Genetic and molecular bases of neurogenesis in Drosophila melanogaster.

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GENETIC AND MOLECULAR Annu. Rev. Neurosci. 1991.14:399-420. Downloaded from www.annualreviews.org by Queens University - Kingston (Canada) on 05/12/13. For personal use only.

BASES OF NEUROGENESIS IN DROSOPHILA MELANOGASTER Jose A. Campos-Ortega

Institut fUr Entwicklungsphysiologie, Universitat zu KOln, GyrhofstraBe 17, 5000 Kaln 41, Federal Republic of Germany Yuh Nung Jan

Howard Hughes Medical Institute, and Departments of Physiology and Biochemistry, University of California San Francisco, California 94143 KEY WORDS:

neurogenesis, drosophila, proneural genes, neurogenic genes

INTRODUCTION

In insects, neurons in the central nervous system (CNS) are generated by progenitor cells called neuroblasts derived from a region of the ectoderm called the neurogenic region or neuroectoderm. In the neuroectoderm of Drosophila melanogaster, neighboring cells take on one of two alternative fates and develop either as neuroblasts or as epidermoblasts (progenitor cells of the epidermis). The neuroblasts move to deeper levels ofthe embryo to build up the central neural primordium, whereas the epidermoblasts remain at the surface to build up part of the epidermal sheath. The peri pheral nervous system (PNS) of insects develops from progenitor cells located within the epidermis. Thus, development of the PNS involves another choice by the epidermal cells between neural and nonneural fates, i.e. to develop as sensory progenitor cells versus nonsensory epidermal cells. As is discussed in this review, many of the molecular mechanisms involved in the cell fate choices are shared in the development of the CNS and PNS. The analysis of the cellular decisions that lead to the formation of the 399 0147-006X/91/0301-0399$02.00

Annu. Rev. Neurosci. 1991.14:399-420. Downloaded from www.annualreviews.org by Queens University - Kingston (Canada) on 05/12/13. For personal use only.

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neural primordia of insects has received considerable attention during the last ten years by developmental neurobiologists. Because its genetics have been well studied, most of the work on this problem has been carried out in Drosophila melanogaster. In this organism, cellular and genetic aspects of the segregation of neuroblasts and epidermoblasts, and of the origin of the progenitor cells of sensory organs, have been investigated and today a fairly comprehensive picture of these processes begins to emerge. Neuro ectodermal cells become committed to their fates by intervening regulatory signals mediated by cellular interactions. Two different groups of genes appear to be required for neural and epidermal development, respectively, by providing the various links of a regulatory signal chain. The sequence of some of these gene products is known, and the molecular analysis of the mechanisms of regulation underlying the cellular decisions under dis cussion has begun. In this review, we discuss cellular, genetic, and molec ular aspects of the separation of progenitor cells for the epidermal and neural lineages. CELLULAR ASPECTS OF NEUROGENESIS Development of the Central Nervous System

The following is a short description of early neurogenesis in the. territory of the trunk [refer to Campos-Ortega & Hartenstein (1985) for a general introduction in Drosophila embryogenesis, and for staging; and to Harten stein & Campos-Ortega (1984), Hartenstein et al (1987), and Technau & Campos-Ortega (1985) for further details]. The segregation of the neuro blasts from the neuroectoderm lasts for approximately three hours (Figure 1) and is discontinuous, proceeding in three discrete pulses that give rise to three subpopulations of neuroblasts, called SI, SIl, and SIll. Roughly 25% (approximately 500) of all cells of the neuroectodermal anlage in the blastoderm stage develop as neuroblasts, whereas the remaining 7 5% (approximately 1500 cells) develop as epidermoblasts. The epidermoblasts remain in the outer layer and ensheath the neuroblasts transiently with tong basal processes, which allow cellular interactions to be continued for some time after the segregation. The processes are later retracted and the epidermoblasts diminish in size to take on an epithelial appearance. Groups of cells of fairly large size, which still have neurogenic capabilities and give rise to neuroblasts, are present in the neuroectoderm until late stage 11. Midway through stage 11 a fourth group of neural progenitor cells appears in each segment that comprises the unpaired median neuroblast (MNB) and a number of small, paired cells called midline precursor cells (MPs) (Bate 1976, Bate & Grunewald 1981, Thomas et aI 1984).

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401

Cell d ivisions that give rise to PNS Cell divisions of the ectoderm

Neuroblast


l-d_ e_la_m_in_at _ I

5

Stage

r-I

Cellularization of blastoderm

Figure 1

6f7

---'+

Cell divisions in CNS ________________

9h

5h

3h

o

_-+

8

9

10

t +L-_-'j

gastulalion

12

11

10h

13

germ band retraction

germ band elogation

The temporal sequence of the development of the CNS and PNS in Drosophila at

25°C. The staging is according to Campos-Ortega & Hartenstein ( 1985).

