The Drosophila central nervous system derives from neural precursor cells, the neuroblasts (NBs), which are born from the neuroectoderm by the process of delamination. Each NB has a unique identity, which is revealed by the production of a characteristic cell lineage and a specific set of molecular markers it expresses. These NBs delaminate at different but reproducible time points during neurogenesis (S1-S5) and it has been shown for early delaminating NBs (S1/S2) that their identities depend on positional information conferred by segment polarity genes and dorsoventral patterning genes. We have studied mechanisms leading to the fate specification of a set of late delaminating neuroblasts, NB 6-4 and NB 7-3, both of which arise from the engrailed (en) expression domain, with NB 6-4 delaminating first. In contrast to former reports, we did not find any evidence for a direct role of hedgehog in the process of NB 7-3 specification. Instead, we present evidence to show that the interplay of the segmentation genes naked cuticle (nkd) and gooseberry (gsb), both of which are targets of wingless (wg) activity, leads to differential commitment to NB 6-4 and NB 7-3 cell fate. In the absence of either nkd or gsb, one NB fate is replaced by the other. However, the temporal sequence of delamination is maintained, suggesting that formation and specification of these two NBs are under independent control.
The specification of unique cell fates as a function of their position within a developing organism is a fundamental process during the development of multicellular organisms. The development of the Drosophila central nervous system (CNS) serves as an ideal model system to elucidate mechanisms that link pattern formation to cell-type specification (Skeath, 1999). The fly CNS is derived from a population of neural stem cells, the neuroblasts (NBs), which delaminate from the neurogenic region of the ectoderm into the interior of the embryo. These NBs are formed in a segmentally repeated pattern (Hartenstein and Campos-Ortega, 1985), each having a unique identity (Doe, 1992), which leads to the formation of a specific set of neurons and/or glial cells (Bossing et al., 1996; Schmidt et al., 1997). In the thoracic and abdominal region, delamination of NBs occurs in five waves, S1-S5, and after S5 each hemisegment contains a subepidermal layer of 30 NBs (Doe, 1992).
The early NBs, delaminating during S1 and S2, form an orthogonal array of four rows (2/3,4,5,6/7) and three columns (medial, intermediate and lateral). Work carried out so far suggests that specification of these NBs is based on a combination of positional information along the anteroposterior (A/P) and the dorsoventral (D/V) axes (reviewed by Bhat, 1999; Skeath, 1999). For example, positional cues provided by segment polarity genes like gooseberry (gsb), wingless (wg) and engrailed (en) establish identities of cell rows along the A/P axis of the neuroectoderm, which is a prerequisite for the formation of specific S1 and S2 neuroblasts within each hemisegment (Bhat, 1996; Bhat and Schedl, 1997; Chu-LaGraff and Doe, 1993; McDonald and Doe, 1997; Skeath et al., 1995; Zhang et al., 1994).
However, about half of the NBs delaminate in the later segregation waves (S3-S5) and acquire a different identity, despite the fact that many originate from positions similar to the early NBs. Additionally, the three-column and four-row arrangement pattern is only transitory during early stages of neurogenesis and is obscured by late emerging neuroblasts (Doe and Goodman, 1985; Goodman and Doe, 1993). As a first step to understand how late delaminating NBs are specified, we have concentrated on studying the function and interactions of segment polarity genes within a specific neuroectodermal region: the En expressing domain. This domain gives rise to row 6 and row 7 NBs and is under the influence of the segmentation genes wg and hedgehog (hh). Wingless, which is a secreted protein, is expressed in row 5 and influences the specification of the fate of NBs in row 5 and in the adjacent rows 4 and 6 of the neuroectoderm (Chu-LaGraff and Doe, 1993). However, the maintenance of En expression in row 7 is also dependent on the Wg signal, so that the question arises as to how row 6 and row 7 NBs become differently specified.
