Defective neuroblast commitment in mutants of the achaete-scute complex and adjacent genes of D. melanogaster

Neuron, Vol. 5, 81-89, July, 1990, Copyright © 1990 by Cell Press

Defective Neuroblast Commitment in Mutants of the achaete-scute Complex and Adjacent Genes of D. melanogaster Fernando Jimdnez* and losd A. Campos-Ortega t *Centro de Biologia Molecular Universidad Autonoma/CSIC Canto Blanco 28049 Madrid Spain f lnstitut for Entwicklungsphysiologie Universit~t K61n Gyrhofstrasse 17 5000 Cologne 41 Federal Republic of Germany

Summary

Loss of function mutations in genes of the achaetPscute complex (ASC) or in the 8ene vnd of D. melanogaster result in neural hypoplasia. Two types of defects contribute to the development of the neural hypoplasic phenotype: a lower than normal proportion of neuroblasts delaminate from the neuroectoderm, and there is abundant cell death in the neural primordium during later stages. In addition, we found that increasing the copy number of ASC wild-type alleles leads to effects opposite to those caused by their deletion. All of these results indicate that the function of these genes is required for the commitment of neuroectodermal cells as neuroblasts and that the loss of these genetic functions causes the cells either to take on an epidermal fate or to die.

Introduction

The achaete-scute gene complex (ASC) of Drosophila melanogaster is located in the subdivision 1B of the X chromosome and comprises four genes, achaete, scute, lethal of scute, and asense (Muller, 1935; Gar- cia-Bellido, 1979; for review see Ghysen and Dambly- ChaudiEre, 1988). A requirement of ASC function for normal development of both the central and the peripheral nervous systems is shown bythe fact that embryos lacking the ASC genes exhibit significant neural hypoplasic defects 0im(~nez and Campos-Ortega, 1979, 1987; White, 1980; Dambly-Chaudi~re and Ghysen, 1987). In addition to the genes of the ASC, the subdivision 1B comprises other genes relevant for neural development, i.e., ventral nervous system condensation defective (vnd; White, 1980) and embryonic lethal, abnormal visual system (elav; Campos et al., 1985; Jim~- nez and Campos-Ortega, 1987). Loss-of-function mutations in vnd result in considerable CNS hypoplasia (White, 1980), similar to that seen upon deletion of the ASC genes, whereas the CNS defects of elav- mu- tants are rather mild 0im~nez and Campos-Ortega, 1987).

The ASC genes are members of a gene family encoding structurally similar proteins characterized by a helix-turn-helix motif (Villares and Cabrera, 1987;

Alonso and Cabrera, 1988; Gonz~Jez et al., 1989); the same motif is present in many other proteins of Dro- sophila (e.g., daughterless [Caudy et al., 1988], twist [Thisse et al., 1988], hairy [Rushlow et al., 1989], several proteins encoded by the Enhancer of split gene complex [Kl~mbt et al., 1989], etc.) and mouse (e.g., myoD [David et al., 1987], inmunoglobulin enhancer binding proteins [Murre et al., 1989a]). It has been shown that some of the proteins encoding the helix-turn- helix motif are capable of forming heterodimers and binding to DNA in vitro and are thus potential tran- scriptional regulators (Murre et al., 1989a, 1989b). The nature of the product encoded by vnd is not yet known.

