity (PCP). Roles of this family members have been reported in a number of different contexts of neural development both in invertebrates and vertebrates. Even so, molecular identities downstream of the family have been poorly understood. Here we show that a LIM domain protein Espinas (Esn) binds to an intracellular domain of Fmi, and that this Fmi-Esn interplay elicits repulsion between dendritic branches of Drosophila sensory neurons.

Among diverse shapes of dendritic arbors of different types of neurons, the “space-filling” type is the one that uniformly covers its two-dimensional receptive field with minimum overlap with each other and it is supported partly by avoidance between branches of the same neuron (self-avoidance). Class IV dendritic arborization neurons in the wild-type Drosophila larvae show this self-avoidance; in contrast, it was disrupted in a fmi mutant, in an esn knockout homozygote, and in the fmi/esn transheterozygote. We found that functional fusion protein Fmi:3eGFP was localized at most of the branch tips of da neurons; and in a heterologous system, assembly of Esn at cell contact sites required its LIM domain and Fmi. We further show that other PCP genes, such as Van gogh/Strabismus (Vang/Stbm) and RhoA, are also necessary for the self-avoidance, and that fmi genetically interact with these loci. We propose that the Fmi-Esn complex, together with the PCP regulators, executes the repulsive interaction between isoneuronal dendritic branches.


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90 Role of a PCP Protein Dachsous in Dendritic Targeting of Olfactory Projection Neurons Misako Okumura1, Masayuki Miura1,3, Takahiro Chihara1,2 1) Dept. Genetics, Grad. Sch. Pharm. Sci., Univ. Tokyo 2) PRESTO, JST 3) CREST, JST

Functional neural network requires correct wiring between axons and dendrites. The

molecular mechanism of axonal guidance has been well studied, but the mechanism of dendritic targeting is largely unknown. During the development of Drosophila olfactory system, the dendrites of projection neurons (PNs) target a single glomerulus out of ~50 glomeruli in the antennal lobe (AL), which is a good model system to study molecular mechanisms of dendritic targeting. Although previous studies suggest that PN dendrites utilize positional information in the AL to target proper glomeruli, it is still largely unknown about the molecules involved in this process. To address this question, we focus on PCP signal molecule, Dachsous (Ds). Ds is an atypical cadherin and plays multiple roles in controlling epithelial planar cell polarity and tissue growth through its concentration difference between cells. To reveal the function of Ds in dendritic targeting, we analyzed dendritic targeting of ds-/- PNs by MARCM system. Interestingly dendrite of ds-/- single PN targeted not only a correct glomerulus but also other glomeruli, demonstrating that Ds is required for dendritic targeting. We will discuss the possible role of Ds during olfactory neural circuit in Drosophila.


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91 Hunting for Genes that Regulate Remodeling and Life-long Maintenance of Dendritic Arbors Kohei Shimono, Takafumi Nomura, Tadao Usui, Tadashi Uemura Graduate School of Biostudies, Kyoto University, Japan

Neurons develop distinctive dendritic morphologies to receive and process sensory or synaptic inputs. Dendritic arbors that are formed in early development are often reorganized and such arbors of many neuronal types are possibly maintained throughout animal life. To investigate underlying mechanisms of this remodeling and maintenance in vivo, we have employed dendritic arborization (da) neurons, which exhibit dramatic dendritic pruning and subsequent growth during metamorphosis, as a model system. We first identified da neurons in the adult abdomen and then clarified developmental basis of these adult neurons by tracing origins of those cells back to the larval stage. We and others also showed that the dendritic arbor of one da neuron, v'ada, exhibited prominent radial-to-lattice transformation in one day after eclosion, and the resultant lattice-shaped arbor persisted throughout adult life.

To conduct a MARCM screening efficiently, we expressed FLP recombinase in sensory organ precursors (SOPs). By using this ‘SOP-FLP’ system, we isolated several mutants that displayed defects in the remodeling and/or the life-long maintenance of dendritic arbors. To identify causative genes for these mutants, we have started employing a next-generation sequencing technique to directly compare whole-genomic sequences of several mutants with each other, in addition to conventional mapping methods. In this poster, we will discuss our on-going screening and mapping.


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92 In Vivo Roles of the Drosophila Homolog of ALS2 Gene Yuta Takayama, Reina Ito, Tadashi Uemura Graduate School of Biostudies, Kyoto University, Japan

Dendrites allow neurons to integrate sensory or synaptic inputs, and the branching

complexity and size of the dendritic arbor limit the number and type of inputs. Dendritic arborization (da) neurons provide a model system to study genetic programs underlying such geometry in vivo. We previously showed that dynein-dependent transport of Rab5-containing early endosomes plays a critical role in controlling spatial disposition of dendritic branching. As for regulations of Rab5 activity, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) have been identified biochemically. Nevertheless little has been shown about how Rab5 activity is controlled in spatiotemporal manners in neurons. To tackle this question, we have been studying Drosophila homologs of human Rab5 GEFs and GAPs. We first examined whether each homolog was able to activate or inactivate Rab5 by expressing each potential GEF or GAP together with a fluorescence resonance energy transfer (FRET) biosensor, Raichu-Rab5, in S2 cells. Fly proteins that exhibited Rab5-GEF activity included a homolog of ALS2/Alsin. Recessive mutations in the ALS2 gene have been linked to juvenile-onset amyotrophic lateral sclerosis. We took advantage of a P-element insertion into the 5’ untranslated region of Drosophila ALS2, and isolated several imprecise jumpers that deleted the coding sequence. Mutants homozygous for each allele were viable and fertile, and we didn’t find any apparent morphological defects in the adults. We are performing genetic and molecular characterization of these alleles and examining cellular phenotypes of da neurons and motor neurons.


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93 Fully Equipped Twin-Spot MARCM for Studying Drosophila Brain Development and Anatomy by Genetic Mosaics Yaling Huang1*, Sam Yu1*, Chih-Fei Kao2*, Barret D. Pfeiffer1, Chun-Hong Chen3, Gerry Rubin1, Tzumin Lee1,2 1) Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn VA 20147, USA 2) UMASS Medical School, Department of Neurobiology, 364 Plantation Street, LRB770Y, Worcester MA 01605, USA 3) Division of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan Town, Miaoli County 350, Taiwan

Twin-Spot MARCM permits comprehensive cell lineage analysis, as it labels sister clones in distinct colors derived after mitotic recombination. The resulting sister clones are homozygous for each of the homologous chromosome arms that recombine. One clone can thus be made homozygous for the mutation(s) placed distal to the site of mitotic recombination. Detailed analysis of such mutant clones is greatly aided by a simultaneous detection of their wild-type sister clones, especially in the complex brain. However, the prototype of Twin-Spot MARCM is genetically cumbersome and applicable only on one chromosome arm, much limiting its power. Here we report a genetically simplified version of Twin-Spot MARCM available on all major Drosophila chromosome arms. We further developed a strategy to co-repress multiple transgenes using a single miRNA transgene. One can therefore express additional markers and/or dominant transgenes specifically in one of the paired sister clones for more sophisticated genetic mosaic studies.


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94 Study of Cell Lineage in Drosophila Retinal Basal Glia Yu Fen Huang1,2, Y. Henry Sun1,2. 1) Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan 2) Departmentof Life Science and Institute of Genome Science, National Yang-Ming University, Taipei, Taiwan

Glial cells play important roles in neuronal development and function. The retinal basal glia (RBG) is a subset of glial cell that originates in the optic stalk in second instar of Drosophila. RBG cells start to migrate into the eye disc as photoreceptor cells (R cell) begin to differentiate. The presence of RBG cells in eye discs is essential for R cell axons to enter the optic stalk. Three main classes of RBG cells have been identified including carpet glia, surface glia, and wrapping glia. According to the “sequential differentiation model” proposed by Silies et al. (J. Neurosci. 27:13130-9), surface glia located at the basal side migrates forward along the carpet glia. Once they reach the anterior margin of carpet glia and contact the neuron, these migratory glia starts to differentiate into wrapping glia and wrap around R cell axons. I am trying to follow the temporal sequence of RBG migration and differentiation. My results will be presented.


139

95 Regulation of Golden Goal Function by Phosphorylation Klaudiusz Mann, Satoko Hakeda-Suzuki, Si-Hong Luu, Menghze Wang, Stephan Ohler, Takashi Suzuki

Max Planck Institute of Neurobiology, Martinsried, Germany

Golden Goal (Gogo) is a single pass transmembrane protein required for axon guidance of photoreceptors in Drosophila. Gogo is required in R8 photoreceptor axons for the recognition of their temporary target layer M1 and then proceeding to their final destination, M3 layer in the medulla; additionally both extracellular and intracellular parts of Gogo are required for its function (Tomasi et al., 2008). Although the cytoplasmic part does not contain any known domains, we show that its middle third part (C2 fragment) is sufficient for Gogo’s cytoplasmic function. We show in vivo a crucial function of a conserved YYD (Tyr-Tyr-Asp) motif within the C2 fragment, as Gogo lacking the YYD motif does not rescue the gogo- phenotype. We postulate that the YYD motif is a phosphorylation site and the dephosphorylated Gogo is the functional form involved in R8 targeting, as the non-phosphorylable form of Gogo rescues the gogo- mutant phenotype whereas the phospho-mimicking form fails to rescue gogo. Moreover, overexpression of constructs mimicking Gogo phosphorylation and Gogo lacking the YYD motif is able to cause stopping of R8 axons at the M1 layer. Thus, we propose that the Gogo phosphorylation status plays a crucial role in the recognition of the M1 layer and proceeding to the M3 layer targeting. We show the phosphorylation of Gogo in vivo using the cell culture system. In our preliminary results we show a genetical interaction between gogo and dinr (Drosophila insulin receptor) suggesting that DInR is a regulator of Gogo phosphorylation status. We will discuss in a more detail the role of DInR in the regulation of Gogo phosphorylation.


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96 Global Control of Neuronal Synapse Positioning in a Single Neuron by Intrinsic Pdm3 Protein Levels in the Central Brain Chien-Kuo Chen1,3, Wei-Hsiang Lin3, Hsun Li3, Wei-Yu Chen2,3, Cheng-Ting Chien1,2,3

1) Institute of Neuroscience, National Yang-Ming University, Taipei, Taiwan 2) Institute of Cell and Molecular Biology, Taipei Medical University, Taipei, Taiwan 3) Institute of Molecular Biology, Academia Sinica, Taipei, Taiwan

In the brain, an inhibitory interneuron elaborates an extremely complex axonal arborization pattern and forms numerous synaptic contacts with many excitatory neurons. It is not clear how neurons control the positioning of all their synapses even within the targeting layer. Here, we show that GABAergic ring (R) neurons form stereotypic concentric patterns of synapses in the ellipsoid body (EB) of the Drosophila central complex. The positions of all

synapses in a single R neuron were registered by cylindrical coordinates (r, ſ, h) to describe their distribution patterns. We characterized synapse distribution patterns of the pdm3 mutant that displays a malformed EB structure and found that the pdm3 gene dosages modulate synapse positioning along the h coordinate. Pdm3 regulates the protein localization of the RacGEF Trio in the EB for synapse positioning. Thus, the intrinsic mechanism involving the transcription factor Pdm3 regulates positioning of all synapses in a single neuron.


141

97 A Genetic Approach for the Organization and Function of the Blood-Brain Barrier in Drosophila

Hiroshi Kanda, Rieko Shimamura, Hideyuki Okano

Department of Physiology, Keio University School of Medicine, Tokyo, Japan

The blood brain barrier (BBB) is a cellular structure in the central nervous system (CNS) that restricts the molecular and cellular exchanges. The pathological disruption of BBB has been implicated in a wide spectrum of neurological disorders. Tight junctions (TJs) and transporters possess the central role to achieve the highly integrated mechanism of the BBB to strictly isolate the vertebrate CNS. TJs restrict the distance between adjacent epithelial cells lining the fine capillaries of the brain microvasculature to form a selective physical barrier. ATP binding cassette (ABC) transporters are known to be utilized for chemoprotection that actively expel lipophilic molecules. In spite of accumulating ex vivo models as well studies using rodents, it has still been less well understood how BBB function is established and maintained.

In Drosophila, “BBB” and epithelial paracellular barriers are provided by a type of glia, subperineurial glia (SPG), that are connected by septate junctions (SJs) that have functional and molecular similarities to vertebrate TJs. It has also been revealed that Drosophila also conserves the xenobiotic exclusion system, which is achieved by fly ABC transporter(s). We thus aimed to address the molecular mechanisms for regulating the BBB functions in vivo using powerful Drosophila genetics. To this end, we have performed a large-scale screen to identify novel genes whose function is required for the integrity of paracellular diffusion barrier in Drosophila BBB. We have screened approximately 50% of Drosophila genes, and identified several candidates whose down-regulation shows the severe dysfunction of BBB. I would discuss about the molecular function of genes identified in the screen.


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98 Evolutionarily Conserved Protein Dogi Regulates Neurite Targeting in the Olfactory System Chisako Sakuma1, Liqun Luo4, Masayuki Miura1,3, Takahiro Chihara1,2,4 1) Dept. Genetics, Grad. Sch. Pharm. Scis, Univ. Tokyo 2) PRESTO, JST, Japan 3) CREST, JST, Japan 4) HHMI, Dept. Biology, Stanford Univ. USA

Genetically defined neuronal morphology is an essential framework for neural network formation. Especially, dendritic and axonal targeting are important processes to wire individual neurons. To reveal the molecular mechanisms of neurite targeting, we performed a genetic mosaic screen in Drosophila olfactory projection neurons (PNs), whose dendrites target a single glomerulus out of ~50 glomeruli in the antennal lobe (AL), and whose axons reach the lateral horn. From the screen, a mutant, doubled glomeruli (dogi), defective in both dendritic and axonal targeting was isolated. Interestingly, dendrite of dogi-/- single PN targeted a wrong glomerulus in addition to a correct glomerulus, resulting in the doubling of target glomeruli. Meanwhile, axons of dogi-/- PNs were significantly shortened and/or misguided. By using genetic mapping methods, we identified a causal gene, dogi, encoding an evolutionarily conserved protein from worm to human. The phenotypes of dogi-/- PNs were rescued by the expression of the full-length dogi transgene, demonstrating that dogi is cell-autonomously required for the dendritic and axonal targeting. Endogenous Dogi protein was expressed in the PN at pupal stage, when the dendritic targeting takes place. Thus, Dogi protein appears to be essential to regulate neurite targeting. Here we present phenotypic analyses of dogi-/- PNs and discuss the possible function of Dogi in neural development.


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99 Concentric Zones, Cell Migration and Neuronal Circuits in the Drosophila Visual Center Eri Hasegawa1, Yusuke Kitada1,2, Takeshi Awasaki3, Tetsuya Tabata2, Makoto Sato1

1) FSO, Kanazawa University, Japan 2) IMCB, University of Tokyo, Japan 3) UMass Med, USA

The Drosophila optic lobe comprises a wide variety of neurons, which form laminar neuropiles with columnar units and topographic projections from the retina, and shares many structural characteristics with mammalian visual systems. However, little is known regarding the developmental mechanisms that produce neuronal diversity and organize the circuits in the primary region of the optic lobe, the medulla.

We found the key features of the developing medulla and novel phenomena that could accelerate our understanding of the Drosophila visual system. The identities of medulla neurons are pre-determined in the larval medulla primordium, which is subdivided into concentric zones characterized by the expression of four transcription factors: Drifter, Runt, Homothorax (Hth) and Brain-specific-homeobox (Bsh). The expression pattern of these factors correlates with the order of neuron production. Once the concentric zones are specified, the distribution of medulla neurons changes rapidly. Each type of medulla neuron exhibits an extensive but defined pattern of migration during pupal development. The results of clonal analysis suggest hth is required to specify the neuronal type by regulating various targets including Bsh and cell adhesion molecules such as N-cadherin, while drifter regulates a subset of morphological features of Drifter-positive neurons. Thus, genes that show the concentric zones may form a genetic hierarchy to establish neuronal circuits in the medulla.

In Hth-positive neurons, Bsh is exclusively expressed in medulla intrinsic Mi1 neurons. We recently found Mi1 neurons switch the neuronal type in bsh mutant clones as well as in hth clones. Possible relationship between hth and bsh will be discussed.


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100 The Drosophila King tubby is a Novel Molecule to Regulate Rhodopsin Cycle Shu-Fen Chen, Seng-Sheen Fan

Department of Life Science, Tunghai University, Taichung 40704 Taiwan.

The tubby (tub) and tubby-like protein (tulp) genes belong to a small family of genes which their function remains unclear. The tub and tulps genes are found in multicellular organisms including both plants and animals. The C-terminal of Tub and Tulps proteins are highly conserved. Mutation of members of this protein family causes disease phenotypes including retinal degeneration and obesity. In this study, we used Drosophila as model system to further investigate the function of tub gene in photoreceptor development and maintenance. Drosophila has only one tulp gene, the king-tubby (ktub). Using immunocytochemistry, we found that Ktub is expressed in the nucleus and the subrhabdomere domain in developing photoreceptor cells. We also found that Ktub expression pattern is light-dependent in the adult. In the dark condition, the Ktub is co-localized with rhodopsin in the rhabdomere. However, the Ktub is translocated from rhabdomere to the cytoplasm upon light activation. Deletion of Ktub in light condition failed to retain the rhodopsin in the rhabdomere domain suggesting that Ktub is required for transportation of rhodopsin. Furthermore, the deletion of Ktub also results in retinal degeneration. Together, these results delimitate the function of Ktub in photoreceptor development and maintenance.


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101 Molecular Characterization of the Neural Sex Determination Factor Fruitless: Phosphorylation, Self-interaction and Expression Gakuta Toba, Daisuke Yamamoto Graduate School of Life Sciences, Tohoku University, Japan

Fruitless (Fru) is a major determinant of sex-specific behaviors in Drosophila. In the nervous system, Fru is male-specifically produced in a subset of neurons. Several lines of evidence indicate that the male-specific production of Fru leads to the development of the male-type structures of neurons, which are responsible for the male-type behavior of the fly. Structurally, Fru belongs to a family of transcription factors that have the BTB/POZ domain and the zinc-fingers. The fru gene produces multiple Fru isoforms with different sets of zinc fingers through alternative splicing. To understand molecular mechanisms underlining the sexual differentiation of the neurons, biochemical characterization of the key protein Fru is essential. Here, we report novel features of Fru that are potentially involved in the molecular mechanism and regulation of Fru activity. Phosphatase treatment of Fru revealed that Fru is phosphorylated in vivo. Phosphorylation of Fru occurred even in the cells without endogenous fru expression, and also in females when a fru transgene was ectopically expressed. Immunoprecipitation assays showed that the Fru isoforms physically interact with each other, suggesting that Fru functions as a protein complex that contains different isoforms of Fru. Temporally controlled RNAi indicated that Fru is produced mostly at the pupal stage, but continues to be present in the adult more than 50 days after eclosion. Biological and biochemical properties of Fru revealed in this study will help understand the molecular mechanism whereby Fru determines the sexual fate of individual neurons.


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102 Dendrite Remodeling in Adult Drosophila Sensory Neurons Kei-ichiro Yasunaga, Kazuo Emoto Department of Cell Biology, Osaka Bioscience Institute, Japan

In response to changes in the environment, dendrites from certain neurons change their shape, yet underlying mechanisms remain elusive. In the Drosophila Class IV dendrite arborization (C4da) neurons, larval dendrites are once completely pruned in the early pupal stage and then adult-specific dendrites regrow to elaborate radial-shaped dendritic trees in the late pupal stage. In this study we performed live-imaging of dendrite dynamics during the regrowth stages and found that the radial arrangement of C4da dendritic arbor is rapidly reshaped to a lattice-like pattern within 24 hr after adult eclosion. This radial-to-lattice reshaping of C4da dendrites was completely blocked by mutations in the GPI-anchored Matrix Metalloproteinase (Mmp) 2. Further genetic studies suggest that epithelial Mmp2 promotes the dendrite reshaping through local modification of the basement membrane upon which C4 da dendrites grow. To gain further insight into molecular mechanisms underlying the dendrite remodeling, we currently perform an RNAi-based screen in C4da neurons. So far, we have screened about 2,000 transgenic RNAi lines and found several interesting candidates involved in a variety of dendrite remodeling events such as dendrite branching, outgrowth, and maintenance. We will discuss how these genes cooperate to regulate dendrite remodeling.


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103 Dual Genome-wide Hunting for Abrupt or Knot/Collier Targets that Control Neuronal Class-Specific Dendrite Morphogenesis Yukako Hattori1, Tadao Usui1, Daisuke Satoh1, Takehiko Itoh2, Katsuhiko Shirahige3, Tadashi Uemura1 1) Graduate School of Biostudies, Kyoto University, Japan 2) Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Japan 3) Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan

Dendritic arbor morphology is a hallmark of cellular diversity in the nervous system, and this diversity supports the differential sampling and processing of sensory or synaptic inputs. Drosophila dendritic arborization (da) neurons are classified into classes I to IV on the basis of branching complexity and arbor patterns. We and other groups have shown that selective expression of the BTB-zinc finger protein Abrupt (Ab) in class I neurons endows its simple and comb-like dendritic pattern, whereas a member of the EBF/Olf-1 family Knot (Kn)/Collier (Col) controls formation of the highly elaborated class IV arbors in a cell-autonomous manner. To explore transcriptional programs governing the class-specific dendritic morphogenesis, we used dual genome-wide approaches to hunt for key targets: binding profiling DamID (DNA adenine methyltransferase identification) and gene expression profiling. Our DamID analysis showed that many of genes, to which Ab and/or Kn preferentially bind, encode components of cytoskeletal organization, cell adhesion, signal transduction, and transport of ions or macromolecules. We modified a previously published protocol to isolate da neurons from third-instar larvae, and analyzed how ab or kn mis-expression altered gene expression profiles. By combining all of these results, we narrowed down candidate targets and then examined RNAi-knocked down phenotypes of those. We will discuss our ongoing study of the transcriptional programs that regulate neuronal class-selective dendrite morphogenesis.


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104 Drosophila cyfip Regulates F-actin Remodeling and is Required for Synaptic Growth and Function Lu Zhao, Dan Wang, Yong Q. Zhang

Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

Synapses are highly specialized intercellular junctions that transmit information between neurons and their targets. Studies on the formation and development of synapses will shed light not only on the physiological neurodevelopment but also the pathogenesis of related neurological diseases. Cyfip, also known as Sra-1, was first identified as a specific Rac1-associated protein. It was later shown to interact with Fragile X mental retardation protein, FMRP, hence the name of Cyfip, cytoplasmic FMRP interacting protein. However, the synaptic function of Cyfip and its regulated actin cytoskeleton has been poorly defined. We report here that lack of cyfip results in dramatically aberrant neuromuscular junction (NMJ) synapses with more numerous and clustered boutons. We further show that cyfip regulates presynaptic actin and genetically interacts with several components of Rho GTPase pathway. FM1-43 dye loading analysis at NMJ synapses revealed an impaired endocytosis in cyfip mutants. Concomitantly, the amplitudes of miniature excitatory junctional potentials are increased in cyfip mutants. Furthermore, electron microscopy revealed a significantly increased size of synaptic vesicles in cyfip mutants. Together, these data demonstrate for the first time that cyfip regulates presynaptic actin cytoskeleton and synaptic endocytosis. We propose that misregulated actin cytoskeleton is at least partially responsible for the endocytic defects in cyfip mutants.


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105 Olfactory Receptor Neurons are Diversified by the Prdm Protein Hamlet that Mediates Chromatin Modification at Notch-Target Loci Keita Endo1, MD Rezaul Karim2, Alena Krejci3, Emi Kinameri2, Hiroaki Taniguchi2, Matthias Siebert2, Kei Ito1, Sarah J. Bray3, Adrian W. Moore2

1) IMCB, Univ. of Tokyo, Japan 2) RIKEN BSI, Japan 3) Dept. of PDN, Univ. of Cambridge, UK

Olfactory sensory system utilizes a diverse array of olfactory receptor neurons (ORNs). In Drosophila, ORN diversity is generated during olfactory lineage elaboration. In developing antenna, sensory organ precursor cell undergoes two rounds of cell divisions and gives rise to two intermediate precursors (IPs), both of which divide once and together produce up to four different ORNs. Differential activation of Notch signal between the two IPs was previously shown to diversify the IPs such that each produces a unique set of ORNs. We now show that differential Notch activation occurs again between the daughter cells of the IPs. Hence, ORNs within a lineage are diversified by the compound outcome of the iterated Notch signaling events. We further show that outcomes of the Notch signals are modified in a context-dependent manner by the Drosophila Prdm Protein Hamlet (Ham). Ham is expressed solely in one of the IPs and its daughter cells, and is required for the fate specification of these daughter cells. Expression analysis of Notch-target genes revealed that ham temporarily suppresses one of the target gene, E(spl)m8, in these daughter cells, making these cells competent to respond to the second differential Notch activation. We further show in cultured cells, that Ham acts by directing locus-specific modifications of histone methylation and histone density, altering the accessibility of the loci to the CSL protein, Su(H), and thereby modifying the responses of the target genes to Notch signal. This illustrates the importance of epigenetic mechanisms that regulate Notch signaling in the generation of ORN diversity.

POSTER: IMMUNITY AND PATHOGENESIS

150

106 Wolbachia Bacteria Reside in Host Golgi Vesicles whose Position is Regulated by Polarity Proteins Kyung-Ok Cho*, Go-Woon Kim, Ok-Kyung Lee

Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Korea

Wolbachia pipientis are maternally inherited symbiotic bacteria that are widespread among most insects including laboratory stocks of Drosophila melanogaster. Wolbachia belong to the Richettsial family responsible for the deadly human diseases such as typhus, Rocky Mountain spotted fever and Q fever. However, Wolbachia bacteria are best known for their ability to induce reproductive alterations in hosts such as male killing, feminization, parthenogenesis, and cytoplasmic incompatibility.