Development of the Peripheral Nervous System

The PNS consists of a variety of sensory organs. In the trunk region, the sensory organs are arranged in three groups, dorsal, lateral and ventral, with a precise pattern (Hertweck 193 1, Campos-Ortega & Hartenstein 1985, Ghysen et al 1986, Hartenstein & Campos-Ortega 1986, Dambly Chaudiere & Ghysen 1986, Bodmer & Jan 1987). In most cases studied in insects, each sensory organ is made of cells that are clonally related, i.e. they are progenies of a common progenitor cell (for review see Bate 1978). This appears also to be the case in the Drosophila embryonic PNS. The study of the pattern of incorporation of the nucleotide analogue BrdU by DNA-replicating ectodermal cells has permitted the reconstruction of the precise sequence of mitotic divisions that produce the sensory organ cells (Bodmer et al 1989). The progenitor cells start dividing during late stage 11, partially overlapping with the third mitotic division of the epidermal primordium ( Figure 1), to give rise to groups of clonally related cells; the members of each clone will then form the different cell types of a sensory organ (Bodmer et al 1989, Hartenstein 1988). Although the mitotic divi sions that will eventually give rise to the cells of the sensory organs take place during embryonic stages 11-1 3 (Bodmer et al 1989), the cor responding progenitor cells are already detectable a couple of hours earlier,

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in stage 10, as they differentiate molecular markers specific for sensory organ precursors and sensory organs in a segmentally specific manner (Ghysen & O'Kane 1989).


Cell Commitment in the Neurogenic Ectoderm

In insects, the decision of the neuroectodermal cells to adopt the epidermal or the neural fate is at least in part mediated by cell-cell interactions. Two pieces of experimental evidence support this argument. Laser ablation experiments carried out in grasshoppers showed that the cells remaining in the neurogenic region after the neuroblasts have segregated are not firmly committed to their fate (Taghert et a1 1984, Doe & Goodman 1985). Under normal circumstances, these cells would develop as epidermoblasts; however, with the neighboring neuroblast ablated they may adopt the neural fate instead. Results of cell transplantations in Drosophila suggest that regulatory signals pass between the cells of the neuroectoderm, thereby causing their commitment to either the epidermal or the neural fate (Tech nau & Campos-Ortega 1986, Technau et al 1988, Campos-Ortega 1988). Two kinds of signals, one with epidermalizing and the other with neural izing character, have been implicated in Drosophila from genetic analy sis (discussed in the following section) and from cell transplantation experiments. GENES AFFECTING NEUROGENESIS

The available evidence strongly suggests that the proteins encoded by two groups of genes provide the molecular basis for the regulatory signals that control the process of neurogenesis. The so-called "proneural" genes, together with the neurogenic genes (abbreviated NG), are required for a proper segregation of neural and epidermal lineages at the development of both the CNS and the PNS (Table 1). "Proneural" Genes

This group of genes is called "proneural" because they define a state that leads to the commitment of cells to become neuronal precursors (neuroblasts in the case of the CNS and sensory organ progenitor cells in the case of the PNS) (Ghysen & Dambly-Chaudiere 1989, Romani et al 1989). It includes genes of the achaete-scute complex CASC), daughterless, and additional genes that have not yet been identified. The Genes of the achaete-scute Complex CASC)

The genes of the achaete-scute complex (ASC) include achaete, scute, lethal of scute, and asense. Their participation in CNS and PNS development

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Table 1


Gene

403

Genes involved in neurogenesis in Drosophila melanogaster Loss of function phenotype

Possible function

Motif found in the gene product

References

ASC'

hypoplasia

HLH

transcriptional regulation

Villares & Cabrera 1 987

da

hypoplasia

HLH

Caudy et al

N

hyperplasia

transmembrane, EGF-like repeats

transcriptional regulation adhesion? signal receptor?

transmembrane,

adhesion?

Viissin et al

EGF-like repeats

signal receptor?

hyperplasia

Dl

E(spl)Cb

hyperplasia

HLH

mam neu

hyperplasia

?

bib

hyperplasia

hyperplasia

"Comprises the genes

transmembrane

achaete (T5), scute (T4), lethal oIscute

transcriptional regulation? ?

transcriptional regulation? channel? transporter?

(T3), and

asense (T l a

1988b Wharton et al 1985; Kidd et al 1 986 1987, Kopczynski et al 1 988 Kliimbt et al 1989

Boulianne et al in prep. Rao et al 1990 or T8). The phenotype is

that of deletion of the whole complex.

b The indicated phenotype is that of the deletion of the whole complex; other data in the table refer to the

transcription units m8

[E(spl)],

m7, and m5. The composition of the

E(spl)C,

however, is not yet completely

understood; besides m8, m7, and m5, the gene complex may include other genes, as for example m3, m4 (Knust

et al 1987c), and m9-m to (Preiss et al 1988, Hartley et al 1988).

has been well documented (Garcia-Bellido & Santamaria 1978, Garcia Bellido 1979, Jimenez & Campos-Ortega 1979, 1987, 1990, Damb1y-Chau dit�re & Ghysen 1987, Ghysen & Dambly-Chaudiere 1988, Gonzalez et al 1989). Embryos packing the ASC have a hypop1asic CNS (Jimenez & Campos Ortega 1979, 1987, Campos-Ortega & Jimenez 1980, White 1980) and lack all sensory neurons, with the exception of those innervating chordotona1 organs and a few multidendritic neurons (Dambly-Chaudiere & Ghysen 1987). Three different, though probably related, processes contribute to the development of the ASC-phenotype in the CNS (Jimenez & Campos Ortega 1979, 1990, Cabrera et a1 1987): ( a ) the complement of neuroblasts is defective, as neurogenesis is initiated by fewer neuroblasts than in the wild-type; (b) the rate of proliferation of neuroblasts is lower than normal; and (c) there is increased cell death in the primordia of the CNS and PNS. With respect to PNS development, Dambly-Chaudiere & Ghysen ( 1987) studied the effects of eliminating particular genes of the ASC on the