Recent work by Ghysen and O'Kane (1988) indicates that the ASC genes are required before or soon after the neural commitment of the progenitors of the PNS. In addition, Bodmer et al. (1989) have found a defective pattern of DNA replication in ASC- mutants, suggesting that some of the progenitor cells of the PNS fail to develop. These findings, along with additional genetic data (Garcia-Bellido, 1979,1981; Garcia Alonso and Garcia-Bellido, 1986; Ghysen and Dambly-Chau- didre, 1987; Romani et al., 1989), suggest that the ASC genes are responsible for the decision of epidermal cells to take on the developmental fate of PNS progenitor cells in both the larva and the imago. The ques- tion arises as to whether the defects observed in the CNS of the ASC- mutants 0imdnez and Campos-Or- tega, 1979,1987; White, 1980) are also due to defective initial commitment of.CNS progenitor cells, rather than to other causes. Indeed, the pattern of transcription of achaete, scute, and lethal ofscute (Cabrera et al., 1987; Romani et al., 1987) correlates well with the process of neuroblast segregation, suggesting that the corresponding gene products may be causally related to the commitment of the neuroblasts; and Cabrera et al. (1987) have already noticed the lack of the median neuroblast in putative achaete-, scute-, and lethal ofscute- embryos. We have used a variety of selective staining techniques to study the early stages of development of the phenotype of ASC- mutants, with particular reference to the segregation of the neuroblasts from the neuroectoderm and the pattern formed by these cells (see Hartenstein and Cam- pos-Ortega, 1984; Doe et al., 1988), with the aim of characterizing the cellular basis of the neural hypoplasia found in embryos lacking the ASC genes. We find that in such embryos neurogenesis is initiated by fewer neuroblasts than in the wild-type, because fewer neuroectodermal cells are committed to the neural fate. In addition, neuroblast proliferation seems to be defective, and there is increased cell death within the neural anlage of the mutants. Similar observations have been made in studies of vndembryos. Finally, we found that additional copies of the wild-type alleles of ASC cause embryonic neural hyperplasia. All of these

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Figure 1. The Pattern of Neuroblasts in a Stage 10 Wild-Type Embryo (A-D) Four planes of focus through the same early stage 10 wild-type embryo to show the pattern of neuroblasts arranged in three rows (arrows). The embryo was stained with an anti-13-gal antibody (stripes encompassing two parasegments) and with MAb43BS, which stains the membrane of all embryonic cells and allows one to distinguish reliably the neuroblasts. The embryo expresses the ftz stripes of 13-galactosidase because it carries a P element-mediated insertion of a ftz promoter-lacZ construct in the elav locus (see Experimental procedures). Same magnification in all pictures; bar in (A), 50 pm.

results together indicate that the ASC genes and vnd are indeed causally involved in the initial decision of the neuroectodermal cells to take on the neural developmental fate.

Results

In our analysis of neurogenesis in ASC- embryos, we have been mainly concerned with the pattern of neuroblasts in thoracic metameres 12 and 13 during embryonic stages 9 to 11 (staging according to Campos- Ortega and Hartenstein, 1985). During these stages, neuroblast segregation takes place in three consecu- tive pulses, resulting in different subpopulations of neuroblasts, SI, SII, and Sill (see Hartenstein and Cam- pos-Ortega, 1984). Neuroblast segregation is a highly dynamic process, and no markers that would label simultaneously and specifically all of the neuroblasts are available; thus, the elaboration of maps compris- ing the entire complement is technically difficult. The best available marker for this purpose is the antibody against the protein encoded by the segmentation gene hunchback (hb), which stains almost all neuroblasts. Within the germ band, the anti-hb antibody stains almost exclusively neuroblasts and their progeny; however, its use as a marker is hampered by the fact that, with the antibody we used, expression of the hb protein in the neuroblasts is detected transiently and not in all of the cells at the same time. Neverthe- less, the use of the monoclonal antibody MAb43B5, which labels cell membranes during the first half of

embryogenesis (F. Jim~nez, unpublished data), either alone or in conjunction with the anti-hb antibody, has provided the best material and allowed the construc- tion of reproducible neuroblast maps. Although MAb- 43B5 stains all embryonic cells, reliable identification of neuroblasts in embryos stained with this antibody is possible based on three criteria (Figure 1): much larger diameter (approximately 10 I~m) than other embryonic cells (approximately 5 I~m), position between the mesoderm and the epidermal primordium, and initial arrangement in three rows.

