Drosophila has four types of sensory elements: external sense organs, multiple dendritic neurons, chordotonal neurons, and photoreceptors. The latter two do not require the proneural genes of the achaete-scute complex (AS-C) in order to develop and function properly, but they do need atonal, a neurogenic gene that behaves in many ways like the genes of AS-C. atonal gets its name from the disruptive effects the gene's mutation has on chordotonal neuron differentiation. Mutants are completely difficient in this lateral sense organ (Jarman, 1995). Early in embryonic development, atonal is expressed in all chordotonal organ progenitor cells, but its later expression is restricted to only a particular set of precursors, through a process of lateral inhibition. Notch does not seem to be required for this process in chordotonal organs, and the mechanism is not well understood. Mutation in atonal also disrupts eye development (Jarman, 1995). Atonal protein is produced in all cells just anterior to the morphogenic furrow. Expression is downstream of hedgehog, an important gene involved in regulating the progression of the furrow. As the furrow progresses, most cells that express atonal in front of the furrow lose that capacity. atonal expression behind the furrow is confined to the R8 progenitors, whose fate atonal determines. atonal is produced within the context of the furrow, which is only rudimentary in atonal mutants. atonal is a neurogenic gene, functioning in the place of achaete-scute complex bHLH genes, both in the eye and in chordotonal organs. The function of Atonal is best illustrated by its role in chordotonal organ development. A scolopidium, the basic unit of chordotonal organs, consists of four cells: a neuron with a single dendrite, the scolopale cell, cap cell and ligament cell. The scolopale cell (a glial cell) forms a sheath around the dendrite, while the cap cell and ligament cell mediate the attachment of the chordotonal organ to the body cell. Expression of atonal is restricted to a subset of atonal-requiring chordotonal precursors, called founder precursors. In atonal mutants, all chordotonal organs are absent except for one scolopidium of Ich5, the abdominal pentascolopidial organ (Ich5 consists of five scolopidia organized in a linear array). This one scolopidium formed in atonal mutants is atonal independent. The atonal independent precursor corresponds to the earliest chordotonal precursor (precursor C1) and corresponds to the P cell which gives rise to the anterior-most scolopidium of Ich5 (zur Lage, 1997). EGF receptor signaling is required in neural recruitment during formation of Drosophila chordotonal sense organ clusters. A total of five neural precursors express atonal in abdominal segments, and this number is too few to explain the formation of the eight scolopidia in each abdominal segment. Of the five precursors, C-1, C2 and C3 contribute to Ich5, C4 migrates slightly anterodorsally and gives rise to v'ch1, the dorsal most scolopidium, which is solitary. C5 contributes to vchAB, a more ventral pair of scolopidia. The remaining precursors require Egf-R signaling for their selection. Signaling by the founder precursors is initiated by atonal activating (directly or indirectly) rhomboid expression in the founder cells. It should be noted that in some developmental processes, rhomboid appears to function in the signal-receiving cells, such as in the patterning of ovarian follicle cells. It is not believed that this is the case in chordotonal-precursor formation, because rho is expressed in precursors that do not require rhomboid function (C1-C5 are formed even in rhomboid mutants). Signaling by these founder precursors, presumably through the EGF receptor ligand Spitz, then provokes a response in the surrounding ectodermal cells, as shown by the activation of expression of the Egf-R target genes pointed and argos. The signal and response then leads to recruitment of some of the ectodermal cells to the chordotonal precursor cell fate. Egf-R hyperactivation by misexpression of rhomboid results in excessive chordotonal precursor recruitment. Argos functions in a feedback mechanism to prevent the excess recruitment of additional ectodermal cells. The increase in the number of scolopidia caused by Egf-R hyperactivation is confined to an enlargement of existing cluster sizes: no new chordotonal clusters are formed. A two step mechanism is postulated for the formation of clusters of chordotonal precursors. In the first step, precursors C1-C5 are selected as founder precursors by the conventional route of proneural gene expression and lateral inhibition. In a distinct second phase, these precursors then signal to adjacent ectodermal cells via the Egf-r pathway, inducing some of them to become chordotonal precursors (secondary or recruited precursors). This two-step process is strongly reminiscent of the way atonal acts in neurogenesis in compound eyes. Here, atonal expression is initially refined by lateral inhibition, until atonal is expressed in only the founding R8 precursor, which then recruits R1-R7 in a mechanism that does not require the activation of atonal in these cells (zur Lage, 1997). The selection of Drosophila sense organ precursors (SOPs) for sensory bristles is a progressive process: each neural equivalence group is transiently defined by the expression of proneural genes (proneural cluster), and neural fate is refined to single cells by Notch-Delta lateral inhibitory signalling between the cells. Unlike sensory bristles, SOPs of chordotonal (stretch receptor) sense organs are tightly clustered. It has been shown that for one large adult chordotonal SOP array (the adult femoral chordotonal sense organ), clustering results from the progressive accumulation of a large number of SOPs from a persistent proneural cluster. This is achieved by a novel interplay of inductive epidermal growth factor- receptor (EGFR) and competitive Notch signals. EGFR acts in opposition to Notch signaling in two ways: it promotes continuous SOP recruitment despite lateral inhibition, and it attenuates the effect of lateral inhibition on the proneural cluster equivalence group, thus maintaining the persistent proneural cluster. SOP