BIOLOGICAL OVERVIEW

escargot is expressed in divergent cells throughout the embryo but it remains primarily ectodermal. Mutations in escargot cause defects in adult structures including the abdominal cuticle, wings and legs (Hayashi, 1993).

The action of ESG as a transcriptional repressor has been documented by measuring the interaction of ESG with E2 boxes, the consensus binding sequence of basic HLH transcription factors. Basic HLH proteins are transcriptional activators and bind very specific nucleotide sequences in gene promoters. ESG binds as a monomer to the E2 box, antagonizing transcriptional activation by Scute and Daughterless (Fuse, 1994). Expression in the neurogenic region and antagonism of Scute and Daughterless suggest that escargot opposes a proneural fate.

ESG regulates the cell cycle in histoblasts. Histoblasts are nests of cells in the abdomen, comprising integral components of the larval epidermis. These cells are precursors of the ectodermal component of the adult abdomen (Hartenstein, 1992).

Abdominal histoblasts in esg mutant larvae undergo endoreplication and become polyploid, preventing the development of adult abdominal epidermis. Endoreplication, which properly occurs in normal development as well as improperly in certain mutations, is the replication of DNA without concommitant cell division. This results in extra thick polytene chromosomes, because the DNA has duplicated beyond the normal diploid amount. Overexpression of esg rescues the abdominal phenotype in esg mutants while at the same time inhibiting normal endoreplication in salivary gland cells (Fuse, 1994).

It has been suggested that ESG represses the activation or somehow restricts the activity of unknown transcription factors responsible for entry into mitosis. Thus Escargot regulates the cell cycle (Fuse, 1994). Subsequent studies reveal that Escargot maintains the level of Dmcdc2, the catalytic kinase subunit of the G2/M cdk heterodimer. In turn, the heterodimer inhibits entry into S and the consequent endoreplication. Dmcdc2 heterodimerizes with cyclin A and cyclin B, both of which are required for entry into mitosis. It would seem that the G2/M cdk heterodimer has two functions: the regulation of entry into mitosis and the prevention of endoreplication (Hayashi, 1996).

Neurogenesis in Drosophila melanogaster starts by an ordered appearance of neuroblasts arranged in three columns (medial, intermediate and lateral) in each side (right and left) of the neuroectoderm. In the intermediate column, the receptor tyrosine kinase Egfr represses expression of proneural genes achaete and scute, and is required for the formation of neuroblasts. Most of the early function of Egfr is likely to be mediated by the Ras-MAP kinase signaling pathway, which is activated in the intermediate column, since a loss of a component of this pathway leads to a phenotype identical to that of Egfr mutants. MAP-kinase activation is also observed in the medial column where escargot (esg) and proneural gene expression are unaffected by Egfr. The homeobox gene ventral nerve system defective (vnd) is required for the expression of esg and scute in the medial column. vnd acts through the negative regulatory region of the esg enhancer that mediates the Egfr signal, suggesting vnd's role is to counteract Egfr-dependent repression. Thus, the nested expression of vnd and the Egfr activator Rhomboid is crucial to subdivide the neuroectoderm into the three dorsoventral domains (Yagi, 1998).

To investigate the involvement of Egfr in neurogenesis, mutant phenotypes of Efgr and its activator rho were examined at various stages of neurogenesis. The dorsoventral subdivision of the neuroectoderm in stage-6 embryos is detectable by expression of esg, which is expressed in the lateral and medial columns but not in the intermediate column. A loss-of-function, temperature-sensitive mutation of Egfr and a null mutation of rhomboid were used for analysis throughout this work. Egfr and rho mutations cause ectopic expression of esg in the intermediate column. Repression of esg in the intermediate column is likely to require a relatively high dose of Egfr signal. To examine the potential role of Egfr in neurogenesis, expression of the proneural genes ac and sc was carried out. These two proneural genes begin expression in the neuroectoderm of stage-7 embryos in a DV pattern of expression similar to that of esg in the previous stage. In Egfr and rho mutant embryos, ac and sc become ectopically expressed in the intermediate column. This phenotype is less penetrant and, occasionally, gaps of ac and sc expression are observed in the intermediate column. Since sc expression was similarly derepressed in Egfr mutant embryos, these phenotypes are likely to represent the near null phenotype of Egfr in the neuroectoderm. These data indicate that, in the intermediate column, the Egfr signal represses not only esg but also proneural genes, which are known to play key roles in neurogenesis. The effect of Egfr on neuroblast formation was monitored by the neuroblast marker Snail. Anti-Sna staining reveals three columns of SI neuroblasts in the control embryo: the intermediate column is distinguishable by the delayed onset of formation and number of Sna-positive cells. In Egfr and rho mutants, Sna-positive neuroblasts in the intermediate position are frequently missing, with a higher frequency of loss in Egfr embryos. In rho mutant embryos, the frequency of the loss of intermediate column neuroblasts is variable among embryos (Yagi, 1998).