Embryos Lacking the ASC Exhibit Defective Patterns of Neuroblasts In the wild-type embryo at early stage 10, the pattern formed by the neuroblasts is fairly precise: per hemi- segment, 8-9 neuroblasts can be readily distinguished in the median row, 5 in the intermediate row, and 4 in the lateral row (see Figures 1-3). In embryos of the same age homozygous for Df(7)sc a57, thus lacking all genes of the ASC, 20%-25% of the neuroblasts are ab- sent. In addition, some of the neuroblasts that succeed in segregating from the neuroectoderm in the mutants are slightly smaller than those in the wild- type. Defects in the precise pattern of neuroblasts are variable among different mutant embryos and between different segments of the same embryo. This variability is difficult to interpret: it may be due to ir- regular displacement of individual neu roblasts within the neural primordium; or, alternatively, it may indicate that the neuroblasts are actually affected at ran-

Defective Neuroblast Commitment 83

L,

Figure 2. Comparison of the Neuroblast Pattern in Stage 10 Wild-Type and DfCI)sc Bs7 Embryos (A) A stage 10 wild-type embryo; (B) a Df(1)sc Bsz embryo of the same age. Both embryos were stained with an anti-I~-gal antibody, for genotype diagnosis, and anti-hb antibody, for selective staining of the neuroblasts. Notice that the mutant has fewer neuroblasts than the wild type. The ftz stripes of I~-galactosidase (arrows) designate the wild-type genotype because the embryo carries a P element-mediated insertion of a ftz promoter-lacZ construct in the elav locus (see Experimental Procedures). Same magnification in both pictures; bar in (A), 50 p.m.

dora and that different types of neuroblasts are missing in different mutants. Yet, despite the variability, it seems that neuroblasts in the medial and lateral rows are preferentially affected in the Df(1)sc BSz embryos, whereas those of the intermediate row appear to be less perturbed. No anti-hb-positive cells, or cells with the characteristics of neuroblasts, are visible within the ectodermal layer; thus, the lower number of neuroblasts present in the mutants is due to commitment of fewer cells than normal to the neural fate, rather than to defects in the process of segregation itself following a normal neuroblast commitment. We found that approximately the same proportion of neuroblasts is missing in Df(1)sc Bs7 stage 11 embryos, when the entire neuroblast complement has segregated (Fig- ure 3A). Due to the general disorganization of the rows after the segregation of the Sill neuroblasts, it is impossible to determine which types are actually missing in the older embryos. A few cells that can be distinguished unambiguously because of their characteristic location, such as the median neuroblast or the midline precursor cell MP2, were found to be ab- sent in all cases studied (data not shown).

Cell Death Contributes to the Production of the Neural Hypoplasia The defects found in the ful ly differentiated Df(1)sc aS7

w i l d t y p e D i l l ) s o B 5 7 ~ d 6 D f ( I ) R T I 8 4

A ) 1 t m l ! m I 1 m 1 1 m

i o 0 ° 8 i

° 8i ' ®° ,

B)

Figure 3. Map of Neuroblasts of Hemisegments 12 and 13 from Stage 10 and Stage 11 Embryos of Different Genotypes (A) Stage 10 embryos; (B) stage 11 embryos. Camera lucida draw- ings of embryos stained with MAb43B5 and anti-engrailed (en) antibodies, m, i, I in (A) refer to median, intermediate, and lateral rows of neuroblasts; the ventral midline is indicated with a bro- ken line. en-expressing neuroblasts, shown to mark the pos- terior margin of the segments, are stippled. The Df(1)RTI84 embryos could not be stained with the anti-en antibody; however, the location of the segmental border in the stage 11 embryos is indicated by arrows representing the tracheal pits. Notice that the mutants have fewer neuroblasts than the wild type and that the neuroblasts are affected differentially in the mutants, i.e., neuroblasts of the lateral and intermediate rows are affected in the Df(7)sc B57 embryo; those of the intermediate row are affected in the vnd 6 embryo.

embryo are more severe than those expected from the lack of 20%-25% of the neuroblast population (Figure 4B); hence, it is likely that other events occurring after neuroblast segregation account for the difference.

