The development of the central nervous system in the Drosophila embryo is initiated by the acquisition of neural potential by clusters of ectodermal cells, promoted by the activity of proneural genes. Proneural gene function is antagonized by neurogenic genes, resulting in the realization of the neural potential in a single cell per cluster. To analyse the relationship between proneural and neurogenic genes, we have studied, in specific proneural clusters and neuroblasts of wild-type and neurogenic mutants embryos, the expression at the RNA and protein levels of lethal of scute, the most important known proneural gene in central neurogenesis. We find that the restriction of lethal of scute expression that accompanies the restriction of the neural potential to the delaminating neuroblast is regulated at the transcriptional level by neurogenic genes.

These genes, however, do not control the size of proneural clusters. Morover, available antibodies do not provide evidence for an hypothetical posttranscriptional regulation of proneural proteins by neurogenic genes. We also find that neurogenic genes are required for the specification of the mesectoderm. This has been shown for neuralized and Notch, and could also be the case for Delta and for the Enhancer of split gene complex. Neurogenic genes would control at the transcriptional level the repression of proneural genes and the activation of single-minded in the anlage of the mesectoderm.

Neuroblasts, the progenitor cells of the central nervous system (CNS) in insects, delaminate from the embryonic neuroectoderm of Drosophila in a stereotyped manner that involves several waves of segregation (Hartenstein and Campos-Ortega, 1984; Doe, 1992). It has been shown, at least for thirteen neuroblasts that segregate in each hemisegment during the first two waves (S1 and S2), that their formation is initiated by the acquisition of neural potential by small groups of ectodermal cells, called proneural clusters (see Campos-Ortega, 1993, for a recent review). Proneural clusters in the embryonic neuroectoderm are defined by the expression of achaete (ac), scute (sc) and lethal of scute (l’sc), genes of the achaete-scute complex (AS-C) (Cabrera et al., 1987; Romani et al., 1987; Martín-Bermudo et al., 1991; Skeath and Carroll, 1992; Ruiz-Gómez and Ghysen, 1993). During segregation, each neuroblast accumulates the highest levels of AS-C proteins, whereas in the remaining cells of the proneural cluster these proteins gradually dissapear. This pattern of expression and the analysis of mutant phenotypes indicates that the function of the AS-C genes is to confer upon cells of the clusters the capacity to become neuroblasts (reviewed by Campuzano and Modolell, 1992; Campos-Ortega, 1993). The restriction of the neural fate to a single cell of a proneural cluster is mediated by neurogenic genes, through an inhibitory process that involves intercellular communication (reviewed by Ghysen et al., 1993). According to Cabrera (1990, 1992), the molecular outcome of the inhibitory process would be the accumulation of an active, unphosphorylated form of the AS-C proteins exclusively in the presumptive neuroblasts. This contrasts with another report which suggests that the control of the AS-C proteins is largely transcriptional (Skeath and Carroll, 1992).

It has been proposed that neurogenic genes constitute a functional cassette that mediates cell interactions in many developmental processes (Ruohola et al., 1991). One such process could be the specification of the mesectoderm in the early embryo, for the analysis of mesectoderm-specific gene expression in different mutants has lead to the suggestion that mesectoderm specification requires an inductive signal from the mesoderm (see Nambu et al., 1993, and references therein). Additional evidence for the induction derives from the ectopic transplantation of mesodermal cells, which results in the expression in the surrounding ectoderm of single-minded (sim), a master regulatory gene for mesectodermal development (Leptin and Roth, 1994; Nambu et al., 1991).

In this paper, we first attempt to clarify the discrepancies existing in the control of proneural gene expression by neuro-genic genes. Our results indicate that neurogenic genes principally regulate l’sc expression at the transcriptional level, without affecting the domains of proneural gene expression. Secondly, we analyse whether neurogenic genes intervene in the transmission of the inductive signal that leads to mesectoderm specification. The results indicate that N and neuralized (neu) are required for the establishment of the mesectodermal fate and suggest also that Dl and members of the Enhancer of split gene complex (E(spl)-C) may be involved in the same process.

Fly strains

The following Drosophila strains were used: wild-type Oregon-R, N55e11, neuIF65, Df(3R)DlFX3, Df(3R)E(spl)RB25.1 (the latter three mutations balanced over a TM3 lacZ chromosome), ovoD1 (Lindsley and Zimm, 1992), P[w+, sim-lacZ] (the sim promoter fused to lacZ, a gift from S. Crews) (Nambu et al., 1991), FRT101 and hsFLP38 (Chou and Perrimon, 1992).

