The peripheral nervous system of mutants of early neurogenesis in Drosophila melanogaster

transcript

Roux's Arch Dev Biol (1986) 195:210-221 Roux's Archives of Developmental Biology �9 Springer-Verlag 1986

The peripheral nervous system of mutants of early neurogenesis in Drosophila melanogaster Volker Hartenstein and Jose A. Campos-Ortega Institut ffir Entwicklungsphysiologie der Universit/it K61n, GyrhofstraBe 17, D-5000 K61n 41, Federal Republic of Germany

Summary. Mutations previously known to affect early neurogenesis in Drosophila melanogaster have been found also to affect the development of the peripheral nervous system. Anti-HRP antibody staining has shown that larval epidermal sensilla of homozygous mutant embryos occur in increased numbers, which depend on the allele considered. This increase is apparently due to the development into sensory organs of cells which in the wild-type would have developed as non-sensory epidermis. Thus, neurogenic genes act whenever developing cells have to decide between neurogenic and epidermogenic fates, both in central and peripheral nervous systems. Different regions of the ectodermal germ layer are distinguished with respect to their neurogenic abilities.

Key words: Peripheral nervous system - Neurogenesis - Mutants - Drosophila

Introduction

The segregation of neural progenitor cells from the undifferentiated ectoderm is one of the first steps in the development of the Drosophila nervous system. The progenitor cells of the central nervous system (CNS), the so-called neuroblasts (Wheeler 1891, 1893), leave the ectoderm soon after gastrulation; most, or all, neuroblasts segregate before hav- ing mitotically divided after formation of the cellular blastoderm (Hartenstein and Campos-Ortega 1984; Technau and Campos-Ortega 1985). In contrast, the progenitor cells of larval epidermal sensory organs, and thus of the peripheral nervous system (PNS), become distinguishable later in embryogenesis, after germ-band shortening, when the remaining epidermal cells are close to terminating their divisions (Hartenstein and Campos-Ortega 1985; Campos-Ortega and Hartenstein 1985a). It has been shown that for a number of insect species the entire set of cells that form a given sensillum (trichogen cell, tormogen cell, sensory neuron(s), glia cell) arise from related mitoses; these cells group together to form a primordial sensory organ, and cytodifferentiation of sensory neuron and supporting cells follows (see comprehensive review of Bate 1978).

Neuroblasts of the ventral cord have been shown to originate from the ventral neurogenic region (Hartenstein and Campos-Ortega 1984, 1985; Technau and Campos-Or-

Offprint requests to: J.A. Campos-Ortega at the above address

tega 1985) where they occur mixed with progenitors of the ventral epidermis and its annexes (e.g. sensilla, salivary glands) in a proportion of approximately one neuroblast to four epidermal, and other, progenitor cells. Some sensory organs originate from the dorsal epidermal anlage, from which no neuroblast for the CNS arises (Campos-Ortega 1983; Hartenstein and Campos-Ortega 1984; Technau and Campos-Ortega 1985). A number of neurogenic mutations have been described, the main defect of which consists in a misrouting of presumptive epidermal progenitors into the neurogenic pathway of development (Lehmann et al. 1983). These mutations define seven complementation groups which have, at least, one feature in common: when the function of any of these genes is completely lost all cells of the ventral neurogenic region, instead of only every fourth, develop as neuroblasts (Jimenez and Campos-Or- tega 1982; Hartenstein and Campos-Ortega 1984). Appar- ently, during normal development these genes provide a sort of genetic switch, which decides how many cells of the neurogenic ectoderm are going to enter the neurogenic pathway. Further study of the phenotype of mutations in neurogenic genes has shown that, besides at early neurogenesis, their function is also required for the development of several different types of imaginal disc cells, for example bristles and ommatidia of the compound eye, where these genes may participate in the decision between neural and epidermal lineages (Dietrich and Campos-Ortega 1984).