In the wild type, the protein encoded by the segmentation gene f ush i tarazu (ftz) is present in the nuclei of a fraction of ganglion mother cells and neurons (Hiromi et al., 1985; Doe et al., 1988) and can therefore be used as a marker for neuroblast progeny. Early stage 11 Df(1)sc as7 embryos, that is to say, before the onset of cell death (see below), exhibit a much lower number of ftz-expressing cells than the expected three-fourths of that found in the wild type (Figure 5), suggesting that the neuroblasts in the mutant neural primordium produce fewer progeny than normal. Analysis of histological sections of increas- ingly aged embryos reveals that many cells degener- ate within the neural primordiu'm of the ASC- mutants, starting midway through embryonic stage 11 and reaching a peak in early stage 12, at the beginning of germ band shortening (data not shown, but pre- viously described and illustrated by Jim~nez and Campos-Ortega, 1979; see also Figure 6). Cell death occurs during the same stages of neurogenesis in the wild type as well, but to a much lower extent. Pycnosis

Neuron 84

Figure 4. Ventral Views of the Ventral Cord of Three Stage 16 Embryos of Different Genotype Stained with the Anti-HRP An- t ibody (A) A wild-type embryo; (B) a Df(1)sc a57 embryo; and (C) a double mutant Df(l)scBSZ; da ~a36 embryo. Notice that the ventral cord fragments and that the phenotype of the double mutant is much more severe than the phenotype of the single mutant. Same magnification in all pictures; bar in (A), 50 p.m.

E ~ C

and shrinkage confer on the dying and dead cells a characteristic dark appearance, which is clearly dis- tinguishable in the microscope. In addition, macro- phages phagocytosing cellular debris are very abundant until embryonic stage 16.

As revealed by staining with anti-horseradish peroxi- dase (HRP) antibodies (Jan and Jan, 1982), the severity of the neural defects of stage 13 mutant embryos (data not shown), i.e., before the sensory afferents have reached the CNS is comparable to that of the fully differentiated mutants (Figure 4B). Thus, defects in the sensorial innervation that occur in these mutants (Dambly-Chaudi~re and Ghysen, 1987) do not contribute in any noticeable manner to the final CNS phenotype.

Interactions of the ASC and daughterless The phenotype of embryos lacking the ASC genes is not completely aneural; many cells in the CNS (lim~- nez and Campos-Ortega, 1979, 1987) and PNS (Dam- bly-Chaudi~re and Ghysen, 1987; Ghysen and Dam- bly-Chaudi~re, 1988) develop normally (Figure 4B). Moreover, the final phenotype of the ASC mutants evolves progressively. As we have seen above, the final phenotype of DfCI)sc B57 embryos results from at least two different factors that act sequentially: defective neuroblast commitment, affecting part (20%-25%) of the entire complement, followed by a massive process of cell death, which includes cells that have segregated from the neuroectoderm (presumptive neuroblasts) and their progeny. The sequential evolution of the incompletely aneural phenotype suggests that other related genes may partially substitute for, or complement, the functions of the ASC genes during early neurogenic stages; the products of those other genes may allow some neuroblasts and their progeny to develop normally and others to perform under some-

what abnormal conditions their first developmental steps before dying.

The gene daughterless (da) may provide a product capable of functionally complementing the genes of the ASC, in view of the similarity of the phenotypes of their respective loss-of-function mutations and chief- ly because the proteins encoded by da and by the ASC genes are structurally (Caudy et al., 1988a, 1988b; Vii- lares and Cabrera, 1987; Alonso and Cabrera, 1988; Gonz,~lez et al., 1989) and functionally (Murre et al., 1989b) similar. Finally, genetic interactions between the ASC genes and da have already been shown to ex- ist with respect to their participation in the control of imaginal sensory organ development (Dambly-Chau-

Figure 5. Comparison of Anti-ftz Staining in Stage 11 Wild-Type and Df(1)sc ss7 Embryos (A) A stage 11 wild-type embryo before the onset of cell death; (B) a Df(1)sc B57 embryo of the same age stained with an anti-ftz antibody. Notice that the number of ftz + cells is much lower in the mutant than in the wild type, suggesting that the mutant neuroblasts have produced fewer progeny. Same magnification in both pictures; bar in (A), 50 p,m.