To further examine the effect of the loss of Egfr signaling on the late events of neurogenesis, the progeny was traced for one of the intermediate neuroblasts, NB4-2. NB4-2 gives rise to the RP2 motor neurons, which can be identified by the expression of Even-skipped (Eve) and its unique position. Loss of RP2 neurons in stage 13 is observed (over half the cases examined) with the frequency of loss slightly higher in Egfr than in rho mutants, reflecting the earlier defect in neuroblast formation in stage 9. It is known that the Ras-MAPK signaling cascade is the major target of Egfr in many tissues. To understand whether Ras-MAPK signaling also mediates the Egfr signal in the neuroectoderm and to determine the relative contribution of each component of the pathway, the expression of esg and sc was examined in embryos lacking one of the Ras-MAPK signaling components. The phenotype of mutants lacking either Sos, Ras1, Draf or Dsor1 was examined. As in wild-type embryos, embryos mutant for any of the four genes examined express esg in three separate domains: procephalic neurogenic region, amnioserosa and neuroectoderm. In all cases, the anterior limit of the procephalic expression and the posterior limit of neuroectodermal expression are expanded to the terminus, consistent with the fact that Ras-MAPK is required for the terminal fate specification controlled by Torso receptor tyrosine kinase. All mutants exhibit specific defects within the neuroectoderm where esg expression is derepressed in the intermediate column. Essentially the same phenotype is also observed with sc expression, suggesting the loss of Ras-MAPK signaling has the same consequence as the loss of Egfr. All four Ras pathway mutants show, qualitatively, the same phenotype in the neuroectoderm. The neuroectoderm phenotype in Ras1 mutants is not rescued by a paternal copy of the wild-type gene, suggesting that a relatively high dose of the Ras signal is required for repression of esg and sc in the neuroectoderm (Yagi, 1998).

Rhomboid (rho) is initially expressed in the medial half of neuroectoderm, but repression of esg, ac and sc transcription by Egfr and Ras-MAPK occurs only in the intermediate column, posing a question as to whether or not the site of MAPK activation and the site of transcriptional repression exactly correspond. The spatial and temporal pattern of MAPK activation has been described by the use of an antibody that specifically reacts with the phosphorylated and activated form of MAPK (diphospho-MAPK=dpMAPK), which shows that dpMAPK is distributed in a broad domain in the neuroectoderm in stage 5-7 embryos. dpMAPK is distributed in an 8- to 10- cell-wide area in the neuroectoderm in stage-5 embryos and becomes restricted to the ventral region at the end of gastrulation. This rapidly evolving pattern of dpMAPK expression made it difficult to determine the exact correlation between distribution of dpMAPK and the DV subdomains in the neuroectoderm. A protocol was used to double label embryos with dpMAPK and antisense RNA probes to study the spatiotemporal relationship between expression of dpMAPK, its activator Rhomboid (Rho) and its downstream target, esg. Initial expression of dpMAPK overlaps with that of Rho in stage-5 embryos; dpMAPK expression remains in this broad domain when Rho expression became restricted to the medial column at gastrulation in stage 6, and finally narrows down to a 2- to 3-cell-wide stripe abutting the stripe of Rho at stage 7. Comparison with the mesodermal marker sna shows that the ventral border of dpMAPK expression abuts the neuroectoderm-mesoderm border. Examination of histochemically stained material reveals a sharp ventral border of dpMAPK expression, which gradually declines in the dorsal direction, resembling the pattern of Rho expression. In Egfr mutant embryos, dpMAPK staining is not detectable. These results demonstrate that MAPK activation in the neuroectoderm is dependent on Egfr and follows the spatial expression pattern of Rho, but persists for some time after termination of Rho transcription. The latter observation may reflect perdurance of Rho or its target protein, Spitz (Spi). Alternatively, a ligand other than Spi, such as Vein, might be activating Egfr. The dorsal limit of dpMAPK expression was determined relative to the three separate columns of neuroectoderm revealed by esg expression. In stage 5, the dorsal limit of dpMAPK reaches halfway within the intermediate column and subsequently retracts to the medial column in stage 6 and 7. These data indicate MAPK is activated at least in the ventral half of the intermediate column of the neuroectoderm when it is required to repress transcription of esg. It is concluded that transcription of esg is repressed by a marginal level of MAPK activation (Yagi, 1998).