Germ-line clones

Female germ-line clones were induced in N55e11/ovoD1 larvae by Xirradiation (Jiménez and Campos-Ortega, 1982), or using the FRT/FLP technique (Chou and Perrimon, 1992), as described by Menne and Klämbt (1994). Virgins of the appropriate genotype were crossed either to wild-type males or to P[w+, sim-lacZ] males, to study sim expression. Heterozygous N55e11/+ female embryos were recognized with an anti-Sex lethal antibody.

Immunohistochemistry

Two classes of anti-L’sc polyclonal antibodies were used. One class is a rat antibody raised against a fusion protein that contains the entire L’sc sequence, except the first fifteen amino acids (Martín-Bermudo et al., 1991). A second class are different antibodies raised against the synthetic peptide DDEELLDYISSWQE, corresponding to the C terminus of the translated sequence of l’sc (Alonso and Cabrera, 1988; Martín-Bermudo et al., 1993). Of this latter class, a rabbit serum was a gift from the late C. Cabrera. In addition, we raised and affinity purified similar antibodies in rats and in one rabbit, following essentially the protocol described by Cabrera (1990). Other antibodies used were: anti-Ac (from J. Skeath and S. Carroll), anti-β-gal (Cappel), anti-Sex lethal (from L. García-Alonso), anti-Hunchback (from P. Macdonald) and anti-Snail (from A. Alberga). Embryos were fixed and stained essentially as described previously (Martín-Bermudo et al., 1991, 1993). Staged embryos were used in most instances, and were collected at 20 minutes (early stage 8), or 35–40 minutes (late stage 8) after the onset of gastrulation at 25°C. In some cases, embryos embedded in Epon were sectioned at 5 μm.

Other procedures

In situ hybridization essentially followed the protocol of Tautz and Pfeifle (1989). After staining, embryos were dehydrated first through an ethanol series, then two times in acetone for a total of 3–5 minutes, transferred to a mixture of acetone:Epon (1:1) and inmediately spread over a microscope slide. Once the acetone had evaporated, the embryos were dissected and mounted in Epon.

Wild-type expression of lethal of scute during early neurogenesis

The detection of AS-C proteins during early neurogenesis with different antibodies led to different hypothesis about the control of AS-C expression (Cabrera, 1990, 1992; MartínBermudo et al., 1991; Skeath and Carroll, 1992). To clarify this issue, we first analysed in detail l’sc RNA and protein expression patterns during wildtype segregation of neuroblasts in stage 8. For simplicity, we restrict our description to the ventral-most region of the neuroectoderm, which gives rise to four S1 neuroblasts (2 –2, MP2, 5 –2 and 7 –1) per hemisegment (Doe, 1992).

l’sc RNA is found at early stage 8 in the proneural clusters of neuroblasts 2 –2, 5 –2 and 7 –1 (Fig. 1A). Neuroblast MP2 arises from a cluster expressing ac and sc but not l’sc (Martín-Bermudo et al., 1991; Skeath and Carroll, 1992; Ruiz-Gómez and Ghysen, 1993). At late stage 8, l’sc transcription has decayed in the neuroectoderm (Fig. 1B) but it is still active in neuroblasts 2–2, 5–2 and 7–1, which have just segregated (Fig. 1C). The pattern of L’sc protein accumulation closely follows that of transcription, as detected either with an antibody raised against the entire protein (Fig. 1D-F; see also MartínBermudo et al., 1991), or with any of those raised against a Cterminal peptide (Fig. 1G-I). These results strongly suggest that the control of the spatial distribution of L’sc is largely transcriptional.

Fig. 1.