In the first descriptions of the embryonic phenotype of the neurogenic mutants under discussion, the embryonic PNS was not studied in great detail (Poulson 1937; Leh- mann et al. 1983). It was concluded from a light microscopic reconstruction of the chordotonal organs of homozygotes for a few alleles that, numerically, this type of sensilla was not very grossly affected. However, neural specific staining techniques applied to the study of these mutants, i.e. anti- HRP antibody (Jan and Jan 1982), have shown us that neurogenic mutations indeed increase the number of all types of embryonic sensilla at the expense of other epidermal cells. Therefore, the neurogenic loci seem to act on the development of the larval and imaginal PNS in a similar way to that in which they act on central neurogenesis. Since the wild-type larval PNS consists of a relatively small number of different sensory organs and nerves, which are located at well-defined positions and, therefore, clearly identifiable (Hertweck 1931; Campos-Ortega and Harten- stein 1985a), it seems better suited for a more subtle assess- ment of the action of the neurogenic loci on neurogenesis

than the CNS itself. In the present report, we deal with several aspects of PNS development based on the phenotype of neurogenic mutations.

Materials and methods

Embryos of Drosophila melanogaster, wild-type (Oregon R) and homozygous for various neurogenic mutant alleles of the genes Notch (N), almondex (amx), master mind (mare), big brain (bib), neuralised (neu), Delta (DO and Enhancer of split (E(spl)) (see Table 1), were stained with the antibody against horseradish peroxidase (anti-HRP), which exhibits a conspicuous affinity for the membranes of both mature and immature neural cells (Jan and Jan 1982). Visualisation of the binding sites was obtained by an HRP- coupled anti-immunoglobulin reacted with the substrate diamino benzidine (DAB).

Staged embryos were dechorionised and devitellinised according to the method of Zalokar and Erk (1977). 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) (pH 7.2) was used as fixative. After devitellinisation, embryos were postfixed for 1 h in the same fixative and washed for 1 h in several changes of 0.1 M PBS buffer containing 0.3% Triton X-100. Anti-HRP (Sigma) was diluted 1:3000 in 0.1 M PBS containing 10% goat serum and 0.1% Triton X-100. After 1 h preincubation in this solution without antibody, embryos were incubated overnight at room temperature in the antibody-containing solution; consecutively the embryos were washed in several changes of 0.1 M PBS over 2 h. This was followed by incubation for 5 h at room temperature in affinity-purified, HRP-coupled goat anti-rabbit IgG (Sigma), diluted at 1 : 50 in 0.1 M PBS containing 10% goat serum and 0.3% Triton X-100. After several washes, first in 0.1 M PBS and then in 0.1 M phosphate buffer (H 7.3), embryos were reacted in DAB (Sigma) diluted at 0.01% in 0.1 M phosphate buffer containing 0.002% of 33% hydrogen peroxide. The reaction was inter- rupted after 1-5 rain by thinning out the substrate with 0.1 M phosphate buffer. The embryos were dehydrated in ethanol (50%, 70%, 90%, 96%, 5 min each; 100%, 15 rain) and propylene oxide (15 min) and left for at least 5 h in a mixture of propylene oxide and Epon. Finally the embryos were individually mounted on glass slides with a nee- dle, oriented and covered with Epon and a coverslip. Poly- merisation of the Epon followed overnight at 60 ~ C.

Temperature shift experiments with the temperature sensitive allele D l 6B3 7 were performed as follows. Flies were allowed to lay eggs on agar plates, which had been brought to a temperature of either 18~ (permissive temperature) or 29 ~ C (restrictive temperature). After 2.5 h development at either of these temperatures, eggs were collected and dechorionised. The embryonic stage of individual embryos was determined by microscopic examination (see Campos- Ortega and Hartenstein 1985 a, for a table of normal development), and the embryos were then allowed to continue development on the surface of destilled water previously brought to a temperature of 18 ~ C or 29 ~ C. Once the ade- quate stage had been determined by microscopic inspection (segregation of most central neuroblasts completed, stage 10 in Campos-Ortega and Hartenstein 1985a), embryos were shifted down or up by transferring them to water of the corresponding temperature. Further development con- tinued until maturation of sensilla was completed. Embryos

were then fixed and labelled with anti-HRP, as described above.

The study of the anti-HRP-stained embryos was performed at 1250-fold magnification. Qualitative and quantitative abnormalities of identified sensilla were recorded in standardised graphs of the PNS (see Fig. 1). At least 8-10 individuals homozygous for each neurogenic allele included in this report were carefully studied.