Why does the high level of dpMAPK in the medial column fail to repress transcription of esg, ac and sc? One possibility is that a factor is present in the medial column that antagonizes or overcomes the events downstream of dpMAPK. A candidate for such a gene is vnd, which is expressed in the medial column in late stage 5 and is required for expression of ac. Expression of esg and sc was examined in vnd null mutant embryos: their expression in the medial column was found to be lost. To understand how vnd controls gene expression in the medial column, a target for vnd was sought in the cis-regulatory regions of an esg enhancer. Expression of esg is regulated by the neurogenic enhancer, which can be divided into two regions, the activator region, which mediates activation in the entire neuroectoderm, and the repressor region, which mediates Egfr-dependent repression. Expression of the esg-lacZ fusion genes was examined in the vnd mutant background. The construct esg-lacZ D1 containing the complete neurogenic enhancer reproduces neuroectodermal expression of esg and is regulated by vnd in the same manner as esg. In contrast, the construct esg-lacZ D5 lacks the repressor region for the Egfr-mediated regulation and is expressed in all three columns. Evidence is provided that vnd does not regulate esg-lacZ D5 and that the target site for vnd regulation is included in the repressor region. vnd is also shown not to be involved in activation of esg or Egfr; rather, it works to counteract the negative effect of Egfr (Yagi, 1998).

Given the results of the present work showing that vnd counteracts the negative regulatory effect of Egfr, a model is proposed for the DV structuring of the neuroectoderm. A gradient of nuclear localized Dorsal protein induces expression of dorsoventrally regulated genes such as dpp, sna, and twi, which determine the extent of the neuroectoderm, and the nested expression domains of rhomboid and vnd. rho determines the domain of MAPK activation, which covers the medial and intermediate columns. vnd is expressed in the medial column where it counteracts the Egfr signal to allow expression of esg. Thus the three columns in the stage 5-6 neuroectoderm are distinguished by unique combinations of activated MAPK and vnd expression. In the lateral column, neither of them are activated or expressed, and esg transcription is activated by default. In the intermediate column, MAPK is activated and represses esg transcription. In the medial column, vnd counteracts activated MAPK to allow the default pathway to activate esg transcription. It is possible that proneural genes are also regulated by the same mechanism. Loss of the Egfr signal leaves two domains, one with and the other without expression of vnd, the pattern likely to be reflected in the appearance of only two neuroblast columns in the later stage. Thus it is proposed that the primary role of Egfr signal in this stage is to define the intermediate domain to the neuroectoderm which is otherwise separated into two domains. It is possible that Egfr signal and vnd have later roles in promoting neuroblast formation in the intermediate and medial columns, respectively (Yagi, 1998).

GENE STRUCTURE

Bases in 3' UTR - 480

PROTEIN STRUCTURE

Amino Acids 470

Structural Domains

escargot encodes a protein with five zinc finger motifs, four of which are C2-H2 class.

date revised: 23 October 98

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