To investigate this, we have chosen the S3 neuroblast NB 6-4 in row 6 and the S5 neuroblast NB 7-3 in row 7 as ‘model’ NBs. Both NBs are missing in embryos mutant for en (Lundell et al., 1996). We show, that in contrast to what was proposed earlier (Matsuzaki and Saigo, 1996) Hh, which is co-expressed in the En domain (Tabata et al., 1992), has no direct role in the formation or specification of any of these NBs. Instead, we provide evidence that Wg is the key player in this process. We show that the activity of the segment polarity gene naked cuticle (nkd), which is a target of the Wg pathway (Zeng et al., 2000), specifically inhibits the other Wg target gene gsb (Li and Noll, 1993; Hooper, 1994) in the posterior En domain, but does not affect en expression itself. The combined expression of gsb and en in the anterior En domain leads to the specification of NB 6-4, while the repression of gsb in the posterior En region is necessary for NB 7-3 identity. Furthermore, our analysis reveals that the mechanisms controlling the timing of delamination of these neuroblasts seem to be independent from those controlling their specification.
MATERIALS AND METHODS
Antibody staining and dissection of embryos were carried out as previously described (Nose et al., 1992; Patel, 1994). The following primary antibodies were used: mouse anti-Invected at 1:3 dilution; rabbit anti-Eagle at 1:1000 dilution; mouse anti-Eagle; rabbit anti-Repo at 1:1000 dilution; rabbit anti-Eyeless at 1:100 dilution; rat anti-Gooseberry-distal at 1:3 dilution; and rabbit anti-β-Galactosidase (Cappel) at 1:1000 dilution.
The following fly strains were used: Oregon R (wild type), hhAC, wgCX4, enE, ptcH84, gsbIIX6 (all described by Bhat and Schedl, 1997), sggm1H FRT101 (a gift from K. Basler), nkd2, Hs-en (both strains from Bloomington stock center), en-Gal4 (a gift from A. Brand), UAS-nkd (a gift from M. Scott), gsbIIX6;nkd2 and UAS-wg;hhAC (a gift from B. Sanson).
Heat shock protocols
Embryos from 1 hour egg layings were collected on apple juice agar (2% agar) plates and aged accordingly at 25°C to the required stage. The heat pulse was then given at 37°C for 20 minutes followed by a recovery phase of 15 minutes at 25°C and again a heat pulse of 20 minutes at 37°C. After this the embryos were aged at 25°C to stage 14-15 and then fixed for immunostaining.
Staging and mounting of embryos
Embryos were staged according to standard morphological markers (Hartenstein and Campos-Ortega, 1985). After antibody staining, the embryos were dissected so that the CNS was exposed and mounted in 70% glycerol in phosphate-buffered saline (PBS).
The analysis of embryos was carried out on a Zeiss Axioplan microscope mainly using Normaski optics. Embryos labelled with fluorescent dyes were analysed with a Leica TCS confocal microscope. Quantitative analysis such as cell counts were made using 63× or 100× oil objectives. Non-fluorescent images were digitally recorded with a CCD video camera. Combination of different focal planes in Figs 2D-F, 4A-F was carried out using Adobe Photoshop 5.1.
We were interested in clarifying how late delaminating neuroblasts in row 7 become specified differently from those in row 6, as both rows are lying within the En domain. For our investigations, we focused on two late neuroblasts in the En stripe: NB 6-4 in row 6 and NB 7-3 in row 7, which give rise to distinct types of lineages (Bossing et al., 1996; Schmidt et al., 1997). NB 6-4 characteristically generates glia that can be identified by the co-expression of eagle (eg) and reversed polarity (repo) (M-CBG and MM-CBG; Ito et al., 1995; Dittrich et al., 1997; Halter et al., 1995), whereas NB 7-3 typically generates serotonergic neurons (Lundell et al., 1996; Dittrich et al., 1997) and can be identified with anti-Eyeless (Ey) or anti-Eg, in combination with anti-En antibodies. As the segmentation genes wg and hh influence the En domain, we analysed the individual role of these genes in detail, in addition to the role of En.