Expression of l’sc in a wild-type embryo, during segregation of S1 neuroblasts, at the RNA (A-C) and protein (D-I) levels. (A) Detail of an early stage 8 embryo. Parallel and adjacent to the midline (marked by a vertical arrow in all pictures), in the region of the neuroectoderm that will give rise to the first four neuroblasts per hemisegment of the medial row (neuroblasts 2-2, MP2, 5–2 and 7–1), l’sc RNA is detected in consecutive groups of about fifteen ectodermal cells, separated by smaller groups of about five non-expressing cells. A group of l’sc-expressing cells is the composite of three individual, but contiguous, proneural clusters of 4–6 cells each (two cells along the anteroposterior axis and two to three cells along the dorsoventral axis). These are the clusters of neuroblasts 5–2 and 7–1 of an hemisegment and of neuroblast 22 of the following hemisegment, respectively. The non-expressing cells are the proneural cluster of neuroblast MP2. The extent of a segment is marked by a large bracket. (B) Shortly after segregation of S1 neuroblasts, l’sc RNA is still found in only 2–3 cells of clusters 2–2 and 5–2 and barely detected in cluster 7–1. (C) At a deeper plane of focus, RNA is detected in neuroblasts 22, 5–2 and 7–1. L’sc protein detection, with either an antibody against the entire protein (D-F), or with an antibody against a Cterminal peptide (provided by the late C. Cabrera) (G-I), yields essentially identical results, although the latter antibody stains more faintly. In both cases, the patterns of protein accumulation in the neuroectoderm, before (D,G), and after (E,H) neuroblast segregation, as well as in the neuroblast layer (F,I), closely correspond to the pattern of transcription. The embryo in E,F, slightly older than those in B,C and H,I, is beginning to express l’sc in the proneural cluster of the median neuroblast at the midline. Anterior is to the top. Scale bar, 20 μm.

Fig. 1.

Expression of l’sc in a wild-type embryo, during segregation of S1 neuroblasts, at the RNA (A-C) and protein (D-I) levels. (A) Detail of an early stage 8 embryo. Parallel and adjacent to the midline (marked by a vertical arrow in all pictures), in the region of the neuroectoderm that will give rise to the first four neuroblasts per hemisegment of the medial row (neuroblasts 2-2, MP2, 5–2 and 7–1), l’sc RNA is detected in consecutive groups of about fifteen ectodermal cells, separated by smaller groups of about five non-expressing cells. A group of l’sc-expressing cells is the composite of three individual, but contiguous, proneural clusters of 4–6 cells each (two cells along the anteroposterior axis and two to three cells along the dorsoventral axis). These are the clusters of neuroblasts 5–2 and 7–1 of an hemisegment and of neuroblast 22 of the following hemisegment, respectively. The non-expressing cells are the proneural cluster of neuroblast MP2. The extent of a segment is marked by a large bracket. (B) Shortly after segregation of S1 neuroblasts, l’sc RNA is still found in only 2–3 cells of clusters 2–2 and 5–2 and barely detected in cluster 7–1. (C) At a deeper plane of focus, RNA is detected in neuroblasts 22, 5–2 and 7–1. L’sc protein detection, with either an antibody against the entire protein (D-F), or with an antibody against a Cterminal peptide (provided by the late C. Cabrera) (G-I), yields essentially identical results, although the latter antibody stains more faintly. In both cases, the patterns of protein accumulation in the neuroectoderm, before (D,G), and after (E,H) neuroblast segregation, as well as in the neuroblast layer (F,I), closely correspond to the pattern of transcription. The embryo in E,F, slightly older than those in B,C and H,I, is beginning to express l’sc in the proneural cluster of the median neuroblast at the midline. Anterior is to the top. Scale bar, 20 μm.

lethal of scute expression in neurogenic mutants

To analyse, at the same level of resolution as above, how the expression of proneural genes is modified in neurogenic mutants, we studied the RNA and protein patterns of l’sc in mutants for neu, Dl, N, and the E(spl)-C. The analysis was restricted to the zygotic lack-of-function situation, except for N. In this case, N embryos were derived from homozygous N female germ line clones, for we found it necessary to eliminate the maternal contribution in order to detect mutant phenotypes during stage 8.

Proneural clusters in the four mutants, revealed by l’sc transcription, are basically normal (Fig. 2A). The only difference from the wild type is that the bilaterally symmetric clusters in neu (Fig. 2A) and N (not shown) embryos tend to fuse at the midline (see below). At late stage 8, l’sc transcription does not become restricted to the neuroblast and persists in entire clusters, which otherwise maintain their initial size (Fig. 2B).

Fig. 2.