recruitment is reiterative because the inductive signal comes from previously recruited SOPs (zur Lage, 1999). The adult femoral chordotonal sense organ arises from a group of some 70-80 SOPs. A developmental analysis of Ato expression has revealed that these SOPs accumulate over an extended period of time in the dorsal region of each leg imaginal disc during the third larval instar and early pupa. The continued expression of Ato implies a sustained requirement for proneural function throughout the process of SOP accumulation. Unusually, Ato is persistently expressed in a group of ectodermal cells identified as the proneural cluster (PNC). From this PNC, cells are funnelled inward into a cavity formed by the folding of the disc. This invagination later becomes visible as a distinctive 2-cell wide intrusion, which is referred to as the 'stalk'. Cells at the deepest end of the stalk undergo shape changes to form an amorphous inner SOP mass. Invaginating cells are characterised by upregulation of Ato expression, a characteristic of SOP commitment. Surprisingly, SOP markers (Ase protein and the A101 enhancer trap line) are not expressed in all the stalk SOPs. Instead, these markers are only apparent in older cells, particularly at the time when they become part of the inner mass (which is therefore referred to as mature SOPs). Despite this, entry into the stalk seems to mark SOP commitment, since both the stalk and the mature SOPs are absent in discs from ato mutant larvae. This apparent intermediate stage may not have a counterpart in external sense organ precursor formation, although there is some evidence for multiple steps between the uncommitted cell and the SOP (the so-called pre-sensory mother cell state). Initially, Ato remains activated in all invaginated SOPs. This extended period of proneural gene expression is unusual since AS-C proneural expression is typically switched off in SOPs shortly after commitment. Later, at approximately 6 hours before puparium formation (BPF), Ato expression is switched off synchronously in the mature SOPs, although expression remains in the stalk SOPs and the PNC. At this point there is very little overlap between Ato and Ase or A101 (zur Lage, 1999). The process of chordotonal SOP formation described above is at odds in several respects with the well-known paradigm of SOP selection for sensory bristles. In the latter, the solitary SOP expresses Delta, which triggers expression in the PNC of genes of the E(spl)-C, thereby preventing further SOP commitment and forcing loss of AS-C expression and neural competence. In the case of the femoral chordotonal organ, newly committed cells from the PNC are in contact with previously committed SOPs in the stalk, but are apparently not receiving (or not responding to) lateral inhibition signals from these to prevent their commitment. Likewise, the presence of committed SOPs does not switch off ato expression in the PNC. Nevertheless, components of the N-Dl pathway are expressed in patterns consistent with lateral inhibition. The newly formed SOPs express Dl, suggesting that they send inhibitory signals, while the PNC expresses mgamma, a member of the E(spl)-C, suggesting that these cells are responding to the Notch-Delta signal. Indeed, mgamma is coexpressed with ato in the PNC throughout the development of the SOP cluster. Chordotonal SOP formation is shown to be sensitive to N inhibitory signaling. Strong activation of N signaling or its effectors can inhibit chordotonal SOP formation. Thus, N signaling has an important role to play: it acts to limit the process of SOP selection from the PNC. Some mechanism, however, must prevent N signaling from completely inhibiting multiple SOP formation (zur Lage, 1999). The progressive accumulation of chordotonal SOPs suggests that a recruitment mechanism could explain the clustering of SOPs. The Drosophila Egfr signaling pathway is involved in a number of recruitment processes in development, and a role for Egfr signaling has been demonstrated in the induction of embryonic chordotonal precursors (zur Lage, 1997). Although there appear to be significant differences in the process of SOP formation in imaginal discs, as compared with the embryo, it was asked whether Egfr signaling is also involved in forming the femoral chordotonal cluster. To address this question, the pathway was conditionally disrupted by expressing a dominant negative form of Egfr protein. Expression of UAS-Egfr DN results in a dramatic loss of chordotonal SOPs in late third instar imaginal leg discs (as judged by Ase protein expression or the A101 enhancer trap line). This demonstrates that Egfr signaling is required for the process of femoral chordotonal SOP formation. In contrast, the appearance of bristle SOPs is unaffected, arguing against the possibility of a nonspecific effect on SOPs in general (zur Lage, 1999). To determine whether Egfr signaling controls SOP number, expression of components of the Egfr pathway that determine the level of signaling was forced, thus resulting in hyperactivation of the pathway. pointed (pnt) is an effector gene that encodes a transcription factor and is activated in cells responding to Egfr signaling. Both rho and pnt are expressed during chordotonal SOP formation. Indeed, forced expression of rho or pnt increases chordotonal SOP formation. Egfr could promote SOP formation by stimulating the commitment of PNC cells or by stimulating proliferation of SOPs. Both functions would be consistent with known Egfr roles, but the current investigations favour the former. Analysis of Ato expression in leg discs in which rho has been misexpressed reveals a large invagination of cells and a smaller PNC. Shrinking of the PNC was confirmed by the reduced extent of mgamma expression. These observations are consistent with an increased rate of SOP commitment upon Egfr hyperactivation. Moreover, this effect is reminiscent of the effect of N loss of function on Ato expression, suggesting that Egfr signaling supplies the mechanism that interferes with lateral inhibition of SOP commitment (zur Lage, 1999). Although it seems that cells of the PNC and stalk are held in a state of mitotic quiescence throughout the time that SOP fate decisions are