En is a key factor for NB 7-3 formation and Hh has no independent role in this process apart from En maintenance
The only factor known so far, to distinguish row 6 from row 7 is Gsb, which is expressed in row 6 neuroectoderm. It is also known that Gsb is a target of the Wg signalling cascade and specifies the identities of neuroblasts in rows 5 and 6 (Skeath et al., 1995). However, as row 7 is also under the influence of the Wg signalling, how is Gsb prevented from being expressed here? One mechanism that could be involved in this process is Hh signalling, because previous work by Matsuzaki and Saigo (Matsuzaki and Saigo, 1996) has postulated that NB 6-4 and NB 7-3 show differences in their dependence on Wg and Hh signalling: NB 6-4, which originates from the anterior En stripe, was missing in wg as well as in hh mutant embryos, whereas NB 7-3, which delaminates around 30 minutes later from the posterior En stripe, always appeared to be present in the absence of Wg or Hh alone, but was no longer found in a wg;hh double mutant. Based on these results, it was proposed that Wg and Hh signalling pathways converge or compensate for each other to specify NB 7-3 fate, while both Wg and Hh are equally important for NB 6-4 formation (Matsuzaki and Saigo, 1996). One important consequence of this would be that Hh could have an autocrine function in specifying NB 7-3. However, as both NBs delaminate from the En-positive neuroectodermal domain and En activity represses ptc (patched; Hidalgo and Ingham, 1990), the only known receptor binding to Hh directly (Marigo et al., 1996), such an autocrine function of Hh would at least need a different receptor. We therefore investigated in more detail the role Hh plays in the formation and/or specification of NB 6-4 and NB 7-3. We chose null mutant alleles of wg and hh for our investigations, whereas previously (Matsuzaki et. al., 1996) a hypomorphic allele of the hh gene was used. We found that the formation of NB 7-3 is affected in both wg and hh single mutants. NB 7-3 is missing in 75% (n=88) of wgCX4 and in 40% (n=202) of hhAC mutant hemisegments counted (Fig. 1D,E). These results also show that the effect on NB 7-3 is more severe in wgCX4 than in hhAC mutant embryos. As En expression is fading away earlier in wgCX4 (∼stage 8) than in hhAC mutant embryos (∼stage10) (Bejsovec and Wieschaus, 1993), we assume that the number of remaining NB 7-3 correlates with the degree of the residual En expression. Indeed, embryos that are deficient for en and inv (invected, a homeobox gene that shows some functional redundancy to en) show that NB 7-3 is missing in 100% (n=50) of the hemisegments counted (Fig. 1C). These results suggest that NB 7-3 formation needs Hh indirectly for the maintenance of En expression via Wg. We confirmed this by analysing hhAC mutant embryos in which Wg was ectopically expressed within the En domain using EnGal4 as a driver of UAS-wg. In these embryos, the dependency of Wg expression on Hh is uncoupled and therefore En expression was rescued (Sanson et al., 1999). In accordance with our hypothesis, these embryos show a very efficient rescue of NB 7-3 to 95% (n=66) of the hemisegments counted (Fig. 1F). Thus, under these conditions NB 7-3 does not need any additional input by the Hh signalling pathway to be formed and specified. We conclude that NB 7-3 normally requires Hh only for maintenance of Wg expression, which in turn leads to En maintenance.
Naked Cuticle activity is essential for NB 7-3 identity
Having shown that Hh is not involved in the differential regulation of Gsb we decided to look at inhibitors of the Wg signalling cascade: naked cuticle (nkd) and shaggy (sgg). Analysis with anti-Ey antibody showed that NB 7-3 specific Ey expression is missing in 83% of sgg mutant (n=92, data not shown) and in 81% of nkd mutant (n=80) hemisegments of embryos at stage 12-13 (Fig. 2B), although Eg-positive cells were always observed at the position of the NB 7-3 cluster using anti-Eg antibody (both NBs 6-4 and 7-3 are Eg positive). This suggests that the fate of NB 7-3 is mispecified (see below). By contrast, NB 6-4 was found to be always present in these mutants, as judged by anti-Eg and anti-Repo antibody staining.