Expression of l’sc in neurogenic mutants during segregation of S1 neuroblasts. (A) At early stage 8, the pattern of transcription in proneural clusters of a neu− embryo is essentially like that of the wild type (Fig. 1A), except for the presence of transcripts in the territory normally occupied by the mesectoderm at the midline (vertical arrow). (B) Unlike the wild type (Fig. 1B), transcription in neu− embryos is fully maintained in the clusters when S1 neuroblasts delaminate at late stage 8. (C-F) At that stage, the L’sc protein is also detected in all cells of the three l’sc-expressing clusters. This is seen in pictures focused at an intermediate plane between the neuroectoderm and the neuroblast layer of embryos mutant for neu (C), Dl (D), N (E) and E(spl)-C (F). Note how proneural clusters clearly fuse at the midline in neu− and N− embryos. Note also that the size of the clusters remains constant throughout stage 8. This can be seen in neu− embryos by comparing the RNA pattern (A) at the proneural cluster stage with the RNA (B) or protein (C) patterns when neuroblasts segregate. Likewise, in all late stage 8 mutants (B-F), the MP2 cluster remains devoid of l’sc expression, indicating that no enlargement of neighbouring l’sc-expressing clusters has taken place. Anterior is to the top. Scale bar, 20 μm.

Fig. 2.

Expression of l’sc in neurogenic mutants during segregation of S1 neuroblasts. (A) At early stage 8, the pattern of transcription in proneural clusters of a neu− embryo is essentially like that of the wild type (Fig. 1A), except for the presence of transcripts in the territory normally occupied by the mesectoderm at the midline (vertical arrow). (B) Unlike the wild type (Fig. 1B), transcription in neu− embryos is fully maintained in the clusters when S1 neuroblasts delaminate at late stage 8. (C-F) At that stage, the L’sc protein is also detected in all cells of the three l’sc-expressing clusters. This is seen in pictures focused at an intermediate plane between the neuroectoderm and the neuroblast layer of embryos mutant for neu (C), Dl (D), N (E) and E(spl)-C (F). Note how proneural clusters clearly fuse at the midline in neu− and N− embryos. Note also that the size of the clusters remains constant throughout stage 8. This can be seen in neu− embryos by comparing the RNA pattern (A) at the proneural cluster stage with the RNA (B) or protein (C) patterns when neuroblasts segregate. Likewise, in all late stage 8 mutants (B-F), the MP2 cluster remains devoid of l’sc expression, indicating that no enlargement of neighbouring l’sc-expressing clusters has taken place. Anterior is to the top. Scale bar, 20 μm.

As in the wild type, the pattern of L’sc protein accumulation closely follows that of transcription in neurogenic mutants. Mutant proneural clusters look essentially normal, as shown by double staining for L’sc and Ac. In the wild-type control (Fig. 3A), S1 clusters occupy the entire ventral-most region of the neuroectoderm, expressing either ac, l’sc, or both. The precise cell-by-cell apposition of those clusters that express either ac or l’sc suggests that in this region a cell never belongs simultaneously to more than one cluster. Proneural clusters in N embryos are also not intermingled (Fig. 3B), indicating that they are not enlarged, at least along the anteroposterior axis. Unlike the wild type, most proneural cells in late stage 8 mutants still bear high levels of L’sc (Figs 2C-F and 3C) and already show the characteristic morphology and patterns of gene expression of neuroblasts (not shown; Jiménez and Campos-Ortega, 1990; Campos-Ortega and Haenlin, 1992). As already noticed for the RNA, the domain of L’sc accumulation is the same as in earlier stages.

Fig. 3.

Double staining of embryos with anti-L’sc and anti-Ac antibodies. (A) An early stage 8 wild-type embryo. (B) An early stage 8 N− embryo. (C) A late stage 8 neu− embryo. In all cases, clusters 2 –2 and 5 –2 express only l’sc (blue), cluster MP2 expresses only ac (light brown), and cluster 7 –1 co-expresses both genes (dark brown). No enlargement of the clusters along the anterior-posterior axis occurs in the mutants. This is best shown by the lack of cells coexpressing l’sc and ac around the border between clusters 2 –2 and MP2 and between clusters MP2 and 5 –2. Whereas in the wild type the two rows of mesectodermal cells (vertical arrow) are devoid of proneural gene expression, many cells occupying equivalent positions express l’sc and/or ac in the mutants. Due to their irregular size, it is not possible to discern whether the mutant clusters are expanded, or just slightly shifted, by one cell diameter towards the midline. Anterior is to the top. Scale bar, 20 μm.

Fig. 3.