Figure 6. Cell Death in vncP Embryos Cross section through Stage 11 (A) and Stage 13 (B) vnd 6 embryos. Cell death in the neural primordium is indicated by arrows. Same magnification in both pictures; bar in (A), 50 I~m.

di~re et al., 1989). Thus, similar interactions may be expected to take place earlier in development, e.g., during neuroblast segregation.

The phenotype of the fully differentiated double mutant DfCl)scBST,'daKX736 embryo is indeed more severe than that of either one of the mutations alone (Figure 4C); thus, additive effects contribute to its production. However, the initial pattern of neuroblasts of the presumptive double mutant is numerically very similar to, or the same as, that of the Df(1)sc 8sz embryo (data not shown). We should point out that the number of neuroblasts in da- embryos is apparently normal (data not shown).

Embryos Lacking the Gene vnd Exhibit Defective Patterns of Neuroblasts The gene vnd is located in the subdivision 1B of the X chromosome, proximal to the ASC (White, 1980). The phenotype of its loss of function is similar to, al- beit not as severe as, that of the deletion for the ASC, essentially consisting of hypoplasia of the CNS. The ventral cord of the vnd mutants is thinner than that in the wild type and is imperfectly condensed; however, there is no obvious fragmentation of the ventral cord, which is a characteristic feature of the phenotype of the ASC- embryos (see Figure 4B; see White, 1980; Jim~nez and Campos-Ortega, 1987). Stage 10 vndembryos lack approximately 20%-25% of their neuroblasts (Figure 3A); stage 11 vndembryos exhibit similar defects (Figure 3B). These results apply to embryos hemizygous for either of two different alleles studied, v n ~ and vnd sT. Although the number of affected neuroblasts is approximately the same in vnd- as in the ASC- (Df(7)sc BsT) embryos, neuroblasts within the intermediate and medial rows are fre- quently affected in the former embryos, and within the lateral and the medial rows in the latter embryos.

An additional observation confirms that essentially nonoverlapping populations of neuroblasts are missing in young embryos lacking the ASC or the vnd gene. Embryos hemizygous for Df(1)RT184, which are both ASC- and vnd-, lack approximately 50% of the neuroblasts, indicating additive effects of both mutations (Figure 3).

As described above with respect to the ASC- phenotype, cell death is also very abundant within the neural primordium of vndembryos (White, 1980; Figure 6). The process of cell degeneration begins slightly earlier in the vnd- than in the Df(1)sc B57 embryos. Cell death starts in the ASC- embryos at mid- stage 11 and reaches a peak of intensity at the beginning of germ band shortening, in early stage 12; in contrast, in the vndembryos cell death starts at the beginning of stage 11, reaching its peak at mid-stage 11. This earlier degeneration might consequently affect younger cells, including cells that have segregated from the neuroectodermal layer but not yet started dividing.

Increased Dosage of the Wild-Type ASC Results in Neural Hyperplasia Loss-of-function mutations in the so-called neurogenic genes, e.g., Notch, Delta, and several others (Poulson, 1937; Lehmann et al., 1981, 1983), cause the phenotype opposite to that of ASC- mutations, i.e., a conspicuous neural hyperplasia resulting from the commitment of more neuroectodermal cells to the neural fate than occurs in the wild type. The neurogenic genes are functionally connected both with the ASC genes and with da. The combination of ASC or da mutations with neurogenic mutations leads to a reduction in the severity of the neural hyperplasia, in that fewer ectodermal cells develop as neuroblasts in the double mutants than in the corresponding neurogenic mutant alone; moreover, the realm of transcrip-

Neuron 86

Table 1. Embryos with Cuticle Defects after increasing the Number of Copies of Wild-Type Alleles of the achaete-scute Complex

Genotype of Parental Animals

Females Males Cuticle Defects

XX/Y;Dp(1;2)sc 79 X/Y 0.7% XX/Y;Dp(1,2)sc 19 X/Y;Dp(1;2)sc 19 9.2% XX/y2Y611;Dp(1;2)sc 79 X/y2y61hDp(1;2)sc ~9 26.3%

tion of ASC genes is markedly increased in neurogenic mutants (Brand and Campos-Ortega, 1988). These observations lend further support to the hypothesis that the functions of the ASC genes are required for normal development of the neuroblasts and suggest that the absence of the genes causes neuroectodermal cells to enter the epidermal pathway of development.