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development of the embryonic sensilla and found that although there are some overlapping effects, particular genes affect particular subsets of sensory organs. The loss-of-function of the asense gene has little effect on adult bristle development, but it has a clear effect on visual ganglia (Gon zalez et al 1 989). The genomic DNA of the ASC has been cloned and characterized by J. Modolell and colleagues (Carramolino et al 1982, Campuzano et al 1985, 1986, Ruiz-Gomez & Modolell 1987, Balcells et al 1988, Gonzalez et al 1989). The genes of the ASC are contained within approximately 8590 kb of genomic DNA (Carramolino 1982, Campuzano et al 1985). The region encodes a number of transcripts, four of which, T5, T4, T3, and T l a (T8), correspond to achaete, scute, lethal o f scute, and asense, respectively (Campuzano et a1 1985, Alonso & Cabrera 1988, Balcells et al1 988, Gon zilez et al 1989). Sequence analysis has shown that the predicted protein products of the four genes share several conserved domains, including the Helix-Loop-Helix (HLH) motif of the myc gene family (Villares & Cabrera 1 987, Alonso & Cabrera 1988, Gonzalez et al 1989, Murre et al 1989a). The spatial distribution of the T3, T4, and T5 transcripts has been studied in sections of staged embryos via in situ hybridization (Cabrera et

al 1987, Romani et al 1987). The distribution is similar but not identical for the three transcripts and shows a high degree of correlation with the processes of neuroblast segregation and the development of sensory organs and stomatogastric ganglia. Since their domains of expression overlap partially, some neuroblasts may contain all three RNAs, and their products, whereas other neuroblasts contain only one or two of them. The pattern of transcription, the correspondence between deletion of ASC genes and defects in particular subsets of sensory organs (Dambly- Chau diere & Ghysen1 987, Ghysen & Dambly-Chaudiere 1988), and that ectopic expression of ASC leads to formation of ectopic sensory organs suggest that local expression of ASC is required for the local decision to form a sensory organ. Cabrera et al (1987) proposed that the ASC genes serve to provide the neuroblasts with specific identities, based on the combination of products expressed in each cell. The daughterless Locus

The gene da has been known for some time to be required for sex deter mination and dosage compensation (Cline 1976, 1980, Lucchesi & Skripsky 1981). Thus, the requirements for da+ for normal neural development was initially a surprising finding (Caudy et al 1988a). Now, however, there are a growing number of examples of a gene's being used multiple times in different developmental processes. da is expressed both during oogenesis and embryonic development. The maternal expression is relevant at early


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405

blastoderm stages for a correct dosage compensation and differentiation in female embryos (Cline 1976, 1980) whereas the zygotic expression is essential for PNS development in both sexes (Caudy et al 1988a). In fact, da is the only gene known to be required for the formation of all sensory organs (Jan et a1 1987; Figure 2). In da mutants, sensory organ precursors never form (Caudy et al 1988a, Ghysen & O'Kane 1989). In contrast to the total absence of PNS, CNS in da mutants is only partially affected. The mutant CNS is smaller than normal, the ventral cord being frequently fragmented in several pieces. Nonneural organs are relatively normal, although some minor defects can be seen in the pattern of muscles and in the gut. The da locus has been recently cloned (Caudy et al 1988b, Cronmiller & Cline 1988). The da gene encodes a single transcription unit with two overlapping RNAs of 3.2 and 3.7 kb. The conceptual translation of the corresponding cDNA sequences reveals the conserved HLH motif. The similarity is particularly high between the da protein and two proteins (E 12 and Ed that bind to the KE2 DNA motif in the immunoglobulin kappa chain enhancer. Murre et al (1989a) showed that the HLH motif plus an adjacent basic domain is sufficient to form dimers and bind to specific DNA sequences. Moreover, members of the HLH protein family, including T3 of ASC and daughterless, are able to form heterodimers, which specifically bind to DNA in vitro (Murre et al 1989b). These findings provide a plausible molecular mechanism for the genetic interaction between the ASC and da observed previously (Dambly-Chaudiere et al 1988). da is probably a ubiquitous transcriptional regulator, whereas ASC products are expressed in regions that approximately correlate with where neuronal precursors appear. An appealing possibility is that da and ASC form heterodimers that bind to DNA and regulate the transcription of their target genes to initial development of neuronal progenitor cells. It has become increasingly clear that HLH proteins play important roles in Drosophila neurogenesis. All of the HLH proteins with known function [including e.g. E12, E47, da, ASC, twist ( Thisse et aI1988), E(spl)C (KUimbt et al 1989; see below), MyoD (Davis et al 1987), and all myogenic genes identified so far] seem to be involved in specifying cell fate. In addition to da, ASC, two other genes affecting sensory organ formation in adult flies, hairy (h, Rushlow et al 1989) and extra maeroehaete (erne, Garrell & Modolell l990, Ellis et aI1990), also encode HLH proteins. Genetic studies suggest that h and erne act as negative regulators of ASC. It is conceivable that both h and erne can form -heterodimers with da and/or ASC such that the beterodimers do not bind DNA or that they have altered tran scriptional regulatory activity. Interestingly, erne shares strong similarity with the HLH mouse protein Id: i.e. they have 80% similarity in the HLH


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CAMPOS-ORTEGA & JAN

b

Figure 2 Drosophila embryonic CNS and PNS as revealed by staining with Mab44Cl l ,

which recognizes all neuronal nuclei (Bier e t al 1988). (a) Wild type. The picture was taken at the focal plane containing the PNS. The CNS is visible but out of focus. (b) A da mutant. Same focal plane as in (a). Notice that the entire PNS is missing. The CNS is present but with ventral cord broken. (c) Both CNS and PNS exhibit severe hypertrophy.