These bacteria are present in astral microtubule-associated vesicular structures in host cytoplasm, but little is known about the identity of these vesicles. We found that Wolbachia are restricted only to a group of Golgi vesicles concentrated near the site of membrane biogenesis and minus-ends of microtubules. These observations raise an interesting possibility that Wolbachia may mark the unique group of Golgi vesicles linked to membrane biogenesis. The Wolbachia vesicles were significantly mislocalized in mutant embryos defective in cell/planar polarity genes such as discs-large, Van Gogh/strabismus, frizzled and disheveled, suggesting that cell/planar polarity genes are required for apical localization of these Golgi vesicles. Furthermore, two of the polarity proteins, Van Gogh/Strabismus and Scribble, were actually present in these Golgi vesicles. Our data demonstrate that establishment of polarity may be closely linked to the precise insertion of Golgi vesicles into the new membrane addition site. The additional finding that localization of Wolbachia vesicles is regulated by genes involved in cell/tissue polarity including Stbm, Dlg, Fz and Dsh also provided a surprising new potential activity for these polarity genes in Golgi localization.


151

107 Studies on Immune Response of Drosophila Malpighian Tubules: A Closer Insight about Regulation of Epithelial Immune Response and Tissue Specific Antimicrobial Peptides Expression Puja Verma, Madhu GowaldasTapadia Cytogenetics laboratory, Department of Zoology, Banaras Hindu University, Varanasi, India

In Drosophila, Innate immune response is the sole mechanism to combat infection. Antimicrobial peptides (AMPs) are important effectors of innate immune response. Fat body is the primary immune tissue which synthesizes antimicrobial peptides. However, epithelial tissue plays a significant role in immune response in both vertebrates and invertebrates. Major disorders such as inflammatory bowl disease, Crohn’s disease and asthma causes by deregulation of epithelial immune defense.Therefore, it is prudent to study immune response in epithelial tissues which are early warning immune tissue. Malpighian tubules (MTs) are simple epithelial tissue which serves as first line of defense. It primarily functions in osmoregulation and detoxification. Apart from this, MTs also play a central role in immune response. To date, Regulation of epithelial immune response and tissue specific AMPs expression are remains to be answered. Here we studied the AMPs expression in MTs at different developmental stages. AMPs expression commences from 3rd instar larvae and persists up to adult. There is developmental variability of AMPs. We also observed the immune competence of MTs which become immune competent from 3rd instar. This is further substantiated by survival assay of different stages of larvae after infection. As Relish regulates AMPs expression in epithelia. We also studied the expression of Relish in MTs at different developmental stages which also varied among developmental stages. All these results show that the immune response in MTs is developmentally regulated.


152

108 Role of Antimicrobial Peptides Over-Expressed in Damaged Gut Cells of Drosophila melanogaster Eun-Young Yun1*, Young-Il Yoon1, Jae-Sam Hwang1, Hye-Min Sin1, Mi-Young Ahn1, Tae-Won Goo1 Department of Agricultural Biology, National Academy of Agricultural Science

Inflammatory bowel disease (IBD) is a group of chronic disorders characterized by the inflammation of the gastrointestinal tract; the exact cause of IBD is unknown. To investigate the cause of IBD by using Drosophila melanogaster, we created a D. melanogaster intestinal damage model by orally feeding the flies with various IBD inducers such as pathogenic bacteria Serratia marcescens, dextran sulfate sodium (DSS), and bleomycin and obtained the survival rate and 50% lethal dose (LD50). After feeding the flies with IBD inducers of LD50, we found that in the damaged gut cells, the number of enteroendocrine cells secreting antimicrobial peptides (AMPs) had increased and the expression level of the AMPs, and upstream signalling of the AMPs (Toll, Pelle, Dorsal and Relish) had up-regulated. We also confirmed that apoptosis and intestinal stem cells (ISCs) had markedly increased in the damaged gut cells. On the other hand, results obtained by using RNA interference (RNAi) lines for the knockdown of various AMP genes showed that the survival rate had increased and apoptosis had decreased in the RNAi lines as compared to wild-type flies. Anti-Toll, anti-Dorsal, and anti-Pelle antibody feeding flies survived longer than normal flies after feeding with DSS. We deduce from the above results that when IBD inducers entered the fly gut, various AMPs were up-regulated for self-defense. Over-expressed AMPs not only function in intestinal protection by destroying harmful microorganisms, but also function in intestinal destruction by attacking their own cellular components. Hence, we suggest that AMPs that are over-expressed by IBD inducer could be a possible cause of IBD.


153

109 Odor-Based Contagious Transmission of Pathogen by Drosophila melanogaster Kiyoshi Okado, Naoaki Shinzawa, Shinya Fukumoto, Hirotaka Kanuka NRCPD, Obihiro University of Agriculture and Veterinary Medicine, Japan

The housefly and flies in general are considered to be mechanical vectors of many kinds of pathogens such as bacteria, protozoa and viruses, whereas the mosquito serves as the biological vector for those pathogens. Mechanical vectors simply convey pathogens and are not essential for their development and life cycle. It has been shown that a large number of bacteria adhere to the surface of the housefly mouthparts, actively proliferate in the minute spaces of the labellum, and accumulate in the crop. In order to uncover the molecular mechanisms of bio-enhanced transmission, a mode of spreading pathogens via excretion of fly feces, we first established a model system for transmission of bacteria using Drosophila melanogaster. A mass of GFP-labeled Escherichia coli located on the center of an agar-plate was freely ingested by Drosophila for 2 hours. Substances excreted in the feces are easily observed as small spot with GFP fluorescence on the surface of agar, showing that flies directly feed E. coli and disseminate them by excretion. The sensory system of insects has evolved the capacity to recognize and discriminate an inordinate number of chemically distinct molecules that signal the presence of food, predators, or mating partners. Flies without antenna that contains a large set of olfactory receptors or deficient for Or83b, which encodes a broadly expressed odorant receptor, showed impaired dissemination of bacteria. While wild type flies showed behavioral responses to attractive odors released from growing E. coli, the Or83b mutants failed to respond to the odors. These results suggest that feeding behavior of fly is stimulated by odors from pathogens and may contribute to the bio-enhanced transmission of infectious diseases such as food poisoning.


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110 Novel Roles of Glycosylation in Drosophila Innate Immunity Miki Yamamoto-Hino1,2, Takako Shibano2, Wakae Awano2, Hideyuki Okano1, Satoshi Goto1,2 1) Keio Univ. 2) Mitsubishi-Kagaku Inst. Life Sc.

Glycosylation plays important roles in various biological processes including development, neural activity and immunity. Drosophila has a variety of innate immunity mechanisms including humoral and cell mediated responses. In humoral response, pattern recognition receptors of Drosophila first recognize glycans and other extracellular molecules of pathogens as non-self. Following this, Toll, Imd and JAK/STAT pathways are activated to produce antimicrobial peptides and stress response factors. Whereas the pathogens’ glycans are essential for the discrimination between self and non-self, it remains unclear whether glycans of Drosophila are involved in the innate immunity. Here, we present that glycosylation controls innate immunity signaling pathways. We knocked out a gene, ashura, which is responsible for glycosylation, and found that ashura mutant exhibits melanotic tumor formation in the larval lymph gland, its hypertrophy and melanotic spot formation under the cuticle. In addition, the number of lamellocyte was markedly increased in ashura as compared to the wild-type. These phenotypes resemble those of Toll10B and hopTum-l, gain-of–function alleles of Toll and JAK, respectively. We further investigated whether these pathways are activated in ashura. Infection-mediated activation of Toll and JAK/STAT pathways induces the expression of drosomycin and totA, respectively. However, without immuno-challenge, ashura flies highly expressed both genes. These results suggest that the signal pathways involved in innate immunity become constitutive activated in ashura and that the glycans synthesized by ashura might negatively regulate Toll and JAK/STAT pathways. This is the first example that the host glycosylation regulates innate immunity.

POSTER: PHYSIOLOGY AND METABOLISM

155

111 A Transgenic RNA Interference Screen for Regulators of Steroid Hormone Biosynthesis in Drosophila Yuko Shimada-Niwa1, Jevgenija Maramzina1, and Ryusuke Niwa1,2

1) Graduate school of Life and Environmental Sciences, University of Tsukuba 2) Initiative for the Promotion of Young Scientists’ Independent Research, University of Tsukuba

Steroid hormones play crucial roles in many aspects of development, growth and reproduction in multicellular organisms. During Drosophila postembryonic development, the principal steroid hormone, ecdysone, is synthesized from dietary cholesterol in a special endocrine organ called the prothoracic gland (PG). Ecdysone biosynthesis in the PG is appropriately controlled in response to several external conditions, such as nutrition, temperature and photoperiod. This adaptive change of ecdysone biosynthesis results in flexible alterations of developmental timing and growth. However, it remains unclear how external information is transmitted to the PG to adaptively control the catalytic activities of the ecdysone biosynthetic enzymes. To uncover essential genes involved in controlling the adaptive regulation of ecdysone biosynthesis in the PG, we conducted a transgenic RNAi screen using the PG-specific GAL4 driver and publicly-available UAS-RNAi strains. We found that developmental arrests were caused by loss of function of several genes encoding transmembrane receptors, including TGF- receptors, neurotransmitter receptors, and cholesterol-sensing proteins. The developmental arrest phenotypes were rescued when the RNAi animals were fed with 20-hydroxyecdysone (20E), suggesting that those receptors are required for ecdysone biosynthesis. In this presentation, functional roles of candidate regulators in ecdysone biosynthesis will be discussed.


156

112 Physiological and Life History Consequences of Starvation Selection in Drosophila Lauren A. Reynolds, Allen G. Gibbs

School of Life Sciences, University of Nevada, Las Vegas, NV 89154, USA

In nature, animals may endure periods of famine to complete their life cycles. At the organismal level, these periods can be survived by increasing energy storage and/or decreasing energy usage. We selected for starvation resistance in replicated populations of Drosophila melanogaster. After 50+ generations of starvation selection, populations survive starvation significantly longer (>10 days) than control populations (~3 days). Starvation-selected populations contain significantly higher amounts of lipid than controls. These resources are acquired during an extended larval feeding period, so that newly eclosed adults immediately contain greater energy stores. Thus, selection for starvation resistance in adult D. melanogaster has significant effects on larval physiology. Use of resource stores were measured with metabolic rates and activity levels. Metabolic rates of adults are higher in starvation-selected populations, likely due to their larger body size. Activity levels of adults are lower in starvation-selected populations allowing for more efficient use of energy stores. Increased starvation resistance has come at a life history cost, starvation-selected populations eclose ~24 hr later and have lower fecundity. Preliminary data suggests that the developmental delay, which allows for a longer feeding period, maybe due to an ecdysone hormone delay.


157

113 Alcohol-Induced Refolding of Prophenol Oxidase in Drosophila melanogaster Nobuhiko Asada*, Kotomi Mita, Eri Sato

Department of Zoology, Okayama University of Science, 700-0005 Japan *: Corresponding author

In the “Post Genome” era, cuticle pigmentation in Drosophila melanogaster is one of the fantastic characters and is rich sources of models of phenotypic evolution. Insect pigmentation differences are often controlled by structural genes and correlate with cascade reaction in the self-defense and with artificial sources as alcohol species. Understanding of the biochemical and evolutionary genetic basis of pigmentation reaction in Drosophila could provide inroads to classic evolutionary problems as phenotypic convergence.

Studies on the high-order structures of given proteins have been widely understood. Phenol oxidase (tyrosinase in the mammal) distributed in hemolymph in many invertebrates and is involved in the precursor, prophenol oxidase. In Drosophila melanogaster, purified prophenol oxidase (folding stasis) is activated with the native prophenol oxidase activating enzyme (PPAE) as limited proteolysis in vivo or conformational change of the protein by the detergents and alcohols especially 2-propanol in vitro (unfolding stasis). This implication marked an effect of the high propensity of 2-propanol to induce conformational change of the prophenol oxidase protein to bind and promote between substrate and protein at the active center of the enzyme. These potentials cause to contribute to the “Bi-Bi system” in the protein science.

Protein “folding-refolding model” of prophenol oxidase in Drosophila melanogaster is expected to give advantage to the applied study for the folding-refolding of the higher-order structure of various proteins.

We appreciated Dr. Yuji Goto in Osaka University, Japan, for his cooperative study This work was financial supported, in part, by The Ryobi Teien Foundation, Okayama, Japan.


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114 Genetic Analysis of Lipid Metabolism Using a Drosophila Model Hiroko Sano1, Kayoko Tsuda1,2, Makoto Umemori1, Reiko Amikura1

1) Ochadai Academic Production, Advanced Interdisciplinary Research Division, Ochanomizu University, Japan 2) Japan Society of the Promotion of Science

Lipid metabolism is a double-edged sword that enables animals to survive starvation and cold but also leads to obesity and fatal coronary artery diseases when it malfunctions. Research using model organisms is expected to uncover the mechanisms of lipid metabolism and offer evidence-based protocols for prevention and treatment of obesity. Recently, it was shown that Drosophila has lipid-metabolizing organs similar to those in humans and evolutionarily conserved lipid-metabolizing genes. With a Drosophila model, we have established a successful genetic screen to find hitherto unidentified lipid regulators. Among the candidate lipid regulators so far identified, we are focused on a putative chromatin regulator and an ATP synthetic enzyme that might be regulating lipid metabolism through providing acetyl groups. Beyond lipid metabolism within the cell, we are also interested in how the animals decide if they store or utilize nutrients. We speculate that the fat body is not only a reservoir of nutrients but also functions as an endocrine organ to coordinate nutritional condition and metabolism and feeding behavior. Aiming at clarifying such endocrine function of the fat body, we have been searching for fat body-derived signals. We expect that our novel approach will uncover molecular and cellular mechanisms of lipid metabolism in the fat body and contribute to the identification of the missing link between calorie intake and the decision by the body to store the extra nutrients as fat. Herein I present recent progress in and future perspectives for my research.


159

115 A Metazoan Ortholog of SpoT Hydrolyzes ppGpp and Functions in Starvation Responses Gina Lee, Jongkyeong Chung Institute of Molecular Biology and Genetics and School of Biological Sciences, Seoul National University, Seoul 151-742, Korea

In nutrient-starved bacteria, RelA and SpoT proteins have key roles in reducing cell growth and overcoming stresses. We identified functional SpoT orthologs in metazoa (named Mesh1, encoded by HDDC3 in human and Q9VAM9 in Drosophila melanogaster) and revealed their structures and functions. Like the bacterial enzyme, Mesh1 proteins contain an active site for ppGpp hydrolysis and a conserved His-Asp (HD)–box motif for Mn2+ binding. Consistent with these structural data, Mesh1 efficiently catalyzed hydrolysis of guanosine 3′,5′-diphosphate (ppGpp) both in vitro and in vivo. Mesh1 also suppressed SpoT-deficient lethality and RelA-induced delayed cell growth in bacteria. Notably, deletion of Mesh1 (Q9VAM9) in Drosophila induced retarded body growth and impaired starvation resistance. Microarray analyses revealed that the amino acid–starved Mesh1 null mutant has highly downregulated DNA and protein synthesis–related genes and upregulated stress-responsible genes. These data suggest that metazoan SpoT orthologs have an evolutionarily conserved function in starvation responses.


160

116 Identification of Small Molecules which Extend Lifespan in Drosophila Using a Versatile Culture System Yukiko Sato1, Toshiro Aigaki1

Department of Biological Sciences, Tokyo Metropolitan University, Japan

The identification of small molecules that extend the lifespan of model organisms are useful for gaining mechanistic insights into aging, and provides opportunities to develop new drugs for age-related human disorders. Drosophila is one of the best organisms to study gene functions in vivo, and it is also useful for chemical biology, because of the short lifespan and availability of human disease models. However, the fly culture system has not been optimized for the testing of small molecules, particularly if the amounts of available chemicals are limited. We designed a small culture system to determine the effects of chemicals on fly lifespan using relatively small amounts of samples. Using our culture system, we found that an antioxidant compound isolated from the leaves of the running bamboo (Sasa senanensis) extends the lifespan of Drosophila. The compound was an inhibitor of NADPH oxidase (Nox). We also provide evidence that the reduction of Nox activity extends lifespan, by feeding Drosophila with other known Nox inhibitors, and by the suppression of Nox expression. Our results suggest that Nox-mediated reactive oxygen stress (ROS) is a novel target for the pharmacological manipulation of lifespan.


161

117 Decreased Ribose-5-Phosphate Isomerase Expression in Neurons Enhances Oxidative Stress Resistance, Increases Lifespan, and Attenuates Polyglutamine Toxicity in Drosophila Ching-Tzu Wang1, Yi-Yun Wang1, Ming-Hao Huang1, Tzu-Kang Sang1, Yi-Chun Chen1, Si-Chih Cho1, Chiou-Hwa Yuh2, Chao-Yung Wang3, Theodore J. Brummel4, Horng-Dar Wang1 1) Institute of Biotechnology and Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan, R.O.C. 2) Division of Molecular and Genomic Medicine, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan, R.O.C. 3) Second Section of Cardiology, Department of Internal Medicine, Chang Gung Memorial Hospital at Linkou, Chang Gung University College of Medicine, Taoyuan, Taiwan, R.O.C. 4) Department of Biology, Long Island University, Brookville, NY 11548, U.S.A.

Aging and age-related diseases can be viewed as the result of the lifelong accumulation of stress insults. The identification of mutant strains and genes which are responsive to stress and can alter longevity profiles provides new therapeutic targets for age-related diseases. Here we reported that a Drosophila strain EP2456, with reduced expression of ribose-5-phosphate isomerase (rpi), exhibits increased resistance to oxidative stress and enhanced lifespan. In addition, the strain also displays higher levels of NADPH. The knockdown of rpi in neurons by double-stranded RNA interference recapitulated the lifespan extension and oxidative stress resistance in Drosophila. This manipulation was also found to ameliorate the effects of genetic manipulations aimed at creating a model for studying Huntington’s disease by overexpression of polyglutamine in the eye, suggesting that modulating rpi levels could serve as a treatment for normal aging as well as for polyglutamine neurotoxicity.


162

118 Metabolomic Analysis of Drosophila Using A Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry Yoshihito Kishita, Yukiko Sato, Manabu Tsuda, Toshiro Aigaki Department of Biological Sciences, Tokyo Metropolitan University, Japan

Energy metabolism is essential for all organisms. Although metabolic pathways have been well characterized, little is known about their homeostatic regulation in response to genetic or environmental perturbation in vivo. Here, we developed a method for metabolomic analysis of Drosophila using a liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOFMS). We used ion pair reagents in the mobile phase to separate negatively charged compounds including metabolites of glycolysis and tricarboxylic acid (TCA) cycle on a C18 reversed-phase column. The method also allows detection of amino acids and fatty acids, allowing a comprehensive analysis of compounds related to energy metabolism. We report metabolomic changes during development and aging of wild-type flies reared on a standard or nutrient-restricted media. We also demonstrate the power of metabolomic analysis to investigate mutant flies that are defective in locomotor activity.


163

119 smb Controls Lifespan through Involving in the Ubiquinone Synthesis in Drosophila Jiyong Liu, Qinghua Wu, Dianlu He, Li Du, Renjie Jiao State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, CAS, Beijing, China

CoQ (ubiquinone) is an essential electron carrier in the mitochondrial respiratory chain of both eukaryotes and prokaryotes. It consists of a hydrophobic isoprenoid tail and a quinone head group. The genes (COQ1-9) involved in CoQ biosynthesis have been characterized in bacteria and yeast while information about these biosynthetic enzymes in other species is poorly available. Many studies indicate that genes involved in CoQ biosynthesis, such as clk-1, the C. elegans COQ7 homologue, play an important role in the aging process.

In this study, we characterized a novel gene, which is named as smb (small boy) following one of its mutant phenotypes, small larvae. Smb contains a UibA prenyltransferase domain and the coding sequence shares 33% identity with the yeast COQ2, which catalyses the condensation of the isoprenoid chain to the p-hydroxybenzoate ring. Constitutive expression of smb rescues the lethality of △COQ2 yeast, indicating that smb is the homologue of yeast COQ2. HPLC results show that the levels of CoQ9 and CoQ10 were significantly reduced in smb heterozygous adult flies. Furthermore, the mean lifespan of males and females heterozygous for smb are extended by 12.5% and 30.8% respectively. Homozygous smb animals exhibit reduced activities of the insulin/insulin like growth factor signaling (IIS) pathway. Taken together, we conclude that smb is an essential gene for Drosophila development, dose reduction of which extends lifespan by altering CoQ biosynthesis.

POSTER: REGULATION OF GENE EXPRESSION

164

120 Genome-wide Identification and Analysis of Direct Targets of the Hox Protein Ultrabithorax in Drosophila Pavan Agrawal1,2, Farhat Habib2, Ramesh Yelagandula1, L. S. Shashidhara1,2

1) Centre for Cellular and Molecular Biology, Hyderabad, India 2) Indian Institute of Science Education & Research, Pune, India

Hox proteins are transcription factors and key regulators of segmental identity along the anterior posterior axis across all bilateral animals. Suppression of wing fate and specification of haltere fate in Drosophila by the Hox protein Ultrabithorax (Ubx) has served as a paradigm for understanding Hox protein function. Despite decades of research mechanism by which Hox proteins select their targets and specify segmental identity remains elusive. We addressed this problem employing ChIP-chip in Drosophila imaginal discs to identify genome wide occupancy of Ubx. We generated polyclonal antibodies against N-terminal region of Ubx lacking homeodomain and tested specificity of the immune sera using western blots, immuno-histochemistry and ChIP-qPCR. From three biological replicates of ChIP-chip we obtained ~500 common DNA fragments, representing ~420 genes, bound by Ubx in vivo. Many of these genes are developmentally regulated, pattern-formation genes e.g. transcription factors or signaling molecules as opposed to realizators genes which were earlier thought to be major categories of Hox targets. Upon sequence analysis we could identify previously known in vitro Ubx motifs in many in vivo Ubx bound sequences suggesting validity of those motifs. We also found many motifs that are similar to binding sites of transcription factors traditionally known to act upstream of Hox proteins. Our results show that Ubx can associate with some of these proteins in haltere discs to regulate downstream targets. We discuss a model where Hox binding sites are plastic and specificity for target selection is achieved by association with various transcription factors in vivo.


165

121 Zelda Coordinates Gene Regulatory Networks in the Early Drosophila Embryos Hsiaolan Liang1, Chungyi Nien1, Shengbo Fu1, John Manak2, Nikolai Kirov1, Christine Rushlow1

1) Department of Biology, New York University, New York, USA; 2) Molecular & Cellular Biology, University of Iowa, Iowa, USA.

Early development consists of a highly choreographed series of events controlled by temporally and spatially regulated genes. In past years, much attention has focused on the gene networks that regulate these processes, but less attention has been paid to how they are coordinated. We previously identified a transcriptional activator Zelda, which binds to CAGGTAG and related sequences that are present in the enhancers of many early-activated genes in Drosophila (ten bosch et al.; De Renzis et al., 2007; Liang, 2010). This discovery hinted at a mechanism for how batteries of early genes are coordinately activated. To address the extent to which Zelda binds and regulates pre-blastoderm genes, we performed genome-wide binding analysis of early embryos. The results combined with our expression studies reinforce and extend our previous conclusion that Zelda is a global activator of the early zygotic genome, uncovering many new Zelda targets involved in body patterning. We demonstrate that Zelda is responsible for the timely activation of Dorsal-Ventral, Anterior-Posterior and terminal patterning genes, which is critical for the initiation of cross-regulatory cascades, and without which leads to dramatically altered positional information in the blastoderm embryo.


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122 The Transcription Factor Network Patterning Drosophila photoreceptors Hui-Yi Hsiao, Robert Johnston, Dave Jukam, Claude Desplan Department of Biology, New York University, USA

In the Drosophila eye, each ommatidium contains eight photoreceptors (PRs) arranged in a trapezoid shape. PR cell fates are specified at late larval stages. After their recruitment, they are defined as outer PRs (R1-R6) vs. inner PRs (R7 and R8), which are located at the center of the trapezoid formed by the outer PRs. Then, during late pupal stages, all PRs undergo terminal differentiation and express specific Rhodopsins (Rh). Even though outer PRs all express Rh1, each of them can be distinguished by a unique transcriptional profile. Although some of the genetic programs controlling outer/inner fate determination are well characterized, many regulatory steps remain unclear. To address the underlying mechanisms, we performed an RNAi screen knocking down all known transcription factors (~950 TFs) and using rh1-GFP to visualize patterning phenotypes of PRs in the adult eye. The expression of UAS-RNAi is controlled spatially and temporally by a combination of two eye-specific GAL4 drivers, ey-Gal4 and lGMR-Gal4.By using rh1>GFP as a readout, couple of genes has been found with a mutant cell fate phenotype showing extra rh1-expressing PRs. Inner rhodopsins are normally expressed in the ectopic rh1-GFP expression PR suggests those candidates function in repression of rh1 in inner PRs rather than in the determination of inner/outer fate. Further study shows that only de-repression of rh1-GFP has been detected in mutant candidates rather than Rh1 protein. The result leads to the explanation that rh1 might be post-transcriptionally regulated.


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123 Modulation of the Innate Immune Response in Drosophila melanogaster by SUMOylation Mithila Handu, Ritika Giri, Girish Ratnaparkhi

Indian Institute of Science Education and Research (IISER), Pune, India

The role of reversible post-translation modifications in rapid transduction of cellular signals has been studied in context of fine-tuning various biological processes. It has been previously shown that phosphorylation and ubiquitination play important roles in elicitation of both cellular and humoral immune responses in Drosophila melanogaster. What is less understood is the role for Ubiquitin-like modifiers and also the relationship between different post-translation modifiers in regulating the innate immune gene regulatory network. With an aim to better understand regulation of the innate immune response, our primary goal is to identify roles for SUMOylation in Host defense in Drosophila melanogaster.

Our approach is two pronged. The proteomics part involves Tandem affinity purification in conjunction with quantitative mass spectrometry to identify changes in SUMOylated states of proteins in response to an infection. In the second part we use loss of function genetics to reduce/knockdown members of the SUMO Cycle. This helps us identify the importance of the SUMO pathway in immune response with respect to regulation of defense genes, hemocyte levels and also susceptibility to infection.

Given the importance of a robust immune response and the widespread nature of SUMOylation, it is likely that many proteins and signal transduction pathways involved in host defense are subject to regulation by SUMOylation. Generating a parts list of such proteins and understanding their regulation by SUMO modification, at individual protein level, as well as network levels will help provide further insights into the mechanisms governing host defense in animals.


