Interaction between Drosophila EGF receptor and vnd determines three dorsoventral domains of the neuroectoderm

The massive number and variety of neuronal cells that constitute the nervous systems of higher metazoans originate from a simple monolayer of cells in the neuroectoderm. In the development of the central nervous system of Drosophila, the first visible sign of neurogenesis is delamination of neuroblasts from the neuroectoderm, a process that follows strict spatial and temporal patterns. Neuroblasts subsequently undergo stem cell divisions in stereotyped patterns to yield neurons and glia cells. The pattern of the first wave of neuroblast delamination in late stage 8 (~4 hours of development) is arranged in four rows along the anterior-posterior (AP) axis and in three columns in a dorsoventral (DV) direction (Skeath et al., 1992). Many of the neuroblasts at the time of delamination already exhibit unique identities distinguishable by specific combinations of gene expression (Doe, 1992), suggesting that determination of neuroblast identity begins in the neuroectoderm. The dorsoventral subdivision of the neuroectoderm is already apparent in late stage 5 (~3 hour of development) when expression of the genes muscle-specific homeodomain (msh, D’Alessio and Frasch, 1996) and ventral nervous system defective (vnd, Jiménez et al., 1995; Mellerick and Nirenberg, 1995) begin in the lateral and medial column, respectively, and escargot (esg) is expressed in both medial and lateral columns (Yagi and Hayashi, 1997). These patterns of gene expression are followed by the expression of the proneural genes achaete (ac) and scute (sc) in the medial and lateral columns in stage 7-8. Primary neurogenesis in vertebrates also occurs in three dorsoventral domains (Chitnis et al., 1995), which are defined by the vertebrate counterparts of msh and vnd (Su et al., 1991; Saha et al., 1993) expressed in an analogous pattern, suggesting that the mechanism of dorsoventral patterning in the CNS has an ancient origin.

Although a great amount of work has demonstrated the importance of segmentation genes in AP patterning of the neuroectoderm (Patel et al., 1989; Skeath et al., 1992; Chu-LaGraff and Doe, 1993; Bhat and Schedl, 1997), little is known about the mechanism of DV patterning. One good candidate for a regulator of DV patterning in the CNS is the receptor tyrosine kinase DER (Drosophila EGF receptor), which was shown to provide DV positional information to the ventral ectoderm in stage 10 (Raz and Shilo, 1993; Kim and Crews, 1993; Golembo et al., 1996; Gabay et al., 1996). Involvement of DER in neuroectoderm patterning in an earlier stage was suggested from the expression of rhomboid (rho), the rate-limiting component of the processing system that produces the active DER ligand Spitz (Spi; Schweitzer et al., 1995) in the presumptive neuroectoderm of stage 5 embryos (Bier et al., 1990). Indeed, analyses of esg (Yagi and Hayashi, 1997) and msh (D’Alessio et al., 1996) expression demonstrated that DER is required for DV patterning of the neuroectoderm. Upon loss of DER function, the intermediate column of neuroectoderm ectopically expressed esg and msh. Analysis of the cis-regulatory region of esg revealed that DER represses transcription of esg through a dedicated negative regulatory element that counteracts a ubiquitous activator acting through a separate positive element (Yagi and Hayashi, 1997).

In many developmental contexts, DER affects cell fate decision by activating the Ras-MAPK pathway (Rogge et al., 1991; Diaz-Benjumea and Hafen, 1994). Components of this universal signaling pathway include Sos, the GDP-GTP exchange factor that activates the GTPase Ras, which triggers a sequential activation of protein kinases Raf (MAPKKK), Dsor1 (DMEK/MAPKK) and MAP kinase (MAPK). Activated MAPK enters the nucleus and modulates gene expression by phosphorylating transcription factors (O’Neill et al., 1994; Rebay and Rubin, 1995). One advantage of having several components in a pathway is to provide potential branch points of the pathway for intricate regulation. One example is the terminal patterning system in the early embryo, in which Torso receptor tyrosine kinase acts through Ras-MAPK to activate transcription of huckebein. In this case, the loss of Raf had a greater effect on huckebein transcription than the loss of Sos or Ras, suggesting that Ras is not the sole activator of Raf in this case (Hou et al., 1995).