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motif and both lack the basic domain that is the putative DNA binding region; Id has been shown to be a negative regulator of certain HLH proteins (Benezra et al 1990). Since the HLH proteins have the ability to form homo or heterodimers and to function as positive or negative regulators, they are capable of generating a high degree of specificity and complexity of regulatory functions.


The Neurogenic Genes

The neurogenic effects of Notch- mutations (N-) were discovered by Poulson as early as 1937. Over 40 years later, Lehmann et al (1981, 1983) described another six loci, the loss of whose functions leads to a phenotype similar to N- mutations. Additional NG genes with predominantly maternal expression were described by Perrimon et al (1984) and LaBonne & Mahowald (1985). Most of the work, however, has been done on the six NG loci with predominantly zygotic expression, i.e. master mind (mam), big brain (bib), neuralized (neu), Delta (Dl), and Enhancer of split [E(spl)], and this review consequently focuses on these zygotic NG loci. Another NG zygotic locus has been recently discovered, although there is still very little known of it (Bier et al 1989). The complete loss of a NO gene function causes cells in the neuro ectoderm that would have been epidermoblasts to become neuroblasts ( Figure 2). Hence, approximately 2000 cells initiate neurogenesis in the NG mutants, instead of only 500 in the wild-type. This leads to embryonic lethality, associated with massive hyperplasia of the CNS and an increase in the number of sensory neurons, with concomitant lack of the entire ventrolateral and cephalic epidermis in the mature embryo (different aspects of the complex phenotypc of the NG mutants are described in Poulson 1937, Wright 1970, Lehmann et a11981, 1983, Jimenez & Campos Ortega 1982, Dietrich & Campos-Ortega 1984, Hartenstein & Campos Ortega 1986). Since most of the NO genes have both maternal and zygotic expression, the complete loss of gene function is only attained when both components are removed (Jimenez & Campos-Ortega 1982, Dietrich & Campos-Ortega 1984). bib is different from the other NG loci, in that no maternal expression can be detected and the phenotypic effects of complete loss of the bib+ function are less severe (Lehmann et al 1983, Dietrich & Campos-Ortega 1984). Besides the neural hyperplasia and the epidermal defects, all remaining embryonic organs of severe NG mutants are also affected to some extent. Thorough embryological analyses, however, have demonstrated that these are not primary defects, but rather morphogenetic consequences of the neural fate adopted by all the neuroectodermal cells (Wright 1970, Lehmann et al 1981, 1983).

408

CAMPOS-ORTEGA

& JAN


Mosaic Analysis of NG Mutants

Two main questions have been addressed by mosaic analysis: Are the NG genes required for adult development? Is the requirement of NG gene function cell autonomous? Genetic mosaicism involving NG mutations has been induced by means of mitotic recombination throughout larval development in cells of various imaginal discs (Dietrich & Campos-Ortega 1984). The main conclusion of that study was that the NG genes, with the exception of bib, are required for normal development of the imaginal epidermal cells. Cell clones homozygous for loss-of-function mutations of the bib locus are morphogenetically normal. At least two interpretations of this latter observation are possible: first, the bib product is not required for imaginal development; or, second, the bib product can diffuse freely and the surrounding heterozygous cells provide the clone of mutant cells with the normal gene product. An additional important conclusion was that NG gene products are unable to diffuse over long distances. The results were compatible with a cell autonomous expression of the NG genes, at least in the imaginal discs. Hoppe & Greenspan (1986 , 1990) studied gynandromorph embryos formed by N+ and N- cells and came to the same conclusion with respect to N in its embryonic function. A different conclusion, however, is drawn from transplanting neuro ectodermal cells from NG mutants into the neuroectoderm of the wild-type (Technau & Campos-Ortega 1987). Following homotopic and isochronic transplantation, single cells from NG mutants into wild-type hosts behave, with the remarkable exception of E(spl) - cells, like wild-type cells, i.e. they give rise to neural, epidermal, and mixed clones in proportions similar to those of the controls. Hence, under the conditions of the transplantation, the NG genes under discussion are not cell-autonomous in their expression. Since cells lacking any of the genes N, bib, mam, neu, or Dl develop normally when surrounded by wild-type cells, the mutant cells are appar ently capable of receiving and processing the epidermalizing signals with the same efficacy as the wild-type cells and, therefore, adopt the epidermal fate in some cases. It follows that the corresponding mutant cells may have normal receptor mechanisms but an abnormal signal source. Hoppe & Greenspan (1990) found that a small patch of homozygous N- cells may develop epidermal histotypes within the ventral epidermis following X-ray-induced mitotic recombination in heterozygous animals; that is, the mutant cells may adopt an epidermal fate when they are surrounded by wild-type neighbors. This result can be interpreted. to mean that the laCK of the N function in a single cell can be compensated for by the surrounding wild-type cells and, thus, taken as evidence for a