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124 A Small Peptide Gene polished rice Participates in Drosophila Ecdysone Signal Pathway Yoshiko Hashimoto, Kaori Niimi, Takefumi Kondo, Yuji Kageyama Okazaki Institute for Integrative Bioscience, Okazaki, Japan

Whole genome analyses have revealed that a substantial population of poly (A)+ transcripts (~500 nt) do not represent apparent ORFs, and are thus presumed to be non-coding RNA. However, a growing body of evidence suggests that some large non-coding RNAs actually encode small peptides. Drosophila polished rice (pri) polycistronically encodes four extremely small peptides (11 or 32 aa) that render full function of this gene in formation of tracheal network and cuticle structures during embryogenesis (Nat Cell Biol., 9, 660-665; Science, 329, 336-339). pri functions, however, remains unclear, although it is known that pri orthologs are widely found in Arthropods.

In this presentation, we will show that pri is a novel member of the ecdysone signaling pathway in Drosophila. pri is an ecdysone-inducible gene and is expressed entirely in imaginal discs at puparium formation. A hypomorphic mutation of pri, which caused developmental arrest during metamorphosis, disturbed the expression of E75A and E74 at the prepupal-pupal transition. In clonal analysis, pri null mutant cells in the eye region caused necrotic ommatidia, reminiscent of flies overexpressing a dominant-negative isoform of EcR (EcR-DN). We found that pri overexpression rescues the necrotic defect induced by EcR-DN, suggesting that pri is the major downstream target of ecdysone signaling. On the other hand, pri overexpression showed synergistic enhancement of EcR-RNAi phenotype, implying that pri functions in the vicinity of EcR. These results demonstrated that pri is a fundamental factor in the ecdysone signaling pathway, and plays important roles in metamorphosis.


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125 JNK/FOXO Signaling Regulates the Expression of Drosophila Peroxiredoxin V during Immune Response Yoon-Seok Suh1,2, Kyu-Sun Lee1,2, Hye-Mi Ahn1,3, Dong-Seok Lee3, Kweon Yu1,2* 1) Aging Research Center, KRIBB, Daejeon 305-806, Korea 2) Functional Genomics program, University of Science and Technology, Daejeon 305-806, Korea 3) College of Natural Sciences, Kyungpook National University, Daegu 702-701, Korea

Innate immunity plays an important role in combating microbial infection in all animals. In the Drosophila gut, Dual oxidase (Duox) produces sufficient amounts of ROS, which is a potential bacterial-killing molecule, to combat the infectious microbes. Simultanesouly, antioxidant systems are activated to eliminate residual ROS for protecting hosts. Here we identified the Drosophila peroxiredoxin V (dPrxV) as an immune-related antioxidant enzyme for maintaining the intestinal redox homeostasis. We found that dPrxV was predominantly expressed in the gut and its expression is induced by oral infection with Erwinia carotovora carotovora (Ecc15) strains. The expression of dPrxV was also increased by the gut-specific overexpression of Duox, whereas it was decreased by knockdown of Duox. Importantly, the induction of the dPrxV expression was mediated by the activation of JNK/FOXO signaling in the gut. These results suggest that JNK/FOXO signaling plays a critical role in regulating the expression of PrxV as an antioxidant system to protect the host gut epithelial cells from oxidative stress.


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126 Transcriptional Regulation of the unpaired3 Gene in Drosophila Hsin-Yi Huang, Yu-Chen Tsai

Department of Life Science, Tunghai University, Taichung, Taiwan

Unpaired3 (Upd3) is a ligand of Janus Kinase/ Signal Transducers and Activators of Transcription (Jak/STAT) signaling in Drosophila. upd3 is expressed in eye-antenna disc, gonad in the larval stages and testis in adult male. The upd3 null mutant is fertile and viable with small eye. upd3 takes part in multiple developmental processes and physiological responses, including eye, gonad development, haematopoiesis and immune response. Upd3 regulates hemocyte homeostasis and midgut homeostasis after septic injury or oral infection in the larval stage. In this study, we focus on the transcriptional regulation of the upd3 gene and further study the upstream signaling of the upd3 gene. The 22.1 Kb genomic regions around the upd3 gene were analyzed. These upd3 genomic fragments were cloned to enhancer-testing vector, pH-Stinger, which contains a GFP reporter. The enhancer-testing constructs were injected into Drosophila and then selected for transgenic lines. The expression patterns of GFP reporter in the transgenic flies were examined in vivo. Preliminary, we found a possible 1.2Kb upd3 enhancer which locates at 1.8Kb downstream of the upd3 gene may regulate eye, intestine cells, posterior signaling center (PSC) in lymph gland and hub in adult male testis. We will further narrow down this enhancer fragment. We predicted the possible transcriptional factor binding sites in upd3 genomic regions. We found multiple Suppressor of Hairless (Su(H)), STAT92E and AP-1 binding sites in 22.1Kb genomic regions. There are two Su(H) and two STAT92E binding sites in the possible 1.2Kb upd3 enhancer fragment. We will mutate the putative Su(H) and STAT92E binding sites in 1.2Kb fragment by site-directed mutagenesis. In the future, we will further analyze whether upd3 is regulated by Notch, JNK, and Jak/STAT signalings in vivo.


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127 Functional Analysis of Drosophila Apt during the Eye Development Qing-Xin Liu1, 2, Kazuho Ikeo2, Yasushi Hiromi3, and Susumu Hirose3, Takashi Gojobori2,

1) Laboratory of Developmental Genetics, Shandong Agricultural University, Taian, Shadong, 271018, China 2) Center for Information Biology and DNA Data Bank of Japan, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan 3) Department of Developmental Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan

In Drosophila, the apontic (apt) gene encodes a bZIP protein that required for the development of trachea, heart, head and neural system. Apt is highly expressed in the cells of the morphogenetic furrow (MF) region. Loss of Apt function causes defects in the eye development. Overexpression of Apt in the eye disc induced abnormal eyes. The targets of apt responsible for these responses have not been identified. To identify apt downstream genes in a comprehensive manner, we used genome-wide oligonucleotide arrays and analyzed differential gene expression in wild-type embryos versus apt mutant embryos. Upon knockout of apt function, expression of 340 genes decreased and 338 genes increased. Many of these genes can be assigned to specific aspects of the tracheal and neural system development. We also discovered apt target genes that are likely to play specific roles in eye morphogenesis.


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128 The Role of Nuclear Actin in the Transcriptional Activity of Proneural Proteins Yu-Ju Chen, Yun-Ling Hsiao, Hsiao-Fong Yeh, Haiwei Pi Department of Biomedicine Sciences, Chang-Gung University, Taiwan

Proneural proteins of basic helix-loop-helix (bHLH) transcription factors are master controllers that initiate and execute neurogenic programs. Although much is known about how the tissue-specific expression of proneural proteins is regulated, it is largely unclear how proneural proteins interact with the transcriptional machinery to activate downstream target gene expression. In order to find out the association components with proneural proteins in the nucleus, I immunoprecipitated the proneural proteins from Drosophila S2 cells and identified the associated nuclear proteins by Mass Spectrometry (MS). I have found that proneural proteins specifically associate with nuclear actin. The interaction between proneural protein and nuclear actin is strictly correlated with the transcriptional activity of proneural proteins, because it required the presence of Daughterless protein, the heterodimeric partner of proneural proteins. And then, I used biochemical approaches to characterize that DNA binding activity of proneural protein is not required for interacting with nuclear actin, and Daughterless itself interacts with nuclear actin. By using reporter assay, I have found that nuclear actin enhances transcriptional activity of proneural proteins, and the activity of transcription correlates with polymerizating activity of nuclear actin. The presence of actin in the nucleus has been well established, and several in-vitro studies have implicated nuclear actin in transcriptional regulation. Furthermore, I used genetic assay and in vivo actin knock down by RNAi to show that Act5C and Act42A regulate ES organ and SOP formation. Mis-expression of nuclear actin induces ectopic SOPs and ES organs formation. The transcriptional activity of phyllopod, the target gene of proneural proteins, is down-regulated in act5c mutant. We therefore propose that nuclear actin acts as a transcriptional co-activator of proneual proteins during ES organ development.


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129 Dual Functions of Proneural Proteins to Regulate cdc25 Transcription in Drosophila Sensory Organ Development Yun-Ling Hsiao, Haiwei Pi Department of Biomedical Sciences, Chang-Gung University, Taiwan

During development, cell-cycle progression is tightly coordinated with differentiation. String/Cdc25 phosphatase initiates G2-M progression and its brief burst of transcription triggers immediate mitotic entry in Drosophila cells. We use Drosophila external sensory (ES) organ as a model to study string (stg) expression in neural development. In ES organ development, sensory organ precursors (SOPs) divide asymmetrically a few times to generate different types of daughter cells that undergo differentiation. In order to detect Stg expression and analyze its transcriptional regulation, we generated C31 integrase-mediated genomic rescue construct which includes the 40 Kb upstream regulatory regions and the stg ORF fused with Flag tag. In sensory organ precursors (SOPs), Stg expression is detected just before mitosis. The highest Stg level is first observed in the mitotic SOPs. Subsequently, we detected high Stg in one daughter cell and low Stg in another daughter cell immediately after cell division, raising the possibility that Stg proteins are asymmetrically segregating to two daughter cells during mitosis.

Studies in our lab and others have shown that proneural proteins Ac and Sc, master proteins of sensory organ development, play a key role in suppressing stg expression before SOP division (Chang et al., 2008). We found that this suppression effect depends on the DNA-binding activity of Ac. To our surprise, we also found evidence to suggest that proneural protein could also positively regulate stg expression through the 2.6 kb cis-regulatory region located from -13 kb to -15.6 kb upstream of the stg. The stg2.6-lacZ is expressed specifically in SOPs, and ectopic expression of Ac drives ectopic stg2.6-lacZ expression. Taken together, our results show dynamic Stg expression in ES organ development and suggest that proneural proteins play a dual role to both positively and negatively regulate stg transcription.


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130 Role of Tws, a B regulatory Subunit of PP2A, in Mitochondrial Fragmentation and Autophagic Response Yu-Chiang Chue, Ming-Tsan Su Department of Life Science, National Taiwan Normal University

Previous studies demonstrated that overexpression of tws induces mitochondrial fission and autophagic response in Drosophila. In addition, fragmented mitochondria were encapsulated by autophagic vacuoles, suggesting that tws might induce autophay indirectly. However, it is equally possible that tws also plays a role in autophay response. The objectives of my research, thus, is to identify the down-stream targets of Tws that modulate mitochondrial dynamics and to investigate whether Tws is directly involved in autophagic response. To search for the down-stream targets of Tws, we plan to conduct mainly genetics and biochemistry approaches. Through literature search we have compiled a list of possible candidates. Using photoreceptor as a model system, we will first test if candidate genes interact with tws genetically. It is expected that gain- or loss-of-function alleles of the candidate genes will either enhance or suppress the rough eye phenotype. In our pilot study, we did observe that some of the candidate genes interact with tws genetically. We also plan to isolated down-stream targets of tws by using phosphor-proteomics approach. The rationale of the study is because Tws is an essential regulatory subunit of Protein phosphatase 2A. It is expected that down- or up- regulation of tws will alter the phosophorylation status of its substrates. Finally, function of the identified genes will be further characterized. To investigate if tws functions in autophagy, loss-of-function of tws mutant will be used. Since autophagic response can be induced in fat bodies of larva during starvation, if tws does play a role in autophagy, it is expect that number of lysosome, an indicator of autophagic response, will not increase in the fat bodies of tws mutant larva in starvation condition.


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131 To Investigate if TATA Box Binding Protein is a Regulator of p53 Dependent Apoptosis in Drosophila Chin Sern Yong, Ming-Tsan Su Institute of Cell Biology and Molecular Biology, National Taiwan Normal University

TATA Box binding protein (TBP) is a general transcription factor that is required for the transcription of virtually all genes in cells. Previous studies reported that inactivation of the murine TBP gene by homologous recombination results in growth arrest and apoptosis at the embryonic blastocyst stage. Moreover, inhibition of TBP expression by injecting antisense morpholino oligos causes developmental arrest and block of epiboly movements in zebrafish embryos at midblastula stage. How TBP regulates animal development and its involvement in apoptosis are elusive. The main objective of my research is to investigate the role of TBP in apoptosis. Since p53, a major pro-apoptotic regulator, interacted with TBP physically, we suspect that TBP may be a component of the intrinsic death pathway. Using genetics approach, we will first dissect the epistatic relationship of TBP and p53. We will also biochemistry approach to exam if TBP modulates function of p53 directly.


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132 drumstick, an Odd Family Gene, Acts Cell Non-autonomously to Specify the Anterior-Most Domain of the Hindgut in Drosophila Sarder N. Uddin1,2, Masahiro Yano1, Ryutaro Murakami1 1) Department of Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Japan 2) Biotechnology and Genetic Engineering Discipline, Khulna University, Bangladesh

An odd family gene drumstick (drm) encodes a zinc finger protein, and is necessary for specifying the small intestine, an anterior domain of the ectodermal hindgut of Drosophila melanogaster. However, mechanisms that specify the small intestine, as well as gene regulatory pathways leading to transcriptional activation of drm, are still unclear. We found that drm is expressed in the regions abutting anterior end of the hindgut primordium, that is, the posterior-most region of the endoderm and in basal portion of the Malpighian tubules, but, not in the prospective region of small intestine. The small intestine failed to form in mutant embryos that lack both the endoderm and Malpighian tubules, but it can develop when either one of the drm-expressing tissues is intact. These results indicate that drm induces the development of small intestine cell non-autonomously, probably through some intracellular signaling. We also found that drm expression in the posterior gut region disappears in tll embryos, and, small intestine failed to develop. On the other hand forced-expression of tll caused expansion of the drm expression in posterior endodrm, resulting in an expanded small intestine. Unexpectedly, byn, which has been recognized as a master gene for the development of hindgut, is not required for the specification of small intestine. Our results demonstrate that drm is activated under the control of tll, and consequently drm acts cell non-autonomously to specify the small intestine in the hindgut primordium.

POSTER: CHROMATIN AND EPIGENETICS

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133 A Novel RNAi Protein that Regulates Heterochromatin Su Jun Lim, Anthony Scott, Dongdong Guo, Shanshan Pei & Willis X. Li Department of Biomedical Genetics, University of Rochester Medical Center

Heterochromatin regulation has been shown to be dependent on the RNA interference (RNAi) machinery. In fission yeast, small RNAs, together with core RNAi components, can recruit chromatin-remodeling factors to facilitate heterochromatin formation. In C. elegans, RNAi can inhibit pre-mRNA levels and be transmitted across generations, suggesting an epigenetic effect. In Drosophila, heterochromatic silencing and HP1 localization are impeded when the RNAi machinery is disrupted. However, the detailed mechanism of the RNAi-dependent heterochromatic silencing remains elusive. To address this, we have conducted a forward genetic screen to seek additional RNAi components. This screen has recovered dCRIF (Drosophila CR-6 interacting factor) as a potential novel RNAi member. Loss-of-function of dCRIF can suppress the RNAi-mediated gene silencing induced by two different RNAi constructs. In addition, immunostaining and co-immunoprecipitation studies suggest that dCRIF protein interacts with Dcr-2 in vivo. Interestingly, besides its function in the RNAi pathway, loss-of-function of dCRIF also suppresses position-effect variegation and disrupts heterochromatin formation. These observations suggest that dCRIF, a new member of RNAi, is involved in transcriptional gene silencing, which is dependent on both RNAi and heterochromatin.


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134 UTX Coordiates with PTIP in the Regulation of Polycomb Target Gene Expression Chengwan Zhang, Wencui Ma, Zehui Hong, Wei Xie, Ming Fang* Institute of life sciences, Southeast University; MOE Key Lab of Developmental Genes and Human Diseases, Nanjing 210096, China

In differentiated cells of a certain developmental stage, Histone modifications are thought to exist in certain combinations, termed Histone code, to maintain, in a long term, the expression status of lineage-specific genes. Little is known on how Histone modifications are regulated in coordinate ways to ensure the proper gene expression patterns. We have previously characterized fly PTIP gene and found it is required for the balance-play of the two Histone methylation forms, tri-methylation at the 4th Lys residue of Histone H3 (H3K4me3) and H3K27me3. H3K4me3 is often associated with actively transcribed gene and H3K27me3 usually confers a repressive gene states. UTX is a recently indentified JmjC family member and a Histone demethylase specific for H3K27. Studies have shown that UTX is a part of the mouse PTIP complex, which also includes MLLs, the homologous proteins of Drosophila Trithorax, thought to confer the H3K4 Histone Methyltransferase activities. We have mapped in detail the physical interaction between UTX and PTIP and found that UTX interacts directly with PTIP via C-terminal BRCTs of PTIP and the N-terminal multi-TPR containing region of UTX. In cultured Drosophila Kc167 cells, we found that both UTX and PTIP are recruited to the PREs and promoter regions of the polycomb targets, such as UBX and pannier. In our efforts toward the characterization of UTX gene function in flies, we have generated several mutant UTX alleles using P-element imprecise excision. The trans-heterozygous mutant fly are mostly pre-pupal lethal. However we found a few escapers possessing a dorsal cleavage phenotype resemble to that of pannier mutant flies, which may indicate the in vivo regulation of ploycomb targets by UTX.


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135 Towards Deciphering the Dual Function of INO80, a Chromatin Remodeling Protein Mohsen Ghasemi1*, Shruti Jain2*, Pratyusha Vavilala2, Shipra Bhatia2, Vani Brahmachari2, Shanti Chandrashekaran1 1) Division of Genetics, Indian Agricultural Research Institute, New Delhi- 110012, India; 2) 2) Dr.B.R.Ambedkar Center for Biomedical Research, University of Delhi, Delhi- 110007, India. *: Equal contribution.

INO80, a member of the SWI⁄SNF family of chromatin remodeling proteins, is unique in combining a DNA dependent ATPase and DNA-binding domain, so far unreported for the SNF2 family. We have identified the consensus binding sequence of DBINO, the DNA binding motif of INO80. Further, our results on the role of INO80 in the regulation of homeotic genes in Drosophila, raise the possibility of INO80 being a recruiter of PcG/Trx complexes on the genome. Among PcG -trxG proteins, only Pleiohomeotic (PHO) is shown to have sequence-specific DNA-binding properties (Klymenko et al., 2006). DNA binding of PHO was achieved through PHO-INO80 complexes and in Drosophila, INO80 always co-purified with PHO in ChIP experiments, strongly suggesting that PHO recruits INO80 to chromatin complexes. We have analysed the distribution of INO80 binding motif in both upstream as well as downstream of genes in the whole genome of Drosophila and find that there are more number of potential binding sites for INO80 than PHO. We provide several lines of evidence including expression pattern and protein localization to show that INO80 can function independent of PHO. This reinforces our recent proposal that INO80 is a dual function protein which can (i) recruit proteins to chromatin remodeling complexes (independently of pho) and (ii) function as an integral component of a multisubunit ATP-dependent chromatin remodeling complex through its ATPase domain.


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136 GAGA Regulates the RNA Polymerase II Pausing Through Multiple Aspects Shih-Ying Tsai, Der-Hwa Huang Institute of Molecular Biology, Academia Sinica, Taiwan

“RNA polymerase II pausing” is a newly proposed mechanism involved in regulating transcription processes. It proposes that NELF (negative elongation factor) and DSIF (DRB sensitivity-inducing factor) help RNA polymerase II paused at 20-50 bp downstream of transcription start site after transcription initiation. When cells require preceding transcription, other regulators such as P-TEFb will be recruited to the paused site to phosphorylate NELF, DSIF and Ser-2 of RNA polymerase II, and allow continuing transcription. The best model to study RNA polymerase pausing mechanism is the Drosophila Hsp70 gene, which can be induced after heat shock. The promoter region of Hsp70 contains GAGA binding and HSF binding sites, which are important for regulating RNA polymerase pausing mechanism. GAGA is encoded by trithroax-like gene, and is a multifunctional protein involved in chromatin regulation, heterochromatin maintenance, and transcription regulation. Moreover, nearly 19% of 6537 promoters in Drosophila have GAGA binding sites. However, it remains unclear about the exact functions of GAGA. In our study, the results show that GAGA may regulate RNA polymerase pausing mechanism. Without GAGA, the RNA polymerase II seems to have reduced pausing, and increased elongation processes. Therefore, we want to further understand the processes of the transcription regulated by GAGA protein.


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137 Hdac1 is Involved in the Maintenance of Epigenetic Code of Heterochromatin Yuh-Long Chang*, Der-Hwa Huang Institute of Molecular Biology, Academia Sinica, NanKang, Taipei, Taiwan

Post-translational modification of histones plays a role in transcriptional regulation in Eukaryotic cells. We have shown that HDAC1 (histone deacetylase 1) is involved in Pc-mediated gene silencing in Drosophila. We found a global increase of acetylated histone level in both euchromatin and centromeric heterochromatin regions in Hdac1 mutant. In addition, it shows higher level of phosphorylated RNA polymerase, reflecting increased transcription. Consistently, a transgene insert in chromo-center region also shows higher level of expression and increased level of acetylation in mutant. Further studies of the specific heterochromatin region with XChIP and variegation assay show HDAC1 is indeed involved in heterochromatin-mediated silencing state. Moreover, methylation states of histone also show significantly changed in either euchromatin and heterochromatin. Those results implicate that the maintenance of epigenetic code are highly fine-tuned by those histone modification enzymes around the whole genome.

POSTER: PATTERN FORMATION

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138 An Integrated Approach to the Alignment and Classification of 3D Neuronal Morphology in the Drosophila Brain Chao-Chun Chuang1,4, Chang-Wei Yeh3,4 , Hsiu-Ming Chang2, Chang-Huain Hsieh4, Ann-Shyn Chiang2,3, Jenn-Kang Hwang1

1) Institute of Bioinformatics and Systems Biology, National Chiao Tung University. 2) Brain Research Center, National Tsing Hua University, Taiwan. 3) Institute of Biotechnology, National Tsing Hua University, Taiwan. 4) National Center for High-Performance Computing, Taiwan.

Characterizing the neuronal morphology of the Drosophila brain has been a challenging problem in contemporary neuroscience. Alignment and clustering of neuronal morphology allows compartmentalization of structures that might reflect a functional link between the neurons. These might include shared input, interaction via local axonal collaterals, and/or shared common targets. However, massive morphology clustering with direct 3D structural comparisons is too difficult to be carried out in a high-throughput screening procedure. In this study, we developed an algorithm to address this specific issue.

First, we reconstructed single neurons into a common standardized 3D framework. Next, we transformed 3D neuronal morphology into a one-dimensional spatial code including its biological features and spatial distribution. Comparison of neuronal morphology with one-dimensional spatial code, which is similar to a protein amino sequence, is much faster than directly comparing the 3D structures of neurons. We applied this algorithm to align and cluster about 16,000 neurons from FlyCircuit database and found that there is sexual dimorphism in neurons expressing the fruitless gene. Also, we found that projection neurons with very similar morphology were formed at different developmental stages. In certain neurons with consistent morphology, we found multiple genes were expressed within. These results corresponded with the actual anatomy atlas, demonstrating our algorithm to be effective and accurate in a high-throughput screening procedure.

In conclusion, we provide a novel approach to integrate anatomy and informatics. The morphology of neurons serves as the criteria for data clustering, which helps validate the functional circuits of the Drosophila brain. Our algorithm can handle massive 3D neuronal image data collected in experiments from different research groups as well as manage bio-images with deep neurological insight.


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139 Mechanical Tension Guides Cell Sorting at Compartment Boundaries in Drosophila Daiki Umetsu1, Katharina Landsberg1, Reza Farhadifar2, Jonas Ranft2, Thomas Widmann1, Thomas Bittig2, Amani Said1, Benoit Aigouy1, Suzanne Eaton1, Frank Jülicher2, Christian Dahmann1 1) Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 2) Max Planck Institute for the Physics of Complex Systems, Dresden, Germany

The formation and maintenance of straight and sharp boundaries separating neighboring groups of cells with different identities is central to building body plans. Lineage restriction is a major strategy to form boundaries by preventing two groups of cells from mixing. These boundaries, called compartment boundaries, often serve as landmarks of tissue development by positioning and stabilizing organizing centers, and therefore are important for morphogenesis. Here, we show by live imaging of the developing Drosophila abdomen that cell proliferation causes prominent cell rearrangements, yet the anteroposterior compartment boundary remains straight despite of cell proliferation. Thus mechanisms are required that counteract cell rearrangements caused by cell division. By analyzing the response to laser ablation of cell bonds in the vicinity of the anteroposterior compartment boundary in developing Drosophila wings, we found that mechanical tension is increased on cell bonds along this compartment boundary as compared to the remaining tissue. Cell bond tension is decreased in the presence of Y-27632, an inhibitor of Rho-kinase whose main effector is Myosin II. Simulations using a vertex model showed that an increased cell bond tension suffices to guide cell rearrangements after cell division to maintain compartment boundaries. These results provide a physical mechanism in which the local increase in Myosin II-dependent cell bond tension directs cell sorting at compartment boundaries.


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140 Global Hexagonalization of Cells by Local Directional Remodeling of Cell Adhesion Surfaces Kaoru Sugimura1,2, Shuji Ishihara3,4 1) iCeMS, Kyoto University, Japan 2) RIKEN BSI, Japan 3) Graduate School of Arts and Sciences, The University of Tokyo, Japan 4) JST PRESTO, Japan

During morphogenesis, mechanical forces trigger a series of deformations to shape an embryo. For instance, recent studies have clarified how changes in the geometry of cells are coordinated via the activity and/or localization of force-generating molecular machineries within a cell (Lecuit and Lenne, Nat Rev Mol Cell Biol 2007; Green and Davidson, Nat Cell Biol 2007; and Fernandez-Gonzalez and Zallen, Sci Signal 2009). On the other hand, it remains unclear how the dynamics of the stress field of a multi-cellular tissue control cellular pattern formation, because methods to quantify dynamics of stress fields of a tissue in vivo are not available.

In this study, we studied mechanical basis of hexagonal packing (the increase of hexagonal cells in the Drosophila wing during the pupal stage; Classen et al., 2005) by using our novel method for estimating the pressure of each cell, the tension of each cell adhesion surface and stress tensor of a group of cells. Our quantification of developmental changes of the stress field within a tissue and of corresponding rearrangements of cells provides a physical mechanism for cell packing: biased external forces acting on the tissue provide the directional information for local remodeling of cell adhesion surfaces which underlies the global hexagonalization. The incorporation of macroscopic (tissue-level) mechanical control represents a new direction in developmental biology.