To substantiate this latter claim further, we have studied the effects of increasing the number of wild- type copies of the ASC and vnd on neurobJast development by using two different duplications that cover different parts of the subdivision 1B: Dp(1;2.)sc 79, com- prising achaete +, scute +, and lethal of scute +, and a special Y chromosome, y2y611, which carries a trans- location from the X chromosome including the whole wild-type ASC as well as vnd +. One additional copy of each achaete +, scute +, and lethal of scute + in the genome of the embryo does not produce any significant effect (see Table 1). However, two copies of the same duplication cause neural hyperplasic defects of variable extent in the ventral cord of approximately 9% of the embryos (Figure 7). These defects are characteristic of weak neurogenicmutants (Lehmann et al., 1983) and consist in local swellings in thoracic and/or abdominal segments of the CNS and various types of epidermal defects, e.g., holes in the ventral

epidermis and fusion of denticle belts (data not shown). The proportion of embryos with neural hyperplasic defects is increased up to 25% by the presence of one additional copy of the ASC and vnd +, using the y2Y611 duplication.

Discussion

The following two observations indicate that the products of the ASC and vnd genes are required for the commitment of neuroectodermal cells as neuroblasts: Loss-of-function mutations in these genes cause the lack of a substantial fraction of the neuroblasts; and duplications carrying additional copies of wild-type alleles of the ASC and of vnd cause defects opposite those of the loss-of-function, that is to say, neural hyperplasia. Since the neuroblasts affected in the loss-of-function mutations are missing from the earli- est stages on and since there is no cell death in the neuroectoderm at these stages, it follows that the corresponding neuroectodermal cells have been committed to a fate different from the neural one. It is probable that some of them are diverted to an epidermal fate. Others succeed to segregate from the neuroectodermal layer as neuroblasts, although some are destined to die at later stages. In the case of the gain of function by duplications, the embryonic epidermis shows a variety of defects, such as holes and fusion of denticle belts, which correlate with local neural hyperplasia and may thus be due to defects in the epidermal commitment of some neuroectodermal cells. Since the present phenotypic defects are identi- cal to those found in weak neurogenic mutants (Leh- mann et al., 1983), it seems plausible that supernu- merary neuroblasts have developed at the expense of epidermoblasts.

Although our observations allow the conclusion that genes of the ASC are required for commitment

Figure Z Ventral Cord of Stage 17 Embryos Stained with the Anti-HRP Antibody (A) A wild-type embryo; (B) an embryo that carries three copies of the ASC by means of DpCl;2)sc 19 and the Y chromosome, y2Y611. Note local hyperplasia of the ventral cord characteristic of weak neu rogenic alleles. Same magnification in both pictures; bar in (A), 50 p.m.


of neuroectodermal cells as neuroblasts, we do not know whether or not all of the genes of the complex play the same role, for in our analysis we have used large deletions or duplications and have not distinguished particular genes. However, previous evidence from the analysis of the terminal phenotypes of partial deletions (Jim~nez and Campos-Ortega, 1987; Brand and Campos-Ortega, 1988) points to the lethal of scute gene as being the key genetic element in central neurogenesis; in its function, however, lethal ofscute interacts with achaete, scute, and asense. Prelim i nary observations on lethal o f scute embryos indicate neuroblast defects that are milder than those found in embryos lacking the entire complex. Hence, the phenotype of the ASC deletion described above probably results from the lack of the genes lethal of scute, achaete, and scute, which have the same temporal pattern of expression (Cabrera et al., 1987; Romani et al., 1987), whereas the lack of asense is probably ir- relevant for neuroblast commitment, since asense is expressed only after the neuroblasts have segregated (Alonso and Cabrera, 1988; Gonz~lez et al., 1989).