Results

The peripheral nervous system of the wild-type Drosophila embryo

In the Drosophila embryo the anti-HRP antibody binds to the whole of the sensory neurons, and to parts of several, if not all, trichogen cells, i.e. anti-HRP stainings show in any sensilhim at least a soma with a clearly distinguishable dendrite directed toward the outside, in most cases sur- rounded by a conspicuous sheath and an axon projecting toward the CNS. In the following, we summarise the major aspects of the composition of the larval PNS in Drosophila after labelling with anti-HRP (cf. Campos-Ortega and Har- tenstein 1985a; Fig. 1). Most sensilla which were defined as such by HRP labelling could be assigned to distinct cuticular specialisations detectable in preparations of the larval cuticle; furthermore, most sensilla have been characterised by scanning and transmission electron microscopy (Kankel et al. 1980; Campos-Ortega 1982; Singh and Singh 1984; Campos-Ortega and Hartenstein 1985a; Hartenstein, unpublished observations).

Three types of sensilla are present in any of the thoracic and abdominal segments of the Drosophila larva: (i) trichoid sensilla, (ii) campaniform sensilla and (iii) chordotonal organs. Trichoid sensilla and campaniform sensilla are innervated by a single sensory neuron each. Chordo- tonal organs comprise one, three or five scolopidia and, therefore, sensory neurons. A fourth type of sensillum, to be classified as basiconical sensillum, only occurs in the three throacic (T1-3) and last two abdominal (A8-9) segments. In T2-3 these special sensilla were called "black organs" by Lohs-Schardin et al. (1979) and "black dots" by Campos-Ortega and Hartenstein (1985a), being innervated by the dendrites of two or three sensory neurons each; basiconical sensilla in A8 and A9 form part of the so-called sensory cones (ttertweck 1931) and exhibit a single dendrite each.

The gnathal segments and the procephalon also contain distinct groups of sensilla. Except for some chordotonal organs, gnathal sensilla are arranged to form complex sensory organs: the hypophysis and labial complex of the labial segment (Hertweck 1931); the terminal and ventral organs of maxillar and mandibular segments; the dorsal organ of the procephalic lobe; the epiphysis and dorsal pharyngeal organ of the labrum. On the basis of their cellular architec- ture terminal, dorsal and ventral organs (which together form the antenno-maxillary complex), and the labial complex, can be considered as arrays of poly-innervated basiconical and campaniform sensilla (Hertweck 1931 ; Kankel et al. 1979; Singh and Singh 1984; Campos-Ortega and Hartenstein 1985a).The cellular organisation of hypophysis, epiphysis and dorsal pharyngeal organ has not been investigated sufficiently to allow their unequivocal classification.

,1 A2 A3 Fig. 1. Schematical representation of the peripheral nervous system of the fully developed Drosophila embryo. Homologies proposed between sensilla of different segments are indicated by corresponding symbols at the same vertical level. Shading labels the groups of sensilla in this and in Figs. 2 and 6 (ventral group: dark shading; lateral group: light shading; dorsal group: blank).The neuropile is vertically hatched and the commissures cross-hatched. Types of sensilla are symbolised as follows: small circles: campaniform sensilla; large circles with central dot: basiconical sensilla; large open circles: campaniform-like, poly-innervated sensilla of procephalic and gnathal territories; circles with triangle: trichoid sensilla; arrowheads: chordotonal organs; arrows: sensory organ of the posterior spiracle (morphologically unclassified). Sensilla of the same type which occur more than once in any of the groups, e.g. campaniform sensilla, have been numbered consecutively from ventral to dorsal, except for trichoid sensilla lh 2. Abbreviations: A1A9: abdominal segments; af: anterior fascicle of segmental nerve; amx: antenno-maxillary complex; csc: caudal sensory cone; dbd: dorsal black dot; dc 1-3: dorsal campaniform sensilla; dcsc: dorsocaudal sensory cone; dh 1-2: dorsal trichoid sensilla; dlsc: dorsolateral sensory cone; dmsc: dorsomedial sensory cone; do: dorsal organ; dpo: dorsal pharyngeal organ; epi: epiphysis; hy: hypophysis; ko: Keilin's organ; LB: labium; lbd: lateral black dot; lbo: labial organ; lc 1-2: lateral campaniform sensilla; lch 1/3/5: lateral (mono-, tri- and pentascolopidial) chordotonal organs; lh 1-2: lateral trichoid sensilla; AID : mandible; MX: maxilla; pch 1 : pharyngeal chodotonal organ; pf: posterior fascicle of segmental nerve; PR: procephalic nervous system; sso : spiracle sensory organ; T1-3: thoracic segments; to : terminal organ; vas: ventral anal sensillum; vbd: ventral black dot; vc 1-5: ventral campaniform sensilla; vch 1: ventral chordotonal organ; vo: ventral organ