Double staining of embryos with anti-L’sc and anti-Ac antibodies. (A) An early stage 8 wild-type embryo. (B) An early stage 8 N− embryo. (C) A late stage 8 neu− embryo. In all cases, clusters 2 –2 and 5 –2 express only l’sc (blue), cluster MP2 expresses only ac (light brown), and cluster 7 –1 co-expresses both genes (dark brown). No enlargement of the clusters along the anterior-posterior axis occurs in the mutants. This is best shown by the lack of cells coexpressing l’sc and ac around the border between clusters 2 –2 and MP2 and between clusters MP2 and 5 –2. Whereas in the wild type the two rows of mesectodermal cells (vertical arrow) are devoid of proneural gene expression, many cells occupying equivalent positions express l’sc and/or ac in the mutants. Due to their irregular size, it is not possible to discern whether the mutant clusters are expanded, or just slightly shifted, by one cell diameter towards the midline. Anterior is to the top. Scale bar, 20 μm.

Neurogenic gene function is required for the specification of the mesectoderm

The mesectoderm forms during gastrulation, when two singlecell wide rows join at the ventral midline. In a stage 8 wildtype embryo these two rows are still devoid of proneural gene expression (Fig. 1), but already express sim (Fig. 4A; Thomas et al., 1988). As shown in Fig. 2, the presumptive mesectoderm appears to be almost absent in N and neu embryos. In addition, defective sim expression suggests that most presumptive mesectodermal cells have not adopted their normal fate (Fig. 4B,C). Slight defects in sim expression are also detected in Dl and in E(spl)-C embryos (Fig. 4D,E). In cross sections, mesectodermal cells are readily observable in the wild type (Fig. 5A), but seem to have adopted a neuroectodermal fate in N (Fig. 5B) and neu embryos (not shown). Comparison of Figs 1A and 2A, or of Fig. 3A and 3B, suggests that the medial proneural clusters of the two mutants might be ventrally enlarged. Alternatively, the position of the entire neuroectoderm may be ventrally shifted by one cell diameter, similar to the more extensive shift that it undergoes when the mesoderm is removed (Rao et al., 1991).

Fig. 4.

The mesectoderm in neurogenic mutants, as determined by lacZ expression under the control of the sim promoter. (A) Detail of the mesectoderm of a stage 8 wild-type embryo, expressing high levels of β-galactosidase. In N− (B) and neu− (C) backgrounds, βgalactosidase is detected at low levels in only a few cells, suggesting a defective specification of the mesectoderm. Similar results have been obtained by in situ hybridization with a sim probe (not shown). In Dl− (D) and E(spl)-C− (E) backgrounds, a few mesectodermal cells fail to express lacZ. Anterior is to the left. Scale bar, 50 μm.

Fig. 4.

The mesectoderm in neurogenic mutants, as determined by lacZ expression under the control of the sim promoter. (A) Detail of the mesectoderm of a stage 8 wild-type embryo, expressing high levels of β-galactosidase. In N− (B) and neu− (C) backgrounds, βgalactosidase is detected at low levels in only a few cells, suggesting a defective specification of the mesectoderm. Similar results have been obtained by in situ hybridization with a sim probe (not shown). In Dl− (D) and E(spl)-C− (E) backgrounds, a few mesectodermal cells fail to express lacZ. Anterior is to the left. Scale bar, 50 μm.

Fig. 5.

Cross sections of early stage 8 embryos double stained with anti-L’sc and anti-Snail antibodies. (A) A wild-type embryo expressing l’sc in nuclei of proneural cells in the ectoderm (brown) and snail in the nuclei of the mesoderm (blue). By this stage, mesodermal expression of snail is decaying, thus the lower and variable intensity of the staining. The two mesectodermal cells at the ventral midline (arrows) are devoid of both l’sc and snail expression. In the N− embryo shown in B, cells at the ventral midline are now expressing l’sc, suggesting that the presumptive mesectoderm has adopted a neuroectodermal fate.

Fig. 5.

Cross sections of early stage 8 embryos double stained with anti-L’sc and anti-Snail antibodies. (A) A wild-type embryo expressing l’sc in nuclei of proneural cells in the ectoderm (brown) and snail in the nuclei of the mesoderm (blue). By this stage, mesodermal expression of snail is decaying, thus the lower and variable intensity of the staining. The two mesectodermal cells at the ventral midline (arrows) are devoid of both l’sc and snail expression. In the N− embryo shown in B, cells at the ventral midline are now expressing l’sc, suggesting that the presumptive mesectoderm has adopted a neuroectodermal fate.