The ASC genes have been proposed by Cabrera et al. (1987) to constitute a subset of a complex network of genes required for the generation of the normal neuroblast pattern. By assuming that other, partially redundant genes are involved in the same process, Cabrera et al. (1987) tried to explain why the domains of transcription of the three ASC genes are more wide- spread than the phenotypic defects actually found in ASC- embryos. The cell death that we found in developing ASC- embryos might be interpreted to support the latter hypothesis. Formally, the abundant cell death within the neural primordium of the mutants may be taken as the manifestation of a new developmental fate adopted by the dying cells in the mutants. Several cell death, determination, and differentiation genes that affect cellular viability by changing the fate of the corresponding cells have actually been identified in Caenorhabditis elegans (Ellis and Horvitz, 1986; for review see Horvitz, 1988). However, as found in the ASC- mutants, cell death may also be interpreted as the result of physiological weakness of some of the neuroblasts, which die eventually after having performed initial developmental steps. This could be due to functional redundancy of the genes involved in neurogenesis, i.e., partial functional re- placement of the ASC by other related genes. We found that similar defective neuroblast commitment followed by cell death occurs in developing vnd mutants as well, suggesting that vnd could be another member of the postulated functional group of genes. Although no molecular data on vnd are yet available, we hypothesize that the vnd protein may be structurally related to the ASC proteins and that its pattern of expression may also be similar.

If other genes partially complement the function of the ASC genes at neuroblast commitment, thus allow- ing for some time development of a number of neuroblasts and their progeny, deletion of the complement-

ing genes along with the ASC should cause more severe neuroblast defects, da is a member of the same gene family as the ASC, and comparisons of mutant phenotypes and protein structure and function have revealed strong similarities (Caudy et al., 1988a, 1988b; Murre et al., 1989b). However, the number of neuroblasts in the double ASC- da- mutants was found to be roughly the same as that in the ASC- mutants. Yet the final phenotype of the double mutant is indeed more severe than that of the single mutants. Thus, we conclude that more pronounced cell death is responsible for the increased phenotypic severity of the double mutants.

Murre et al. (1989b) have recently demonstrated the formation of heterodimers between the lethal ofscute and da proteins, which are active in an in vitro transcription system. These observations suggest that the same heterodimers may function in vivo as regulato- ry proteins, acting by binding to enhancer sequences in the DNA. The model presupposes that lethal of scute, which is expressed in the neuroectoderm (Cab- rera et al., 1987; Romani et al., 1987), is the tissue- specific element; however, this element would depend for its action on da, which is ubiquitously expressed (M. Brand and J. A. Campos-Ortega, unpublished data). If the interaction of lethal ofscute with da applies to the other ASC proteins as well, the same set of neuroblasts would be controlled by da and the ASC; it follows, thus, that similar neuroblast defects would be expected in da- and in ASC- mutants. However, we find the same number of neuroblasts in da- embryos as in the wild type. Although the current data do not convincingly explain this result, three possible expla- nations are as follows: the maternal expression of cla provides the embryo with enough gene product during early stages of neurogenesis; another related protein can substitute da; or the proposed functional community between the ASC proteins and da oper- ates after early neurogenesis.

Experimental Procedures

Fly Stocks and Crosses DfCI)sc 8s7 is an interstitial deletion obtained from E. Grell that removes achaete, scute, lethal of scute, and asense and the com- plementation group EC4. Df(7)RTI84 is a terminal deletion that is both ASC- and vnd-. For the studies with vnd, we used the strong alleles vnd 6 and vndST; and for da, we used the allele da sx~, recovered by M. Brand.