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141 The Drosophila dRYBP Mutation Enhances the Pleiohomeotic Mutant Phenotypes during Embryogenesis and in the Adult Hyunpyo Shim, and Sang-Hak Jeon Department of Biology Education, Seoul National University, Korea

Polycomb Group(PcG) proteins functions in maintaining silenced state of many genes including Homeotic genes. The one of PcG proteins Pleiohomeotic(Pho) has a DNA binding motif. So it might be have important role of maintaining repressed state of gene expression. But pho mutant showed weak homeotic mutant phenotypes. This indicated that there are other factors which interact with Pho and co-regulate gene expression by redundant pathway.

Recent studies showed that Drosophila dRYBP protein might be interact with Pho and had a role of recruiting other proteins to form polycomb complex to repress gene expression. From this we thought that dRYBP might regulate homeotic gene expression with Pho and consequently show homeotic phenotypes. But we found dRYBP mutants had no striking effect on those phenotypes. So we made dRYBP and pho double mutant. We found that dRYBP and pho double mutant showed more serious homeotic mutant phenotypes than pho single mutants, especially in female flies. We also observed the expression of homeotic genes in double mutants and we could find anteriorly expanded UBX and ABD-B expression patterns indicating that two genes might regulate homeotic gene expression by redundant pathway. From these results we suggested that dRYBP mutation enhances the pho mutant phenotypes during embryogenesis and in the adult.


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142 Identifying Zelda-Regulated Early Zygotic MicroRNAs Shengbo Fu1, Chung-Yi Nien1, Hsiao-Lan Liang1, John Manak2, Christine Rushlow1

1) Department of Biology, New York University, New York, USA; 2) Molecular & Cellular Biology Program, The University of Iowa, Iowa, USA.

The maternal to zygotic transition (MZT) is a conserved pivotal process in which the transcribed zygotic genome gradually replaces maternally loaded products to control embryonic development (Schier et al., 2007; Baroux et al., 2008). Previously, we identified that Zelda is a key activator of the early zygotic genome during the MZT of Drosophila. Zelda specifically binds to TAGteam sites (Liang, et al. 2008), which are over-represented in the upstream regions of early zygotic genes (ten Bosch et al., 2006; De Renzis et al., 2007). By comparing the expression profiles of wild-type and zelda mutant embryos, we demonstrated that many maternal transcripts are up-regulated in zelda mutants. Moreover, expression of the miR-309 cluster, the first Drosophila zygotic factor shown to be involved in maternal transcript degradation (Bushati et al, 2008), is eliminated in zelda mutants, TAGteam sites present in the miR-309 enhancer region suggest its activation by Zelda may be direct. Together, these hinted that Zelda can function in maternal mRNA degradation by activating zygotic microRNAs (miRs).

To further study the activation and functions of miRs during the MZT, we examined the expression profiles of wild-type and zelda mutant embryos using whole genome tiling arrays, and validated several Zelda-regulated miR candidates by in situ hybridization. We also studied the enhancers of these early microRNAs to understand the transcriptional regulation of microRNAs by Zelda. This study will help us to understand the functions of early zygotic microRNAs during MZT.


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143 Atypical Cadherins Dachsous (Ds) and Fat (Ft) Regulate Collective Cell Migrations during Development and Regeneration in Drosophila Amit Kumar, Priya Srivastava, Satish Sasikumar, Pradip Sinha

Indian Institute of Technology Kanpur, India

Collective cell migration involves movement of interconnected cells and is important for animal morphogenesis. Dorsal closure during embryogenesis, thorax closure during metamorphosis, or healing of epithelial wounds are all examples of collective cell migration. While reorganization of cytoskeleton is a necessary prelude for collective cell migration, mechanisms regulating such cytoskeletal reorganization en masse are yet to be understood. It is also believed that the triggers for directed and collective cell migration are generated at the interface of cells with differential adhesive properties and/or tension. Here we show that the atypical cadherin Dachsous (Ds) and its binding partner Fat (Ft) regulate collective cell migration during epithelial morphogenesis. we have observed that both Ds and Ft localize at the Leading Edges (LEs) formed at the interface of differentially adhesive cells in Drosophila imaginal epithelia and at embryonic lateral epithelium during dorsal closure as well as healing epithelial wounds. We further show that Ft/Ds serve as global regulators of collective cell migration, a role reminiscent of their function during planar cell polarity (PCP). Our results provide a novel role for global PCP regulators in the control of collective cell migration during animal morphogenesis.


188

144 Drosophila Long-Chain Acyl-CoA Synthetase Acts Like a Gap Gene in Embryonic Segmentation Yi Zhang1,2, Yang Zhang1, Yu Gao, Xi Zhao1,2, Zhaohui Wang1* 1) Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beichen Xilu #1, Beijing 100101, P.R. China. 2) Graduate School, Chinese Academy of Sciences. 19 Yuquan Road, Beijing 100039, P.R. China. *: Correspondence:

Long-chain acyl-CoA synthetases (ACSLs) convert long chain fatty acids to acyl-CoA esters, the activated forms participating in diverse metabolic and signaling pathways. dAcsl is the Drosophila homolog of human ACSL4 and their functions are highly conserved in the processes ranging from lipid metabolism to the establishment of visual wiring. In this study, we demonstrate that both maternal and zygotic dAcsl are required for embryonic segmentation. The abdominal segmentation defects of dAcsl mutants resemble those of gap gene knirps (kni). The central expression domain of Kni transcripts or proteins was reduced whereas the adjacent domains of another gap gene Hunchback (Hb) were correspondingly expanded in these mutants. Consequently, the striped pattern of the pair-rule gene Even-skipped (Eve) was disrupted. We propose that dAcsl plays a role in embryonic segmentation at least by shifting the anteroposterior boundaries of two gap genes.


189

145 The Redundant Roles of Palmitoylations in Wingless Secretion through Affecting Wingless Interaction with Wntless Yihui Wu1*, Xiaofang Tang2*, Xinhua Lin1,2 1) State Key Laboratory of Biomembrane and Membrane Biotechnology, and Key Laboratory of Stem Cell and Developmental Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China 2) Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, and the Graduate Program in Molecular and Developmental Biology, University of Cincinnati College of Medicine, Cincinnati, OH, 45229, USA *: These authors contributed equally to this work.

The Wnt family members are secreted glycolipoproteins which play essential roles in many developmental processes and in adult homeostasis. By mutating the relevant amino acids, we have investigated the in vivo roles of palmitoylation and glycosylation on the main Drosophila Wnt member, Wingless (Wg). We show that glycosylation is dispensable for Wg secretion and signaling. However, two palmitoylation sites, Cysteine 93 (C93) and Serine 239 (S239) play distinct roles for Wg activity. We show that S239 is essential for Wg signalling activity. Our data argue that palmitoylation at S239 is likely to be required for effective interaction of Wg with Frizzled2 receptors.

Importantly, when both palmitoylation sites are mutated, Wg fails to reach the plasma membrane when examined in wing discs and in cultured cells, suggesting a redundant role of two palmitoylations in Wg secretion. Moreover, we propose that impaired interaction with Wls is one mechanism underlying the secretion defect of this double palmitoylation mutant.


190

146 Genetic Screens of Mutants on the Second and Third Chromosomes to Identify Genes Involved in the Left-Right Asymmetric Development of the Embryonic Gut in Drosophila Mitsutoshi Nakamura1, Naotaka Nakazawa1, Kiichiro Taniguchi1, Takashi Okumura1, Reo Maeda1, Ryo Hatori1, Akira Ishio1, Ayumi Ozaki1, Kenji Matsuno1,2 1) Dept. Biol. Sci / Tec., Tokyo Univ. Sci. 2) Res. Ins. Sci / Tec., Tokyo Univ. Sci.

Although bilateral animals appear left-right (LR) symmetrically on the outside, their internal organs often show directional and stereotypical LR asymmetry. If the LR axis is not formed correctly during embryogenesis, these embryos develop with laterality defects, such as LR inversion. In vertebrates, the mechanisms of LR symmetry breaking and the downstream signal activation responsible for the LR asymmetric development are understood well. However, in invertebrates, mechanisms of LR specification and LR asymmetric development are largely unknown.

To address this issue, we have been studies the LR asymmetric development of the Drosophila embryonic gut, which shows stereotypical LR asymmetry. To identify the genes involved in the LR asymmetric development of the embryonic gut, we performed a genetic screen. About 4,500 mutants on the second and third chromosomes, predicted to cover about 80%; of Drosophila genes, were induced by ethyl metanesulfonate, and LR defects in homozygote of each mutant were screened. From these screens, we isolated 31 mutants that showed various LR detects in the embryonic gut. We classified these mutants into 5 types on the basis of their LR defects in three parts of the embryonic gut, the foregut, midgut, and hindgut. Type 1 : the laterality of the foregut was randomized. Type 2 : the laterality of the midgut and hindgut was synchronous by inverted. Type 3 : the laterality of the anterior midgut was randomized, or this organ became bilateral. Type 4 : the laterality of the posterior midgut was randomized, or this organ became bilateral. Type 5 : the laterality of the hindgut was randomized, or this organ became bilateral. Further genetic characterizations of these mutants will be presented.


191

147 Exploring Molecular Functions of Atypical Cadherins Dachsous and Fat that Affect Morphogenesis at Multiple Levels from Cell to Organ Masaki Arata1, Kousuke Mouri1, Toshiyuki Harumoto1, Yuzo Watanabe1, Motoki Saito1, Tadashi Uemura1, 2 1) Graduate School of Biostudies, Kyoto University, Japan 2) CREST, JST

Normal development and homeostasis of individual organs depend on functional connections between cells, tissues, and organs. One such connection is exemplified by planar cell polarity (PCP) in various developmental contexts; for example, epidermal cells in the Drosophila wing sense the proximal-distal (P-D) axis of the wing and generate wing hairs at the distal cell vertexes. Two of the evolutionary conserved PCP regulators are atypical single-pass transmembrane cadherins Dachsous (Ds) and Fat (Ft). Intriguingly, ds or ft mutations cause pleiotropic defects, including foreshortening of the wing along the P-D axis, misoriented cell divisions, hair formation at ectopic cell vertexes (a PCP phenotype), and abnormal geometry of individual cells. Although both atypical cadherins belongs to one upstream branch of the Hippo signaling pathway that is a key regulator of tissue growth, molecular functions of Ds and Ft are still largely unknown, except for Ds-Ft heterophilic binding between adjacent cells. To dissect roles of the atypical cadherins further at the molecular level, we have been searching for their novel binding partners. We first generated transgenic flies that express eGFP-tagged Ds or Ft, and we showed that those tagged proteins were functional by rescue and/or overexpression experiments. Then we immunoprecipitated those proteins from staged samples by using an anti-GFP antibody, analyzed the precipitates by mass spectrometry, and screened for the potential binders. In addition to our molecular hunting, we are observing behaviors of Ds:eGFP and Ft:eGFP in live pupal wings.


192

148 Ubx and abd-A in the Visceral Mesoderm of the Midgut of Drosophila Regulate Regional Differentiation of the Adjacent Endoderm through the Action of Dpp Yumiko Harada1, Masahiko Arishige2, Lily Shimooka3, Ryutaro Murakami2,3 1) Grad. Sch. Sci. Eng., Yamaguchi Univ., Japan 2) Grad. Sch. Med., Yamaguchi Univ Japan 3) Dept. Biol. Chem., Fac. Sci., Yamaguchi Univ. Japan

The midgut of Drosophila consists of endodermal epithelium and visceral mesoderm, and transiently forms four chambers in middle stages of development. Several Hox genes, Scr, Antp, Ubx, and abd-A, are expressed in this antero-posterior order in the visceral mesoderm of midgut. Ubx is known to activate dpp in the visceral mesoderm of 2nd midgut chamber, and secreted Dpp protein induces expression of lab in the adjacent endoderm. Another HOX gene abd-A is expressed in visceral mesoderm of the 3rd and 4th chambers. We found that abd-A in the visceral mesoderm is required for expression of several region-specific endodermal genes in the 3rd and 4th chambers. However, double mutant embryos deficient for both abd-A and Ubx unexpectedly restored expression of these endodermal genes, suggesting that abd-A allows activation of these posterior endodermal gene by suppressing inhibitory signal(s) emanated under the control of Ubx. In abd-A mutant embryos, expression of Ubx and its target dpp in the visceral mesoderm, as well as expression of endodermal genes of the 2nd chamber, expanded posteriorly. Ectopically-expressed Dpp repressed posterior endodermal genes. In dpp mutant embryos, overall morphology of the midgut is strongly affected, but, expression of a posterior-most endodermal genes remained. These results demonstrate that Dpp, a signaling factor activated by Ubx in the visceral mesoderm, elicits anterior-posterior pattern of gene expression along the midgut endoderm both by activating endodermal genes of 2nd chamber andrepressing endodermal genes specific to the 3rd and 4th chambers.


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149 Formation of Tarsal Segments by the Temporal Regulation of Transcription Factor Genes during Leg Development Kouhei Natori, Tetsuya Kojima Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Japan

The numbers of tarsal segments of insect legs are divergent among species but its

underlying mechanism remains elusive. During development of the Drosophila leg, which has five tarsal segments (ta1-ta5), two transcription factor genes, BarH1 and BarH2 (collectively referred to as Bar), is initially expressed in the region destined to ta3-ta5 in early 3rd instar. By late 3rd instar, however, regions with no, weak and strong Bar expression appear within the initial Bar domain, thereby creating ta3, ta4 and ta5, respectively. Here, we investigated the roles of transcription factor genes, rotund (rn), nubbin (nub) and apterous (ap), in Bar regulation and found the followings: At early 3rd instar, nub is regulated by EGFR signal and expressed in the broad region including whole tarsus region. Growth of the tarsus region and/or reduction in the level of EGFR signal causes progressive restriction of nub-expressing region from that completely including the initial Bar domain to the future ta5 region. This leads to progressive derepression of rn, which in turn progressively represses Bar in the proximal region of its initial domain and thus, creating the ta3. From mid 3rd instar onward, the progressive Bar repression by the progressive expansion of rn expression is counteracted by the initiation of ap expression in the Bar domain. This leads to the formation of the ta4. Thus, timing of nub restriction and ap initiation appears fundamental to forming ta3 and ta4, and might be one of the factors for the difference in the numbers of tarsal segments among species.


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150 The Cohesin-Subunit SMC3 is Required for Both Planar Cell Polarity and Cell Packing Kousuke Mouri1, Shin-ya Horiuchi1, Tadashi Uemura1,2 1) Graduate School of Biostudies, Kyoto University 2) CREST, JST

Planar cell polarity (PCP) is the asymmetric organization of cells within the plane of the epithelium. This polarity is perpendicular to apical-basal polarity and is seen in epithelial tissues of many animals. To date, underlying mechanisms of PCP have been well studied in the Drosophila wing, where epidermal cells sense the tissue cue along the proximal-distal (P-D) axis of the wing and produce single wing hairs at distal cell edges. Some of proteins called “core group proteins”, including Frizzled and Flamingo, assemble into asymmetric proximodistal complexes that specify the wing hair position. In addition to core group members, several other genes controlling PCP signaling are identified. However, the overall mechanism of PCP signaling is still unclear.

To uncover the mechanism of PCP signaling, we have been hunting for novel mutations on X chromosome that affect the orientation of wing hairs through a mosaic screening. We focused on one mutant that exhibited a severe misorientation phenotype and showed that the responsible gene encodes the SMC3 subunit of the cohesin complex. Cohesin has been originally identified as a regulator of sister chromatid cohesion; and it has been more recently shown that it regulates gene expression and development in organisms from yeast to human. In contrast to the localization of Fmi at proximodistal cell borders in the wild type, Fmi was mislocalized at anterior-posterior borders or distributed at all borders in SMC3 mutant clones. Furthermore, the Fmi level was elevated at many of those borders. In addition to the PCP defect, the cell size became irregular and epidermal cell packing appeared to be impaired. We will report our ongoing analysis of the pleiotropic phenotypes of the cohesin mutant.

POSTER: GAMETOGENESIS AND ORGANOGENESIS

195

151 A Paternal Imprint Essential for the Inheritance of Telomere Identity in Drosophila Guanjun Gao1,2, Yan Cheng1, Natalia Wesolowska1, Yikang S. Rong1* 1) LBMB, NCI, NIH, Bethesda, MD 20892 2) School of Life Sciences, Tsinghua University, Beijing, China *: Corresponding author

Chromatin remodeling during sperm maturation could erase epigenetic landmarks on the paternal genome creating a challenge for its re-establishment upon fertilization. Here we demonstrate that selective retention of a chromosomal protein in mature sperm protects the identity of paternal telomeres in Drosophila. The ms(3)k81 (k81) gene was a duplication of hiphop that encodes a telomeric protein. While HipHop protects telomeres in somatic cells, K81 is produced exclusively in males and localizes to telomeres in post-mitotic cells, including mature sperm. In embryos fathered by k81 mutants, the maternal supplies fail to re-establish a protective cap on paternal telomeres leading to their fusions. These fusions hinder the segregation of the paternal genome, and result in haploid embryos with maternal chromosomes. The functional divergence between hiphop and k81 manifests not only in their expression patterns but also in the protein functions that they encode. Through swapping of the two coding regions, we show that K81 can replace HipHop for somatic protection while HipHop cannot replace K81 in the germline to specify telomere identity, as HipHop ectopically expressed in testis is removed from chromatin during sperm maturation. HipHop lacks a short motif in K81 that is essential for K81 to survive the remodeling process. We show that the combined functions of HipHop and K81 are likely fulfilled by the single ancestral hiphop locus in other Drosophila species, supporting that the evolutionary process of subfunctionalization was responsible for the preservation of the hiphop-k81 duplicate.


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152 Ball-and-Socket Joints in the Insect Leg: Development and Evolution Reiko Tajiri1, Kazuyo Misaki2, Shigenobu Yonemura2, Shigeo Hayashi1 1) Laboratory for Morphogenetic Signaling, RIKEN Center for Developmental Biology, Japan 2) Electron Microscopy Laboratory, RIKEN Center for Developmental Biology, Japan

Both in the internal skeleton of vertebrates and in the exoskeleton of arthropods, joints connect neighboring skeletal elements and allow movement. Joints in which the articular surfaces of reciprocal shapes fit each other and form an interlocking structure underlie efficient and controlled motion. Mechanisms for precisely regulating skeletal morphology in joints during development was unclear.

We have investigated the cellular mechanisms controlling the morphogenesis of the joints in the fly tarsus (distal leg). Each tarsal joint comprises two reciprocally shaped cuticles (exoskeletal element secreted by the epidermis): a ball and a socket. During development, the ball-shaped cuticle forms first and subsequently acts as the “mold” for sculpting the socket. We have shown that the conjunction of cell fate specification and cell shape changes in the epidermis drives the joint morphogenesis: distinct cell populations are assigned to secrete the ball and the socket, and the cells extensively move their apical surfaces while secreting cuticles.

Abnormal joint structures caused by conditional loss-of-function of Notch indicated its involvement in the morphogenetic processes. Interestingly, we find similar structures in the tarsal joints of primitive insects such as bristletails and mayflies. I would like to discuss how cell fate specification and shape changes are regulated downstream of Notch, and how these processes have been acquired during evolution.


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153 gll is Involved in Maintaining Germ Cells in Drosophila Testis Qing Geng1,2, Shaowei Zhao1,2, Di Chen1,2, Zhaohui Wang1 1) Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China. 2) Graduate School, Chinese Academy of Sciences, China.

The testis of Drosophila Melanogaster has proved its quality to be a supreme system to study spermatogenesis, where mammals and flies exhibit remarkable resemblance. Many questions pertained to the field, however, still remain unaddressed. In our clonal-analysis based EMS screen to identify more factors involved in the early developmental stages during this process, we obtained an interesting mutant line, which led to a dramatic germ cell loss phenotype when mutant germ cell clones were generated within an otherwise heterozygous testis. We designated the mutant as gll (germ line loss). The possibility of general cell lethality is excluded, given that the mutant was homozygous viable with normal fertile testis. Additionally, the testis containing minute clones resulted in abundant functional germ cells, indicating a potential non-autonomous role of gll. We examined the male gonads carrying germ line gll-/- clones and did not find germ cell loss phenomenon up to 3rd instar larval stage. At present, we are tracking the gene down and trying to reveal how gll contributes to the homeostasis of adult germ line cells and elucidate the molecular mechanisms underlying this intriguing phenotype.


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154 Tracheal Development in Drosophila Visual System Wei-Chen Chu1,2, Yuan-Ming Lee1,3 and Yi Henry Sun1,2,3

1) Institute of Molecular Biology, Academia Sinica, Taiwan 2) Graduate Institute of Life Sciences, National Defense Medical Center, Taiwan 3) Department of Life Sciences and Institute of Genome Sciences, National Yang Ming University, Taiwan

Drosophila eye is a highly specialized neuronal system, and its neuronal activity should require lots of oxygen. The oxygen can be transported by trachea in insects. However, the distribution and development process of trachea in the Drosophila visual system have not been studied. To address this issue, we study the adult tracheal pattern, tracheal development process and its biological role in eye. These may provide a link between visual system and respiratory system. We have determined the retinal tracheal pattern and established the 3D model. And we also found the critical developmental stage for the trachea in Drosophila visual system. It has been shown that branching morphogenesis of the Drosophila tracheal system in embryo depends on the FGFR/ FGF (Fibroblast Growth Factor/ Fibroblast Growth Factor) signaling pathway. Tracheal cells specifically express the FGFR (Breathless, Btl) that receives the FGF ligand Branchless (Bnl) signal. Bnl acts as a guidance molecule controlling tracheal cell migration. We have also found that btl/ bnl signaling affected tracheal growth in eye. There is an ERG defect in the flies with less retinal trachea that created by overexpression of domain-negative Btl in trachea. These data suggest that the trachea in the Drosophila eye may be important for normal eye function. We speculate that as the eye grows in size, hypoxia will develop and induce tracheal ingrowth. This system may be used to study the role of hypoxia response in eye development, perhaps similar to mammalian angiogenesis.


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155 Argonaute 1 Regulates Germ Cell Division and Oocyte Determination in Drosophila melanogaster Ghows Azzam, Ji-Long Liu

MRC Functional Genomics Unit, Department of Physiology, Anatomy and Genetics, University of Oxford,Oxford, OX1 3QX, United Kingdom

Argonaute 1 (Ago1) is a member of the Argonaute/PIWI protein family that involves in small RNA-mediated gene regulation. In Drosophila melanogaster, Ago1 plays a specific role in microRNA biogenesis and functions. Previous studies have demonstrated that Ago1 regulates the fate of germline stem cells. However, the function of Ago1 in other aspect of oogenesis is still elusive. Here we study the function of Ago1 in developing egg chambers. We found that Ago1 protein is enriched in the oocytes and also highly distributed in the cytoplasm of follicle cells. Clonal analysis of multiple ago1 mutant alleles has shown that significantly many mutant egg chambers contain only 8 nurse cells without an oocyte. Our results suggest that Ago1 play a role in cystoblast division and oocyte determination.


200

156 Control of Drosophila Female Germline Stem Cell Niche Formation by Insulin Signals Chun-ming Lai, Hwei-Jan Hsu

Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan, R.O.C.

Stem cells are a small group of cells embedded in tissues with the capacity to produce differentiated cells for replenishing lost cells in fast-turnover or damaged tissues, thereby maintaining tissue homeostasis. Stem cells reside in the stem cell niche, a specialized microenvironment, which provides both physical contact and tissue-intrinsic signals to control stem cells. Stem cells and tumor cells share similar features of self-renewal and proliferation; interestingly, a hypothesis of a latent tumor cell niche for tumor initiation is also proposed. The regulation of the stem cell niche itself, however, is poorly understood. The Drosophila ovary is an excellent system to study stem cell biology, because of its ease of manipulation and well-characterized germline stem cells (GSCs) and their niches. The GSC niche formed by terminal filament cells and cap cells houses two to three GSCs. We have previously shown that insulin/insulin-like growth factor (IGF) signals mediate the effect of diet to directly control GSC niche cell survival. It is unclear, however, if insulin/IGF signaling also controls the formation of the GSC niche. To examine this, we diminished the expression of the insulin receptor in the somatic cells of developing ovaries using UAS-RNAi lines driven by specific GAL4 drivers. Compared to the controls, flies that grew from insulin receptor-suppressed larvae produced fewer progeny two days after eclosure and thereafter. In addition, we also observed that those flies carry small ovaries which are composed of fewer ovarioles, functional units of ovaries, suggesting that insulin/IGF signaling controls the formation of terminal filament cells known to play a role in subdividing the developing ovary into ovarioles. If insulin signaling controls cap cell formation and GSC recruitment, and the mechanisms underlying these processes will be further investigated. Nevertheless, our results have provided new insights into the role of nutritional inputs on stem cell niche formation, and that may eventually reveal therapeutic intervention for cancer.


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157 Mitotic Cell Rounding Accelerates Invagination of the Drosophila Tracheal Placode Takefumi Kondo, Shigeo Hayashi Laboratory for Morphogenetic Signaling, Riken Center for Developmental Biology, Japan

Cell shape change during morphogenesis is governed by interphase cytoskeletons. Once cells enter mitosis, cytoskeletons are extensively reorganized into mitotic apparatus. Therefore, it is thought that mitotic entry is not permitted because it is not compatible with extensive demand for cytoskeletons to drive cell shape changes. From the other point of view, mitotic entry leads a massive cell shape change, resulting in a round shape. Whether mitotic cell rounding plays an active role in morphogenesis is unknown.

During the course of the study of Drosophila tracheal placode invagination, we found that this morphogenetic event proceeds in two distinct phases. First, the placode formed a wave of circular Myosin concentration and constricted the apices of central cells, which underwent slow invagination. In the second phase, speed of invagination was suddenly increased. This acceleration was accompanied by entry of one of the central cells into mitosis and rounding at the basal side, resulting in the fast basal movement of the apical surface of the placode. The genetical or pharmacological block of mitotic entry and cell rounding inhibited transition to the second phase. On the other hand, colchicine, which prevents spindle formation and cell division, but not cell rounding, did not interfere the phase transition. These results suggest that cell rounding accompanied by mitosis actively drives fast invagination of tracheal placode.