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409

nonautonomous expression of N. In this experiment, however, there were fewer epidermal clones recovered than in controls. Since the progenitors of the mutant cells are heterozygous, i.e. they have a copy of the N+ allele, another interpretation of the result is thus possible, namely that some of the homozygous N- cells develop an epidermal fate because of still having wild-type gene product derived from their heterozygous mother cells. Hence, with respect to the autonomy of expression of the N gene, the available evidence is contradictory. In striking contrast to the results with cells lacking N, bib, mam, neu, or Dl, only neural clones develop upon transplantation of cells lacking the E(spl) locus. Therefore, E(spl) is the only NG locus with cell autonomous expression under the conditions of the transplantation experiment. According to the same lines of reasoning as before, this result is interpreted to mean that the E(spl)- cells cannot react to the epidermalizing signal. Thus, the E(spl) locus is a good candidate to encode protein(s) related to the different steps from the receptor to the nucleus, e.g. receptor molecules themselves, "second messengers," or transcription factors. Functional Interactions Between the Neurogenic Loci

The similar phenotype caused by the loss of any of the NG gene functions strongly suggests that their products may contribute to serve the same overall function and, indeed, a large number of observations show that the NG loci interact functionally (Campos-Ortega et al 1984, Dietrich & Campos-Ortega 1984, Vassin et al 1985, de la Concha et a1 1988, Shepard et al 1989, Brand & Campos-Ortega 1989, Xu et al 1990). Although the NG genes exhibit fairly pleiotropic expression, all of the NG loci tested, with the exception of bib, are involved in the same developmental pathway. The data, however, emphasize that eloser functional relationships are maintained by particular loci, e.g. E(spl) with Dl, E(spl) with N, N with Dl, N and Dl with mam, and neu with E(spl). Epistatic relationships exist between five of the NG, whereas bib appears to be independent from the others ( Figure 3). These results are consistent with the notion that the five loci are links of a chain, or network of epistatic interactions, the last link of which comprises the genes of the E(spl)C (de la Concha et al 1988). There is as yet no direct indication about the molecular level at which the functional interactions mentioned above take place; however, some guesses are possible. Two Dl alleles have been recovered as suppressors of spl (Brand & Campos-Ortega 1989). Since the suppression of the spl phenotype by Dl is allele specific, the interaction between both genes is likely to be at the level of the proteins. Furthermore, the sequence of th7 putative products of genes of the E(spl)C and neu

410

CAMPOS-ORTEGA & JAN T

T

MU �

T

rneu lQM!

I

T

�lY.

L, �

� Dl

:!:

t ......:1

_-____

EWill

l


Eoider mogenesis Figure 3

The figure illustrates the network of genetic relationships of the NG genes. This diagram is based on formal arguments derived from transmission genetics and gives no indication concerning the molecular level at which the proposed interactions take place. Positive or negative signs reflect the kind of functional influences, i.e. repression or activation, assumed to be exerted by one gene product upon the next one. The data suggest that the E(sp/) locus provides an epidermalizing signal to the neuroectodermal cells. Although bib takes part in the process of neuroblast segregation, its function is apparently independent of the other six genes (from de la Concha et a1 1 988, modified).

suggests that interrelationships involving these genes are likely to take place at the level of transcriptional regulation. The Notch Locus

Mutations at the N locus display a large variety of phenotypic traits (discussed by Wright 1970); however, the results of a thorough genetic analysis carried out over many years by W. Welshons (1965, 1974; Wel shons & Keppy 1975, 1981) and molecular analysis (Artavanis-Tsakonas et al 1983, 1984, Kidd et al 1983, 1986, Kelley et al 1987, Hartley et al 1987, reviewed in Artavanis-Tsakonas 1988) indicate that N mutations affect a single gene, rather than a gene complex. The Nlocus contains a poly A + -RNA of 10.5 kb (Artavanis-Tsakonas et aI198 3), which is temporally regulated according to a complex pattern (Artavanis-Tsakonas et a11983, 1984, Kidd et aI1983). In situ hybridization has shown that during early embryonic stages, the N transcript is ubiquitously expressed (Hartley et al 1987). From stage lion, NRNA is still present in most of the embryonic cells, although concentrated on the periphery of the eNS, in cells that probably correspond to the neuroblasts. Antibodies raised against fusion proteins uncover a similar distribution of the N protein (Kidd et a1 1989, Johansen et al 1989). These data suggest that the function of the N locus is likely to be required for other processes in addition to neurogenesis. The sequence of the 10.5 kb poly A + RNA has been established (Whar ton et al 1985b, Kidd et al 1986); conceptual translation of the sequence