In this work, we investigated the role of DER in DV patterning of neuroectoderm and in neurogenesis. We show herein that DER is required to repress transcription of proneural genes and to promote neuroblast formation in the intermediate column of the neuroectoderm. A loss of Ras-MAPK signaling causes a phenotype identical to that of DER mutants, suggesting that MAPK is a sole downstream effector of DER in this case. The spatial and temporal pattern of MAPK activation suggest that MAPK is activated, but failed to affect transcription of its target genes in the medial columns, where vnd is expressed. We show that vnd counteracts the negative effect of the DER-MAPK signal in the medial column to allow the medial-column-specific gene expression.

Most of the fly strains used are described in Flybase (1994) and sources were as follows: flb^IF26from the Tübingen Stock Center; rho^del1from Dr H. Okano; Draf^X1, Dsor1^Gp158(Tsuda et al., 1993), ovo^D1FRT101 and FLP³⁸from Dr Y. Nishida; Dras1^e2ffrom Dr Y. Hiromi; Sos^e46, Dras1^c40b and Gap1^BQ from Dr M. Okabe; msh^{delta 68} and UAS-msh from Dr A. Nose; Kr-Gal4 and da-Gal4 from Dr B.-Z. Shilo; vnd⁶and flb^1K35from Dr J. Skeath; ovo^D1FRT40A, ovo^DFRT2A and FRT82B ovo^D1from Bloomington Stock Center. Mutant chromosomes were balanced over one of the following balancers carrying lacZ expression vectors to distinguish mutant embryos: FM7 ftz-lacZ (From Dr Yasushi Hiromi), CyO ftz-lacZ, TM3 Ubx-lacZ, or TM6B abd-A lacZ. esg-lacZ D1 and D5 were described in Yagi and Hayashi (1997).

In situ hybridization was performed as previously described (Tautz and Pfeifle, 1989; Yagi and Hayashi, 1997). The following DNA fragments were used as probes; ac and vnd from Dr J. Skeath, sc from Dr C. Cabrerra, rho from Dr H. Okano, sna from Dr N. Fuse and msh from Dr A. Nose. For identification of mutant embryos, lacZ antisense riboprobe was mixed in the hybridization solution to detect lacZ expression from the marked balancers.

Immunostaining was performed as described by Hayashi et al. (1993). Rabbit anti-Sna antibody was obtained from Dr R. Reuter, mouse monoclonal Anti-Eve, from Dr Y. Hiromi, and anti-dpMAPK, from Sigma Chemicals and used as described by Gabay et al. (1997b). Identification of mutant embryos was carried out by staining them simultaneously with anti-β-galactosidase and Cy3-conjugated secondary antibody. Signals were detected with a Vectastain ABC Kit (Vector Lab). TSA indirect system (NEN) was used for further enhancement of the signals.

Embryos fixed for anti-dpMAPK staining (Gabay et al., 1997b) were used for double labeling. Antibody staining and in situ hybridization were carried out according to Goto and Hayashi (1997). The dpMAPK signal was enhanced by the use of TSA-indirect (NEN) and Streptavidin-Cy2 (Amersham). Embryos were examined under a confocal microscope (MRC1024, Bio-Rad).

Germline clonal analysis was done by the FLP-DFS technique (Chou and Perrimon, 1992). Mutant females were mated to males carrying mutant chromosomes balanced over one of the blue balancers to distinguish embryos with or without paternal gene products.