202

158 Investigation of the Germ Plasm Localization Mechanism of Vasa Protein Szu-Chieh Wang1, Gee-Way Lin3, Chun-Che Chang3*, Ming-Der Lin1,2*

1) Department of Life Science, Tzu-Chi University, Hualien, Taiwan 2) Department of Molecular Biology and Human Genetics, Tzu-Chi University, Hualien, Taiwan 3) Department of Entomology, National Taiwan University, Taipei, Taiwan *: Authors of correspondence

Vasa is a highly conserved protein in metazoan and essential for germline development. Aphid is one of the most destructive insect pests that transmit viruses and pathogens through their sucking mouthparts. The understanding of germ cell formation in Acyrthosiphon pisum might help us in pest control. Acyrthosiphon pisum Vasa (ApVasa) has been considered as a germ cell marker and is localized in germ plasm as well as its Drosophila homolog. In Drosophila, the germ plasm localization of Vasa is Oskar (Osk) dependent. In contrast, no Drosophila osk homolog has been found in aphid genome. How does ApVasa localize to germ plasm remains to be elucidated. Here, we use Drosophila egg chamber as a model system for analyzing the functional domains of ApVasa. We first constructed GFP-tagged ApVasa fusion protein to examine the subcellular localization of ApVasa. In Drosophila egg chamber, ApVasa is not able to localize in the posterior end of the oocyte as dmVasa. Though sequence comparison, we reasoned that the lack of homologous N-terminal Osk binding domains might be responsible for the failure of germ plasm localization of ApVasa in Drosophila oocyte. To uncover the role of ApVasa in germ plasm localization and germ cell determination, we performed domain swapping and rescue experiments to analyze functional domains of ApVasa. Surprisingly, we found that the N-terminal Osk interaction domain of dmVasa is not sufficient to localize Vasa and the C-terminal domain of dmVasa plays an essential role in directing its germ plasm localization.


203

159 Brinker Directly Represses Wingless Target Gene Expression in Drosophila Lin Yang, Fei Meng, Caili Bi, Wei Xie, Ming Fang* Institute of life sciences, Southeast University; MOE Key Lab of Developmental Genes and Human Diseases, Nanjing 210096, China

Developmental signaling pathways, such as Transforming Growth Factor (TGF-)/ Decapentaplegic (Dpp) and Wnt/Wingless (Wg) pathways, are evolutionally conserved and play essential roles in many aspects in animal development. These pathways exert their functions mainly by the regulation of pathway-specific target gene expression. Recently, however, accumulated evidence suggests that in many cases they act cooperatively in directing cell differentiation and fate determination in animal tissue growth. It remains largely unknown for the molecular mechanism underlying such pathway crosstalk. In this report, we show that Brinker, a transcription factor that negatively regulates Dpp target genes, also play a direct repressing role in Wg target gene, such as naked cuticle (nkd). We have identified a putative Brinker site in the regulatory sequence of nkd. We demonstrate experimentally that Brinker directly binds to this sequence stretch in vitro and in vivo. In cultured Drosophila cells, Brinker represses nkd expression together with Groucho, a known co-repressor in the Wg signaling pathway. Furthermore, our ChIP analysis shows that the physical occupation of Brinker in nkd is dramatically decreased in embryo at stages when Wg begin to take action. Finally, we found that ectopic Dpp signaling enhances nkd expression in vivo. Taken together, these data suggest that Brinker might act in both Wg and Dpp signaling and may serve as a node for the crosstalk between the two pathways. We acknowledge the funding support from Natural Science Foundation of China (30771068, 90608019 and 31070826) to M.F.

POSTER: CELL BIOLOGY AND SIGNAL TRANSDUCTION

204

160 Modulation of JNK Signaling Pathway by MYC Oncoprotein Jiuhong Huang, Lei Xue Shanghai Key Laboratory for Signaling and Diseases, School of Life Science and Technology, Tongji University, 1239 Siping Road, Shanghai 200092, China

c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) family that is activated by sequential protein phosphorylation through a MAP kinase cascade. The JNK signaling pathway is involved in regulation of many cellular events, including proliferation, differentiation, growth, transformation and cell death. In this work, we have performed a genetic screen for modifiers of the JNK- mediated cell death phenotype and identified dMyc as a negative regulator of the JNK signaling pathway. Our genetic epistatic analysis indicates dMyc modulates JNK signaling downstream of dTAK1 and Hep, but upstream of JNK. In addition, dMyc collaborates with dMax to activate the transcription of puc, which encodes a phosphatase that dephospharylates JNK and inhibits JNK activation. Finally, the human homolog c-myc1 could inhibits JNK signaling in both Drosophila and mammalian cells, suggesting this function of Myc has been highly conserved in evolution.


205

161 Auxilin is Required for Formation of Golgi-Derived Clathrin-Coated Vesicles during Drosophila Spermatogenesis Xin Zhou1, Lacramioara Fabian2, Julie A. Brill2, Henry C. Chang1 1) Purdue University, West Lafayette, IN 47907, USA 2) The Hospital for Sick Children, Toronto, ON M5G 1L7, CANADA

In Drosophila testes, male germ cells undergo drastic morphological changes before they mature into long, thread-like sperm. The transformation from round to elongated spermatids requires extensive membrane biosynthesis and remodeling, although the mechanism by which membrane addition during sperm development is achieved or regulated is not well understood. To further understand this process, we analyzed the phenotypes of mutations in Drosophila auxilin (dAux), a regulator of clathrin function, in spermatogenesis. Like partial loss-of-function Clathrin heavy chain mutants, dAux mutant males are sterile and produce no mature sperm. The reproductive defects of dAux males were rescued by male germ cell-specific expression of dAux, indicating that auxilin function is required autonomously in the germ cells. Furthermore, deletion analysis suggests that the ability of dAux to bind clathrin and the Hsc70 is essential for sperm formation. dAux mutant spermatids show a deficit in forming the plasma membrane during elongation, which likely disrupts the subsequent migration of investment cones during individualization. In wild-type germ cells, GFP-tagged clathrin localized to clusters of vesicular structures near the Golgi. These structures also contained the Golgi-associated clathrin adaptor AP-1, suggesting that they were Golgi-derived. In contrast, in dAux mutant cells, clathrin localized to abnormal patches surrounding the Golgi and its co-localization with AP-1 was disrupted. Together, our data suggest that dAux participates in forming these Golgi-derived clathrin-positive vesicles, which transport lipids to sustain the plasma membrane increase necessary for spermatid differentiation.


206

162 Tracking the Collective Migration of Border Cells David Doupé, Adam Cliffe, Weimiao Yu, Pernille Rørth Institute of Molecular and Cell Biology, Singapore

Collective cell migration plays important roles in both normal development and invasive behaviour of cancer cells1. The border cells of the Drosophila egg chamber are a well-studied example of collective cell migration. At stage 9 of oogenesis this group of around six to eight cells delaminates from an epithelium and migrates through the egg chamber between the nurse cells to the oocyte, carrying a pair of non-migratory polar cells2. The tools of Drosophila genetics2 combined with the ability to track their migration live3,4 have made the border cells a powerful system in which to study collective cell migration. Previous studies have shown that their migration can be divided into two discrete phases: the first, guided by PVR (PDGF and VEGF related receptor) signalling, is characterized by highly directional movement of an elongated cluster; the second is guided by epidermal growth factor receptor (EGFR) signalling and is associated with tumbling motion and less directional migration3. Migration has generally been analysed using 2D projections of border cell clusters labelled with cytoplasmic fluorescent protein, this approach has proved informative but has two main limitations. First compression of a 3D process into 2D results in a loss of spatial information, and second this method does not reveal cell-cell interactions within the cluster. Here we use membrane tagged fluorescent proteins to allow quantitative tracking of cluster migration in 3D. Using different fluorescent proteins to label individual cells or the whole cluster reveals the cell-cell interactions within the cluster and also at the edge of the cluster where border cells contact the nurse cells.

1Rørth, Annu. Rev. Cell Dev. Biol., 2009 2Montell, Nat. Rev Mol. Cell Biol., 2003 3Bianco et al., Nature, 2007 4Prasad and Montell, Dev. Cell, 2007


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163 Systematic Dissection of Myofibril Assembly in the Adult Drosophila Heart Annette Hellbach, Frank Schnorrer Muscle Dynamics, Max-Planck-Institute of Biochemistry, 82152 Martinsried, Germany

Complex organisms require continuous circulation of their internal body fluids for life. This is achieved by a rhythmically beating muscle housing regularly assembled myofibrils, the heart. In Drosophila pupae, the five-chambered adult heart is built by remodeling of the larval heart. During this remodeling, four new heart chambers form and a large number of regularly spaced circular myofibrils assemble in every cardiomyocyte. These myofibrils are of key importance for regular heart contractions in the adult fly. Interestingly, heart remodeling is completed without any cell divisions or recruitment of new cardiomyocytes, thus all adult cardiomyocytes were already part of the larval heart.

In order to systematically identify genes controlling heart morphogenesis and heart function, we performed a genome-wide, heart-specific RNAi screen in combination with a longevity assay and identified 1,444 genes with a putative role in heart. To single out genes that instruct heart remodeling and execute myofibrillogenesis during pupal stages, we focused on transcription factors and cytoskeletal proteins from this primary positive heart gene list for detailed analysis. In total, we assayed 274 genes following heart-specific knockdown using fluorescently labeled myofibrils in intact adult hearts. We quantified heart chamber shape and diameter, myofibril number and regularity, as well as sarcomere number and length. We functionally assigned the genes into different phenotypic categories and hence have identified regulators and effectors of adult heart chamber morphogenesis, myofibril assembly and sarcomere formation.

As heart function as well as its patterning principles are conserved from Drosophila to vertebrates, we expect that our results will be of wide significance for heart biology.


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164 Homo-Dimerization of Hippo Kinase is Essential for its Autophophorylation and Activity in the Hippo Pathway

Yun-Yun Jin, Lei Zhang

Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, P.R. China

The Hippo (Hpo) pathway controls organ size by regulating the balance between cell

proliferation and apoptosis. The activation of Hpo (MST1/2 in mammals) plays an important role in triggering pathway kinase cascade phosphorylation and inhibition of the transcriptional activity of Yorkei (Yki) and Scalloped (Sd).

Despite conservation of Hpo function, little is known about the mechanism of the regulation of Hpo kinase activity. Here we find the homo-dimerization of Hpo protein is critical for its activity. We provide evidence in vitro and in vivo that its activity related dimerization is not only in C terminal SARAH domain, but also in N terminal kinase domain. We define Thr195 as its autophophorylation site, which is critical for kinase activity. Our data shows that, HpoI634D mutation destroy the dimerization of SARAH domain, lead to partially decrease its autophosphorylation level and kinase activity. However, in kinase domain, the HpoM242E and Hpo∆238-246 deletion break Hpo N terminal dimerization, then block Thr195 autophosphorylation and kinase activity totally. Furthermore, we show that the autophosphoryltion depends on dimerization, and dimerization promotes autophosphorylation and kinase activity.

Beside dimerization and autophosphorilation, we found there may existing extra mechanism(s) regulating Hpo activity. In vivo rescue experiments by MARCM show even kinase dead Hpo has pathway activity. Finally, using membrane tether forms of Hpo, we found both wild type and kinase dead Hpo display high pathway activity in vitro and in vivo. Our observations suggest the mechanism of Hpo activation through dimerization and membrane localization.


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165 Ter94 Specific Involves in the Process of Cubitus Interruptus Protein Incomplete Proteasomal Degradation Zhao Zhang and Yun Zhao Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China

The Hedgehog (Hh) family of morphogens plays an important role in developmental patterning and organogenesis in animals from Drosophila to human. Diverse upstream Hh signals are mediated by the activity and the proteolysis of Cubitus Interruptus (Ci) transcriptional factor. In the presence of Hh, un-phosphorylated full-length Ci (Ci155) enters the nucleolus to active Hh downstream genes expression, high level Hh signaling also promotes completely degradation of excess Ci155 by Cul3Hib E3 ligase. In the absence of Hh, phosphorylated Ci155 undergoes an incomplete proteasomal degradation to generate a truncated form (Ci75) by Cul1Slimb E3 ligase, Ci75 acts as an repressor role. Zinc fingers and other sequence form a protection signal that prevents Ci from the complete proteasomal degradation, but the road between the E3 ligase and proteasome is still not clear. Here we show that an AAA ATPase protein Ter94 specific involves in the process of Ci incomplete proteasomal degradation by Cul1Slimb E3 ligase. In Drosophila eye disc, loss of Ter94 only results in elevation of Ci155 in the anterior part which Ci155 is mainly degraded by Cul1Slimb E3 ligase. The decrease of Ter94 blocks the degradation of Ci155 by Cul1Slimb but not Cul3Hib E3 ligase. Ter94 binds with Cul1Slimb complex in S2 cell. Taken together, these results suggest that Ter94 may play some role between Cul1Slimb E3 ligase and proteasome in the Ci75 formation process.


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166 Investigating the Mechanisms of Wnt/Wg Secretion Varun Chaudhary1 Julia Gross, Michael Boutros German Cancer Research Center (DKFZ), Division Signaling and Functional Genomics and University of Heidelberg, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany.

Wnt/Wingless (Wg) are hydrophobic secreted glycoproteins that are required for variety of developmental processes and are highly conserved from fly to human. In Drosophila wing imaginal discs, a gradient of Wg protein is observed up to 20 cells away from the secreting cells. Wg is both required for activation of long-range and short-range target gene expression. How this gradient for a hydrophobic Wg molecule is established has been a long standing question. Different hypotheses have been proposed to explain how Wnt/Wg could travel in the extracellular space for generation of the gradient, for example Wg could be secreted on lipoprotein particles and/or on secreted vesicles. Nevertheless, the mechanism by with Wnt/Wg proteins are secreted is poorly understood. Furthermore, a complete set of proteins involved in various aspect of Wnt/Wg trafficking still remains to be identified.

We have previously identified the transmembrane protein Evenness interrupted (Evi/Wls) as a core component of the Wnt signaling pathway that is strictly required for the secretion of Wnt/Wg. Evi shuttles Wnt/Wg from the Golgi to the plasma membrane and is then recycled by the retromer complex. However it remains unclear, how and when Evi releases Wnt/Wg into the extracellular space.

We are interested in understanding how Wnt/Wg proteins are secreted and finding other factors that might be required for their secretion. We are addressing these questions using both cell culture and Drosophila in vivo RNAi screening approaches. A focused RNAi library subset was chosen, containing genes with functions in trafficking, such as Rab-protein family member, exocyst complex genes, retromer and others. Here, we present our RNAi screening data along with the candidates that are under further detailed investigation. Moreover, we also present preliminary data on novel functional roles of Evi in Wnt/Wg secretion.


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167 Function of a Neurogenic Gene, pecanex in Notch Signaling Tomoko Yamakawa1, Takeshi Sasamura1, Maiko Kanai1, Emiko Suzuki2, Mark E. Fortini3, Kenji Matsuno1 1) Dept. of Biological Sci./Tec, Tokyo Univ. of Science, Japan 2) Gene Network Laboratory, National Institute of Genetics, Japan 3) Dept. of Biochemistry and Molecular Biology, Kimmel Cancer Center, Thomas Jefferson Univ., USA

Notch signaling is an evolutionarily conserved mechanism that regulates a broad spectrum of cell-specification through local cell-cell interaction. The homozygous mutant flies of pecanex (pcx) are viable, but pcx homozygous females mated with the pcx mutant males produce embryos that show a Notch–like neurogenic phenotype, suggesting that pcx encodes a component of Notch signaling. Pcx is a multipass membrane protein. However, its biochemical functions are still unknown.

To understand the roles of pcx in Notch signaling, we examined the expression of two target genes of Notch signaling, Enhancer of split and single-minded in pcx homozygote lacking its maternal contribution. The expression of these genes was decreased in these embryos compared with those of wild-type, indicating that Pcx is required for Notch signaling. Epistatic analysis involving pcx mutant and the overexpression of other Notch signaling components demonstrated that Pcx acts upstream of a membrane tethered form of activated Notch and downstream of a full-length Notch.

We found that Pcx protein was specifically localized to the endoplasmic reticulum (ER). In addition, ER was enlarged in the embryos homozygous for pcx lacking its maternal contribution. Furthermore, hyper-induction of the unfolded protein response (UPR) suppressed the neurogenic phenotype and ER enlargement caused by the absence of pcx.

Taken these results together, we speculate that the reduction in N signaling in embryos lacking pcx function might be attributable to defective ER structure and functions, which are compensated by up-regulation of the UPR.


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168 Regulation of the Atg1 Complex by TOR and PKA Signaling in Drosophila Autophagy Yu-Yun Chang, Thomas P Neufeld

Department of Genetics, Cell biology and Development, University of Minnesota, Minneapolis, Minnesota, USA, 55414

Autophagy provides eukaryotic cells with a means of degrading defective macromolecules

and organelles, and has recently been implicated in a wide range of human diseases including neurodegenerative diseases and cancer. A large number of genes with conserved roles of autophagy have been identified in several multicellular organisms; however, concepts about the regulation of autophagy are still based largely on models from yeast, in which target of rapamycin (TOR) acts upstream to inhibit autophagy by down-regulating the assembly and activity of an Atg1/Atg13/Atg17 kinase complex. Although orthologs of the Atg1 kinase have been shown to be critical for autophagy induction in Drosophila and mammalian cells, the regulatory mechanisms have remained unclear.

We have identified Drosophila Atg13 that interacts with Atg1 cooperatively to induce autophagy in response to starvation and under the control of TOR signaling. While yeast Atg1 and Atg13 have also been shown to serve as direct substrates for protein kinase A (PKA), which acts to inhibit autophagy in S. cerevisiae, PKA is essential for autophagy in Drosophila. Moreover, overexpression of a wild-type PKA catalytic subunit, but not a kinase-inactive form, induces autophagy in well-fed animals, indicating that the catalytic activity of PKA is critical for autophagy induction. Our observations further suggest that PKA regulates autophagy by protecting Atg1 and Atg13 from proteasome-dependent degradation as well as by down-regulating TOR signaling activity. The interaction of PKA and AKT suggests that PKA may play a novel role to regulate TOR through AKT and PKA acts as a key signal to direct the induction of autophagy by relieving the TOR-dependent inhibition of Atg1/Atg13.


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169 Analysis of the Function of AIP1 and Cofilin in Actin Dynamics during Drosophila Bristle Morphogenesis Jing Wu, Jiong Chen Model Animal Research Center, Nanjing University

Drosophila bristle (macrochaetes) is a good model system to study regulation of actin

filament dynamics. During bristle development, forked and fascin crosslink actin filaments and a series of modules attach end to end to form actin bundles. In wild type, the actin bundles are highly ordered and are parallel to each other. Previous studies were mainly focused on regulators of actin filament bundling (fascin and forked) or assembly (profilin and Arp2/3). Little is known of how actin filaments disassembly is regulated during bristle morphogenesis. Here, we show that the major actin depolymerization and severing factor, cofilin [encoded by twinstar(tsr)], and its cofactor AIP1[encoded by flare(flr)] are both required for bristle morphogenesis. In tsr mutant and flr mutant bristles, actin bundles are disorganized in a way similar to actin defects from loss of functions in such key actin dynamics regulators as Arp2/3 and Capping protein. This result prompts us to examine if the actin treadmilling process (of which cofilin, Arp2/3, and capping protein all are major regulators) plays important roles in actin bundles assembly, and experiments are currently undergoing to test this hypothesis. In addition, a genetic interaction was uncovered between singed (encoding fascin) and tsr, suggesting a likely functional link between actin bundling and actin disassembly.


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170 Functional Analysis of Akt Signaling Pathway in Drosophila Border Cell Migration Di Kang, Juntao Zuo, Lijun Zhang, Jiong Chen Model Animal Research Center, Nanjing University

Akt is one of the most frequently activated protein kinases in human cancer. Tumor promoting activity of this protein in human cancer has been well established by many studies. An equally important component of tumor progression is the propensity of cancer cells to migrate and invade. In this context however, relatively little information exists for roles of Akt in directly regulating cancer cell migration or invasion. Here, we use border cells as a model to investigate the function of dAkt in cell migration. Genetic mosaic analyses of dAkt1[KD] (a kinase-dead allele of dAkt) show that border-cell migration is disrupted. In addtion, the homozygous mutant clones lose Eya (eye absent) expression, which appears to be

independent of fate change because of unchanged Fascin and Fasciclin Ⅲ expression. The migration defect phenotype is further confirmed by knocking down dAkt by RNAi in border cells. Furthermore, we examined several key components of Akt signaling in border cells by RNAi knock-down, including PI3K, Pten, PDK1, TSC1 and TSC2. We found that disruption of any of them will impede cell migration to various extent, indicating that Akt signaling is vital for border cell migration. Moreover, we found that single cell overexpressing dAkt within an otherwise wild type border cell cluster is more likely to be the leading cell during the course of migration of border cells, whereas single cell with dAkt knockdown is more likely to be at the lagging end of the migrating cluster. Taking together, these results suggest a vital role for Akt signaling in the collective migration of border cells.


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171 Analysis of the Roles of the Rho GTPases during Drosophila Eye Development Hanshuang Pan, Juntao Zuo, Jiong Chen Model Animal Research Center, Nanjing University, China

Drosophila eye development can be divided into three parts: cell proliferation at early larval stage, cell fate determination at late larval stage, and terminal differentiation at pupal stage. We analyzed the function of small Rho GTPase family members Rho, Rac and Cdc42 during Drosophila eye development and found that each member plays distinct roles during different stages. Notably, Rho and Rac seem to be required for cell proliferation, as the larval eye imaginal discs are severely reduced in size by expression of their RNAi or dominant negative forms. Cdc42 is likely involved in ommatidial cluster formation in the late larval stage, as its dominant negative form results in a moderate disruption of ommatidial cluster alignment posterior to the mophogenetic furrow. Interestingly, during early and mid- pupal development, the photoreceptor adherens junctions are disrupted when Rho function is blocked, but the apical marker aPKC is not as much affected. Furthermore, we found that the rhabdomere of photoreceptors are not elongated properly in the proximal-distal axis. As a result, rhabdomeres of adult eyes exhibit a severely disorganized structure. Together, these results suggest Rho is required for the remodeling of photoreceptors’ adherens junction, which is crucial for retinal elongation during pupal eye development.


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172 Border-Cell Migration Requires Integration of Spatial and Temporal Signals by the BTB Protein Abrupt Anna C.-C. Jang1,3, Yu-Chiuan Chang1, Jianwu Bai2, Denise Montell1

1) Department of Biological Chemistry, Johns Hopkins School of Medicine, 725 North Wolfe Street, Baltimore, Maryland 21205-2185, USA. 2) Department of Genetics,Harvard Medical School, 77 Avenue Louis Pasteur, Boston,Massachusetts 02175, USA. 3) Institute of Biotechnology, National Cheng Kung University, Taiwan

During development, elaborate patterns of cell differentiation and movement must occur in the correct locations and at the proper times. Developmental timing has been studied less than spatial pattern formation, and the mechanisms integrating the two are poorly understood. Border-cell migration in the Drosophila ovary occurs specifically at stage 9. Timing of the migration is regulated by the steroid hormone ecdysone, whereas spatial patterning of the migratory population requires localized activity of the JAK–STAT pathway. Ecdysone signalling is patterned spatially as well as temporally, although the mechanisms are not well understood. In stage 9 egg chambers, ecdysone signalling is highest in anterior follicle cells including the border cells. We identify the gene abrupt as a repressor of ecdysone signalling and border-cell migration. Abrupt protein is normally lost from border-cell nuclei during stage 9, in response to JAK–STAT activity. This contributes to the spatial pattern of the ecdysone response. Abrupt attenuates ecdysone signalling by means of a direct interaction with the basic helix–loop–helix (bHLH) domain of the P160 ecdysone receptor coactivator Taiman (Tai). Taken together, these findings provide a molecular mechanism by which spatial and temporal cues are integrated.


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173 Heat Stress Induces the Co-aggregation of Drosophila P-body Components with Stress Granules in Nurse Cells Szu-Jing Huang1, Ming-Der Lin1,2*

1) Department of life science, Tzu-Chi University and 2) Department of Molecular Biology and Human Genetics, Tzu-Chi University

Regulation of mRNA translatability and stability are critical for the cell survival in stress conditions. In mammalian cells, stress granules and P-bodies are distinct RNP granules with different functions. Stress granules are responsible for mRNA storage in stress conditions while P-bodies are for 5’ to 3’ mRNA degradation. In Drosophila, it has not been proved whether P-bodies and stress granules possess distinct function or not. Our previous results indicate that dDcp1, a P-body component, aggregates around nurse cell’s nuclei in heat stressed egg chamber. We hypothesize that Drosophila P-bodies may possess mRNA storage function as mammalian stress granules in heat stress. To address whether heat stress can induce the co-aggregation of Drosophila P-body components with stress granules in nurse cells, we constructed GFP-tagged Drosophila homologs of mammalian canonical stress granule components including PABP, Rasputin, Trip1 and Rox8. Similar to mammalian stress granule, GFP-tagged stress granule components are able to congregate in nurse cell cytoplasm in response to heat stress. Our data also indicate that Drosophila stress granule components could colocalize with Pacman. These results imply that dDcp1 containing P-bodies change its contents and coaggreate with stress granules in response to heat stress.


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174 Functional Analysis of Drosophila Protein Tyrosine Phosphatases in Development Dong-Yuan Chen1, Meng-Yen Li1,2, Yu-Tsen Lin1, Hsueh-Yen Ku1, Kay-Hooi Khoo1,2,3, Tzu-Ching Meng1,2, Guang-Chao Chen1,2

1) Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan 2) Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan 3) National Core Facility for Proteomic Research, Academia Sinica, Taipei, Taiwan.

Accumulated evidence has indicated that the regulation of reversible tyrosine phosphorylation plays a crucial role during development. Among 15 classical PTPs identified in Drosophila genome, 8 are predicted to be non-transmembrane PTPs (NT-PTPs). To investigate the role of these NT-PTPs in development, we have analyzed the gene expression pattern and phosphatase activity of each NT-PTP. In addition, we have applied both genetic and biochemical approaches to investigate the biological function of them. Our recent findings have demonstrated that PTP61F regulates actin organization and is involved in the proper development of central nervous system. Moreover, loss-of-function analysis revealed that the BRO1 domain-containing tyrosine phosphatase dHD-PTP is required for cell survival and plays an important role in maintaining border cell clustering during oogenesis. Our results not only provide novel insights into physiological function of Drosophila PTPs, but also shed light on unexpected role of them in the regulation of various signaling pathways.