NEUROGENESIS

4 11

reveals the N product as a putative transmembrane protein of 2703 amino acids. Its extracellular domain consists mainly of 36 cysteine-rich tandem repeats with homology to various proteins of mammals, among them the epidermal growth factor (EGF). Several N alleles, e.g. split and Abruptex alleles, have been shown by DNA sequencing to correspond to single amino acid exchanges, in particular EGF-like repeats (Hartley et al 1987, Kelley et al 1987). There are also three copies of another cysteine- rich repeated motif in the extracellular part, called the Notch repeats (Wharton et aI1985b), and a homopolymeric repeat consisting of glutamine residues, called the opa repeat in the putative intracellular domain (Wharton et al 1985a). Antibodies have confirmed the location at the membrane of the N protein (Kidd et al 1989, Johansen et aI1989). The structure of the N protein is compatible with its participation in cell communication processes, as suggested by embryological and genetic data. It is remarkable that the protein encoded by the lin-12 gene of C. elegans, which is known to control several developmental cell decisions in the nematode (Greenwald et a1 198 3, Sternberg & Horvitz 1984), exhibits overall similarity to the putative N protein (Greenwald 1985, Yochem et al 1988). On the basis of a thorough analysis of eye morphogenesis in mutants carrying any of various N alleles, including spl, Cagan & Ready ( 1989) conclude that the N protein plays a permissive rather than an instructive role during eye development, as it is required in decisions of every cell type; according to this hypothesis, N would facilitate intercellular communication by a general process such as cell adhesion. Whether or not the N protein exerts a similar function during embryonic development is unknown. The Delta Locus

Dlmutations express a large variety of phenotypic traits and, in particular, they exhibit a complex pattern of heteroallelic complementation com patible with the notion that Dlmay be a complex locus (Vassin & Campos Ortega 198 7, Alton et al 1988). The Dl locus spans a stretch of approxi mately 25 kb of genomic DNA (Vassin et al 1987, Kopczynski et aI1988). The transcriptional organization of the locus is not yet completely worked out (see, however, Kopczynski et a1 1988 and Kopczynski & Muskavitch 1989). In situ hybridization to embryonic tissue sections shows a striking dis tribution of the major Dl5.4 kb RNA transcript (Vassin et aI1987). Two main aspects of its very complex expression pattern should be emphasized: 1. Dl RNA is expressed in territories with neurogenic capacities, like the neuroectoderm or the anlagen of sensory organs.

412


2.

CAMPOS-ORTEGA & JAN

After an initial phase, during which it is abundantly transcribed in all cells of such territories, the RNA becomes restricted to the cells that adopt the neural fate, e.g. the neuroblasts or the cells forming sensory organs, and persists in those cells for some time.

Two of the regions where Dl is transcribed are not known to have neuro genic abilities. One is the mesodermal layer, where Dl is transiently tran scribed during stages 9 to 10 (Kopczynski & Muskavitch 1989; D. Godt and J. A. Campos-Ortega, unpublished); the other region is the anterior half of the hindgut, where a high concentration of Dl RNA is present throughout embryogenesis. Sequences of the putative proteins encoded by the 5.4 kb Dl transcript have been deduced (Knust et al 1987a, Vassin et al 1987, Kopczynski et al 1988); they show some similarity to the N protein (refer to Wharton et al 1985b, Kidd et al 1986). The sequences indicate a transmembrane protein with a number of features, among them a putative signal peptide, five potential glycosylation sites, and an extracellular domain comprising 9 EGF-like repeats. In view of their homology to the EGF, the repeats encoded by N and Dl might conceivably be cleaved from the cell membrane and diffuse through the intercellular space. Data from genetic mosaics, however, indi cate that the products of both Nand Dl are unlikely to diffuse over long distance (Dietrich & Campos-Ortega 1984, Hoppe & Greenspan 1986). Anti-Nantibodies indicate that the Nprotein is indeed a stable component of the cell membrane (Kidd et aI1989). It is more probable that Nand Dl mediate protein-protein interactions between neighboring, rather than dis tant, cells. The results of transplanting mutant cells (Technau & Campos Ortega 1987) suggest that both proteins act at the side of the signal source. The Enhancer of split Locus

The E(spl) locus was discovered by means of the mutation E(spl)D (re covered by L. M. Green, quoted in Lindsley & Zimm 1985; see Welshons 1956), which considerably enhances the phenotype of spl (an allele of Notch). Several mutations, including E(Spl)D, have been mapped to a 3436 kb stretch of genomic DNA (Knust et a1 1987c, Preiss et a1 1988) deleted in the variant Df(3)E(splt·A7!, and have been molecularly characterized to some extent. The region comprises 10 different transcription units and encodes 11 transcripts (one of the units encodes two overlapping RNAs) that have been called ml to mIl, in the proximo-distal direction. P element-mediated transformation with the m8 gene derived from E(Spl)D animals demonstrates that defects in the coding region of m8 are respon sible for the enhancement of the spl phenotype (Klambt et al 1989; K.


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Tietze and E. Knust, personal communication). Thus, the gene E(sp/) corresponds to m8. An increasingly large body of experimental evidence (Knust et al 1987c, Ziemer et al 1988, KUimbt et al 1989; H. Schrons, E. Knust, and J. A. Campos-Ortega, unpublished observations) suggests that E(sp/) is one of a cluster of several related genes, to which the name Enhancer of split complex [E(sp/)C) has been given (Campos-Ortega & Knust 1990). The following two arguments support E(sp/) as a likely part of a gene complex. First, DNA sequencing (Klambt et a1 1989) has uncovered extensive hom ology among the putative proteins encoded by m8, m7, and m5, thus substantiating the hypothesis that various products of the complex perform similar functions. In particular we want to emphasize a conserved domain located at the amino-terminal ends of the putative protein products of m8, m7, and m5 that exhibits sequence similarity to the HLH motif of other transcriptional regulators (Murre et al 1989b; see above). Second, the RNAs encoded by the transcription units m4, m5, m7, and m8 show a very similar spatial distribution during embryogenesis (Knust et al 1987c), thereby suggesting functional community for the four genes. Thus, we propose that these four genes are members of the E(sp/)C. Transformation experiments have been used by Preiss et al (1988) to present evidence that the RNAs m9-mlO are related to neurogenesis. Hartley et al (1988) determined the sequence of the protein encoded by the overlapping transcripts m9-mlO and showed that it is similar to the p subunit of transducin, a G protein of mammals. The neuralized Locus