To investigate the involvement of DER in neurogenesis, we examined mutant phenotypes of DER and its activator rho in various stages of neurogenesis. As previously shown (Whiteley et al., 1992, Yagi and Hayashi, 1997), the dorsoventral subdivision of the neuroectoderm in stage-6 embryos is detectable by expression of esg (Fig. 1A), which is expressed in the lateral (arrowhead) and medial (arrow) columns but not in the intermediate column. A loss-of-function, temperature-sensitive mutation of DER (flb^IF26) and the null mutation of rho (rho^del1) were used for analysis throughout this work. flb and rho mutations caused ectopic expression of esg in the intermediate column when cultured at 25°C (Fig. 1B,C). This phenotype was highly penetrant and was also observed in flb embryos cultured at 18°C. No further enhancement of this phenotype was observed in flb^IF26 embryos cultured at 29°C (Yagi and Hayashi, 1997) where the mutants show null phenotypes in the epidermis (Clifford and Schüpbach, 1992) and in null flb^IK35 embryos (data not shown), suggesting that repression of esg in the intermediate column requires a relatively high dose of DER signal. To examine the potential role of DER in neurogenesis, we studied expression of the proneural genes ac and sc, which begin expression in the neuroectoderm of stage-7 embryos in a DV pattern of expression similar to that of esg in the previous stage (Fig. 1D,G). In flb and rho embryos, ac and sc become ectopically expressed in the intermediate column (Fig. 1E,F,H,I). This phenotype was less penetrant and, occasionally, gaps of ac and sc expression were observed in the intermediate column. Since sc expression was similarly derepressed in flb^IK35 embryos, these phenotypes are likely to represent the near null phenotype of DER in the neuroectoderm. These data indicate that, in the intermediate column, the DER signal represses not only esg but also proneural genes, which are known to play key roles in neurogenesis. Effect of DER on neuroblast formation was monitored by the neuroblast marker Snail (Sna, Fig. 1J-L). Anti-Sna staining revealed three columns of SI neuroblasts in the control embryo, in which the intermediate column is distinguishable by the delayed onset of formation and number of Sna-positive cells (Fig. 1J, white arrowhead). In flb and rho mutants, Sna-positive neuroblasts in the intermediate position were frequently missing (Fig. 1K,L, white arrowhead), with higher frequency in flb embryos. In rho mutant embryo, frequency of the loss of intermediate column neuroblasts was variable between embryos.

To further examine the effect of the loss of DER signaling on the late events of neurogenesis, we traced the progeny of 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 (Fig. 1M). Loss of RP2 neurons in stage 13 was observed (over half the case examined) with the frequency of loss slightly higher in flb than in rho (Fig. 1N,O, white arrowhead), reflecting the earlier defect in neuroblast formation in stage 9. The variability in the flb phenotype observed in stage 9 and 13 might be due to the hypomorphic nature of the flb^IF26 allele used, or to a limited requirement for DER in the late stage of neurogenesis.

It is known that Ras-MAPK signaling cascade is the major target of DER in many tissues (Rogge et al., 1991; Tsuda et al., 1993; Diaz-Benjumea and Hafen, 1994; Hsu and Perrimon, 1994). To understand whether Ras-MAPK signaling also mediates the DER signal in the neuroectoderm and to determine the relative contribution of each component of the pathway, we examined the expression of esg and sc in embryos that lacked one of the Ras-MAPK signaling components. We studied the phenotype of mutants lacking either Sos, Ras1, Draf or Dsor1. To remove the maternal store of those gene products, we used the FLP-DFS technique developed by Chou and Perrimon (1992). As in wild-type embryos, embryos mutant for any of the four genes examined expressed 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 were expanded to the terminus, consistent with the previous report that Ras-MAPK is required for the terminal fate specification controlled by Torso receptor tyrosine kinase (Hou et al., 1995; Tsuda et al., 1993; Fig. 2). The terminal-loss phenotype was stronger in Draf and Dsor1 mutants than in Sos and Ras1 mutants, confirming the results of previous analysis of huckebein expression (Hou et al., 1995; Tsuda et al., 1993). In the trunk, initial subdivision into amnioserosa, dorsal ectoderm, neuroectoderm and mesoderm appeared normal, based on the expression pattern of esg in amnioserosa and in neuroectoderm. This observation suggests that global DV patterning by the gradient of Dorsal protein (Roth et al., 1989; Rushlow et al., 1989; Steward, 1989) does not require Ras-MAPK. All mutants exhibited specific defects within the neuroectoderm where esg expression was derepressed in the intermediate column. Essentially the same phenotype was also observed with sc expression, suggesting the loss of Ras-MAPK signaling has the same consequence as the loss of DER. In contrast to the case of the terminal-loss phenotype, all four mutants showed qualitatively the same phenotype in the neuroectoderm. The neuroectoderm phenotype in Ras1 mutants was 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. We also examined embryos mutant for Gap1^BQ, the negative regulator of Ras, but found no significant change in esg and sc expression (not shown). In later stages, the mutant embryos showed defects in germ band extension and many embryos showed defects in gastrulation, precluding further analysis of neurogenesis.