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175 Drosophila FMRP is Required for DNA Damage Response and Maintaining Genome Integrity Wei Liu1, Xiao-lin Bi2, Yong Q. Zhang1

1) Key Laboratory for Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China 2) Key Laboratory for Biological Effects of Nanomaterial and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, China

Fragile X syndrome (FXS) is the most common form of inherited mental retardation caused by the loss of fragile X mental retardation protein FMRP. Although increased chromosomal breakpoints and elevated carcinogen-induced genome instability in cultured cells from FXS patients have been observed for decades, the exact role of FMRP in the maintenance of genome stability is unclear. Here, we show that Drosophila dfmr1 mutants are hypersensitive to genotoxic stresses such as γ-irradiation and exposure to mutagens, indicating that dfmr1 is critical for genome stability. To elucidate the underlying mechanisms, we examined the roles of dfmr1 at cell cycle checkpoints and apoptosis. We found that loss of dfmr1 leads to defective G2/M checkpoint in larval brains upon DNA damages. Cyclin B, a crucial regulator for G2/M transition, is up-regulated in dfmr1 mutants. dFMRP, a transcript-specific translational regulator, specifically associates with Cyclin B mRNA, indicating that dFMRP regulates the execution of G2/M transition by repressing Cyclin B translation. In addition, we found significantly increased apoptosis upon γ-irradiation in dfmr1 mutants in a p53-dependent manner. Moreover, loss-of-heterozygosity assay revealed that loss of dFMRP leads to higher rate of genomic instability induced by irradiation. Together, we unveil a novel function of dfmr1 in the maintenance of genome integrity, probably through controlling of G2/M DNA damage checkpoint.


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176 GOLPH3 Modulates the Retrograde Trafficking of Tumor Suppressor Exostosin Proteins in Golgi Complex Wei-Ling Chang, Che-Wei Chang, Yu-Yun Chang, Hsin-Ho Sung, Chung-Hao Chen, Tze-Bin Chou* Institute of Molecular and Cellular Biology, National Taiwan University, Taipei, Taiwan

The exostosin (EXT) family proteins encode glycosyltransferases required for

glycosaminoglycan (GAG) chain polymerization in the biosynthesis of heparan sulfate proteoglycans (HSPGs). Mutation of EXT1 and EXT2 in human causes autosomal dominant hereditary multiple exostoses (HME). Till now, how these EXT enzymes are transported in the Golgi complexs is not clear. We first demonstrated that Rotini (Rti), the Drosophila GOLPH3, regulates the retrograde trafficking of fly EXTs within the Golgi complex. The reduction of Rti shifts the steady-state distribution of fly EXTs to trans-Golgi. These accumulated EXTs tend to be degraded and their re-entrance toward the route for polymerizing GAG chains is disengaged. Conversely, fly EXTs are mislocalized toward ER/cis-Golgi when Rti is over-dosed. Both conditions make less proper HSPGs and morphogen signal transduction. Further, in human osteosarcoma cell, GOLPH3 is confirmed to modulate the dynamic retention of EXT1/2 in Golgi complex in an evolution conservative manner.


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177 Ecdysone Signaling is Required for Proper Rearrangement of Cells and Its Functions in Malpighian tubules of Drosophila melanogaster Naveen Kumar Gautam and Madhu Gwaldas Tapadia

Cytogenetics Lab, Department of Zoology, Banaras Hindu University, Varanasi, India

Drosophila Malpighian tubules (MTs) are functional homologue of human kidney which function in removal of toxic components from haemolymph and maintain homeostasis. Drosophila renal system or MTs development has close similarities to human renal development. During human renal development, interactions between the epithelial mesonephric duct and the bordering metanephric mesenchyme guides to the formation of renal tubules. Similarly, in Drosophila, MTs originated from ectodermal epithelial buds and the surrounding mesenchymal mesoderm which are recruited to growing tubule and undergo mesenchymal-to-epithelial transition. So, mature tubules are made up of ectodermal principal cells (PCs) and mesodermal stellate cells (SCs) which are functionally distinct. PCs are responsible for cations transport in lumen while SCs are involved in chloride conductance and aquaporin mediated water transport. Thus, dual origin of tubule cells serves as an excellent system to study the mechanisms underlying the cellular rearrangements, intercalation of different cell types and its function.

Although the larval MTs do not seem to be affected by ecdysone during metamorphosis, our present studies suggest that ecdysone signaling play a key role in development and functioning of MTs. Disruption of ecdysone signaling in PCs and SCs leads to improper arrangement of PCs and SCs in MTs. Disruption also causes disorganization of cytoskeleton element and derangement of cell adhesion and cell junction protein. Further we observed that disruption of ecdysone signaling in PCs up regulates the expression of (Na+/K+)-ATPase which elevates Na+ concentration in MTs while disruption in SCs down regulates the DRIP expression in MTs.


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178 A Role for Soluble Inositol Polyphosphates in Cell Proliferation, Cell Division and Apoptosis in the Development of Drosophila Man-kin Marco Tsui1,2, Man-ying Tin2, Andy Seeds3, John D. York2

1) Present address: Developmental Signaling Unit, OIST, Japan 2) HHMI, Duke University, USA 3) Janelia Farm, HHMI, USA

Phosphoinositides are important signaling molecules regulating cell proliferation, vesicular trafficking, actin organization and hedgehog signaling in Drosophila. Phosphoinositides can be hydrolyzed by phospholipase C into inositol trisphosphate (IP3), a soluble second messenger. IP3 can be further phosphorylated to form higher inositol polyphosphates (IPs,) such as IP4, IP5 and IP6, via the sequential actions of the evolutionally conserved kinases Ipk2 and Ipk1. IPs have been shown to be involved in nuclear processes such as chromatin remodeling and mRNA export in yeast. Deletion of either IPK2 or IPK1 is embryonic lethal in mice, indicating IPs are essential in higher organisms but the role(s) IPs play in metazoan development remains unclear. Here we report that loss of fly Ipk2 results in defects including pupal lethality and abnormalities of the eyes, wings and body. The imaginal discs of the mutant exhibit ectopic apoptosis and slower DNA replication. A number of signaling pathways including JNK and ERK signaling are mis-regulated in the mutant, suggesting that IPs are regulators of proliferation signals. We further showed that IP6 binds to and regulate the activity of a protein complex which is thought to be essential for mitotic chromosome integrity. Consistent with this, the ipk2 null mutant larval brain shows defective mitotic chromosome morphology. In summary, we demonstrated that inositol polyphosphates are essential for proliferation of mitotic tissues in flies, and play a role in maintaining proper mitotic chromosome structures, probably by regulating various signaling pathway and the functions of a novel IP6 binding protein in flies.


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179 Receptor Protein Tyrosine Phosphatase 52F – An Ecdysone Inducible RPTP Involved in the Degradation of Ubiquitinated Proteins Abirami Santhanam1,2,3 , Guang-Chao Chen1,2,3, Tzu-Ching Meng1,2,3

1) Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. 2) Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan. 3) Taiwan International Graduate Program, Academia Sinica, Taipei, Taiwan.

Studies have shown a positive relationship between estrogen/progesterone stimulation and the expression of a receptor protein tyrosine phosphatase (PTP) LAR during breast cancer development. However it remains elusive whether PTP-mediated phosphotyrosine (pTyr) signaling is a critical event in the context of steroid hormone action. To shed light into the interplay between them we began our studies using the fruit fly Drosophila melanogaster as a model. It has been shown clearly that the steroid hormone ecdysone regulates key developmental processes during fly metamorphosis. Analysis by microarray data mining suggested that PTP52F, a receptor PTP, may be affected by ecdysone action since its level was increased significantly during the pupa formation (PF) stage in which the ecdysone pulse kicks in. This result was confirmed by the quantitative real-time PCR method. Furthermore, using Drosophila Kc167 embryonic cells, we showed a positive relation between ecdysone stimulation and inducible expression of PTP52F. We adopted a substrate trapping strategy combined with mass spectrometry based analyses to identify physiologically relevant substrates of PTP52F. Our results showed that transitional endoplasmic reticulum ATPase 94 (TER94), ortholog of human Valosin Containing Protein (VCP) was a potential substrate of PTP52F. Based on preliminary data, we propose that Tyr800 of TER94 is phosphorylated by Src kinase, and dephosphorylated by PTP52F. Interestingly the phosphorylation status of tyr800 seems to control the degradation of ubiquitinated proteins as well as the stability of TER94. Our study may reveal a novel regulatory role of a PTP in steroid hormone signaling involved in development.


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180 Atg1-Mediated Myosin-II Activation Regulates Autophagy in Drosophila and Mammalian Cells Hong-Wen Tang1, 2, Yu-Bao Wang1, Shiu-Lan Wang1, Mei-Hsuan Wu1, Shu-Yu Lin1, 3, Guang-Chao Chen1,2

1) Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan. 2) Institute of Biochemical Sciences, College of Life Science, National Taiwan University, Taipei, Taiwan. 3) NRPGM Core Facilities for Proteomics and Glycomics, Academia Sinica, Taipei, Taiwan.

Autophagy is a highly conserved, degradative process which is involved in many developmental and pathological conditions such as cancer and neurodegeneration. The formation of double-membrane degradation vesicles, autophagosomes, is the central part of autophagy, whereas the underlying mechanism is not well understood. Among the autophagy-related genes identified, Atg1 is found to be the key regulator for autophagy induction. We have previously identified a link between Atg1 and the cytoskeletal protein paxillin in Drosophila, and paxillin-deficient cells exhibited defects in autophagosome formation, suggesting a role of Atg1 in regulating actin organization and autophagy. In this study, we find that overexpression of Atg1 induces the phosphorylation dependent activation of the actin-associated motor protein myosin II. A novel myosin light kinase (MLCK)-like protein, spaghetti-squash activator (Sqa), is identified as a link between Atg1 and the observed actomyosin activation. Sqa interacts with Atg1 and is a substrate of Atg1. Significantly, myosin II inhibition or depletion of Sqa compromised the starvation-induced formation of autophagosomes both in Drosophila and in mammalian cells. Our findings provide evidence of a link between Atg1 and the control of autophagosome formation through the myosin II motor protein.


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181 DTestican Regulates the Extracellular Distribution of Wingless Yung-Heng Chang1,2, Y. Henry Sun1,2

1) Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan, Republic of China 2) Institute of Molecular Biology, Academia Sinica, 115, Taipei, Taiwan, Republic of China

Morphogens are extracellular signalling molecules involved in regulating growth and pattering of tissues or organs. They form a concentration gradient from the producing source and activate different target genes at different concentrations within the gradient. Without proper distribution of these extracellular signalling molecules, developmental defects in many aspects occur. The concentration gradient of a morphogen is regulated by the combination of many factors that some acts in the intracellular space and others functions on the cell membrane or extracellular environment. In the transporting of Wingless (Wg) from producing region to the target cells, some mechanisms are reported to involve that include diffusion and active transcytosis. In an ectopic screening, the Drosophila DTestican (DTestican) is found to regulate the extracellular movement of Wg. DTestican encodes one secreted heparan sulfate glycoprotein (HSPG). HSPGs are well known to regulate the activity of extracellular signalling molecules in mammal or Drosophila by playing as co-receptor or helping the transport of these molecules. DTestican is found to have the ability in helping the extracellular movement of Wg by forming a physiological complex and changing the moving speed of Wg. Without proper expression of DTestican in embryo or wing disc, it causes accumulation of Wg in the producing cells and changes the gradient activity of Wg. In this situation, the target gene patterns of Wg are reformed and then causes denticle fusion and ectopic wing margin bristles similar to Wg signaling activity defects. Importantly, DTestican is involved in helping the Wg transport on the apical surface. This is a missing puzzle that Wg moves faster on the apical surface when the active transcytosis is blocked. Our results indicate DTestican can form a protein complex with Wg and changes the distributing speed of Wg in the extracellular space. It suggests that the DTestican plays as an extracellular shuttle to regulate the extracellular movement of Wg.


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182 Drosophila ADI1, a Methionine Salvage Pathway Enzyme, Regulates Fasciclin III Expression in Follicle Cells He-Yen Chou1, Yen-Hsien Lee1, Shu-Heng Zeng1, Yu-Hung Lin1, Wei Chiang1, Pei-Yu Wang1, Ying-Hao Wen1, Guan-Lin Shiu1, Wei-Hao Li1, Chau-Ting Yeh2, Li-Mei Pai1 1) Department of Biochemistry and Molecular Biology, Chang Gung University, Taipei, Taiwan 2) Liver Research Unit, Chang Gung Memorial Hospital, Chang Gung University School of Medicine, Taipei, Taiwan

Drosophila aci-reductone dioxygenase 1 (DADI1), an enzyme involved in methionine salvage pathway (MTA cycle), shares homology with bacterial aci-reductone dioxygenase (ARD/ARD’) in the cupin family. Recently, a human ARD orthologue (hADI1) has been demonstrated to exert additional functions, including regulation of cellular migration and viral entry. We use ovary follicular epithelium as a model to study DADI1 function in Drosophila. We found that DADI1 is required for the accumulation of Fasciclin III (FasIII), cell adhesion molecule, at dorsal follicle cells. Reduced accumulation of FasIII was found in protein-null mutants and RNAi knockdown experiment. The FasIII accumulation defect in Dadi1 mutant cells could be rescued by expressing wild-type DADI1 but not DADI1 enzyme-dead mutant. Moreover, analysis of Dadi1 mutant egg chambers revealed that cell shape was changed in dorsal follicle cells, and boundary between floor and roof cells was unclear cells. Using the same approach, we further observed that the constriction of dorsal follicle cell fold into tubular structures was delayed. These findings suggest DADI1 involved in tissue remodeling through modulating FasIII expression. The immunostaining result showed that DADI1 colocalized with cis-Golgi protein DGM130. In addition, DADI1 genetically interacts with DGM130 and γcop

(COPⅠsubunit), suggesting that a possible role of DADI in Golgi function. In sum, DADI1 may control membrane protein FasIII expression through regulating protein trafficking in Golgi.


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183 Study the Interaction of Ligands and Receptors of Drosophila Jak/STAT Pathway Po-Kai Wang, Shin-Han Lin, Yu-Chen Tsai Department of Life Science, Tunghai University, Taichung, Taiwan

The Janus kinase(Jak)/signal transducer and activator of transcription(STAT) pathway regulates immune response and hematopoiesis in mammals. Mis-regulation of Jak/STAT signaling may induce cancer or immune diseases. The Jak/STAT pathway is evolutionarily conserved. There are four Jaks and seven STATs in mammals. Drosophila only has one Jak and one STAT. Therefore, Drosophila is a simple and non-redundant system to study Jak/STAT pathway in vivo. The Jak/STAT pathway regulates multiple developmental processes, including segmentation of embryos, eye development, hematopoiesis, immune response and stem cell renewal. Three ligands, Unpaired (Upd), Upd2, Upd3 and two receptors, Domeless (Dome) and Domelike/CG14225, were identified in the Drosophila Jak/STAT signaling. The expression patterns of Upd-likes are overlapping or adjacent during embryonic, eye and testis development. It’s still unclear how such a simple Jak/STAT signaling can mediate multiple distinct developmental processes. We proposed different combination of Upd-likes and Dome receptors may have different affinity and induce different biological effects. We checked interaction of ligands in S2 cells by co-immunoprecipitation. We found Upd-likes can form homodimers and heterodimers. We will further study whether different combination of Upd-likes and Dome receptors causes different Jak/STAT activity and biological effects. We found over-expression of different combination of Upd-likes and Dome receptors caused different effects in fly eyes. This study may help us further understand the molecular mechanism of ligand-receptors interaction in Drosophila Jak/STAT pathway.


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184 An RNAi Screen for New Epithelial to Mesenchymal Transition Factors, using Wing Disc Eversion Rosemary Manhire-Heath1,2, Joy Thompson2, Robert Saint2, Michael Murray2

1) Research School of Biology, The Australian National University, ACT, Australia 2) Department of Genetics, The University of Melbourne, VIC, Australia

Epithelial to Mesenchymal Transitions (EMTs), the process wherein epithelial cells dissociate and become migratory, play key roles in development and tumour metastasis. During Drosophila development, EMTs occur during the formation of the mesoderm and endoderm, and in the delamination of neuroblasts from the epidermis. Molecular pathways regulating these EMTs, however, are not well defined.

We are screening for new EMT factors using an event well suited to large scale RNAi screens: wing disc eversion. During eversion, cells of the peripodial epithelium lose apico-basal polarity and adherens junctions, breakdown the basement membrane, and invade and migrate over larval cells. Disc eversion requires activation of the JNK and TGFbeta pathways, and expression of matrix metalloproteinases. When eversion is disrupted adult flies exhibit phenotypes which are easily scored, ranging from mild clefts at the thoracic midline, to internalised wings and missing thoracic tissue. Using UAS RNAi libraries we are screening for genes that, when knocked down in the peripodial epithelium, disrupt eversion.

We have screened the majority of kinases/phosphatases, as well as putative downstream targets of genes/pathways associated with more mesenchymal behaviour such as Twist, the JNK pathway and Asense. To date we have identified genes implicated in the JNK, Wnt, PI3K and TGFb signalling pathways, basement membrane breakdown, cytokinesis, cell polarity, and regulation of cell motility. We are currently focusing on genes in which JNK pathway activation is normal, but other aspects of the EMT, such as the acquisition of F-Actin rich motile protrusions, are defective.


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185 The Role of Drosophila Endophilin B in Oskar Regulated Endocytosis Yi-Chen Tsai1, Wei Chiang1, Wei-How Li1, Yu-Wei Chang1, Tsubasa Tanaka2, Akira Nakamura2, Yi-Chen Li1, I-Ching Chu1, Li-Mai Pai1 1) Institute of Biomedical Sciences, Chang Gung University, Tao-Yuan, Taiwan. 2) Riken Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan

The Endophilin is a highly conserved protein family among many species. All Endophilins are composed of an N-terminal N-BAR domain and a C-terminal SH3 domain. Endohpilin B is reported to possess multiple function involving in various cellular mechanisms, such as autophagy and tumor suppression. However, it is still unknown about the function of Endophilin B in Drosophila. Here we show that Drosophila Endophilin B is expressed in germarium and early to middle oogenesis. By immunostaining, the Drosophila Endophilin B is colocalizes with Oskar at the oocyte posterior pole. We also find that the posterior D-EndoB depends on Oskar. In Drosophila, Oskar determines the polarity of the oocyte and the development of the germ cells. Recent studies report that the endocytic level and yolk content were reduced in the oskar mutant oocyte. In our study, D-Endophilin B does not affect the pole plasm assembling but is required for maintaining the endocytic activity in the oocyte by FM4-64 incorporation assay. Furthermore, the eggs produced by endoB54/endoB54 mutant females are decreased and the yolk content in D-endoB mutant oocyte is also less than that in wild-type. When exogenously expressing the D-EndoB in germline, the reduced endocytic level in the mutant oocyte is recovered and also rescues the defect of egg production. In sum, we demonstrate that D-Endophilin B plays a role in regulating fertility through yolk uptake by endocytosis. Our latest discovery shows that the numbers of germline stem cell in the germarium are reduced in endoB54 homozygous mutant. It implies the possibility which D-EndoB is involved in the maintenance gremlins stem cell.


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186 Fibroblast Growth Factor Signalling Controls Successive Cell Behaviours during Mesoderm Layer Formation in Drosophila Ivan B.N. Clark1, Villo Muha1, Anna Klingseisen1, Maria Leptin2, Arno Müller1

1) College of Life Sciences, University of Dundee, Dundee, Scotland, U.K. 2) Institut für Genetik, Universität zu Köln, Germany.

Drosophila mesoderm layer formation from an invaginated epithelial primordium involves a transition to a mesenchymal state (EMT) and the dispersal of cells away from the point of internalisation in a Fibroblast-Growth-Factor (FGF) - dependent fashion. FGF-dependent epithelial-mesenchymal transitions and cell migration also contribute to the establishment of germ layers in vertebrates, but a comprehensive demonstration of the cellular activities that FGF controls to mediate these events has not been addressed in much detail. We show that FGF plays multiple subcellular roles at successive stages of mesoderm morphogenesis in Drosophila. FGF-signalling is first required for the mesoderm primordium to lose its epithelial polarity. An intimate, FGF-dependent contact is established and maintained between the germ layers through mesoderm cell protrusions that extend deep into the underlying ectoderm epithelium. Finally, FGF directs distinct, hitherto unrecognized and partially redundant protrusive behaviours during later mesoderm spreading. Cells first move radially towards the ectoderm, and then switch to dorsally directed movement across its surface. The radial protrusions are differentially dependent on the small GTPase Cdc42 and required for normal mesoderm layer formation. Thus during mesoderm layer formation FGF signalling contributes to the initiation of EMT and directs differential protrusive activity during mesoderm-ectoderm attachment and dorsal migration. The roles of cell adhesion and small GTPases of the Rho family will be discussed.


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187 Identification and Analysis of the Poly(A) Nuclease Complex as a Novel Activator of ASK1 using Drosophila Genetics Ryo Hatanaka, Yusuke Sekine, Kohsuke Takeda, Hidenori Ichijo

Cell Signaling, Grad. Sch. Pharmaceut. Sci., Univ. of Tokyo, Japan

ASK1 is a member of the MAP3K family, which activates both the MKK4/MKK7-JNK and

MKK3/MKK6-p38 MAPK cascades and constitutes important signaling pathways in various types of stress responses. Although ASK1 is activated by various stresses, signaling components that serve as activators of the ASK1-MAPK cascades have not been fully understood. Here we identified such components by taking advantage of Drosophila genetics. Drosophila ASK1 (DASK1) is an ortholog of mammalian ASK1, which also activates the Drosophila JNK and p38 pathways. When a constitutively active form of DASK1 was expressed along the dorsal middle line of flies using the GAL4/UAS system, a marked increase in melanization was observed in the thorax of adult flies. By using this phenotype as a visible marker for DASK1 activation, we carried out a misexpression screening to explore novel genes that regulate the activation and/or function of ASK1. From this screening, we identified an mRNA deadenylation-related gene Poly(A) nuclease 3 (Pan3) as a potential activator of DASK1. Genetic interaction analyses revealed that Pan3 induced melanization in a Dp38 pathway-dependent manner. Pan3 is known to form the complex with the exoribonuclease Pan2 and Poly(A) binding protein (PABP) and to function in deadenylation of mRNA as a poly(A) nuclease both in yeast and mammalian cells. In Drosophila S2 cells, Pan3 bound to both Pan2 and PABP. In addition, expression of Pan2, Pan3, or PABP strongly activated DASK1, suggesting that the Pan complex may serve as an activator of DASK1. We are currently exploring the stress conditions in which the Pan complex plays a role as an activator of ASK1.


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188 A Genome-Wide RNAi Screen Identifies Core Components of the G2/M DNA Damage Checkpoint Shu Kondo, Norbert Perrimon Department of Genetics, Harvard Medical School, USA

The DNA damage checkpoint is one of the first pathways activated in response to DNA damage. Checkpoint activation temporarily arrests the cell cycle to provide time for DNA repair. The DNA damage checkpoint is mediated by the evolutionarily conserved ATM/ATR kinase cascade. The mechanism by which DNA damage activates the ATM/ATR kinases is not fully understood. We conducted a genome-wide RNAi screen in Drosophila cells to identify novel genes required for the G2/M checkpoint. We identified ~60 genes that could be classified into particular biological functions including DNA repair, DNA replication, cell cycle control, chromatin regulation and RNA processing. The screen identified previously unrecognized roles of two DNA damage response genes, mus101 and mus312, which was confirmed by in vivo mutant analysis. Our results suggest that the DNA replication preinitiation complex, which includes MUS101, and the MUS312-containing nuclease complexes, which are important for DSB repair, also function in the G2/M checkpoint. Our results provide insight into the diverse mechanisms that link DNA damage and the checkpoint signaling pathway.


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189 Tousled-like Kinase Regulates the Morphogenesis of Germline and Follicle Cells in Drosophila Shu-Yu Huang, Gwo-Jen Liaw, Jenn-Yah Yu

Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taiwan

The Tousled-like kinase (Tlk) is highly conserved among plants and animals. In plants, tlk mutations cause random loss of floral organs, suggesting that tlk is important for cell proliferation. In mammalian cells, TLK is activated during S phase of the cell cycle. TLK phosphorylates the Histone chaperon anti-silencing function 1 (ASF1) and participates in the control of chromatin replication, cell cycle, and DNA repair. In Drosophila, Tlk has also been shown to participate in chromatin assembly, nuclear division, and cell survival. These results demonstrate that Tlk plays crucial role in regulating cell division. However, whether Tlk is involved in other cellular functions remains unknown.

Here, we used the Drosophila ovary as a model to study Tlk functions in germline and follicle cells in vivo. In germline cells, tlk mutant cysts did not form a disc-like shape during early oogenesis in the germarium. These abnormal-shaped cysts were not properly wrapped by follicle cells. In follicle cells, the cell morphology was also affected in tlk mutant clones at late stages of oogenesis. The microtubule pattern was abnormal and β-tubulin was up-regulated in tlk mutant follicle cells. The cell adhesion molecule DE-cadherin was increased in tlk mutant cells. We further examined if the apical-basal cell polarity is affect in tlk mutant follicle cells. Atypical protein kinase C (aPKC), a key regulator for the cell polarity, was increased in tlk mutant cells. Discs Large (Dlg) is a cell polarity marker localized to the basal side. In tlk mutant cells, Dlg is mis-localized to the apical side. Together, our results showed that tlk is involved in cell morphogenesis, cell adhesion, and apical-basal polarity.