We selected nkd mutants for further analysis, as Nkd (like Gsb) is a target of the Wg signalling cascade and is thought to establish a negative feedback loop by downregulating the Wg signal (Zeng et al., 2000). As a first step, we tested whether Gsb was derepressed in nkd mutations in regions from where NB 7-3 normally delaminates. Indeed, we found that while in the En domain of wild-type embryos, only row 6 NBs and NB 7-1 expressed Gsb (Skeath et al., 1995), in nkd mutants, the Gsb-expressing neuroectodermal region was broadened. As a result the more lateral row 7 NBs also expressed Gsb, which must include NB 7-3 (Fig. 3). Because, in this situation, row 7 is similar to row 6, it could have the ability to give rise to an additional ectopic NB 6-4. Staining of nkd mutant embryos with the glia specific anti-Repo antibody in combination with anti-Eg antibody indeed revealed an additional NB 6-4-like fate in 54% (n=40) of hemineuromeres counted (Fig. 2F). Co-expression of these markers is characteristic for NB 6-4 derived cells. To ensure that this is not due to secondary effects of the nkd mutation, we ectopically expressed Gsb in the En domain using the UAS/Gal4 system, which yielded the same result as nkd mutations: a replacement of NB 7-3 by an ectopic NB 6-4 in 52% (n=40) of the hemisegments (Fig. 2E).
That Gsb expression acts a switch between row 6 and row 7 identity in the En-positive neuroectoderm is additionally supported by earlier work analysing the role of Gsb in the CNS (Matsuzaki and Saigo, 1996; Patel et al., 1989), where it was shown that in hemineuromeres of gsb mutant embryos an additional NB 7-3 fate was formed. Taking this result further, we confirmed that this additional NB 7-3 fate is at the cost of NB 6-4, which is converse to the situation in nkd mutants. Analysis of gsb mutant embryos with anti-Ey antibody showed that a duplicated NB 7-3 was formed in 70% (n=82) of the hemisegments counted (Fig. 4D) and NB 6-4 markers were missing in 100% of hemisegments counted (data not shown). Additionally, in the absence of both Nkd and Gsb, ectopic NB 7-3 was found in 76% of hemisegments (n=86) (Fig. 4E). Similarly, 40% of the hemisegments (n=80) showed duplicated NB 7-3 fate when Nkd was ectopically expressed in the En domain using the UAS/Gal4 system (Fig. 4F). Thus, we conclude that NB 7-3 formation, as opposed to formation of NB 6-4, requires the absence of Gsb, which is inhibited by Nkd function. Taken together the above results suggest that row 6 and 7 neuroectoderm can potentially produce NBs with the same identities, and that the differential effects of Wg signalling are responsible for bringing about the different fates of the two late NBs from this region.
NB 6-4 and NB 7-3 specification is independent of time of NB formation
The above results show that in nkd mutants, an extra NB 6-4 is formed in the position of NB 7-3. As NB 6-4 normally delaminates earlier than NB 7-3, the question arises as to when the duplicated NB 6-4 delaminates. In wild-type embryos, NB 6-4 delaminates during S3 (stage 10), followed by NB 7-3 in S5 (stage 11) from the En domain (Broadus et al., 1995; Doe, 1992). Therefore, embryos either mutant for nkd or expressing ectopic gsb in the En domain (EnGal4::gsb) of stage 10 and stage 11 were examined with anti-Eg antibody to look for the timing of NB duplication. In wild-type embryos at stage 10, Eg is detected only at the position of NB 6-4 and never at the position of NB 7-3 (Fig. 5A). At stage 11, Eg-positive cells are visible in the En domain at the position of NB 7-3, as well as NB 6-4. (Fig. 5C). Surprisingly, in EnGAL4::gsb embryos this temporal sequence is maintained: Eg is first detected at the position of NB 6-4 (Fig. 5B) and later at the position of NB 7-3 (Fig. 5D). Therefore, the ectopic NB 6-4 is delaminating at S5, at the time NB 7-3 would normally appear. Conversely, in gsb mutants, an extra NB 7-3 is formed at the cost of NB 6-4. This NB 7-3 is detected by anti-Ey antibody staining in embryos mutant for gsb at stage 10 (Fig. 4B), whereas in wild-type embryos, no Ey-positive cell is present in the En domain at this stage (Fig. 4A). Thus, this ectopic NB 7-3 delaminates at the time of NB 6-4. We conclude that, with respect to NB 6-4 and NB 7-3, the timing of NB formation appears largely independent of NB specification, and that the segmentation genes nkd and gsb are essential to bring about the specification of the two NB fates investigated.