rho is initially expressed in the medial half of neuroectoderm (Bier et al., 1990), but repression of esg, ac and sc transcription by DER and Ras-MAPK occurred only in the intermediate column, posing a question as to whether the site of MAPK activation and the transcriptional repression exactly correspond or not. 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 showed that dpMAPK is distributed in a broad domain in the neuroectoderm in stage 5-7 embryos (Gabay et al., 1997a,b). The result was confirmed here, which showed that dpMAPK is distributed in an 8- to 1-cell-wide area in the neuroectoderm in stage-5 embryos and becomes restricted to the ventral region at the end of gastrulation (Fig. 3A-C,M). 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. We therefore used the protocol of Goto and Hayashi (1997) to double label embryos with dpMAPK and antisense RNA probes to study the spatiotemporal relationship between expression of dpMAPK, its activator rho and its downstream target, esg. Initial expression of dpMAPK overlapped with that of rho in stage-5 embryos (Fig. 3D); dpMAPK expression remained in this broad domain when rho expression became restricted to the medial column at gastrulation in stage 6 (Fig. 3E), and finally narrowed down to a 2- to 3-cell-wide stripe that abutted the stripe of rho at stage 7 (Fig. 3F). Comparison with the mesodermal marker sna showed that the ventral border of dpMAPK expression abutted the neuroectoderm-mesoderm border (Fig. 3G-I). Examination of histochemically stained material revealed a sharp ventral border of dpMAPK expression, which gradually declined in the dorsal direction, resembling the pattern of rho expression (Fig. 3M). In flb^IF26 mutant embryos cultured at 25°C, dpMAPK staining was not detectable (data not shown). These results demonstrate that MAPK activation in the neuroectoderm is dependent on DER 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 (Schnepp et al., 1996) might be activating DER.

We next determined the position of the dorsal limit of dpMAPK expression relative to the three separate columns of neuroectoderm revealed by esg expression. In stage 5, the dorsal limit of dpMAPK reached halfway within the intermediate column (Fig. 3J) and subsequently retracted to the medial column in stage 6 and 7 (Fig. 3K,L). 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. We conclude that transcription of esg is repressed by a marginal level of MAPK activation.

Why did 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 was shown to be required for expression of ac (Skeath et al., 1994). We examined expression of esg and sc in vnd null mutant embryos and found that their expression in the medial column was lost (Fig. 4A,B). To understand how vnd controls gene expression in the medial column, we searched for a target for vnd in the cis-regulatory regions of esg enhancer. Expression of esg is regulated by the neurogenic enhancer, which was divided into two regions, the activator region, which mediates activation in the entire neuroectoderm, and the repressor region, which mediates DER-dependent repression (Yagi and Hayashi, 1997; Fig. 4F). We thus examined expression of the esg-lacZ fusion genes in the vnd mutant background. The construct esg-lacZ D1 containing the complete neurogenic enhancer reproduced neuroectodermal expression of esg and was regulated by vnd in the same manner as esg (Fig. 4C,D; Yagi and Hayashi, 1997). In contrast, the construct esg-lacZ D5 lacks the repressor region for the DER- mediated regulation and was expressed in all three columns (Fig. 4E; Yagi and Hayashi, 1997). We collected embryos from a cross between vnd⁶/FM7ftz-lacZ females and y w; esg-lacZ D5 males, and detected lacZ expression. Although half of ftz- lacZ⁻ embryos were expected to be vnd⁻, no alteration of esg- lacZ D5 expression pattern was observed in 23 embryos scored. We thus conclude that vnd does not regulate esg-lacZ D5 and that the target site for vnd regulation is included in the repressor region.