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190 Verification of Collective Guidance Model in Drosophila Border Cell Migration Mikiko Inaki, Smitha Vishnu and Pernille Rørth Institute of Molecular and Cell Biology, Singapore

Cell migration is a critical event in development as well as in pathological conditions. In

both cases, cells often migrate as a group, not alone. The guidance mechanisms for group cell migration are largely unknown. Groups of cells could use the same mechanisms as single cells to migrate, with each cell having polarized signals and reacting individually. It is also possible for group cells to respond in collective mode, with the whole group acting as one big cell. In this model the front part of the cluster receives higher signals than the back and as a whole, the cluster judges the migration direction. Importantly, this model does not required localized signal within each cell. To verify the latter hypothesis, we used border cell of the Drosophila ovary as a model system and designed an experiment. First we removed all the external guidance information by using either dominant-negative or mutant guidance receptors or ligands. We then introduced one uniformly activated cell in the cluster, by expressing chimera receptors. The chimera receptors have human extracellular domain and fly intracellular domain and do not react with fly ligands, but can activate down-stream signaling by auto-dimerization upon over-expression. We examined the position of the activated cell and the direction of the cluster migration and found a correlation between them. We also observed changes in the direction of the cluster migration when one cell of the cluster was stimulated by photo-activatable Rac, a down-stream component of guidance receptors, in the same background. These results support the collective guidance model in group cell migration.


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191 Identification and Characterization of Novel Planar Cell Polarity Genes in Drosophila melanogaster Tomonori Ayukawa1, Juergen A. Knoblich2, Takehiko Sasaki1,3 and Masakazu Yamazaki1 1) The Global COE program, Akita University School of Medicine, Japan 2) Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Austria 3) Department of Pathology and Immunology, Akita University School of Medicine, Japan

Planar cell polarity (PCP) signaling is an evolutionarily conserved mechanism that orchestrates cell behaviors within the plane of the epithelium. In Drosophila, each of wing cells is polarized along the proximal-distal axis and produces a single hair at the distal vertex of each cell. The PCP core proteins, such as transmembrane proteins Frizzled, Flamingo/Starry night and Strabismus/van Gogh, are localized at proximal and/or distal cell cortex in the wing, and this asymmetry prefigures the site of hair formation. It has been proposed that several mechanisms, including intercellular communication of epithelial cells and directional microtubule-based transport of Frizzled, play crucial roles in the polarized distribution of the PCP core proteins. However, it still remains unclear how the polarized distribution is regulated.

We previously carried out a genome-wide RNAi screen to study the development of the sensory bristles on the Drosophila notum and identified genes potentially involved in PCP (Nature 458, 987-992, 2009). To identify novel general PCP regulators that function in multiple tissues, we rescreened the PCP related genes obtained in the primary screen by knocking down their expression in the wing, and then categorized the genes into distinct groups based on phenotypic differences. We are currently investigating whether these novel PCP genes affect the polarized localization of the PCP core proteins and how these regulate the PCP pathway.

POSTER: TECHNIQUES AND FUNCTIONAL GENOMICS

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192 National BioResource Project – Drosophila NBRP Drosophila consortium: Masatoshi Yamamoto1, Ryu Ueda2, Masayoshi Watada3, Muneo Matsuda4

1) Drosophila Genetic Resource Center, Kyoto Institute of Technology, Japan 2) Genetic Strains Research Center, National Institute of Genetics, Japan 3) Faculty of Science, Ehime University, Japan 4) Graduate School of Medicine, Kyorin University, Japan

The National BioResource Project (NBRP) supported by MEXT was started as 5-year-project in 2002 to construct the framework for systemic collection, preservation, and distribution of bioresources, with a focus on those that required strategic development by the national government. This project resulted in the development of 25 categories of bioresources and a center for information on the resources. In 2007, the government launched the current second phase of the NBRP to enhance the bioresource framework by increasing value-added genomic resources, developing preservation technologies and adding new collections, while maximizing the potential of existing resources.

The fly community in Japan, at application of this grant, proposed a consortium of 4 centers to develop a cooperative activity to cover all of Drosophila wild-type and mutant strains, which are increasing in number along with a recent development of genome-wide mutant libraries in addition to a huge collection from a long history of fly genetics. The consortium now houses 36,000 fly strains and provides over 40,000 of them every year to researchers all over the world. A portal site, “DGRC” (http://www.dgrc.jp/flystock/index_e.html), has been established to facilitate users select their favorite flies on the web. An introduction and 2010 activity of NBRP-Drosophila will be presented.


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193 Functional Profiling of Rab GTPases in the Drosophila Nervous System Chih-Chiang Chan1,*, Shane Scoggin3,, Dong Wang1, Smita Cherry1, Michael Buszczak3,#, and P. Robin Hiesinger1,2,#

. 1) Department of Physiology 2) Green Center for Systems Biology 3) Department of Molecular Biology, Univ. of Texas Southwestern Medical Center, Dallas, TX; *: Co-first authors; #: Co-corresponding authors

Rab GTPases are master regulators of intracellular membrane trafficking in all cells. Neurons have high and specialized demands on membrane trafficking both during development (wiring-specific extensive arborizations) and function (neurotransmitter release). Here we test the hypothesis that neuronal Rab GTPases are key regulators of neuron-specific membrane trafficking. In order to systematically profile the entire gene family, we have developed a streamlined vector platform for ends-out homologous recombination based on P[acman], BAC recombineering and PhiC31 transgenesis. Using this technology we have generated a comprehensive set of 38 genomic rab-Gal4 transgenic flies for 25 rab genes. Surprisingly, our expression profiling shows that half of all rab GTPases in Drosophila are either neuron-specific or strongly enriched in neurons with highly variable expression patterns in the brain. Subcellular localization profiling of all neuronal Rab proteins revealed that all neuron-specific rabs encode synaptic proteins. The rab-Gal4 lines presented here are designed to easily generate molecularly defined null alleles using an optimized approach to ends-out homologous recombination. We provide the protocols and three knock-ins to quantitatively evaluate the efficiency of our technique. Our findings and tools provide a powerful approach to systematically study neuron-specific membrane trafficking in a brain in vivo. Furthermore, the streamlined Gal4 knock-in method presented here is applicable to all genes.


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194 A Novel Method for Mosaic Gene Expression with Cre-loxP System in Drosophila Embryos Naotaka Nakazawa1, Kiichiro Taniguchi1, Takashi Okumura1, Reo Maeda1 and Kenji Matsuno1,2 1) Department of Biological Science and Technology, Tokyo University of Science, Japan 2) Research Institute for Science and Technology, Tokyo University of Science, Japan

Many techniques have been developed for studying the development of Drosophila melanogaster. Mosaic analysis is a powerful tool to assess the functions of genes in a subset of the cells in individual organism. Although some methods for mosaic analysis have been developed, it is still difficult to generate mosaic cells in the most of Drosophila embryonic tissues.

Here, we report a novel method to generate mosaic cells during early embryogenesis using a modified Cre / loxP system. In this method, we constructed a novel cassette of loxP combined with Actin5C promoter and Gal4 cDNA, designated as pAct5C-loxP-gypsy- loxP-Gal4 (pAloxg-Gal4), and generated transgenic fly carrying this construct (Aloxg-Gal4). In these flies, Gal4 expression is suppressed by gypsy insulator. When site-specific recombination between the two loxP sites is induced by Cre, the removal of gypsy insulator results in the activation of Gal4 expression.

After the recombination, we detected the Gal4 expression using UAS-Redstinger. The clonal expression of UAS-Redstinger was detected in almost 100% of embryos from embryonic stage 10. Using Aloxg-Gal4 in a mutant background, we can easily make mosaic embryos composed of mutant cells with and without the overexpression of a gene disrupted in this mutant. In these mosaic embryos, phenotypes of cells homozygous for the mutant were rescued in the mutant cells overexpression the gene responsible for the mutation. Thus, our novel system is useful to study the cell-autonomy of the gene function during the embryogenesis.


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195 New Site-Specific Recombinases for Drosophila Genomic Manipulation Barret D. Pfeiffer, Aljoscha Nern, Gerry Rubin Janelia Farm Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn VA 20147, USA

Site-specific DNA recombinases are widely used in multicellular organisms to manipulate the structure of genomes and, in turn, to control gene expression. The use of site-specific recombinases has been limited by the availability of recombinases with high activity, distinct site-specificity, and low toxicity. In Drosophila, the most widely used recombinase is FLP, encoded by the Saccharomyces cerevisiae 2µm plasmid. However, there has been no equally good second recombinase. Through stepwise mutagenesis the specificity of the FLP recombinase has been modified to recognize altered FRT sites, but some cross-reaction still remains. Cre, encoded by the bacteriophage P1, functions in Drosophila, but exhibits obvious toxicity. In this report we describe the characterization of four other recombinases encoded by the 2µm circle-like plasmids of other yeasts. We compare the properties of these novel yeast recombinases to FLP, Cre and Dre in Drosophila. We demonstrate four non-cross-reacting pairs of recombinases and target sites that have low toxicity and high activity.

POSTER: EVOLUTION AND QUANTITATIVE GENETICS

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196 Parthenogenesis of Drosophila albomicans Chia-Chen Chang1, Hwei-yu Chang1,2

1) Department of Entomology, National Taiwan University, Taipei 106, Taiwan 2) Research Center for Biodiversity, Academia Sinica, Nankang, Taipei 115, Taiwan

Although parthenogenesis is uncommon in Drosophila, a parthenogenetic D. albomicans strain in our lab was used to explore how the genetic elements related to parthenogenesis can be maintained when parthenogens coexist with their sexual relatives in natural populations, and how diploidy is restored during automixis. We examined the reproductive characteristics of this strain. While a bisexual female produces hundreds of offspring in a month, a single parthenogenetic female of D. albomicans produces on average only 35 offspring. As compared to bisexual flies, the parthenogenetic reproduction of D. albomicans is not only low but also delayed. A parthenogenetic female can mate with a bisexual male without any difficulty. Although 69% of the F1 females can still conduct parthenogenesis, the average number of offspring produced by them is remarkably reduced, i.e., only 1.5 in one month. Despite the fact that the parthenogenetic reproduction is low and even much lower after mating, the persistence of the parthenogenetic capability in a bisexual population is likely due to that a high percentage of their sexual offspring can still perform parthenogenetic reproduction. The mechanisms of restoring diploidy after meiosis were determined by two molecular markers on the 3rd chromosome. One of them is near the centromere, the other one near the tip and the distance between them is over 50 centiMorgans. With these two markers three types of mechanisms including central fusion, terminal fusion and gametic duplication can be distinguished and percentages can be estimated. Our results revealed that the diploid of parthenogenetic offspring was formed mainly (ca. 92%) by gametic duplication with a small portion by terminal fusion.


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197 Association between Color and Behavior in a Natural Population of Drosophila melanogaster Aya Takahashi1,2, Toshiyuki Takano-Shimizu1,2,3 1) Department of Population Genetics, National Institute of Genetics, Japan 2) Department of Genetics, Graduate University for Advanced Studies (Sokendai), Japan 3) Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Japan

Natural variations of ecological traits within a species have a potential to cause reproductive isolation as a byproduct. Variation in pigmentation intensity in adult Drosophila melanogaster is one of these traits that are likely to be involved in thermal regulation, and is also reported to be associated with non-random mating. The molecular basis of this variation has been attributed to the gene ebony, which codes for an enzyme in the melanin synthesis pathway. The lower expression of this gene in developing epidermis results in darker pigmentation in thoracic and abdominal segments. The changes in cis-regulatory region of the gene are shown to be responsible for the expression level variation in epidermis. This enzyme is also expressed in the nervous system such as in glia cells of brain and functions in the neurotransmitter metabolism pathway.

We quantified the expression levels of this gene in alleles sampled from a natural population. Interestingly, a negative correlation was observed between the allelic expression level in brain and that in developing epidermis of the body. This result suggests that effects of the cis-regulatory mutations of this gene on its transcription in these tissues are not independent. We also found that knocking-down this gene specifically in glia cells changes the circadian activity pattern and affects the timing of mating. Thus, pigmentation and behavior may have had close association during the course of evolution through non-independent transcriptional control of this gene between epidermis and brain.


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198 Differential Gene Expression at the Early Stage of Neo-Sex Chromosome Evolution in Drosophila albomicans Ching-Ho Chang1, Shu Fang2, Chau-Ti Ting1,3, Hwei-yu Chang2,4 1) Institute of Ecology and Evolutionary Biology, National Taiwan University, Taiwan, ROC 2) Biodiversity Research Center, Academia Sinica, Taiwan, ROC 3) Department of Life Science & Institute of Zoology, National Taiwan University, Taiwan, ROC 4) Department of Entomology, National Taiwan University, Taiwan, ROC

In the XY sex determination system, X chromosome remains gene-rich while Y chromosome has lost most but sex determination factors. The degeneration of Y-linked genes is caused by accumulation of deleterious mutations as a consequence of recombination inhibition between sex chromosomes. However, very little is known at the early stage of sex chromosome evolution. To address this question, we compared sequence divergence and expression differences between neo-X and neo-Y chromosomes of Drosophila albomicans in which a pair of autosomal arms fused to X and Y chromosomes only 60 thousand years ago. Among 2587 neo-sex linked transcripts collected by mRNA-seq, 12.8% showed reduced gene expression in neo-Y linked alleles whereas only 3.0% in neo-X linked alleles. In contrast, both neo-X and neo-Y alleles remain under strong evolutionary constraint at the amino acid substitution level. These results suggest that reduced expression could evolve prior to the coding sequence degeneration on the neo-Y chromosome in D. albomicans. It implies that changes at the expression level of Y-linked alleles are the first step of the neo-sex chromosome evolution.


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199 Genome Features of a Drosophila Strain Adapted to Dark Environment Minako Izutsu1, Osamu Nishimura1, Yuzo Sugiyama1, Kiyokazu Agata1,2, Naoyuki Fuse1

1) Laboratory for Biodiversity, Global COE Program, 2Laboratory for Molecular Developmental Biology, Kyoto University, Japan

The Laboratory of Ethology, Kyoto University has maintained a Drosophila melanogaster strain in a constant dark condition for 56 years, 1400 generations. We designate this fly strain as " Dark-fly " and utilize it to investigate molecular mechanisms underlying environmental adaptation. We initially examined the heritability of Dark-fly in dark and light conditions. Under mating competition with the wild type strains, Dark-fly exhibited about 5% higher heritability in a dark condition, indicating that Dark-fly is adaptive in dark conditions. To address the molecular nature of the adaptation, we performed whole genome sequencing for Dark-fly and identified 240,000 SNPs compared to the sequence of Oregon-RS strain, a control strain. The majority of SNPs (90.9%) were located in the non-coding regions and the remaining 9.1% were mapped into the gene coding regions. Among them, 2.0% were classified as non-synonymous SNPs which alter the amino acid sequence of gene products. Using a statistical analysis tool, the DAVID software, we identified 21 Gene Ontology families, in which the Dark-fly non-synonymous SNPs are preferentially concentrated. Interestingly, 3 out of 21 families consist of glycosyltransferases which catalyze adding a glycosyl group moiety to diverse substrates, such as xenobiotics. From further analysis of the Dark-fly genome, we detected 40 genes carrying nonsense mutations which gain a stop codon within the protein sequence. These include genes encoding olfactory receptors, an enzyme involved in eye pigmentation and a light-receptor. Although we need further experiments to clarify the biological meanings of these mutations, our results revealed unique features of Dark-fly genome.


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200 Differential Gene Expression between Two Behavioral Races of Drosophila melanogaster Wei-Chin Ho1, Shun-Chern Tsaur2, Wan-Ju Shen3, Chau-Ti Ting1, 4, Shu Fang3 1) Department of Life Science, National Taiwan University, Taiwan, R.O.C. 2) Department of Life Sciences & Institute of Genome Sciences, National Yang-Ming University, Taiwan, R.O.C.

3) Biodiversity Research Center, Academia Sinica, Taiwan, R.O.C. 4) Institute of Zoology & Institute of Ecology and Evolutionary Biology, National Taiwan University, Taiwan, R.O.C.

Two behavioral isolated races of Drosophila melanogaster, Z and M, have been

demonstrated to be a good model to study the genetic basis of racial differentiation. A series of genetic analyses showed that the behavioral isolation is mainly contributed by several fragments on two major autosomes. However, so far only desat2 has been identified to be involved in this racial differentiation. Change in desat2 expression results in pheromonal difference between the two races, and loss of desat2 expression in the M race might be adaptive when D. melanogaster migrated to temperate zones. To systematically identify more differentially expressed genes (DEGs), we performed RNA-seq to examine the expression differences between the Z (from Zimbabwe and Malawi) and M (from France, USA and Malawi) races. Among the 12775 genes expressed in female heads, 63 show significantly higher expression levels in the Z race, and 75 have higher expression levels in the M race. These DEGs are disproportionally distributed on three major chromosomes (12%, 49% and 39% for the X, second and third chromosomes, respectively). Interestingly, several genes upregulated in the Z race are related to different sensory functions such as light and pheromone detection, whereas genes upregulated in the M race are enriched in lipid metabolic processes. These results not only provide the genetic basis of behavior isolation but also suggest the driving forces underlying racial differentiation.


245

201 Deficiency Mapping of the Genomic Regions Associated with Effects on Developmental Stability in Drosophila melanogaster Kazuo H. Takahashi1, Yasukazu Okada2, Kouhei Teramura2, Masahiro Tsujino1 1) Research Core for Interdisciplinary Sciences, Okayama University 2) Graduate School of Environmental Science, Okayama University

Developmental stability is the tendency of morphological traits to resist the effects of developmental noise, and is commonly evaluated by examining fluctuating asymmetry (FA)—random deviations from perfect bilateral symmetry. Molecular mechanisms that control FA have been a long-standing topic of debate in the field of evolutionary biology and quantitative genetics. In this study, we mapped genomic regions that are known to have effects on the mean and FA of morphological traits, and characterized the sex and trait specificity of those regions. A collection of isogenic deficiency strains established by the DrosDel project was used for deficiency mapping of genome regions responsible for FA. We screened 435 genome deficiencies or approximately 64.9% of the entire genome of Drosophila melanogaster to map the region that demonstrated a significant effect on FA of morphological traits. We found that 406 deficiencies significantly affected the mean of morphological traits, and 92 deficiencies increased FA. These results suggest that several genomic regions have the potential to affect developmental stability. They also suggest the possibility of the existence of trait-specific and trait-non-specific mechanisms for stabilizing developmental processes. The new findings in this study could provide insight into the understanding of the genetic architecture underlying developmental stability.


246

202 Biogeography of Drosophila in East and Southeast Asia Hsiao-Ting Huang, Fu-Guo Robert Liu

Department of Life Sciences, National Central University, Taiwan

This project used nuclear and mitochondrial genes to study the biogeography of fruit flies

from five species, Drosophila albomicans, formosana, immigrans, melanogaster, and simulans, in East and Southeast Asia in order to understand an evolutionary scenario causing enrichment of endemic species in these areas. Based on genetic and phylogenetic analyses suggest each of the five studied species experienced very different evolutionary patterns in the East and Southeast Asia. For instance, D. albomicans may derive from multiple original since their last common ancestors. D. formosana may form different species between Southeast Asia and Taiwan population. D. immigrans may also under process of endemic speciation in Taiwan. D. melanogaster stays a giant population in East and Southeast Asia and nearby islets. About D. simulans, it still remains mystery in this area, due to lack of samples. Thus, due to ecological or geographic unique, Taiwan (Formosa) may play a great potential role on the evolutionary stage. Therefore, for flying-insects, a whole island conservation scale may need to be considered.

POSTER: OTHERS

247

203 Ehime-Fly Provides Reliable and Repeatable Stocks for Evolutionary Studies of Asia-Pacific Drosophila Researchers Masayoshi Watada Department of Biology, Faculty of Science, Ehime University, Japan

During the first five years of the National Bio-Resource Project (NBRP), Ehime-fly collected, kept and provided 549 wild-type strains of 57 Japanese species of Drosophila. These species consists of four genera (Chymomyza, Scaptodrosophila, Colocasiomyia and Drosophila) and three subgenera (Dorsilopha, Sophophora and Drosophila) of the genus Drosophila. All strains of the stocks are available for foreign Drosophila researchers as well as Japanese. Some Drosophila species other than D. melanogaster have specific characters such as diapause, parthenogenesis and hybrid fertility between species etc., not found in D. melanogaster. Comparative genomic studies seem to become more important and interesting to both melanogaster and non-melanogaster researchers.

For the next five years (2007-2011), Ehime-fly will collect, keep and provide over 1,000 strains of 100 species of Drosophila, consisting of not only Japanese Drosophila but also non-Japanese species (mostly Asian species, including African and American species). Some new genera and species groups will be collected and supplied to Drosophila researchers for the study of evolution. For the reliable use by Drosophila researchers, Ehime-fly checks basic strains (one strain for each species) by DNA sequences of mitochondrial DNA and male external reproductive organs in order to conform species identification. Ehime-fly continues to provide a reliable and repeatable resource and expand our services for the Asian-Pacific fly community.

POSTER: OTHERS

248

204 Binding Specificity of Two Odorant-binding Proteins, OBP57d and OBP57e Eriko Harada1,2, Toshiro Aigaki1, Takashi Matsuo3

1) Department of Biological Sciences, Tokyo Metropolitan University, Japan 2) JSPS research fellow 3) Laboratory of Applied Entomology, University of Tokyo, Japan

Odorant-binding proteins (OBPs) are extracellular proteins found in insect chemosensilla, where they participate in chemosensation of odors, tastes, and pheromones. OBP genes share several structural characteristics, and form a large gene family in an insect genome. Among more than 50 OBP genes in Drosophila, Obp57d and Obp57e were shown to be involved in the evolution of host-plant preference of D.sechellia. The two OBP genes arose by tandem duplication of an ancestral OBP gene at the early stage of the melanogaster species group evolution. Because functional constraint on each amino-acid for non-synonymous substitution is different between Obp57d and Obp57e, the two OBPs are suggested to have functionally differentiated from each other. Behavioral analyses of the D. melanogaster knockout mutants showed that both Obp57d and Obp57e participate in taste perception of fatty acids. Nevertheless, there was no qualitative difference between the Obp57dKO and Obp57eKO flies in the behavioral response as far as to the tested fatty acids, which are limited to a narrow range regarding their chain length (C6 - C9) because of the methodological difficulties. Here, to examine the function of these OBPs more directly, ligand specificity was examined by in vitro binding assay using recombinant OBPs. Wide range of compounds was screened for their ability of binding to OBP57d and OBP57e. According to these results, the evolutionary history of Obp57d and Obp57e was reconstructed at their molecular-function level.

POSTER: OTHERS

249

205 Unphosphorylated STAT and Heterochromatin Protect Genome Stability Shian-Jang Yan1, Su Jun Lim1, Song Shi1, Pranabananda Dutta1, Willis X. Li1,2

1) Department of Biomedical Genetics, University of Rochester Medical Center, USA 2) Department of Medicine, University of California, San Diego, USA Heterochromatin is a form of highly compacted chromatin associated with epigenetic gene silencing and chromosome organization. We have previously shown that unphosphorylated nuclear signal transducer and activator of transcription (STAT) physically interacts with heterochromatin protein 1 (HP1) to promote heterochromatin stability. To understand whether STAT and heterochromatin are important for maintenance of genome stability, we genetically manipulated the levels of unphosphorylated STAT and HP1 [encoded by Su(var)205] in Drosophila and examined the effects on chromosomal morphology and resistance to DNA damage under conditions of genotoxic stress. Here we show that, compared with wild-type controls, Drosophila mutants with reduced levels of unphosphorylated STAT or heterochromatin are more sensitive to radiation-induced cell cycle arrest, have higher levels of spontaneous and radiation-induced DNA damage, and exhibit defects in chromosomal compaction and segregation during mitosis. Conversely, animals with increased levels of heterochromatin exhibit less DNA damage and increased survival rate after irradiation. These results suggest that maintaining genome stability by heterochromatin formation and correct chromosomal packaging is essential for normal cellular functions and for survival of animals under genotoxic stress.

POSTER: OTHERS

250

206 The Effect of Heat Shock Protein 27 and Autophagy 7 on the Regulation of the Eye Morphology, Starvation, and Lifespan in Drosophila

Shih-Fen Chen1§, Ming-Lun Kang1§, Chun-Pu Lin2, Horng-Dar Wang1, 2

1) Institute of Biotechnology, National Tsing Hua University 2) Department of Life Science, National Tsing Hua University §: Equal contribution.

Aging is a process due to the imbalanced homeostasis with accumulated damages to macromolecules under external environmental stresses. Heat shock proteins (Hsps) refold denatured proteins or facilitate the degradation of protein aggregates upon stress (Calderwood, Murshid et al. 2009). In addition, autophagy also removes damaged protein to maintain cellular homoeostasis, and loss of autophagy results in cumulated cellular damage. Our previous study indicate that over-expression of hsp27 improves starvation resistance, partially rescues hid-induced apoptosis, and extends lifespan in Drosophila (Liao, Lin et al. 2008). However, the interaction of hsp27 with autophagy remains unknown. Here, we demonstrate that the decreased hsp27 expression in fly eyes results in the rough eye phenotype, and it can be rescued by the co-expression of autophagy gene 7 (atg7). However, the rough eye phenotype derived from atg7 knockdown cannot be rescued by hsp27 co-expression. To investigate the possible connection of hsp27 and atg7 to apoptosis, we co-express the different combination of the over-expression and knockdown of hsp27 and atg7 with apoptosis genes in fly eyes. Like hsp27, the expression of atg7 improves the small eye phenotype by the dominant-negative rasN17expression. However, atg7 has no effect on the improvement of the hid-induced small eye like hsp27 does. Similar to hsp27, elevated atg7 level exhibits starvation resistance and lifespan in fly. Our study provides the first evidence that over-expression of atg7 prolongs lifespan and atg7 functions in the downstream of hsp27 in Drosophila.

