mechanism of early neural stem cell lineage specification in the mouse epiblast

429ABSTRACTS / Developmental Biology 306 (2007) 427–435

transgenic embryos expressing stabilized β-catenin in retinalprogenitors and showed that proneural gene expression wasnormal but proneural target genes and markers of differen-tiated retinal neurons were lost, suggesting a block inproneural function. Sox2, normally downregulated as progeni-tors differentiate, persisted in the undifferentiated cells,suggesting that Sox2 may prevent retinal neuron differentia-tion. Consistent with this, overexpression of Sox2 mimickedthe effects of Wnt signaling on gene expression, and the cellsdifferentiated as non-neural Müller glia. Inhibition of Notchsignaling reversed the ability of both Wnt/β-catenin and Sox2to suppress neuronal differentiation and proneural target geneexpression. We conclude that Wnt/β-catenin regulates theprocess of retinal progenitor differentiation via Sox2, which isrequired for proneural gene expression but inhibits proneuralfunction through Notch signaling. We propose that differentia-tion does not proceed until Sox2 is downregulated throughfeedback inhibition of Wnt signaling. NIH EY14954 andWellcome Trust.

doi:10.1016/j.ydbio.2007.03.455

Program/Abstract # 386Sbt1 is required downstream of proneural bHLH factorsfor neurogenesis in the developing retinaMonica L. Vetter, M.A. Logan, M.R. Steele, I. Al-Diri,W. Chen, C. Dooley, B. MooreDept of Neurobiol & Anat, U of Utah, Salt Lake City, UT, USA

Proneural basic helix–loop helix (bHLH) transcriptionfactors are key regulators of retinal neurogenesis, and theyfunction by activating the expression of target genes thatexecute a program of neuronal differentiation within progeni-tors. In a previous screen for proneural target genes weidentified a novel gene called sbt1 (shared bHLH target 1).The purpose of this study is to investigate the expression andfunction of sbt1 and determine whether it plays a role inregulating retinal neurogenesis. sbt1 is conserved acrossvertebrate species and encodes a novel protein with noconserved functional motifs. In situ hybridization analysisshowed that sbt1 is transiently expressed in late proliferating orearly differentiating cells in both the Xenopus and mouseretina. Epitope-tagged SBT1 protein localizes to both theplasma membrane and the nucleus in Xenopus animal caps,and the N-terminal region is required for membrane localiza-tion. Inhibition of SBT1 translation in Xenopus retinalprogenitors by injection of morpholino into cleavage-stageblastomeres prevented or delayed retinal neuron differentia-tion. Conversely, overexpression of either mouse or Xenopussbt1 by RNA injection promoted differentiation of early bornretinal neurons, and also enhanced the ability of proneuralbHLH factors to promote retinal neurogenesis. We concludethat sbt1 is expressed in retinal progenitors as they initiateneuronal differentiation, and appears to function as aconserved component of the neuronal differentiation program

downstream of proneural bHLH factors during retinaldevelopment.

doi:10.1016/j.ydbio.2007.03.456

Program/Abstract # 387Examining early retinal progenitor multipotency by Mash1misexpression in the Math5-lineageRobert B. Hufnagel, Malgorzata Quinn, Nadean L. BrownDivision of Developmental Biology, Cincinnati Children’sHospital Research Foundation and Departments of Pediatricsand Ophthalmology, University of Cincinnati College ofMedicine, Cincinnati, OH, USA

Proneural basic helix–loop–helix (bHLH) transcriptionfactors influence neuronal determination and fate specificationthroughout the developing nervous system. In the mouse retina,bHLH factors are required for the normal development of theseven major cell types, six neuronal and one glial. Math5 is thefirst proneural bHLH factor expressed in the embryonic retinaand is required for the development of retinal ganglion cells(RGCs), the first retinal neuron specified. Mash1 expressioninitiates three days later and is required for the proper generationof bipolar neurons, specified last. To test the multipotency ofretinal progenitors in the Math5-lineage, we performed a geneswap experiment to generate a Math5Mash1 knock-in allele,wherein the endogenous Math5 coding sequence was replacedwith Mash1 by homologous recombination. As a result, Mash1is precociously expressed in the Math5-lineage in these mice.The Math5Mash1 knock-in allele also expresses an IRES-dsRedreporter to distinguish those progenitors that misexpressMash1. Adult Math5Mash1/Mash1 mice lack RGCs and opticnerves, indicating that Mash1 cannot rescue the Math5phenotype. We have assessed these mice embryonically forfate alterations in the Math5-lineage, namely the precociousdifferentiation of bipolar neurons, and for the relative distribu-tion of the seven retinal neuronal and glial cell types.

doi:10.1016/j.ydbio.2007.03.457

Program/Abstract # 388Mechanism of early neural stem cell lineage specification inthe mouse epiblastLan Dang, Vincent TropepeDept. of Cell & Systems Biol., Univ. of Toronto, Toronto, ON,Canada

Mouse definitive neural stem cell (NSC) lineages are derivedfrom a population of primitive neural stem cells (PrNSC) in theepiblast or from embryonic stem cells (ESC) in vitro, yet detailson the signaling and transcriptional mechanisms that control thislineage transition are lacking. Data from chick and Xenopus invivo experiments suggest that FGF and Wnt signaling play a