Control of l’sc expression by neurogenic genes

In the wild-type CNS, the proneural proteins become restricted from a cluster of cells to a single delaminating neuroblast. As for ac (Skeath and Carroll, 1992), we have shown that the restriction of l’sc is mainly accomplished at the transcriptional level. In neurogenic mutants, the transcriptional repression of l’sc fails to occur (see also Brand and Campos-Ortega, 1988) and all proneural cells accumulate high levels of L’sc protein, as is the case for ac (Skeath and Carroll, 1992; Ruiz-Gómez and Ghysen, 1993). A different mode of control of l’sc activity was proposed by Cabrera (1992), using an antibody against a C-terminal peptide of L’sc that contains a putative tyrosine phosphorylation site (Cabrera, 1990). With that antibody, L’sc was apparently detected only in neuroblasts, leading to the postulation that two forms of the protein exist. One form, accumulating in neuroblasts, would be unphosphorylated and functionally active. The other form, present in every cell that transcribes l’sc, would be phosphorylated and inactive. Accordingly, the anti-peptide antibody was found to stain a larger number of cells in Dl and N embryos, suggesting that neurogenic genes control l’sc at the posttranscriptional level (Cabrera, 1990, 1992). Our observations that proneural cells are similarly stained with the two available classes of anti-L’sc antibodies would suggest that both states of phosphorylation of L’sc, if present, would coexist in proneural cells. However, our observations are equally compatible with the possibility that antibodies against the C-terminal peptide cannot discriminate between different protein forms. In this context, it has been shown that neither the deletion of the C-terminal region, nor the substitution of the tyrosine in this domain by a residue that cannot be phosphorylated, have pronounced influence on the proneural activity of L’sc assayed in imaginal discs (Hinz et al., 1994).

Our analysis of embryos double stained for Ac and L’sc demonstrate, as already noted (Brand and Campos-Ortega, 1988; Ruiz-Gómez and Ghysen, 1993), that neurogenic genes do not play a role in the emergence of proneural clusters in the embryo, except for a possible slight ventral enlargement of the medial clusters in Nand neumutants. However, the possibility that the domains of proneural gene expression later become enlarged in neurogenic mutants has been a controversial issue (Brand and Campos-Ortega, 1988; Campos-Ortega, 1993; Ruiz-Gómez and Ghysen, 1993). In this regard, our results also support the view that, in neurogenic mutants, the domains of proneural gene expression in the CNS do not become enlarged at the stage when S1 neuroblasts normally delaminate.

Neurogenic genes and mesectoderm specification

As indicated in the Introduction, several lines of evidence suggest that mesectodermal specification partially requires an inductive signal from the mesoderm. Our observation that N is required for the activation of sim (see also Menne and Klämbt, 1994) and for the early repression of proneural genes in the presumptive mesectoderm suggests that mesectoderm induction involves cell communication. We have also found that neu participates in the same process, raising the possibility that the family of neurogenic genes participate in the specification of the mesectoderm, in a way analogous to their role during neuroblast formation (de la Concha et al., 1988). Thus, expression of neu and Dl in the mesoderm (Boulianne et al., 1991; Price et al., 1993; Kopczynski and Muskavitch, 1989; Haenlin et al., 1990) would correlate with their participation in the emission of the inductive signal. Similarly, specific expression of four E(spl)-C genes in the mesectoderm (Knust et al., 1992) suggests that they function to implement the reception of the signal. Maternal expression of Dl and of m3, a fifth E(spl)-C gene (Kopczynski and Muskavitch, 1989; Haenlin et al., 1990; Knust et al., 1987), could explain the lack of a strong mesectodermal phenotype in zygotic Dl and E(spl)-C mutants.

We thank S. Campuzano, A. Martínez Arias and J. Modolell for helpful comments on the manuscript; and A. Alberga, S. Crews, L. García Alonso, P. Macdonald, A. Martínez Arias and J. Skeath for antibodies and fly stocks. M. D. M.-B. and A. C. were predoctoral fellows of Ministerio de Educación y Ciencia. This work was supported by grant PB90-0082 from DGICIT to F. J. and an institutional grant from Fundación Ramón Areces.