POSTER: OTHERS

251

207 Control of Transit-Amplifying Divisions and Organ Size by DSmurf in Drosophila Male Germline Development Pao-Ju Chang1, Chang-Che Hsieh1, Margaret T. Fuller2, Haiwei Pi1 1) Department of Biomedical Sciences, ChangGung University, TaoYuan, Taiwan 2) Department of Developmental Biology, Stanford University School of Medicine, Beckman Center B300, 279 Campus Drive, Stanford, CA 94305-5329, USA. In germline stem cell (GSC) lineages, the spermatogonia undergo four rounds of transit-amplifying (TA) division before differentiation. We find that DSmurf, encoding a HECT domain ubiquitin E3 ligase, is required in germ cells to restrict the TA division. 24% of the spermatocyte cysts have 32 cells in dsmurf mutant. Furthermore, dsmurf activity is required to control the size of testis; testis size is increased 1.8 fold in smurf mutants. Studies in both fly and mammalian cells have shown that DSmurf acts as negative regulator of TGF-β signaling that mediates degradation of TGF-β type I receptor and R-SMAD. It is known that pMad (phosphorylating R-SMAD) is detected only in GSCs. We find that pMad levels are not only increased but also expended to 2-cell cysts in dsmurf mutant. The genetics analysis also shows that reduction of mad activity suppresses dsmurf mutant phenotype. Thus, our results suggest that DSmurf restricts TA division, at least in part, through inhibiting pMad levels in the early spermatogonia cells. In-situ hybridization experiment and analysis of dsmurf-GFP reporter expression show that dsmurf expression reaches the highest level in cells entering spermatocyte differentiation program. dsmurf expression in differentiating spermatocyte absolutely depends on Bam, the key regulator mediating the transition from TA proliferation to spermatocyte differentiation. In future, we will study the function of DSmurf in spermatogonia to spermatocyte transition.

POSTER: OTHERS

252

208 Projectome Analysis of the Lineage-Dependent Neural Circuits in the Drosophila Brain Masayoshi Ito, Keita Endo, Kei Ito IMCB, The University of Tokyo

To analyze the complex information processing system of the brain, it is important to look at the whole neural network rather than focusing on just a few parts of seemingly interesting neurons. Towards understanding the entire neural projections (so called projectome) in the Drosophila brain in a systematic manner, we investigated the projection patterns of each clonal unit, which is a characteristic set of neural circuits derived from a single neuroblast. We visualized the structure of each clonal unit using the MARCM technique, and recorded the projection patterns and the distributions of presynapses of the clonal units on a database. By comparing different samples by registration to the standard brain, we have identified 94 clonal units out of the expected ca. 100 units. First, we checked the relationship between cell body positions and innervation areas. We found that clonal units with a same cell body site project to various brain regions and oppositely, clonal units with various cell body sites project to the same brain region. Next, we investigated how clonal units overlap to each other. We found that the specific brain regions receive projection from a lot of clonal units and in these regions, clonal units partly overlapped to each other. Most part of brain regions received projections from both sides of clonal units and these projection areas were roughly overlapped. The linage-based projectome analysis would thus provide important points of views of the constructions principles of the brain.

POSTER: OTHERS

253

209 Nak Regulates the Localization of Specialized Clathrin Sites in Higher-Order Dendrites to Promote Local Dendrite Growth Wei-Kang Yang1, 2, Yu-Hui Peng1, Hsun Li1, Hsien Suo1, Cheng-Ting Chien1,2*

1) Institute of Molecular Biology, Academia Sinica, Taipei, 115, Taiwan 2) Institute of Molecular Medicine, National Taiwan University, Taipei 106, Taiwan

During development, dendrites arborize in a field several hundred folds of their soma size, a process regulated by intrinsic transcription programs and cell adhesion molecule (CAM)-mediated dendrodendritic interaction. However, underlying cellular machineries that govern distal higher-order dendrite extension remain largely unknown. Here, we show that Nak, a clathrin adaptor-associated kinase, promotes higher-order dendrite growth through endocytosis. In nak mutants, the number and length of higher-order dendrites are reduced, which are phenocopied by compromising clathrin-mediated internalization. Nak genetically interacts with genes involved in endocytosis, colocalizes with clathrin puncta and is required for localization of clathrin puncta in distal dendrites. Furthermore, these clathrin structures preferentially localize to branching points and dendritic tips that are undergoing active growth. We present evidence to show that the Drosophila L1-CAM homolog Neuroglian is a relevant cargo of Nak-dependent internalization, suggesting that localized clathrin-mediated endocytosis of CAMs facilitates the extension of nearby higher-order dendrites.

SPEAKER AND AUTHOR INDEX

254

Name Abstract Number

Adachi-Yamada, Takashi T20, 22, 23, 24, 32

Agata, Kiyokazu 50, 199

Agrawal, Pavan 120

Ahn, Hye-mi 125

Ahn, Mi-Young 108

Aigaki, Toshiro 116, 118, 204

Akagawa, Hiromi 29

Akashi, Hiroshi P13

Akimoto, Saori 77

Amikura, Reiko 114

Amita, Sehgal P7

Ando, Mai 42

Ando, Tetsuya 42

Arama, Eli 30

Arata, Masaki 147

Asada, Nobuhiko 113

Asaoka, Miho 11

Aso, Yoshinori 60

Aughey, Gabriel T11

Awano, Wakae 110

Awasaki, Takeshi 99

Ayukawa, Rie 29

Ayukawa, Tomonori 191

Azzam, Ghows T11, 155

Bai, Jianwu T18, 172

Banerjee, Utpal P2

Bao, Hong 79

Baumgardt, Magnus 19

Belenkaya, Tanya T21

Berger-Mueller, Sandra T6

Bergmann, Andreas T2

Berman, Sara 84

Betsumiya, Aya T3

Bhatia, Shipra 135

Bi, Caili 159

Bittig, Thomas T17, 139

Bivik, Caroline 19

Bohm, Rudolf 79

Boo, George 41

Bossuyt, Wouter 1

Botella, Jose A. 66

Boulianne, Gabrielle L. 80

Bousquet, Francois 36

Boutros, Michael 166

Brahmachari, Vani 135

Bray, Sarah J. 105

Brill, Julie 161

Brown, Joel 26

Buszczak, Michael 193

Carlson, John 57

Chan, Chih-Chiang 193

Chan, Edwin 82

Chan, Priscilla 82

Chandrashekaran, Shanti 135

Chang, Che-Wei 176

Chang, Chia-Chen 196

Chang, Ching-Ho 198

Chang, Ching-Jin 75

Chang, Chun-Che 158

Chang, Henry 161

Chang, Hsiu-Ming 138

Chang, Hwei-yu 196, 198

Chang, Kai, Chen T1

Chang, Wei-Ling 176

Chang, Yu-Chiuan T18, 172

Chang, Yuh-Long 137

Chang, Yung-Heng 181

Chang, Yu-Yun 168, 176

Chaudhary, Varun 166

Chen, Chiao-lin 1

Chen, Chien-Kuo 96

Chen, Chung-Hao 176

Chen, Chun-Hong 93

Chen, Dong-Yuan 174

Chen, Guang-Chao 174, 179, 180

Chen, Hanqing 20

Chen, Hsi-Ju 6

Chen, Jiong 169, 170, 171

Chen, Kangni T11

Chen, Linyi 52

Chen, Nan-Yow 51

Chen, Ruey-Hwa 86

Chen, Wei-Yu 96


255

Chen, Yi-chun T8,39, 117

Chen, Yu-Ju 128

Cheng, Tat-Cheung 82

Cheng, Ying-Ju 86

Cherry, Smita 193

Chiang, Ann-Shyn P3, 44, 46, 51, 52, 53, 64, 85, 138

Chiang, Wei 182,185

Chien, Cheng-Ting 86, 88, 96

Chien, Ju-Yi 75

Chihara, Takahiro 83, 90, 98

Chin, An-Lun 53

Chklovskii, Dmitri 54

Cho, Kyung-Ok 16, 106

Choe, Joonho P5

Choi, Kwang-Wook 63, 73, 74

Chou, He-Yen 182

Chou, Hsiao-His 13

Chou, Tze-Bin 176

Christiansen, Audrey T2

Chu, Chen-Wei 44, 64

Chu, Kuang-His T26

Chu, Li-An 44, 46, 51, 64

Chu, Wei-Chen 154

Chuang, Chao-Chun 138

Chueh, Tung-Hung 51

Chung, Hui-Min 26

Chung, Jongkyeong 115

Clark, Ivan 186

Cliffe, Adam 162

Cooper, Emilie 26

Cui, He 80

Deivasigamani, Senthilkumar 68

Delandre, Caroline 77

Desplan, Claude P4, 122

Deussen, Zillah T11

Dickson, Barry J. 64

Ding, Tian T2

Doupe, David 162

Drummond-Barbosa, Daniela 2

Ejima, Aki 59

Emoto, Kazuo 102

Endo, Keita 105

Epstein, Daniel T7

Fabian, Lacramioara 161

Fan, Yun T2

Fang, Ming 134, 159

Fang, Shu T26, 198, 200

Farhadifar, Reza T17, 139

Feinstein-Rotkopf, Yael 30

Ferveur, Jean-Francois 36

Fore, Taylor 79

Fortini, Mark E. 167

Fu, Chien-Chung 46

Fu, Shengbo 121, 142

Fukawa, Naoya 12

Fukumoto, Shinya 109

Furlong, Eileen P10

Furukubo-Tokunaga, Katsuo 42

Fuse, Naoyuki 50, 199

Gajewski, Kathleen 1

Gan, Guangming 80

Gao, Fuying 77

Gao, Guanjun 151

Gao, Yu 144

Garcia-Alvarez, Gisela T1

Gautam, Naveen 177

Geng, Qing 153

Gerber, Bertram 39

Geshwind, Daniel 77

Ghasemi, Mohsen 135

Giangrande, Angela P6

Gibbs, Allen 112

Gibbs-Bar, Liron 30

Giri, Ritika 123

Goda, Nobuhito 12, 40

Goh, Lihui 3, 14, 67

Gojobori, Takashi 127

Gong, Zhefeng 47, 49, 62

Goo, Tae-Won 108

Goodnight, Lindsey 79

Goto, Satoshi 110

Gross, Julia 166

Gui, Hongxing T13

Guo, Aike 37


256

Guo, Chao 62

Guo, Dongdong 133

Haberman, Adam T7

Habib, Farhat 120

Haenfler, Jill T4

Hakeda-Suzuki, Satoko T6, 95

Halder, Georg 1

Hamaratoglu, Fisun 1

Handu, Mithila 123

Hanyu-Nakamura, Kazuko T12

Hara, Yusuke 29

Harada, Eriko 204

Harumoto, Toshiyuki P12, 147

Harvey, Kieran 15

Hasegawa, Eri 99

Hashimoto, Yoshiko 124

Hatakeyama, Masatsugu T16

Hatanaka, Ryo 34, 187

Hatori, Ryo T15, 146

Hattori, Yukako 103

Hayashi, Shigeo 152, 157

He, Bin T25

Heck, Bryan T13

Hellbach, Annette 163

Hiesinger, Peter Robin T7, 193

Hime, Gary 7

Hirai, Keiichiro 29

Hiromi, Yasushi 11, 127

Hirose, Susumu 127

Ho, Wei-Chin T26, 200

Hong, An-Kuo 46

Honjo, Ken 42

Horiuchi, Shin-ya T6, 89, 150

Houot, Benjamin 36

Hsiang-Wen, Shih 85

Hsiao, Hui-Yi 122

Hsiao, Yun-Ling 128, 129

Hsieh, Chang-Huain 138

Hsu, Hwei-Jan 2, 156

Hsu, Tun-Chieh 55

Huang, Hsiao-Ting 202

Huang, Jiuhong 160

Huang, Ming-Hao T8,117

Huang, Shu-Yu 189

Huang, Szu-Jing 173

Huang, Yaling 93

Huang, Yi-Chun 88

Huang, Yu Fen 94

Huang, Zhenxing 14

Hwang, Jae-Sam 108

Ichijo, Hidenori 34, 187

Igaki, Tatsushi T3, 10

Ikeo, Kazuho 127

Imano, Takao 23

Inaki, Mikiko 190

Ip, Tony P8

Ishihara, Shuji 140

Ishio, Akira 146

Ishitsuka, Ayano 29

Ito, Kei 105

Itoh, Takehiko 103

Iwabuchi, Kikuo 29

Iwamura, Masashi 29

Izutsu, Minako 50, 199

Jain, Shruti 135

Jang, Anna C.-C. T18, 172

Jeon, Sang-Hak 69, 78, 141

Jiang, Shih-Sheng T10

Jiao, Renjie 20, 119

Juang, Jyh-Lyh T10

Kageyama, Yuji 124

Kai, Toshie P9, T14

Kalifa, Yossi 30

Kanai, Mai 12

Kanai, Maiko 167

Kanda, Hiroshi 97

Kang, Di 170

Kang, Jongkyun 63

Kanuka, Hirotaka 109

Kao, Chih-Fei 87, 93

Kao, Hsiu-Hua 86

Kao, Jui-Chun 87

Kaplan, Yosef 30

Karim, MD Rezaul 76, 105


257

Karlsson, Daniel 19

Keene, Alex C. 61

Khoo, Kay-Hooi 174

Kim, Go-Woon 106

Kim, Jaeseob 63

Kim, Sang Hee 78

Kimura, Ken-ichi 38

Kinameri, Emi 105

Kirov, Nikolai 121

Kirsanov, Kirill 28

Kishita, Yoshihito 118

Kitada, Yusuke 99

Klingseisen, Anna 186

Knight, David 80

Knoblich, Juergen A 191

Kobayashi, Tetsuya P12

Kokuryo, Akihiko 23, 32

Komori, Hideyuki 8

Kondo, Shu 188

Kondo, Takefumi 124, 157

Konrad, Basler P11

Kraut, Rachel 41, 71

Krejci, Alena 105

Ku, Hsueh-Yen 174

Kumar, Amit 143

Kunnapuu, Jaana T16

Kwon, Jae Young 72

Lai, Chun-ming 156

Lai, Jason Sih-Yu 44, 52, 64

Landsberg, Katharina T17, 139

Lau, Kwok-Fai 82

Lee, Cheng-Yu T4, 8

Lee, Dong-Seok 125

Lee, Gina 115

Lee, Hong 81

Lee, Kyu-Sun 125

Lee, Ok-Kyung 106

Lee, Ting-Kuo 51

Lee, Tzumin T5, 87, 93

Lee, Yen-Hsien 182

Lee, Young Eun 56

Lee, Yuan-Ming 45, 154

Leptin, Maria 186

Lesovaya, Ekaterina 28

Li, Changqing 20

Li, Hoi-Yeung 82

Li, Hsun 96

Li, Meng-Yen 174

Li, Wei-Hao 182

Liang, Hsiao-Lan 121, 142

Liaw, Gwo-Jen 13, 189

Lim, Su Jun 133,205

Lin, Gee-Way 158

Lin, Guonan 17,

Lin, Ming-Der 158, 173

Lin, Nianwei 31

Lin, Shuping 3, 14

Lin, Shu-Yu 180

Lin, Wei-Hsiang 96

Lin, Xinhua T21, 145

Lin, Yen-Yin 46

Lin, Yu-Hung 182

Lin, Yu-Jing 86

Liu, Chang 60

Liu, Fu-Guo 202

Liu, Jiangqu 48, 62

Liu, Ji-Long T11, 155

Liu, Jiyong 119

Liu, Li 47, 49, 62

Liu, Pei-Ling 51

Liu, Qing-Xin 127

Liu, Tsung-Ho 46

Liu, Wei 175

Long, Manyuan P14

Lou, Shyn-Jie 64

Lu, Zhiyuan 54

Lundin, Erika 19

Luo, Liqun 83, 98

Luu, Si-Hong 95

Ma, Wencui 134

MacDonald, Ryan 19

Maeda, Maki 50

Maeda, Reo T15, 146, 194

Manak, John 121, 142


258

Manhire-Heath, Rosemary 184

Mann, Klaudiusz T6, 95

Matise, Michael T13

Matsubara, Daisuke 89

Matsuda, Kazuki T12

Matsuda, Rie 38

Matsuda, Shinya T16

Matsuno, Kenji T15, 146, 167, 194

Matsuo, Takashi 204

Matsuoka, Shinya 11

Matsuzaki, Fumio 21

Meinertzhagen, Ian T7, 54

Meng, Fei 159

Meng, Tzu-Ching 174, 179

Milton, Claire 15

Minami, Ryunosuke 23, 32

Misaki, Kazuyo 152

Mishra, Dushyant 39

Mita, Kotomi 113

Miura, Masayuki 83, 90, 98

Miyawaki, Atsushi 10

Mochizuki, Megumi 59

Montell, Denise T18, 172

Moore, Adrian 76, 77, 105

Mouri, Kousuke 147, 150

Muha, Villoe 186

Mukhopadhyay, Bibhash 74

Muller, Arno 186

Murakami, Ryutaro 132, 148

Murray, Michael 184

Murugaiyah, Vikneswaran 65

Nakagoshi, Hideki 23, 32

Nakamura, Akira T12, 185

Nakamura, Mai T3

Nakamura, Mitsutoshi 146

Nakao, Keiko 25

Nakazawa, Naotaka T15, 146, 194

Natori, Kouhei 149

Navarro, Juan A. 66

Nern, Aljoscha 54, 195

Neufeld, Thomas 168

Newfeld, Stuart T16

Nien, Chung-Yi 121, 142

Niimi, Kaori 124

Nishimura, Osamu 50, 199

Niwa, Ryusuke 111

Nojima, Tetsuya 36

Nomura, Takafumi 91

Novo, Michael 31

Ohler, Stephan 95

Ohsawa, Shizue T3, 10

Ohta, Nao 21

Ojwang, Audrey 79

Okada, Yasukazu 201

Okado, Kiyoshi 109

Okano, Hideyuki 25, 97, 110

Okumura, Misako 90

Okumura, Takashi 22, 24, 146, 194

Orihara-Ono, Minako 25

Osborne, Kathleen 41

Oswald, Matt 71

Ou-Yang, Chih Hsin 13

Pai, Li-Mei 182

Paik, Donggi 56

Pan, Hanshuang 171

Pan, Rong-Long T10

Park, Jeong Ho 72

Park, Joong-Jean 56

Park, Ki Seok 78

Pei, Shanshan 133

Pek, Jun Wei T14

Peng, Qionglin 49

Peng, Xinyun 79

Perrimon, Norbert K2, 82, 188

Pfeiffer, Barret 93, 195

Pi, Haiwei 88, 128, 129

Quinn, Leonie 7

Ranft, Jonas T17, 139

Ratnaparkhi, Anuradha 68

Ratnaparkhi, Girish 68, 123

Reina, Ito 92

Ren, Jing 37

Reynolds, Lauren 112

Rong, Yikang 151


259

Rorth, Pernille 162, 190

Rubin, Gerry 93, 195

Rushlow, Christine 121, 142

Ryu, Kumbok 69

Saint, Robert 184

Saito, Motoki 147

Sakata, Rumi 23, 32

Sakuma, Chisako 98

Sang, Tzu-Kang T8,117

Sano, Hiroko 114

Santhanam, Abirami 179

Sasaki, Takehiko 191

Sasamura, Takeshi 167

Sasikumar, Satish 143

Sato, Chiaki 38

Sato, Eri 113

Sato, Makoto 99

Sato, Yoshitaka T3

Sato, Yukiko 116, 118

Satoh, Daisuke 70, 103

Sawamura, Kyoichi T24

Scheffer, Lou 54

Schmitt, Linda 39

Schmuker, Michael 39

Schneuwly, Stephan 66

Scoggin, Shane 193

Scott, Anthony 133

Seeds, Andy 178

Sekine, Sayaka 83

Sekine, Yusuke 34, 187

Senoo-Matsuda, Nanami 12, 40

Shashidhara, L. S. 120

Shaw, Pang-chui 82

Shen, Wan-Ju T26, 200

Shibano, Takako 110

Shih, Chi-Tin 51

Shih, Hsiang-Wen 85

Shih, Meng-Fu 51, 52

Shim, Hyunpyo 141

Shimada, Yuko P12

Shimada-Niwa, Yuko 111

Shimamura, Rieko 97

Shimmi, Osamu T16

Shimono, Kohei 89, 91

Shinzawa, Naoaki 109

Shirahige, Katsuhiko 103

Shiu, Guan-Lin 182

Sidorov, Roman 28

Siebert, Matthias 105

Sin, Hye-Min 108

Sinha, Ashesha 15

Sinha, Pradip 143

Siwanowicz, Igor 60

Somers, Gregory T1, 7

Son, Wonsuk 16

Song, Yingying 5

Sousa-Nunes, Rita T1

Srivastava, Priya 143

Stopfer, Mark 58

Su, Chih-Ying 57

Su, Ming-Tsan 33, 35, 43, 55, 75, 130, 131

Sudo, Tatsuya 29

Sugie, Atsushi 84

Sugimura, Kaoru 10, 140

Sugiyama, Yuzo 199

Suh, Yoon-Seok 125

Sun, Mingkuan 80

Sun, Y.Henry T22, 45, 81, 94, 154, 181

Sung, Hsin-Ho 176

Sung, Ying Ying 4, 14

Suyama, Ritsuko 70

Suzuki, Emiko 167

Suzuki, Takashi T6, 84, 95

Sweeney, Sean 71

Tabata, Tetsuya 99

Tajiri, Reiko 152

Takahashi, Aya 197

Takahashi, Kazuo 201

Takahashi, Takashi 29

Takano-Shimizu, Toshiyuki 197

Takayama, Kojiro 42

Takayama, Yuta 92

Takeda, Kohsuke 34, 187

Takeda, Koji 22


260

Takemura, Masahiko T20

Takemura, Shinya 54

Takino, Kyoko 10

Tanaka, Daisuke 42

Tanaka, Nobuaki 58

Tang, Chiou-Yang 75

Tang, Hong-Wen 180

Tang, Xiaofang 145

Taniguchi, Hiroaki 105

Taniguchi, Kiichiro T15, 22, 23, 32, 146, 194

Tanimoto, Hiromu 60

Tao, Chunyao 1

Tapadia, Madhu G. 177

Teng, Yan 62

Teo, Lin Shin 71

Teramura, Kouhei, 201

Tetsuya, Kojima, 149

Thompson, Joy 184

Thor, Stefan 19

Tin, Man-Ying+A654 178

Ting, Chau-Ti T26, 198, 200

Toba, Gakuta 101

Togane, Yu 29

Tong, Xin T13

Toriya, Masako 25

True, John T23

Tsai, Chih-Cheng T13

Tsai, Pei-I 86

Tsai, Shih-Ying 136

Tsaur, Shun-Chern T26, 200

Tsogtbaatar, Orkhon 16

Tsoi, Ho 82

Tsuda, Kayoko 114

Tsuda, Manabu 118

Tsui, Man Kin 178

Tsujimoto, Keita 50

Tsujimura, Hidenobu 29

Tsujino, Masahiro 201

Tsutsumi, Kanako 40

Tu, Pang-Hsien 75

Turner, Glenn C 52

Uddin, Sarder 132

Ueda, Hiroki P12

Ueda, Ryu 68, 192

Uemura, Tadashi P12, T6, 70, 89, 91, 92, 103, 147, 150

Ulvklo, Carina 19

Umemori, Makoto 114

Umetsu, Daiki T17, 139

Urushizaki, Akira 38

Usui, Tadao T6, 89, 91, 103

Vavilala, Pratyusha 135

Verma, Hemant 68

Verma, Puja 107

Vishnu, Smitha 190

Vitaladevuni, Shiv 54

Vuong, Linh, Thuong 74

Wang, Cheng Wei T22

Wang, Chen-Hui 17

Wang, Ching-Tzu T8,117

Wang, Dong 193

Wang, Hongyan T1

Wang, Horng-Dar T8,117

Wang, Hsiang-Yu 43

Wang, Hui T13

Wang, Lan-Hsin 9

Wang, Manyu 86

Wang, Menghze 95

Wang, Pei-Yu 182

Wang, Shiu-Lan 180

Wang, Szu-Chieh 158

Wang, Xing-Jun 27

Wang, Yi-Yun T8,117

Wang, Yu-Bao 180

Wang, Yu-Chiun T19

Wang, Yu-Chun 35

Wang, Zhaohui 18, 144, 153

Wang, Zhi Qiang 27

Warner, Margaret 79

Watada, Masayoshi 192, 203

Watanabe, Takeshi 34

Watanabe, Yuzo 147

Welch, William 79

Wen, Ying-Hao 182

Weng, Mo T4


261

Wesolowska, Natalia 151

Widmann, Thomas T17, 139

Wieschaus, Eric T19

Williamson, W. Ryan T7

Wilson, Harry 75

With, Sheila Irene 80

Wohland, Thorsten 71

Won, Jong-Hoon 16

Wong, Eric 82

Wu, Chan 18

Wu, Chia-Lin 52

Wu, Di 4

Wu, Jheng-Jian 51

Wu, Jing 169

Wu, June-Tai 88

Wu, Mei-Hsuan 180

Wu, Ming-chin 46

Wu, Ronald 82

Wu, Shih-Cheng T10

Wu, Shiuan-Tze 44

Wu, Yihui T21, 145

Xi, Rongwen 17

Xie, Wei 80, 134, 159

Xing, Guanglin 80

Xu, Na 17

Xu, Tian P1, 10

Yakubovskaya, Marianna 28

Yamakawa, Tomoko 167

Yamamoto, Daisuke 36, 38, 101

Yamamoto, Masa-Toshi D1,192

Yamamoto-Hino, Miki 110

Yamazaki, Masakazu 191

Yan, Shian-Jang 205

Yang, Fu 17

Yang, Hsun-Ti 52

Yang, Jin 67

Yang, Lin 159

Yang, Sheng-An 33

Yang, Xiaohang 3, 4, 5, 14, 67

Yasunaga, Kei-ichiro 102

Yeh, Chang-Wei 138

Yeh, Po-An 75

Yeh, Shu-Dan T23

Yelagandula, Ramesh 120

Yenisetti, Sarat 66

Yeom, Eunbyul 73

Yonemura, Shigenobu 152

Yoon, Young-Il 108

York, John 178

Yoshiura, Shigeki 21

Yoshiyama, Naotoshi T16

Yu, Hung-Hsiang T5, 87

Yu, Jenn-Yah 6, 189

Yu, Kweon T9, 125

Yu, Sam 93

Yu, Weimiao 162

Yuan, Liudi 80

Yuh-Nung, Jan K1

Yun, Eun-Young P8, 108

Zeng, Shu-Heng 182

Zhang, Bing 79

Zhang, Can 31

Zhang, Chengwan 134

Zhang, Lijun 170

Zhang, Peng T21

Zhang, Yang 144

Zhang, Yi 144

Zhang, Yongqing 104, 175

Zhang, Zhao 165

Zhao, Lu 104

Zhao, Xi 144

Zhou, Lei 31

Zhou, Xin 161

Zhou, Xiu 4, 67

Zhou, Yanqiong 47, 62

Zuo, Juntao 170, 171

net abstract final new - researchmap

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