being made, BrdU is incorporated in the older (mature) SOPs. The experiments so far have indicated that Egfr signaling affects SOP commitment from the PNC. To determine more precisely the spatial patterning of Egfr activity required for SOP clustering and N antagonism, the expression patterns of key components of the pathway were characterized. Localized expression of rho appears to play a central role in spatial restriction of Egfr activity in cases where Spi is the ligand; in these cases it appears to mark the cells that are a source of signaling. During development of the femoral chordotonal organ, rho is expressed in a very restricted pattern: RHO mRNA is only detected in the SOPs, becoming confined in the late third instar larva to the youngest SOPs at the top of the stalk. To identify the cells responding to rho-effected signaling, an antibody that detects the dual-phosphorylated (activated) form of the ERK MAP kinase (dp-ERK) was used. In leg imaginal discs, dp-ERK is detected in a confined area corresponding to the uppermost (youngest) stalk SOPs. Thus, like rho, dp-ERK is expressed in the newly formed stalk SOPs. Double labelling for RHO RNA and dp-ERK confirms this, but also suggests that the overlap in expression is not complete: dp-ERK is detected above the uppermost rho-expressing cells of the stalk, probably in one or a few cells of the proneural cluster as they funnel into the stalk. This suggests that Egfr promotes SOP commitment as a consequence of direct signaling from previous SOPs to overlying PNC cells. Since rho expression is itself activated upon SOP commitment, this process occurs cyclically: the newly recruited SOPs are in turn able to signal to further overlying PNC cells. That is, recruitment is reiterative. Egfr signaling via Spitz has been shown to help to maintain neural competence by attenuation of Notch directed lateral inhibition. The opposing forces of Notch and Egfr signaling are thought to be played out through direct Notch and Egfr signaling between the epidermal proneural cells, which bear Notch, and the SOP, which sends inhibitory signals through the Delta ligand, and stimulatory signals through the Spitz ligand (zur Lage, 1999). Reiterative recruitment alone cannot entirely explain the accumulation of SOPs. Such an accumulation also relies on the persistence of the competent pool of PNC cells from which SOPs can be recruited. For AS-C PNCs, this does not occur, because the mutual inhibition required for continued competence is unstable and resolves quickly to a state of lateral inhibition once the SOP emerges from the PNC. This results in rapid shutdown of AS-C expression and hence competence within the PNC. It is possible that the members of E(spl)-C that are expressed in the PNC (notably mgamma and mdelta) are less aggressive inhibitors of proneural gene expression than the E(spl)-C members expressed in AS-C PNCs (m5 and m8). The results obtained in the femoral SOP suggest, however, that Egfr has a role to play in maintaining the PNC by partially attenuating lateral inhibition on a PNC-wide scale. Thus, the PNC is not completely shut off by inhibition from SOPs, but instead kept in check, allowing continued mutual inhibition and maintenance of competence but not allowing general SOP commitment. Since neither rho nor dp-ERK are detected in the PNC as a whole, this function of Egfr could be indirect and achieved through partial attenuation of Dl signaling from the stalk SOPs themselves. The trans- or auto-activation of EGFR signaling between the stalk SOPs (as suggested by the co-expression of dp-ERK and rho) might be an indicator of this function. It is also possible, however, that Egfr signaling is direct and that the dp-ERK antibody is not sensitive enough to detect expression in the PNC cells (zur Lage, 1999). The neurogenetic role of lethal of scute resembles that of the other three proneural gene members in the achaete-scute complex (achaete, scute and asense). This overview will examine instead the role of lethal of scute in the specification of muscle progenitors. Muscle development takes place in two phases. First, the pattern of muscle development is laid down by allocation of founder cells, specific cells in the mesoderm, each one serving as a founder for a unique muscle. Second, founder cells recruit neighboring myoblasts to form the syncylial precursors of mature muscle by fusion. Initially l'sc expression is widespread in cell clusters, but becomes allocated to muscle founder cells through the action of the Notch pathway. The expression of lethal of scute in mesoderm is transient, occuring in twist expressing cells. From late stage 9 until stage 12, there are at least 19 clusters of l'sc expressing cells in each hemisegment. In each cluster, one cell accumulates higher levels of l'sc than the other cells in the cluster. It is this single cell, allocated from a cluster of cells, that moves to a position close to the ectoderm and eventually becomes a muscle founder cell. Genes coding for three transcription factors, (nautilus, S59 and msh1) are each expressed in small groups of cells destined to differentiate into muscle cells. Another transcription factor, MEF2, is required for myosin expression and the fusion of myoblasts. None of these are selector genes initiating muscle fate. Genes with a decisive role in myogenesis, similar to members of the MYO-D family in vertebrates, have not been found in Drosophila. In neurogenic mutants, the domains of mesodermal S59 expression are expanded. This suggests the Notch pathway is involved in restricting the expression of l'sc and S59 to single cells, the same way it functions in neuroblast differentiation. l'sc is not the only factor involved in founder cell specification. In some instances no l'sc is found in the founder cell, and l'sc mutation does not completely upset muscle specification. Founder cell specification is easily comparable to neuroblast specification. The role of l'sc in muscle specification is analagous to the role of achaete-scute genes in neural growth. Specification of muscle founder cells is one of the many different processes involving the Notch pathway, a significant proportion of which involve proneural genes (Carmena, 1994).