To ask whether vnd affects the pattern of DER activation, we examined expression of rho in vnd mutants but found no change in its expression pattern in stage 5-7 embryo (data not shown). Similarly, vnd expression pattern was not altered in flb and rho mutants (flb^IF26, flb^IK35, rho^del1) in stage 6 embryo (data not shown; Gabay et al., 1996), suggesting DER and vnd do not cross-regulate each other in this stage. These results indicate that vnd is not involved in activation of esg or DER, but rather in counteracting the negative effect of DER.

Expression of msh starts at stage 6 in the lateral column of the neuroectoderm and is required for proper cell fate determination of neuroblasts formed in the lateral column in later stages (Isshiki et al., 1997). Upon loss of DER function, expression of msh expands to the intermediate column (D’Alessio and Frasch, 1996), suggesting that msh is negatively regulated by DER. To determine whether msh is involved in lateral fate determination in the neuroectoderm in the pregastrulation stage, we examined the effect of a loss and a gain of msh function on expression of esg. We did not observe any alteration of esg expression in msh null mutants or in embryos expressing msh under the control of the Krüppel or daughterless enhancer up to stage 8 (data not shown). We therefore conclude that, while msh is regulated by DER, msh by itself does not contribute to the subdivision of the neuroectoderm in the pregastrulation stage.

DER signaling initially occurs in a broad domain in the neuroectoderm in stage 5-6 embryos (Gabay et al., 1997a,b; this work) and later becomes restricted to the midline, where it continues to affect the patterning in the ventral ectoderm (Raz and Shilo, 1993; Kim and Crews, 1993; Golembo et al., 1996, Gabay et al., 1996) and CNS (Skeath, 1998). The relationship between genes involved in DER signaling also changes with time (Gabay et al., 1996). Here we will limit our discussion to the early (stage 5-6) function of DER in neurogenesis.

Insect neuroblasts are selected from a group of equipotential cells in the neuroectoderm and acquire a specific fate according to their position (Doe, 1992). In order to obtain a fixed number of neuroblasts with a specific repertoire of cell identity, it is essential that the neuroectoderm be subdivided into domains in a reproducible manner, so that a single neuroblast is selected from each of the domains. Failure in this subdivision process is expected to cause misspecification or a loss of neuroblasts. In this work, we investigated the mechanism of DV patterning in the neuroectoderm by studying the function of DER. Upon a loss of DER function, the intermediate column neuroectoderm was incorrectly specified as evidenced by ectopic expression of esg and proneural genes ac and sc (Fig. 1B,C,E,F,H,I), leading to a failure in the formation of intermediate column neuroblasts (Fig. 1K,L,N,O). The activated form of MAPK was detectable in the intermediate column neuroectoderm in stage 5-6 embryos (Fig. 3), but was not detectable at this position or in intermediate column neuroblasts later in stage 7. From these observations, we propose that DER acts in the neuroectoderm to provide the positional information necessary to specify neuroblasts that are formed at a later stage.