430 ABSTRACTS / Developmental Biology 306 (2007) 427–435

major role in neural induction and may mediate the PrNSC toNSC lineage transition. Using mouse ESC, we show that thesesignals do not affect neural induction in vitro and that neuralinduction under these conditions is independent of FGF andWntsignaling. To determine if these pathways control mouse NSCdevelopment in vivo, we expose the 7.0 dpc mouse epiblast toFGF and Wnt signaling using an embryo microinjection andculture assay. We find that early neural gene expression ispromoted by FGF and blocked with exogenous Wnt. Thus thesefactors are key players that modulate mouse neural geneexpression in vivo. To determine whether these factors mediatethe lineage transition, 7.0 dpc mouse embryos will be treatedwith Fgf8b, Wnt3a, or BMP4, cultured and assayed for clonalNSC colony-formation at the headfold stage. Also to decipherthe early transcriptional mechanism that control this lineagetransition, we are testing whether transient overexpression ofdownstream target of FGF signaling are sufficient to enhance thePrNSC to NSC transition in the epiblast in vivo. Altogether theseexperiments will reveal whether these signaling pathways play acritical role in the PrNSC to NSC lineage transition in the mousesimilar to other vertebrates.

doi:10.1016/j.ydbio.2007.03.458

Program/Abstract # 389Temporal patterning determines visceral motoneuronsubtypes generated from Nkx2.2+ progenitors in thehindbrainJoanna M. Applequist 1, Mattias Karlen 1, Elisa Jordi 2,Thomas Perlmann 2, Johan Ericson 1

1 Dept of Cell and Molecular Biology, Karolinska Institute,S-171 77 Stockholm, Sweden2 Ludwig Institute of Cancer Research, Karolinska Institute,S-171 77 Stockholm, Sweden

The establishment of cell diversity in the developing CNS isgoverned by spatial and temporal patterning mechanisms.While insight has been obtained into spatial mechanisms, less isknown about how different neuronal subtypes can be generatedfrom a common population of progenitors over time. We haveexamined the generation of visceral motor neuron (vMN)subtypes in the caudal hindbrain. vMN are generated from acommon Nkx2.2+ progenitor domain, and we find that thesecells acquire at least three distinct subtype identities that projecttheir axons to distinct peripheral targets. We provide evidencethat the identity of vMN subtypes is predicted by the birth-dateof neurons, implying a temporal mechanism in vMN subtypespecification. In vitro assays indicate that this mechanism isinitiated at the neural progenitor cell stage, and that subtypespecific properties are established independent of any contactbetween vMNs and their peripheral target cells. Moreover, wefind that the orphan nuclear receptor Nurr1 is expressed in lateborn vMNs, and that in mice lacking Nurr1 function, late bornvMNs adopt an axonal projection pattern typical of early bornneurons. These data reveal a temporal mechanism in vMN

subtype specification in the hindbrain, and suggest that Nurr1 isinvolved in manifesting the functional properties of late bornvMNs and in suppressing traits of early born cells.

doi:10.1016/j.ydbio.2007.03.459

Program/Abstract # 390The role of NF1 in Schwann Cell development and tumorformation and the influence of steroid hormones andmetabolitesTherese M. Roth 1, Poornapriya Ramamurthy 1, Fumi Ebisu 1,Kate F. Barald 1,2

1 Cell & Dev. Biol., U-M, Ann Arbor, MI, USA2 Dept. of Neuroscience, U-M, Ann Arbor, MI, USA

The Neurofibromatosis Type 1 (NF1) gene functions as atumor suppressor. One known function is to turn off thep21ras pathway by hydrolyzing active rasGTP to inactiverasGDP. Loss of neurofibromin (the protein product of theNF1 gene) in this autosomal dominant disorder is associatedwith tumors of the PNS. The major affected cell type is theSchwann cell (SC). NF1 tumors tend to increase in size andnumber during times of high hormonal activity, includingpregnancy. We have developed an in vitro system fordifferentiating mouse embryonic stem cells that are NF1wild type (+/+), heterozygous (+/−), or null (−/−) into SC-likecells. These SC-like cells, regardless of their NF1 status,express SC markers, support and preferentially direct neuriteoutgrowth, and are capable of expressing myelin protein. NF1null and heterozygous SC-like cells proliferate at anaccelerated rate compared to NF1 wild type; this growthadvantage can be reversed using a MEK inhibitor, whichblocks the ras pathway that is constitutively active in NF1mutant cells. NF1 mutant cells also express higher levels ofsteroid hormone receptors than NF1 WT cells, suggestinggreater hormone responsiveness. A hormone metabolite thathas been found to kill many types of tumor cells is cytotoxicto NF1 malignant tumor cells, while inhibiting proliferation inother cell lines tested. This metabolite or its derivatives couldprovide new avenues for therapy.

doi:10.1016/j.ydbio.2007.03.460

Program/Abstract # 391Differential effects of the TGF-beta superfamily memberson dopaminergic phenotype inductionEleni Roussa, Oliver Oehlke, Belal Rahhal, Michael Wiehle,Kerstin KrieglsteinDept. of Neuroanatomy, Univ Goettingen, Goettingen, Germany

The experimental control of progenitor cells differentiationtowards particular types of neurons, such as dopaminergic neu-rons, is dependent on identification of intrinsic and extrinsic brain