GENE STRUCTUREcDNA clone length - 1067 Bases in 5' UTR - 46 Exons - one Bases in 3' UTR - 268

PROTEIN STRUCTUREAmino Acids - 257 Structural DomainsLethal of scute has a basic helix-loop-helix domain, and an C-terminal acidic domain (Cabrera, 1988 and Martin-Bermudo, 1993). A PEST domain is located just C-terminal of the bHLH domain. Deletion of the N terminal amino acids up to the basic domain, or deletion of the C terminal domain including the PEST domain, still leaves a functional protein (Hinz, 1994). Evolutionary Homologies The lin-32 gene of C. elegans codes for an Achaete-Scute homolog, sufficient for specification of neuroblast fate (Zhao, 1995). Chicken Achaete-Scute homolog (CASH-1) is one element in a multiple parallel pathway involving notochord or floor plate-derived signals for the specification and development of chick sympathetic neurons (Groves, 1995). A Xenopus Achaete-Scute homolog, XASH-3, when dimerized with the promiscuous binding partner XE12, specifically activates the expression of neural genes in naive ectoderm (Ferreiro, 1994). Xenopus Achaete-Scute homologs XASH-1a and XASH-1b appear in defined regions of the developing central nervous system. The pattern of expression of the Xenopus genes is modified by the cyclops mutant (Allende, 1994). The study of achaete-scute (ac/sc) genes is a paradigm to understand the evolution and development of the arthropod nervous system. The ac/sc genes have been identified in the coleopteran insect species Tribolium castaneum. Two Tribolium ac/sc genes have been identified -- 1) a proneural achaete-scute homolog (Tc-ASH) and 2) asense (Tc-ase), a neural precursor gene that reside in a gene complex. These genes reside 55 kb apart from each other and thus define the Tribolium ac/sc complex. Focusing on the embryonic central nervous system it is found that Tc ASH is expressed in all neural precursors and the proneural clusters from which they segregate. Through RNAi and misexpression studies it has been shown that Tc-ASH is necessary for neural precursor formation in Tribolium and sufficient for neural precursor formation in Drosophila. Comparison of the function of the Drosophila and Tribolium proneural ac/sc genes suggests that in the Drosophila lineage these genes have maintained their ancestral function in neural precursor formation and have acquired a new role in the fate specification of individual neural precursors. These studies, however, do not support a role for Tc-ASH in specifying the individual fate of neural precursors, suggesting that the ability of ac and sc to separately regulate this process may represent a recent evolutionary specialization within the Diptera. Furthermore, it is found that Tc-ase is expressed in all neural precursors, suggesting an important and conserved role for asense genes in insect nervous system development. This analysis of the Tribolium ac/sc genes indicates significant plasticity in gene number, expression and function, and implicates these modifications in the evolution of arthropod neural development (Wheeler, 2003). The work presented in this paper together with studies on ac/sc gene function in Drosophila provide strong evidence that serial duplications of proneural ac/sc genes in the dipteran lineage led to the diversification of proneural ac/sc gene function in Drosophila. In Drosophila, ac and sc carry out functions distinct from l'sc in specifying the individual fate of the MP2 precursor. Tc-ASH can function in Drosophila as a proneural gene but like Drosophila l'sc fails to specify efficiently the MP2 fate in the CNS. Together these results suggest the ability of ac and sc to specify MP2 fate in Drosophila arose after the divergence of Drosophila and Tribolium. These data provide an example whereby a subset of duplicated genes has evolved a new genetic function while the entire set of duplicate genes has retained the ancestral function (Wheeler, 2003). In addition to functional changes, the generation of multiple proneural ac/sc genes in the insects was paralleled by modifications to the expression profiles of these genes. In Anopheles (a basal dipteran), and Tribolium a single proneural ac/sc gene is expressed in all CNS proneural clusters. In more derived Diptera the presence of multiple ac/sc genes allows for more complex proneural ac/sc gene expression patterns. For example, Ceratitis contains two proneural ac/sc genes, l'sc and sc; l'sc is expressed in all CNS proneural clusters while sc is expressed in a subset of these clusters. In Drosophila, ac and sc are expressed in the identical pattern of proneural clusters and their expression is largely complementary to that of l'sc. The sum of proneural ac/sc expression in each species then marks all CNS proneural clusters despite differences in the expression pattern of individual proneural ac/sc genes. Thus, in Drosophila, the complete expression pattern of proneural ac/sc genes is divided between the largely complementary expression profiles of ac and sc relative to l'sc. The division of labor between proneural ac/sc genes in Drosophila has resulted in mutually exclusive expression patterns for ac and sc relative to l'sc in proneural clusters like MP2. This spatial separation of proneural gene expression probably facilitated the potential for ac and sc to acquire developmental functions distinct from l'sc (Wheeler, 2003). Together this work and that of others on arthropod ac/sc genes highlights the utility of studying ac/sc genes in elucidating the genetic basis of the development and evolution of arthropod nervous system pattern. These studies illustrate the dynamic nature of ac/sc gene number, expression and function over a relatively short evolutionary time. Based on this, future work on ac/sc genes in additional arthropod species should continue to provide insight into the molecular basis of the evolution of arthropod nervous system development (Wheeler, 2003).