The ectopic expression of esg, ac and sc in the neuroectoderm of DER mutant suggests that the intermediate column of neuroectoderm acquired characteristics of either the lateral or medial column. D’Alessio and Frasch (1996) reported that msh expression in the lateral column expands into the intermediate column. Gabay et al. (1996) and this work reported that expression of vnd in the medial column in the blastoderm stage did not change in flb mutants. This suggests that intermediate column in DER signal mutants is lost and is replaced by expanded lateral column. On the contrary, we previously observed that, in Star (s) and spi mutants, where DER activity is mildly reduced, both medial and lateral expression of esg expanded into the position of the intermediate column (Yagi and Hayashi, 1997). These results suggest that a loss of DER function leads to a loss of the cell fate of the intermediate column, which is replaced by that of the lateral and medial columns. Such expanded lateral (and medial) columns give rise to a single column of neuroblasts in a timing and pattern characteristic of lateral (and medial) SI neuroblasts. We currently do not understand why the expanded lateral column neuroectoderm produced only one set, instead of two, of neuroblasts despite expression of proneural genes ac and sc.

A variety of evidence suggests that a high dose of MAPK activation is required to correctly specify the intermediate column neuroectoderm in stage 5-6. First, embryos of the temperature-sensitive allele of DER (flb^If26) cultured at 18°C showed a fully penetrant phenotype of ectopic expression of esg in the intermediate column. Second, paternal copies of the genes of the Ras-MAPK pathway (Sos, Ras1, Draf, Dsor1) failed to rescue the phenotype. Third, removal of rho was sufficient to derepress esg, ac and sc expression in the intermediate column, although vein, encoding a putative ligand of DER, is also expressed in the neuroectoderm in stage 6 and can potentially activate DER (Schnepp et al., 1996). In addition, loss of Sos, Ras1, Draf or Dsor1 showed the same severity of phenotype in the neuroectoderm (Fig. 2), in contrast to their effect on terminal patterning where loss of Draf or Dsor1 had a greater effect compared to the loss of Sos or Dras1. This would indicate that the levels of Sos and Ras1 that are insufficient to fully promote the terminal patterning are sufficient to shut down the DER signal and repress esg and sc. We have shown that expression of dpMAPK forms a gradient, the highest level being detectable at the ventral side, with a reduction in the dorsal direction, to cover the ventral half of the intermediate column of the neuroectoderm, reflecting the graded expression pattern of its activator rho (Bier et al., 1990). This indicates that transcription of esg, ac and sc is repressed by a marginal level of activated MAPK. Insufficient activation of DER or Ras-MAPK would be expected to restrict the domain of MAPK activation, which leads to a failure to repress esg, ac and sc in the intermediate column. We suggest that a high dose of DER signaling is required to expand the domain of MAPK activation to cover the intermediate column of the neuroectoderm.

In contrast to the near absolute requirement for DER for repression of esg, ac and sc, neuroblast formation in the intermediate column was not completely eliminated by flb^IF26or rho^del1mutations. One possibility is that an additional DER activator such as the putative DER ligand Vein (Schnepp et al., 1996), appears slightly later than rho to stimulates DER to partially rescue the phenotype of flb^IF26or rho^del1. This idea is consistent with our previous observation in spi and S mutants (Yagi and Hayashi, 1997). In these mutants, initial expression of esg in the intermediate column neuroectoderm was partially derepressed till stage 6, but returned to the normal pattern in stage 7, suggesting delayed onset of an additional DER activation partially complemented the defect.

pointed (pnt) and yan are known to encode transcription factors that are targets of the MAPK pathway in the eye and ventral ectoderm (O’Neill et al., 1994; Gabay et al., 1996), and the expression pattern of pnt and yan in stage 5-6 showed a gradient in the neuroectoderm (Klämbt, 1993; Gabay et al., 1996). However, zygotic loss of either of the genes did not alter the pattern of esg expression (Yagi and Hayashi, 1997). Therefore either maternal products of pnt and yan, or some unknown factor act as a downstream transcription factor of MAPK in the early neuroectoderm.