Late ectopic En expression induces an ectopic NB 7-3 fate in row 3 neuroectoderm
As the above results suggest that the prerequisite for NB 7-3 fate specification is En in absence of Gsb expression, we examined embryos with ectopic En expression in order to see whether we could induce ectopic NB 7-3 cells outside of the normal En domain. A heat pulse given to Hs-en embryos just before the delamination of NB 7-3 (i.e. 5-6 hours after egg laying at 25°C) results in an ectopic NB 7-3 formation in 20% (n=100) of the hemisegments, based on anti-Eg antibody staining (Fig. 6C). This ectopic NB 7-3 is also able to give rise to characteristic progeny cells. We detected additional serotonergic neurons in 20% (n=68) of the hemineuromeres counted in first instar larvae (Fig. 6H). The ectopic NB 7-3 seems to be formed at the cost of the Eg-positive NB 3-3 of row 3, as we find a loss of the Eve-positive EL cells, which are progeny of NB 3-3 (Fig. 6E; Schmidt et al., 1997). No additional NB 7-3 cluster is seen when the heat pulse is given between 4 and 5 hours after egg laying at 25°C (Fig. 6B), although the CNS is very malformed. Additionally, we analysed ptc mutant embryos, as a derepression of En in an ectopic stripe is seen in such mutants (DiNardo et al., 1988). Ptc, which is a receptor for Hh, is expressed in rows 2-5 of the neuroectoderm (Bhat, 1996). Ptc activity represses En expression (Hidalgo and Ingham, 1990), which results in a mutually exclusive gene expression pattern with respect to these two genes. We found that a NB 7-3 like fate was formed ectopically in 50% of ptc mutant hemineuromeres (n=60, see also Patel et al., 1989), which is similar to the ectopic expression experiments using Hs-en. Again this seems to be at the cost of NB 3-3, as 60% of the EL cells are missing in these mutant embryos as well (Fig. 6F). This is in accordance with the observation that the ectopic En stripe in ptc mutant embryos is in the region where the row 3 NBs delaminate (data not shown). Taken together these results suggest that in wild type, Ptc represses En expression in row 3 neuroectoderm, thereby enabling the specification of late delaminating row 3 NBs. In the absence of Ptc function, at least some of these neuroectodermal cells acquire row 7 identity, owing to the presence of En and the absence of Gsb.
The impact of segmentation genes and D/V patterning genes has been well studied for the early segregating neuroblasts (S1 and S2). The expression of these genes defines three dorsoventral columns and four rows in anteroposterior direction, thus creating a Cartesian coordinate system that assigns unique fates to individual NBs as a function of their position in the neuroectoderm (Bhat, 1999; Skeath, 1999). This pattern is partially obscured by the late segregating NBs and the progeny of early NBs (Doe and Goodman, 1985; Goodman and Doe, 1993). As a consequence, late delaminating NBs might face a very different situation, considering that substantial morphogenetic movements take place. Therefore we asked how segmentation genes interact to confer unique identities on late delaminating NBs and whether NB formation and specification is tightly linked in this case. We selected a pair of late segregating NBs in the En domain, namely the S3 neuroblast NB 6-4 in row 6 and the S5 neuroblast NB 7-3 in row 7 for our analysis.