REGULATIONPromoter Structure Thirty nucleotides upstream from the start of transcription is an unconventional TATA box (TATTTAAA). Seven CANNTG motifs (E-boxes), putitive binding sites for bHLH transcription factors, are located in this upstream region 200 to 1000 bp above the start site. These local upstream elements could function in l'sc autoregulation or activation by Achaete or Scute (Martin-Bermudo, 1993). Transcriptional Regulation Snail represses l'sc transcription in the presumptive embryonic mesoderm (Kosman, 1991). Elements regulating l'sc are scattered throughout 75 kb between achaete and asense. These elements activate l'sc in specific proneural clusters and as a consequence, also in their corresponding neuroblasts (Martin-Bermudo, 1993). Short gastrulation prevents Decapentaplegic from suppressing neurogenesis laterally in the blastoderm embryo. It is possible to exacerbate defects in sog mutants by increasing the level of DPP. The earliest neuroectodermal marker affected in sog mutants with a double dose of dpp is rhomboid, which is normally expressed in lateral stripes 8-10 cells wide in wild-type embryos but rapidly narrows to stripes 4-6 cells across in sog mutants with elevated DPP. Similarly l'sc expression is reduced in sog mutants with elevated DPP. A striking feature of the affects of DPP on neural suppression and dorsalization is that neuronal suppression is induced by a lower threshold of DPP activity than is dorsalization. Much less DPP is required to suppress expression of neuroectodermal genes than is required to activate dorsal markers. For example, brief submaximal heat induction of heat shock dpp in a wild type sog background leads to nearly maximal suppression of lethal of scute, scratch and snail expression during germ band extension, but there is no detectable ectopic expression of zerknllt in the neuroectoderm (Biehs, 1996). The segmented portion of the Drosophila embryonic central nervous system develops from a bilaterally symmetrical, segmentally reiterated array of 30 unique neural stem cells, called neuroblasts. The first 15 neuroblasts form about 30-60 minutes after gastrulation in two sequential waves of neuroblast segregation and are arranged in three dorsoventral columns and four anteroposterior rows per hemisegment. Each neuroblast acquires a unique identity, based on gene expression and the unique and nearly invariant cell lineage that this expression produces. Little is known as to the control of neuroblast identity along the DV axis. The Drosophila Egfr receptor (Egfr) has been shown to promote the formation, patterning and individual fate specification of early forming neuroblasts along the DV axis. Molecular markers identify particular neuroectodermal domains, composed of neuroblast clusters or single neuroblasts, and show that in Egfr mutant embryos (1) intermediate column neuroblasts do not form; (2) medial column neuroblasts often acquire identities inappropriate for their position, while (3) lateral neuroblasts develop normally. Active Egfr signaling occurs in the regions from which the medial and intermediate neuroblasts will later delaminate. The concomitant loss of rhomboid and vein yields CNS phenotypes indistinguishable from Egfr mutant embryos, even though loss of either gene alone yields minor CNS phenotypes. These results demonstrate that Egfr plays a critical role during neuroblast formation, patterning and specification along the DV axis within the developing Drosophila embryonic CNS (Skeath, 1998). In a screen to identify mutations that disrupt embryonic CNS development, one P element mutation, l(2)03033, was identified that causes a loss of essentially all Eve-positive RP2/RP2 sib neurons. This P element maps to cytological position 57F1-2 in the right arm of the second chromosome and is known to be inserted within the Egfr locus. To verify that lesions in Egfr result in a nearly complete loss of RP2 motoneurons, three additional Egfr mutants were obtained, including the Egfr null allele, flb 1K35Egfr allele (Skeath, 1998). The first phase of CNS development, as gastrulation commences, involves the activation of the Ac-S proneural genes in a precise pattern of proneural clusters. To investigate whether Egfr regulates As-C expression in the neuroectoderm, the expression patterns of the achaete (ac) and lethal of scute (lsc) genes were followed in Egfr mutant embryos. Loss of Egfr causes specific defects to the DV registration of ac and lsc gene expression in the neuroectoderm; however, no defects to the AP registration for either ac or lsc gene expression were found. In wild-type embryos during stages 8/9, ac is expressed in the medial and lateral, but not intermediate, clusters of rows 3 and 7; lsc is expressed in the medial and lateral, but not intermediate, clusters of row 7 and in the medial, intermediate and lateral clusters of rows 1 and 5. A single neuroblast subsequently forms from each proneural cluster. In Egfr mutant embryos, ac gene expression expands into the intermediate column in rows 3 and 7 and lsc expression expands into the intermediate column in row 7; lsc is expressed normally in rows 1 and 5. The lateral limits of ac and lsc gene expression in the neuroectoderm are unaltered in Egfr mutant embryos. The changes to the DV registration of ac and lsc gene expression in Egfr mutant embryos suggest that neuroectodermal cells in the intermediate column change their fate. Both ac and lsc are normally expressed in the medial and lateral columns in the affected rows, thus the phenotype is consistent with intermediate cells acquiring either a lateral or a medial fate. msh-1, which is expressed exclusively in the lateral column, expands into the intermediate column in Egfr mutant embryos. In this context, it appears that ac and lsc expression expand from the lateral column into the intermediate column in the absence of Egfr (Skeath, 1998). The maternal Dorsal nuclear gradient initiates the differentiation of the mesoderm, neurogenic ectoderm and dorsal ectoderm in the precellular Drosophila embryo. Each tissue is subsequently subdivided into multiple cell types during gastrulation. This study