The genetic features of the neu locus are still largely unknown. Increasing the number of copies of the neu+ locus causes the production of a few embryos with neural hypoplasic defects, reminiscent of those found among the progeny of females triploid for E(sp/)+ (Knust et aI1987b). Increasing the ploidy of N+ or D/+ does not cause such embryonic defects, thereby suggesting particular relationships between neu and E(spl) (see below). neu also has a maternal component of expression (Dietrich & Campos Ortega 1984). The neu locus has been recently cloned, and it gives rise to a 4.0 kb transcript that is present throughout embryogenesis (G. L. Bou lianne et aI, in preparation). The neu RNA is temporally and spatially regulated during embryonic deVelopment according to a complex pattern. Particularly interesting is that after the segregation of lineages, neu tran scripts continue to be present in the neuroblasts, but not in the epidermo blasts, thus suggesting a role for neu in the former cells. Sequencing of neu eDNA clones has uncovered a protein with a potential DNA-binding motif of 43 amino acids at its carboxy terminus, encompassing three potential

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alpha helices. The first potential alpha helix shows similarity to the first alpha helix of the homeobox motif, whereas the other two helices are similar to the helix-tum-helix structure identified in the DNA-binding domain of the lambda or the gal repressor. The persisting location of neu RNA in the neuroblasts and the structure of its protein suggest that it may play a role in transcriptional regulation during the initial steps of lineage segregation.


The master mind Locus

Flies heterozygous with any amorphic mam mutation exhibit various defects of the wings, particularly notching at the posterior margin and delta-shapcd widenings of the tips of the veins (Lehmann et al 1981, Niisslein-Volhard et al 1984; H. Schrons, U. Wetter, D. Weigel, U. Dietrich, and 1. A. Campos-Ortega, unpublished). These phenotypic traits are remarkably similar to those of heterozygotes for N or Dl mutations. Very little is known about the molecular organization and expression of the mam locus. Several mam mutations have been mapped to a stretch of 45-60 kb of genomic DNA that contain a large number of copies of two repetitive sequences (Weigel et al 1987, Yedvobnick et al 1988). Yedvob nick et al (1988) have shown that one of the repeats corresponds to opa (Wharton et al 1985a) and the other to the RS-repeat. The 45 kb stretch encodes two major overlapping RNAs, of 5.0 and 3.9 kb approximately, which show the expected temporal regulation, i.e. strong maternal expres sion and zygotic expression during 3-8 hr of embryonic development. The bib Locus

The gene big brain is unique in at least four respects. First of all, bib is the only NG gene that is not expressed during oogenesis. Second, its lack of function does not produce the same severe form of the embryonic NG phenotype, but rather an intermediate form of neural hyperplasia. Third, its function seems to be superfluous during development of imaginal sen sory organs. Fourth, the function of hih is independent of the function of the other NG genes (Lehmann et al 1983, Dietrich & Campos-Ortega 1984, de la Concha et al 1988, Rao et al 1990). As yet, very little is known about the genetic structure of the bib locus; however, its DNA has been cloned recently (Rao et a1 1990) and found to comprise two RNAs of 3.4 and 3.1 kb that are spatially regulated. In the blastoderm and during germ band elongation, expression of bib matches thc ectodermal layer. After lineage segregation, bib continues to be expressed in the epidermoblasts as well as in the mesodermal derivatives. Sequencing of cDNA clones uncovers a putative transmembrane protein of 700 amino acids, with six hydrophobic stretches that could span the cell membrane. This protein

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415

exhibits considerable similarity to the major intrinsic protein of bovine fiber cell membrane, soybean nodulin-26, and E. coli glycerol facilitator. The function of the bib protein remains mysterious. Its putative structure suggests that it may be involved in cellular interactions; since from several genetic analyses bib has been shown to be functionally independent from the other six major NG loci, its function is likely to involve a different pathway.


CONCLUSIONS

Albeit still tentative, the main conclusion to be drawn from the current data (Brand & Campos-Ortega 1988) is that the proteins encoded by the NG genes, the "proneural" genes (ASC, da, etc), form a regulatory net work that permits the neuroectodermal cells to take on the neural or the epidermal fate. The genetic regulation responsible for the devclopment of neuroblasts appears to be achieved by the proteins encoded by the "proneural" genes, and for the development of epidermoblasts at least at ventral levels, by the proteins encoded by the E(spl)C. Cellular interactions necessary for the regulatory signals to be passed from one cell to another seem to occur at the cell membrane. At the cells' membrane, the cellular interactions are mediated by the Dl and N proteins, perhaps at the EGF like repeats present in the extracellular domains of both proteins. The question of the nature of the receptor molecule for the postulated signal is still open. Transplantation of NG mutant cells into the neuro ectoderm of the wild-type argues against the notion that N acts as a receptor. As we mentioned above, however, the implications of this evi dence are still uncertain, since under different experimental conditions N cells may behave differently. Thus, we must await additional experiments to resolve this issue. Although only the E(spl) locus behaves autonomously in the transplantation, and, thus, as though it encodes functions related to the reception of the signals, none of the products from genes of the E(spl) region whose putative sequence has been determined so far resembles a receptor-like protein. Nevertheless, the determined sequences suggest transcriptional regulators [the proteins encoded by m5, m7, and m8, with the HLH motif (Kliimbt et aI1989)]; together with the results of the genetic analysis, which locates the function of the E(spl)C at the end of the epidermalizing pathway, these data are in agreement with the results of transplanting NG mutant cells. The data available on the structure of the neu protein suggest that it may serve regulatory functions as a DNA binding protein. The genetic analysis shows that neu is hypostatic to Dt, N, and E(spl), thus suggesting that neu may regulate the transcription of the latter genes. The results of