Segment polarity genes separate NB 6-4 and NB 7-3 fates
In the En domain Wg plays a role both in NB formation and NB specification (Chu-LaGraff and Doe, 1993). The homeodomain transcription factor En is a prerequisite for the formation of the NBs 6-4 and 7-3, because in its absence both NBs fail to form (Lundell et al., 1996; Matsuzaki and Saigo, 1996). As Wg signalling is necessary for maintaining En expression (Hidalgo and Ingham, 1990), it is also essential for the formation of these two NBs. In addition Hh is co-expressed in the En domain, but we found no evidence for a direct function of Hh with respect to the formation and specification of these NBs, as opposed to a previous report (Matzuzaki and Saigo, 1996). En maintains Hh expression in rows 6 and 7, and Hh in turn is essential for Wg expression in row 5, thereby constituting a maintenance loop (Bejsovec and Wieschaus, 1993; Heemskerk et al., 1991; Hidalgo, 1991). Thus, for late NBs in row 6 and 7, the expression of En is crucial and Hh is required to maintain En expression via Wg. However, for the separate specification of NB 6-4 and NB 7-3, differential regulation of two Wg targets, nkd and gsb, is essential (Fig. 7).
Wg is a diffusible molecule expressed in row 5 and acts on neighbouring rows, which include rows 6 and 7 (Chu-LaGraff and Doe, 1993). However, row 6 differs from row 7 as it expresses gsb, which is, as stated above, a target of Wg signalling (Fig. 7A). The fact that row 7 does not express gsb, despite being under the influence of Wg raises the question of how this differential regulation is brought about. In this work we have shown that Nkd is essential for this regulation. Recently, Nkd has been identified as a negative regulator of the Wg signal transduction pathway, itself being a target of this pathway (Zeng et al., 2000). We have found that in the absence of Nkd, Gsb is derepressed, owing to Wg hyperactivity in row 7, leading to the generation of an ectopic NB 6-4 like fate (Fig. 7C). Thus, the distinct identities of NB 6-4 and NB 7-3 are brought about by the interplay of Gsb and Nkd. For NB 6-4 specification, Gsb is an essential factor. In the absence of Gsb NB 6-4 fails to be specified (Matsuzaki and Saigo, 1996; Skeath et al., 1995) and instead takes the identity of NB 7-3 fate (Fig. 7B). Conversely, for NB 7-3 specification, a Gsb-free environment, which is created by the activity of Nkd, is essential. In summary, NB 6-4 needs the expression of Gsb and En, whereas NB 7-3 needs En but the absence of Gsb.
However, the fact that gsb as well as nkd are targets of Wg signalling makes it difficult to explain why gsb is repressed by nkd only in the posterior region of the En stripe. The posterior En domain is further away from the Wg source than the anterior En domain and therefore should receive a lower signalling input when compared with the anterior region. As a consequence, this should lead to higher Nkd activity in the anterior En cells, leading to a stronger Gsb repression in this region – the opposite of what we observe. A careful analysis of the expression pattern on the transcriptional level did not give any obvious clues to solve this apparent paradox (data not shown). We confirmed that during early germ band extension (stage 8-9) nkd transcription is nearly ubiquitous with higher RNA levels in the two to four cell rows posterior to the En stripe (Zeng et al., 2000). At late phase of germ band extension, nkd expression is most abundant anterior to the En stripe and lower just posterior to the En-stripe (stage 10-11; Zeng et al., 2000). No significant difference between the anterior and posterior En domain could be detected (data not shown). One explanation for the differential regulation of gsb could be that, owing to earlier pair rule gene activity of paired (Bouchard et al., 2000), the level of Gsb protein at the time of NB 6-4 delamination in the anterior En region is high enough to override repression by Nkd activity. Alternatively, a direct differential regulation of the two Wg targets that is due to the different levels of Wg signalling could be responsible for the observed regulatory differences. It could be that the regulation is such that the amount of Wg signalling within the En stripe causes a relatively homogenous level of nkd expression in this region. At the same time, the transcriptional activation of gsb could be more sensitive to Wg signalling levels, resulting in a very strong activation, especially near to the Wg-expressing cells. As a result, the relatively low Nkd activity in the whole En stripe might be able to inhibit gsb expression in the region of low gsb activation only: the posterior En domain. A hint that a differential regulation of Wg targets indeed exists comes from the Wg-dependent En regulation: it seems that a lower Nkd activity is sufficient to repress gsb but not to inhibit en expression. This conclusion was drawn from our finding that overexpression of nkd within the En stripe using an EnGal4 driver line led to a selective repression of gsb with no obvious effect on en expression itself. Clearly, additional work has to be carried out to clarify these points.