Alonso
,
M. C.
and
Cabrera
,
C. V.
(
1988
).
The achaete-scute gene complex of Drosophila melanogaster comprises four homologous genes
.
EMBO J
.
7
,
2585
2591
.
Boulianne
,
G. L.
,
de la Concha
,
A.
,
Campos-Ortega
,
J. A.
,
Jan
,
L. Y.
and
Jan
,
Y. J.
(
1991
).
The Drosophila neurogenic gene neuralized encodes a novel protein and is expressed in precursors of larval and adult neurons
.
EMBO J
.
10
,
2975
2983
.
Brand
,
M.
and
Campos-Ortega
,
J. A.
(
1988
).
Two groups of interrelated genes regulate early neurogenesis in Drosophila melanogaster
.
Roux’s Arch. Dev. Biol
.
197
,
457
470
.
Cabrera
,
C. V.
,
Martínez Arias
,
A.
and
Bate
,
M.
(
1987
).
The expression of three members of the achaete-scute gene complex correlates with neuroblast segregation in Drosophila
.
Cell
50
,
425
433
.
Cabrera
,
C. V.
(
1990
).
Lateral inhibition and cell fate during neurogenesis in Drosophila: the interactions between Scute, Notch and Delta
.
Development
110
,
733
742
.
Cabrera
,
C. V.
(
1992
).
The generation of cell diversity during early neurogenesis in Drosophila
.
Development
115
,
893
901
.
Campos-Ortega
,
J. A.
and
Haenlin
,
M.
(
1992
).
Regulatory signals and signal molecules in early neurogenesis of Drosophila melanogaster
.
Roux’s Arch. Dev. Biol
.
201
,
1
11
.
Campos-Ortega
,
J. A.
(
1993
).
Early neurogenesis in Drosophila melanogaster
. In
The Development of Drosophila melanogaster
(ed.
M.
Bate
and
A.
Martínez Arias
), pp.
1091
1129
.
Cold Spring Harbor, NY
:
Cold Spring Harbor Laboratory Press
.
Campuzano
,
S.
and
Modolell
,
J.
(
1992
).
Patterning of the Drosophila nervous system: the achaete-scute gene complex
.
Trends Genet
.
8
,
202
207
.
Chou
,
T. B.
and
Perrimon
,
N.
(
1992
).
Use of a yeast site-specific recombinase to produce female germline chimeras in Drosophila
.
Genetics
131
,
643
653
.
de la Concha
,
A.
,
Dietrich
,
U.
,
Weigel
,
D.
and
Campos-Ortega
,
J. A.
(
1988
).
Functional interactions of neurogenic genes of Drosophila melanogaster
.
Genetics
118
,
499
508
.
Doe
,
C. Q.
(
1992
).
Molecular markers for identified neuroblasts and ganglion mother cells in the Drosophila central nervous system
.
Development
116
,
855
863
.
Ghysen
,
A.
,
Dambly-Chaudière
,
C.
,
Jan
,
L. Y.
and
Jan
,
Y. N.
(
1993
).
Cell interactions and gene interactions in peripheral neurogenesis
.
Genes Dev
.
7
,
723
733
.
Haenlin
,
M.
,
Kramatscheck
,
B.
and
Campos-Ortega
,
J. A.
(
1990
).
The pattern of transcription of the neurogenic gene Delta of Drosophila melanogaster
.
Development
110
,
905
914
.
Hartenstein
,
V.
and
Campos-Ortega
,
J. A.
(
1984
).
Early neurogenesis in wildtype Drosophila melanogaster
.
Roux’s Arch. Dev. Biol
.
193
,
308
325
.
Hinz
,
U.
,
Giebel
,
B.
and
Campos-Ortega
,
J. A.
(
1994
).
The basic-helix-loop-helix of Drosophila lethal of scute protein is sufficient for proneural function and activates neurogenic genes
.
Cell
76
,
77
87
.
Jiménez
,
F.
and
Campos-Ortega
,
J. A.
(
1982
).
Maternal effect of zygotic mutants affecting early neurogenesis in Drosophila
.
Roux’s Arch. Dev. Biol
.
191
,
191
201
.
Jiménez
,
F.
and
Campos-Ortega
,
J. A.
(
1990
).
Defective neuroblast commitment in mutants of the achaete-scute complex and adjacent genes of D
.
melanogaster. Neuron
5
,
81
89
.
Knust
,
E.
,
Tietze
,
K.
and
Campos-Ortega