investigates the formation of the mesectoderm within the ventral-most region of the neurogenic ectoderm. Previous studies suggest that the Dorsal gradient works in concert with Notch signaling to specify the mesectoderm through the activation of the regulatory gene sim within single lines of cells that straddle the presumptive mesoderm. This model was confirmed by misexpressing a constitutively activated form of the Notch receptor, NotchIC, in transgenic embryos using the eve stripe2 enhancer. The NotchIC stripe induces ectopic expression of sim in the neurogenic ectoderm where there are low levels of the Dorsal gradient. sim is not activated in the ventral mesoderm, due to inhibition by the localized zinc-finger Snail repressor, which is selectively expressed in the ventral mesoderm. Additional studies suggest that the Snail repressor can also stimulate Notch signaling. A stripe2-snail transgene appears to induce Notch signaling in 'nave' embryos that contain low uniform levels of Dorsal. It is suggested that these dual activities of Snail -- repression of Notch target genes and stimulation of Notch signaling -- help define precise lines of sim expression within the neurogenic ectoderm. It is proposed that Snail functions as a gradient repressor to restrict Notch signaling. In precellular embryos, the initial snail expression pattern is broad and extends into the future mesectoderm. During cellularization, the pattern is refined and snail expression is lost in the mesectoderm and restricted to the mesoderm. The early, broad snail pattern might create a broad domain of potential Notch signaling by repressing components of the Notch pathway, such as Delta and lethal of scute. After cellularization, Notch signaling is blocked in the presumptive mesoderm by sustained, high levels of the Snail repressor. However, Notch can be activated in the mesectoderm because of the loss of Notch inhibitors repressed by transient expression of the Snail repressor. According to this model, the dynamic snail expression pattern determines both the timing and limits of Notch signaling (Cowden, 2002). Targets of Activity L'SC activates Enhancer of split and HLH-M5 of the Enhancer of split complex, and possibly dorsal as well (Hinz, 1994). Protein Interactions Extramachrochaete forms inactivating heterodimers with L'SC as it does with AC and SC (Cabrera, 1994). Daughterless is required as a dimerization partner for L'SC function (Hinz, 1994). Classical genetics indicates that the achaete-scute gene complex (AS-C) of Drosophila promotes development of neural progenitor cells. To further analyze the function of proneural genes, the effects of Gal4-mediated expression of lethal of scute, a member of the AS-C, were studied during embryogenesis. Expression of lethal of scute forces progenitor cells of larval internal sensory organs to take on features of external sensory organs. Normally, these cells are committed to this fate independent of AS-C activity. Surprisingly, overexpression of l'sc does not result in supernumerary neural cells. Supernumerary neural cells can be induced ectopically only if daughterless is overexpressed, either alone or together with lethal of scute: cells of the amnioserosa and the hindgut then express neuronal markers. Cells of the proctodeal anlage, which normally lack neural competence, acquire the ability to develop as neuroblasts following transplantation into the neuroectoderm. Activated Notch prevents the cells of the neuroectoderm from forming extra neural tissue when they express an excess of proneural proteins. Under the present conditions, lateral inhibition is thus dominant over the activity of proneural genes (Giebel, 1997).

DEVELOPMENTAL BIOLOGY Embryonic See the embryonic expression pattern of l(sc) at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.Like achaete and scute, l'sc is expressed in very specific subsets of cells in neuroblasts of the ventral neurectoderm (Martin-Bermudo, 1993). Gene expression in the ventral neuroectoderm is discussed at the achaete-scute complex site. As with achaete and scute, l'sc is expressed in every neurogenic region of the fly, including the cephalic and gnathal regions. After stage 9, l'sc is expressed in the mesectodermal, central and peripheral nervous system anlage, as well as the stomatogastric nervous system and the optic lobes (Cabrera, 1990). In head midline structures, in particular the optic lobe and stomatogastric nervous system, there may be a late phase of EGFR signaling (as assayed by the expression of aos and activated ERK) whose significance is not yet known. EGFR signaling could be involved in modifying the inhibitory feed-back loop between neurogenic and proneural genes that exists in other neurectoderm cells. In the head midline neurectoderm, regulation of proneural and neurogenic genes has to be different. Thus, instead of a short burst of proneural gene expression in proneural clusters that is resolved into expression in individual neuroblasts, proneural genes are expressed for a long period of time; at the same time, the expression is never restricted to single neuroblasts. Since genes of the E(spl) complex are expressed in the same cells that express lsc, the inhibitory loop between E(spl)-C and proneural genes must be interrupted at some level. It is possible that Egfr signaling is causing the interruption of this inhibitory loop. Based on genetic studies of Notch and Egfr signaling in the compound eye, it has been speculated that one of the consequences of Egfr activation (which ultimately is required for all ommatidial cell types to differentiate) is to inhibit N signaling, since constitutively active N inhibits ommatidial cell differentiation by preventing response to differentiative signals. However, the same effect could be achieved if Egfr signaling, similar to what is proposed here for the midline neurectoderm, interrupts the inhibition of proneural genes by E(spl). Although this would not prevent N signaling, it would cancel the effect of N signaling on downregulating proneural genes and thereby keep cells in a state of competency to respond to signals (Dumstrei, 1998). The expression