Row 3 has the potential to generate a late row 7 neuroblast
Besides row 6 neuroectoderm, row 3 neuroectoderm also has the potential to generate an ectopic NB 7-3. It has been shown previously that in embryos mutant for ptc, neuroectodermal cells in the area of row 3 begin to express En and additional serotonergic neurons can be found in these mutant embryos, which suggests the presence of an ectopic NB 7-3 like fate (Patel et al., 1989). We now show, additionally, that when En is ubiquitously expressed, only row 3 has the ability to give rise to an ectopic NB 7-3 fate. In all cases, this occurs at the cost of row 3 NBs such as NB 3-3. We think that this might reflect that row 3 neuroectoderm, which is right in the middle of the segment, represents something like a ‘ground state’ in the neuroectoderm: in this area neither Hh nor Wg signalling may take place. Therefore the decision to specify late row 3 or late row 7 NBs seems to be only dependent on the absence or presence of En, respectively.
Temporal aspects of NB specification
Previous work has indicated that genes expressed in proneural clusters are involved in specifying the individual fates of NBs that develop from these clusters (Chu-LaGraff and Doe, 1993; Matsuzaki and Saigo, 1996; Skeath et al., 1995). Our finding that NB 6-4 and NB 7-3 can be mutually transformed while the sequence of birth does not change suggests that the mechanism for the timing of late NB delamination is independent from mechanisms that regulate NB identity. This might be reminiscent of early NBs. Initiation of S1 NB formation requires the activity of proneural genes that have been shown to be dependent on pair-rule genes (Skeath et al., 1992). The identity of the NBs delaminating from these clusters, however, is dictated by the activity of segment polarity genes (Chu-LaGraff and Doe, 1993; Skeath et al., 1995). Thus, the control of proneural gene expression that enables NB formation and the control of segmentation genes conferring NB identity occurs in parallel. At later stages, pair-rule gene expression vanishes and can no longer be responsible for NB formation (Skeath et al., 1992). How is NB formation regulated in the following segregation waves? One possibility is that after the first segregation wave, NB formation and identity are more tightly linked; the finding that specific NBs like NB 4-2 are sometimes not transformed but missing in wg mutant embryos (Chu-LaGraff and Doe, 1993) seems to support this idea. However, our finding that the transformed NB 6-4 and NB 7-3 are delaminating according to the ‘old identity’ shows that, at least in these cases, NB formation and specification is independent. Our results favour the idea that the timing of the formation of proneural clusters within the neuroectoderm is generally independent of the segment polarity genes investigated here. This does not exclude permissive functions, such as those of En, which enable the proneural cluster formation as such. According to this hypothesis, intrinsic or extrinsic factors present in the position of the proneural cluster at the time of delamination govern the identities of the NBs. This might be not only true for the positional regulation of NB identity but also for the determination of NB identity along the temporal axis. Indeed, heterochronic transplantation experiments recently performed in our laboratory (Berger et al., 2001; in the same issue) strongly support the possibility that one or more extrinsic factors exist that lead to stage specific NB identities. It will be a challenge for the future to identify these factors, and to investigate whether similar mechanisms exist in higher organisms.
We thank Dr R. Holmgren for generously providing the anti-Gooseberry-distal antibody, Dr M. Frasch for the anti-Eve antibody, Dr Marc R. Freeman for the mouse anti-Eagle antibody and Dr U. Waldorf for providing the anti-Eyeless antibody. We are grateful to Drs K. Basler, A. Brand, B. Sansons and M. Scott for providing fly stocks. We also thank C. Berger, Dr L. Meadows and Dr A. Prokop for their critical comments on this manuscript. A special thanks to C. Rickert for the help with the images and to Dr O. Vef for the help with genetics. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to J. U. (UR 42/3-2).