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the transplantation experiments with mutant cells indicate a role for neu at the signal source; the spatial pattern of expression suggests that neu may be involved in transcriptional regulation within the neuroblasts. Finally, bib seems to act on neurogenesis through a pathway different from that of the other NG genes, and the structure of the putative protein suggests that it is related to membrane-mediated cell interactions.


ACKNOWLEDGMENTS

We would like to thank Lily lan, Elisabeth Knust, and Paul Hardy for constructive criticisms on the manuscript. The research reported here was supported by grants of the Deutsche Forschungsgemeinschaft (DFG) to I.A.C.-O. and the Howard Hughes Medical Institute to Y.N.J. Literature Cited Alonso. M. c., Cabrera. C. V. 1 988. The achaete-scute gene complex of Drosophila melanogaster comprises four homologous genes. EMBO J. 7: 2585-91 Alton, A. K., Fechtel, K., Terry, A. L., Meikle, S. 8., Muskavitch, M. A. T. 1 988. Cytogenetic definition and morphogenetic analysis of Delta, a gene affecting neuro genesis in Drosophila melanogaster. Gen etics 1 1 8: 235-45 Artavanis-Tsakonas, S. 1988. The molecular biology of the Notch locus and the fine tuning of differentiation in Drosophila. Trends Genet. 4: 95-100 Artavanis-Tsakonas, S., Muskavitch, M . A. T., Yedvobnick, B. 1 983. Molecular clon ing of Notch, a locus affecting neuro genesis in Dorsophila me lanogaster . Proc. Natl. Acad. Sci. USA 80: 1 977-8 1 Artavanis-Tsakonas, S., Grimwade, B. G., Harrison, R. G., Makopoulou, K., Mus kavitch, M. A. T., Schlesinger-Bryant, R., Wharton, K., Yedvobnick, B. 1984. The Notch locus of Drosophila melanogas ter: A molecular analysis. Dev. Genet. 4: 233-54 Balcclls, L. 1., Modolcll, J., Ruiz-Gomez, M . 1988. A unitary basis for different Hairy wing mutations of Drosophila melano gasler. EMBO J. 7: 3899-3906 Bate, C. M. 1976. Embryogenesis of an insect nervous system. 1. A map of the thoracic and abdominal neuroblasts in Locusta migratoria. J. Embryol. Exp. Morphol. 35: 1 07-23 Bate, C. M . 1978. Development of sensory systems in arthropods. In Handbook of Sensory Physiology, cd. M. Jacobson, 9: 153. Berlin/New York/Heidelberg: Springer Verlag

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Genetics of early neurogenesis in Drosophila melanogaster.

Genetic and molecular analysis of maternal information in region 32 of Drosophila melanogaster.

Genetic and molecular analysis of six tumor suppressor genes in Drosophila melanogaster.

The suppressor of forked locus in Drosophila melanogaster: genetic and molecular analyses.

Molecular and genetic organization of the antennapedia gene complex of Drosophila melanogaster.

Genetic and biochemical aspects of trehalase from Drosophila melanogaster.

Developmental genetic analysis of Contrabithorax mutations in Drosophila melanogaster.

Dissecting the Genetic Architecture of Behavior in Drosophila melanogaster.

Genetic manipulation of cyclic AMP levels in Drosophila melanogaster.

Buffering of Genetic Regulatory Networks in Drosophila melanogaster.

Transposition rates of movable genetic elements in Drosophila melanogaster.

Genetic Architecture of Micro-Environmental Plasticity in Drosophila melanogaster.

Genetic analysis of the 5S RNA genes in Drosophila melanogaster.

Genetic determinism of the cellular immune reaction in Drosophila melanogaster.

Genetic variation in male-induced harm in Drosophila melanogaster.

FlyVar: a database for genetic variation in Drosophila melanogaster.

Molecular characterization of a putative peroxidase gene of Drosophila melanogaster.

Molecular, genetic, and cellular bases for treating eosinophilic esophagitis.

Molecular cloning of an olfactory gene from Drosophila melanogaster.

Genetic and molecular analysis of new female-specific lethal mutations at the gene Sxl of Drosophila melanogaster.

Molecular genetic analysis of Drosophila rDNA arrays.

The genetic basis for variation in resistance to infection in the Drosophila melanogaster genetic reference panel.

Two genetically and molecularly distinct functions involved in early neurogenesis reside within the Enhancer of split locus of Drosophila melanogaster.

Large-scale assessment of olfactory preferences and learning in Drosophila melanogaster: behavioral and genetic components.