of the proneural gene lethal of scute is required for the development of the majority of the procephalic neuroblasts. lethal of scute expression patterns correspond to many of the identifiable 23 groups of neuroblasts in the developing brain. l'sc expression in the procephalic neurectoderm is controlled in partially overlapping domains of the neuroectoderm. Loss of function of a given head gap gene results in the absence of l'sc expression in its domain, followed by the absence of neuroblasts that would normally segregate from this domain (Younossi-Hartenstein, 1997). Neuroblasts delaminate from the procephalic neurectoderm in a stereotyped spatiotemporal pattern that is tightly correlated with the expression of l'sc. The pattern of neuroblasts was reconstructed by using the marker asense; similar to its expression in the ventral neuroblasts, asense labels all brain neuroblasts. seven-up, expressed in specific subsets of neuroblasts making up approximately one-third of the total, is also used as a marker. For most, if not all, of these clusters the number of neuroblasts and the time of onset of svp expression are absolutely invariant (Younossi-Hartenstein, 1996). Effects of Mutation or Deletionlethal of scute mutants may reach adulthood (Martin-Bermudo, 1993). This attests to the many redundancies or biological fail safe mechanisms created by the duplication of function in proneural genes. klumpfuss shows genetic interactions with achaete, scute, lethal of scute and asense. l'sc is able to activate klu expression, but apparently only in the wing disc. There appears to be only a weak influence of the AS-C genes on klu expression, restricted to the wing area of the wing disc. However, the overall expression pattern of klu is largely independent of proneural genes. The assumption that SOPs enter apoptosis in klu mutants is supported by the observation of abundant cell death in other developing organs of klu mutants, like the legs. At certain bristle positions, such as that of the anterior sternopleura, klu is required during early bristle development immediately after proneural gene function, in order to allow a particular epidermal cell to develop as a SOP. It is suggested that klu is required only for initiation of bristle development, being downregulated once specification takes place (Klein, 1997). During Drosophila embryogenesis, mesodermal cells are recruited to form a stereotyped pattern of about 30 different larval muscles per hemisegment. The formation of this pattern is initiated by the specification of a special class of myoblasts, called founder cells, that are uniquely able to fuse with neighbouring myoblasts. The COE transcription factor Collier plays a role in the formation of a single muscle (muscle DA3[A] in the abdominal segments; DA4[T] in the thoracic segments T2 and T3). Col expression is first observed in two promuscular clusters (in segments A1-A7), corresponding to two progenitors and then their progeny founder cells, but its transcription is maintained in only one of these four founder cells, the founder of muscle DA3[A]. It is proposed that specification of the DA3[A] muscle lineage requires both Col and at least one other transcription factor, supporting the hypothesis of a combinatorial code of muscle-specific gene regulation controlling the formation and diversification of individual somatic muscles (Crozatier, 1999). Following establishment of the promuscular clusters, specification of the progenitors is controlled by lateral inhibition, a cell-cell interaction process mediated by the neurogenic genes Notch (N) and Delta (Dl)). In both N and Dl mutant embryos, promuscular Col expression is initiated normally but fails to become restricted to a single cell per cluster, similar to observations previously made for the expression of lsc. As a consequence, a hyperplasic expression of Col is observed from stage 11. Since it is expressed in promuscular clusters and segregating muscle progenitors, lsc has been proposed to play a role in muscle progenitor selection similar to the role of achaete and scute in neuroblast specification. However, in embryos lacking lsc activity, selection of the Col-expressing progenitors occurs normally at stage 11 and muscle DA3[A] forms as in wild type (Crozatier, 1999). In the embryonic ventral neuroectoderm of Drosophila the proneural genes achaete, scute, and lethal of scute are expressed in clusters of cells from which the neuroblasts delaminate in a stereotyped orthogonal array. Analyses of the ventral neuroectoderm before and during delamination of the first two populations of neuroblasts show that cells in all regions of proneural gene activity change their form prior to delamination. Furthermore, the form changes in the neuroectodermal cells of embryos lacking the achaete-scute complex, of embryos mutant for the neurogenic gene Delta, and of embryos overexpressing l'sc, suggest that these genes are responsible for most of the morphological alterations observed (Stollewerk, 2000). Almost all neuroectodermal cells are larger than the cells of the dorsal epidermal anlage (DEA). In comparison with the cells of the DEA in early stage 8 embryos the dorsoectodermal cells of mid-stage 8 embryos are clearly smaller. A comparison of the neurogenic region in early and mid-stage 8 embryos shows that the medial and intermediate regions of the ventral neuroectoderm (VNE) do not increase further in size whereas the lateral region enlarges considerably during this time. Due to these morphological changes the VNE can now be subdivided in relation to the cell sizes into three longitudinal regions on both sides of the midline: medial, intermediate, and lateral regions. In contrast to the cells of the medial and lateral regions, which now have approximately the same average values, the intermediate cells are smaller. Only 20% of all cells in the intermediate region are larger than the average, whereas 63% of the medial and 64% of the lateral cells exceed the average value. Most of the enlarged cells have a cuboidal shape. In every hemisegment the apical surfaces of two to four cells in the medial and lateral regions are very