,
J. A.
(
1987
).
Molecular analysis of the locus Enhancer of split of Drosophila melanogaster
.
EMBO J
.
6
,
4113
4123
.
Knust
,
E.
,
Schrons
,
H.
,
Grawe
,
F.
and
Campos-Ortega
,
J. A.
(
1992
).
Seven genes of the Enhancer of split complex of Drosophila melanogaster encode helix-loop-helix proteins
.
Genetics
132
,
505
518
.
Kopczynski
,
C. C.
and
Muskavitch
,
M. A. T.
(
1989
).
Complex spatiotemporal accumulation of alternative transcripts from the neurogenic gene Delta during Drosophila embryogenesis
.
Development
107
,
623
636
.
Leptin
,
M.
and
Roth
,
S.
(
1994
).
Autonomy and nonautonomy in Drosophila mesoderm and morphogenesis
.
Development
120
,
853
859
.
Lindsley
,
D. L.
and
Zimm
,
G. G.
(
1992
).
The genome of Drosophila melanogaster
.
San Diego, USA
:
Academic Press
.
Martín-Bermudo
,
M. D.
,
Martínez
,
C.
,
Rodríguez
,
A.
and
Jiménez
,
F.
(
1991
).
Distribution and function of the lethal of scute gene product during early neurogenesis in Drosophila
.
Development
113
,
445
454
.
Martín-Bermudo
,
M. D.
,
González
,
F.
,
Domínguez
,
M.
,
Rodríguez
,
I.
,
Ruiz-Gómez
,
M.
,
Romani
,
S.
,
Modolell
,
J.
and
Jiménez
,
F.
(
1993
).
Molecular characterization of the lethal of scute genetic function
.
Development
118
,
1003
1012
.
Menne
,
T. V.
and
Klämbt
,
C.
(
1994
).
The formation of commisures in the Drosophila CNS depends on the midline cells and the Notch gene
.
Development
120
,
123
133
.
Nambu
,
J. R.
,
Lewis
,
J. O.
,
Wharton
,
K. A.
and
Crews
,
S. T.
(
1991
).
The Drosophila single-minded gene encodes a helix-loop-helix protein that acts as a master regulator of CNS midline development
.
Cell
67
,
1157
1167
.
Nambu
,
J. R.
,
Lewis
,
J. O.
and
Crews
,
S. T.
(
1993
).
The development and function of the Drosophila CNS midline cells
.
Comp. Biochem. Physiol
.
104A
,
399
-
409
.
Price
,
B. D.
,
Chang
,
Z.
,
Smith
,
R.
,
Bockheim
,
S.
and
Laughon
,
A.
(
1993
).
The Drosophila neuralized gene encodes a C3HC4 zinc finger
.
EMBO J
.
12
,
2411
2418
.
Rao
,
Y.
,
Vaessin
,
H.
,
Jan
,
L. Y.
and
Jan
,
Y. N.
(
1991
).
Neuroectoderm in Drosophila embryos is dependent on the mesoderm for positioning but not for formation
.
Genes Dev
.
5
,
1577
1588
.
Romani
,
S.
,
Campuzano
,
S.
and
Modolell
,
J.
(
1987
).
The achaete-scute complex is expressed in neurogenic regions of Drosophila embryos
.
EMBO J
.
6
,
2085
2092
.
Ruiz-Gómez
,
M.
and
Ghysen
,
A.
(
1993
).
The expression and role of a proneural gene, achaete, in the development of the larval Drosophila nervous system
.
EMBO J
.
12
,
1121
1130
.
Ruohola
,
H.
,
Bremer
,
K. A.
,
Baker
,
D.
,
Swedlow
,
J. R.
,
Jan
,
L. Y.
and
Jan
,
Y. N.
(
1991
).
Role of neurogenic genes in establishment of follicle cell fate and oocyte polarity during oogenesis in Drosophila
.
Cell
66
,
433
449
.
Skeath
,
J. B.
and
Carroll
,
S. B.
(
1992
).
Regulation of proneural gene expression and cell fate during neuroblast segregation in the Drosophila embryo
.
Development
114
,
939
946
.
Tautz
,
D.
and
Pfeifle
,
C.
(
1989
).
A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback
.
Chromosoma
98
,
81
85
.
Thomas
,
J. B.
,
Crews
,
S. T.
and
Goodman
,
C. S.
(
1988
).
Molecular genetics of the single-minded locus: A gene involved in the development of the nervous system of Drosophila
.
Cell
53
,
133
141
.