Embryos hemizygous for ASC- or vnd could be identified unambiguously because they were derived from parental animals carrying an X chromosome with a P element-mediated insertion of a ftz promoter-/acZ construct in the elav locus (kindly provided by Y. Hiromi). The elav locus is located adjacent to the ASC and vnd genes within the subdivision 1B (Campos et al., 1985), in a region in which meiotic recombination is negligible. The parental females were heterozygous for this latter chromosome with the corresponding ASC or vnd mutation, and the males carried the ftz promoter-lacZ insertion in their X chromosome; hence, with the exception of the ASC or vnd mutants, all progeny expressed I~-galactosidase with the characteristic ftz pattern, as revealed with an anti-l~gal antibody. In the cross to obtain double mutant embryos Df(1)scSS~da ~6, the parental flies carried the same X chromosomes and a conventional sec-

Neuron 88

ond chromosomal balancer, i.e., without a P element-mediated lacZ insertion. Thus, the ASC- embryos could be identified unambiguously; one-fourth of them are expected to be da Kx~36 as well. Over 30 such ASC- embryos were examined, of which the- oretically 7-8 should have been double mutants.

Immunocytochemistry Dechorionated embryos were placed on Petri dishes, covered with water, and observed with a stereo microscope. Between 0 and 15 rain following the beginning of gastrulation, the embryos were transferred to a Petri dish lined with moist paper. They were allowed to continue development at 25°C for 80 min (for early stage 10 collections) or for 150 min (for early stage 11 collections) and were processed further for antibody staining.

For the analysis of neuroblast segregation, staged embryos were fixed for 20 min in heptane saturated with 4% parafor- maldehyde in 0.1 M PIPES, 2 mM EGTA, 1 mM MgSO4 (pH 6.95). The vitelline envelopes were removed as described by Mitchison and Sedat (1983). Following an overnight incubation with anti-en (provided by M. Wilcox), anti-hb (two different antibodies provided by H. J~ickle and P. Macdonald, respectively), or anti-ftz (provided by W. Gehring) antibodies in PBGT (PBS, 10% goat serum, 0.3% Triton X-100), embryos were washed with PBGT, reacted with the appropriate biotinylated secondary antibody, and stained with Vectorlabs Vectastain ABC kit using diaminobenzidine and nickel and cobalt ions (Lawrence et al., 1987). In a second step, embryos were incubated overnight with rabbit anti-~-gal (Cappel) and with the mouse MAb43B5 (which binds to cell membranes; F. Jimbnez, unpublished data) in PBGT, washed, and incubated with HRP-labeled anti-rabbit and biotin- labeled anti-mouse secondary antibodies. The staining for the anti-rabbit antibody was developed with diaminobenzidine, lacZ + and lacZ- embryos were separated 'from each other, and the anti-mouse antibody was revealed with Vectastain ABC kit and diaminobenizidine. Embryos of different ages Were stained with affinity-purified anti-HRP antibodies (following the pro- tocol of Bodmer and Jan [1987]) for general morphology of the CNS and PNS. After staining, the embryos were dehydrated and embedded in Epon. Before mounting, the embryos were split into dorsal and ventral halves and the gut was removed to permit a better orientation and to reduce the background staining of the preparations.

Histology Staged embryos were fixed for 10 min with glutaraldehyde- heptane, devitellinized mechanically, and incubated in an X-Gal solution as described by Hiromi et al. (1985). Mutant embryos, i.e., those that did not stain for I~-galactosidase, were separated from their wild-type siblings, and both groups were processed further for light microscopy using conventional histological techniques.

Acknowledgments

We are grateful to Simone Guth for expert technical assistance with the histology; W. Gehring, H. J~ickle, R Macdonald, and M. Wilcox for antibodies; Michael Brand for discussions; and Paul Hardy for critical reading of the manuscript. This work was sup- ported by grants from the Direcci6n General de Investigaci6n Cientifica y Tdch~ica (PB87-0433-CO2-02) to F.J. and from the Deutsche Forschungsgemeinschaft (SFB 243) to J. A. C:O. In addition, F. J. acknowledges the institutional grant from the Funda- cfon Areces to the Centro de Biologfa Molecular (Madrid).

Received February 21, 1990; revised April 19, 1990.

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Defective neuroblast commitment in mutants of the achaete-scute complex and adjacent genes of D. melanogaster