small (12-16 m2) but expand basally to cover an area of 65-80 m2. One or two cells of this shape are also located in the intermediate regions but are smaller basally (48-58 m2) than the medial and lateral cells. The number and position of these cells suggest that they correspond to the delaminating neuroblasts; this was confirmed by staining the embryos with anti-Hunchback antibody, an early marker for neuroblasts (Stollewerk, 2000). During delamination of the SI neuroblasts the neuroectodermal cells gradually decrease in size, with the exception of a few cells located close to the midline. The cells that remain enlarged are either elongated perpendicularly to the midline or have a rounded appearance. Basally, between neuroectoderm and mesoderm, large round cells are located that lose contact with the apical surface at about 60% EL. On the basis of their position and arrangement, as well as the analysis of embryos stained for Hunchback, these cells can be identified as the SI neuroblasts. Before delamination of the SII neuroblasts, cells in the intermediate region of the neuroectoderm increase in size. Most of the SII neuroblasts delaminate from this region, whereas only a few neuroblasts arise from the medial region, where enlarged cells can also be detected. After delamination of the SII neuroblasts the enlarged cells shrink once again, as revealed by double staining with anti-Hunchback antibody and phalloidin. Cells in all regions of the VNE increase in size again prior to delamination of the SIII neuroblasts. Thus, the VNE of wild-type embryos becomes morphologically distinguishable from the DEA shortly before delamination of the SI neuroblasts. At this point the cells of the DEA have already divided, and about two-thirds of all cells in the medial and the lateral regions have become enlarged so that the DEA and the VNE are clearly distinguishable due to differences in cell size. In addition, almost all cells of the intermediate region increase in size prior to delamination of the SII neuroblasts. These data are at odds with claims that only the neuroblasts enlarge prior to delamination, both in grasshopper and Drosophila (Stollewerk, 2000). Is there a correlation between the activity of the ASC genes and the observed morphological changes? The results presented indicate that the ASC genes are not the only ones responsible for the morphological changes that occur before delamination of the SI neuroblasts. Although the number of enlarged cells corresponds closely to the number of cells that express the ASC genes at this time point, the lack of the ASC does not result in all cells remaining the same size. Whereas in the medial region of the VNE of Df (1)260-1 embryos (that is, those lacking the ASC) only about 50% of the cells are smaller in size than in the wild type, and the lateral region is most strongly affected in comparison to the medial and intermediate regions. Therefore the enlargement of the neuroectodermal cells depends to a varying degree on the activity of the ASC genes and is additionally influenced by other factors. However, a clear correlation can be seen prior to delamination of the SII neuroblasts. At this time almost all cells of the intermediate region increase in size, which coincides with the expression of l'sc in this region. Furthermore, analysis of the VNE of embryos lacking the ASC reveals that the intermediate cells do not become enlarged prior to delamination of the SII neuroblasts, suggesting that the observed morphological changes are due to the activity of the ASC genes at this point. In addition, the shrinkage of the cells that had enlarged during delamination of the SI and SII neuroblasts is correlated with the decrease in ASC gene expression in the VNE at these time points (Stollewerk, 2000). Analysis of wild-type and Delta mutant embryos also suggests that the ASC genes are important for the maintenance of the morphology of the neuroectodermal cells. Despite the fact that the total area of the intermediate region does not change significantly between early and mid-stage 8, cell size changes can be detected in this region shortly before delamination of the SI neuroblasts. While 20% of the intermediate cells remain larger than the average, the cells that had an average cell size in the VNE of early stage 8 embryos now split into groups of smaller cells. The fact that the number of cells that remain larger than the average corresponds to the number of cells that express the ASC genes in the intermediate region suggests that the proneural genes are required to keep these cells enlarged. This view is confirmed by analyses of the VNE of Delta mutant embryos. In Delta mutant embryos all cells of a proneural cluster continue to express the proneural genes and become neuroblasts. This altered gene expression causes all cells of a proneural cluster to remain enlarged until proneural gene expression is turned off (Stollewerk, 2000). A correlation between increase in cell size and ASC gene expression has also been shown by the analysis of embryos labeled for ac protein and embryos overexpressing l'sc. Area measurements reveal that 85% of all cells that express ac are enlarged in these embryos. The fact that not all ac-expressing cells are larger than the average at the time point analyzed may be due to the rapidity of the morphological changes (enlargement and shrinkage) that occur immediately before and during delamination of the neuroblasts. A clear influence of a proneural gene on the cell sizes in the VNE can be seen in embryos overexpressing l'sc: 45% more cells become enlarged in the intermediate region in comparison to the wild type. Only a minor increase in the numbers of enlarged cells can be seen in the medial and lateral regions, because two-thirds of these cells already express proneural genes. In addition, the high proneural gene activity in the VNE of embryos overexpressing l'sc causes the future neuroblasts to change their morphologies: they expand not only their basal but also their apical surfaces. These data clearly show that the ASC genes have an influence on the morphologies of the neuroectodermal cells (Stollewerk, 2000).