Here we consider possible regulatory mechanisms of DV patterning of the neuroectoderm based on the current results as well as on our previous results from an analysis of cis-acting elements in the esg promoter (Yagi and Hayashi, 1997). Our previous study showed that all aspects of esg expression in the neuroectoderm are reproducible by the action of its neurogenic enhancer, which is composed of activator and repressor regions. The activator region potentially promotes transcription of esg throughout the circumference of stage 5-6 embryos, but is normally repressed in the dorsal ectoderm and amnioserosa by decapentaplegic (dpp), and in the mesoderm by sna and twist (twi). The repressor region is the target of DER-dependent repression in the intermediate column. Identification of separate cis-acting elements for activation and repression excludes a possibility of DER acting directly upon transcription factors activating transcription. It is also unlikely that dpp, sna or twi acts at a distance to affect esg expression within the neuroectoderm, since the dorsal and ventral borders of the intermediate column defined by the expression of esg were not altered in any of these mutants (Yagi and Hayashi, 1997). To explain the subdivision of the neuroectoderm by DER, we previously proposed that a combination of opposing gradients of DER ligand and receptor creates a peak of DER activity in the intermediate position (Yagi and Hayashi, 1997). This model, however, proved not to be correct because this and other works showed that the peak of DER activity detected with anti- dpMAPK antibody is located in the medial column (Gabay et al., 1997a,b, this study).

Given the results of the present work showing that vnd counteracts the negative regulatory effect of DER, we propose a model shown in Fig. 5A. Gradient of nuclear localized Dorsal protein induces expression of dorsoventrally regulated genes such as dpp, sna, twi, which determines the extent of the neuroectoderm, and the nested expression domains of rho 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 DER 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 repress 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 DER 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 we propose that the primary role of DER signal in this stage is to define the intermediate domain to the neuroectoderm which is otherwise separated into two domains. It is possible that DER signal and vnd have later roles in promoting neuroblast formation in the intermediate and in the medial column, respectively.

This study revealed a new aspect of the regulatory hierarchy between DV pattering genes. Based on expression timing and interaction, we grouped the four genes expressed in the neuroectoderm into 2 classes (Fig. 5B). Maternal DV genes instruct the formation of the nuclear Dorsal protein gradient (Chasan and Anderson, 1993), which controls expression domains of zygotic class 1 genes in stage 5-6 embryos (rho, Ip et al., 1992; vnd, Mellerick and Nirenberg, 1995). Expression of class 2 genes are regulated by class 1 genes (esg, this study; msh, D’Alessio and Frasch 1996). Class 1 gene rho and vnd do not regulate each other’s expression, and class 2 gene msh does not regulate esg expression (this study). We infer that msh does not regulate class 1 genes, because class 1 gene-dependent expression of esg was unaltered in msh mutants. Later in stage 8, proneural genes starts to be expressed in a class 1 gene- dependent manner (Skeath et al., 1994; this study). The role of class 2 genes on proneural gene expression is likely to be in later stage of neural cell specification. msh was shown to affect neuroblast formation and identity (Isshiki et al., 1997). Role of esg in neurogenesis is less clear at this moment but it was shown that Esg modulates Scute-dependent transcriptional activation in tissue culture cells (Fuse et al., 1994) and inhibits neurogenesis when expressed in proneural cells (N. Fuse and S. H., unpublished result). Use of early neuroectodermal markers enabled us to recognize the earliest role of DER in development and the regulatory hierarchy between zygotic DV genes. Further analyses should reveal a link between the flow of DV positional information and determination of position specific characters of the neuroblast.

We thank Drs Y. Hiromi, Y. Nishida, H. Okano, A. Nose, J. Skeath as well as Bloomington Stock Center and Tübingen Stock Center for fly stocks, Drs J. Skeath, C. V. Cabrera, M. Okabe, H. Okano, N. Fuse, A. Nose for DNA fragments, Drs R. Reuter and Y. Hiromi for antibodies. We are grateful to Drs Y. Hiromi, M. Okabe and Jim Skeath for comments on the manuscript. We also thank Jim Skeath for discussion and communication of his results prior to publication. This work was supported by grants from Japan Society for the Promotion of Sciences (Research for the Future) and from Program for Promotion of Basic Research Activities for Innovative Biosciences.