Sensory neurons form clusters in the cleft between epidermis and somatic muslces; clustering of sensory neurons is constant , al lowing classification of sensilla according to topological criteria. In each of T1-3 and A 1 - 7 segments, three clusters - dorsal, lateral and ventral - can be distinguished on either side o f the embryo (Fig. 1).

1. The dorsa l cluster contains the dorsal t r ichoid and campani form sensilla (dh 1, dh 2, dc 1-3 of thoracic segments; dh 1, dc 1-2 of abdomina l segments). In T2 and T3 the dorsal cluster addi t ional ly contains a tr iscolopidial chordotona l organ (dch 3), in T1 a basiconical sensillum (dba).

2. The lateral group contains neurons of the lateral trichoid and compani form sensilla (lh 1, lc 1-2 of thoracic segments; lh 1-2, lc i of abdomina l segments). In the lateral cluster of abdomina l segments, there is a lateral pentascolopidial chordotona l organ (lch 5) and a monoscolopid ia l chordotona l organ (lch 1) (Fig. 2A). In TI the lateral cluster contains a lateral t r iscolopidial chordo tona l organ (lch 3); in T2 and T3, however, there is a basiconical sensillum (lbd) instead. In our foregoing study (Campos-Ortega and Hartenste in 1985a) the ventra lmost o f the three trichoid sensilla of abdomina l segments (lh 2) was assigned

to the ventral group and designated vh 1. However, this sensillum has been shown by Ghysen, using a different antibody to study the larval PNS of Drosophila, to actually belong to the lateral cluster (A. Ghysen, unpublished per- sonal communicat ion) .

3. The ventral cluster is split up into several small nar- rowly spaced, yet separate groups of cells. In the abdominal segments the ventral cluster comprises five campani form sensilla (vc 1-5), and two monoscolopidia l chordotona l organs (vch / ) .The ventral cluster in T1-3 contains a basiconical sensillum (vbd), two campani form sensilla (vc 1-2), an ar ray of three tr ichoid sensilla (so-called Keil in 's organ, ko), and a monoscolopidia l chordotona l organ (vch 1).

Sensory neurons in A8-9 , the gnathal segments and the procephalon are similarly arranged in clusters. There is one cluster each for the labial complex (lbo), hypophysis (by), terminal organ (to), ventral organ (vo), dorsal organ (do), epiphysis (epi) and dorsal pharyngeal organ (dpo). InA8 and A9 addi t ional clusters are present beneath each of the so-called sensory cones (Hertweck 1931) [dorsomedial (dmsc), dorsolateral (dlsc), dorsocaudal (dcsc) and caudal (csc)], which comprise a large tr ichoid sensillum and a basiconical sensillum each. In addi t ion to the cells innervat ing

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Fig. 2A, B. Photographs illustrating the sensilla of an abdominal hemisegment in the Drosophila wild-type embryo (A) and a neurogenic mutant (bib xD~ (B). The drawings show the corresponding sensory organs. Anti-HRP antibody staining. Note increase in the number of otherwise well differentiated sensilla in the lateral group of the mutant embryo (light shading). For symbols and abbreviatons, see legend to Fig. I

these sensilla the clusters contain the following neurons. The dorsolateral cluster in A8 and the dorsocaudal cluster in A9 contain four more neurons innervating a triscolopidial and a monoscolopidial chordotonal organ (Ich 3, lch 1). Another four neurons occur in the dorsomedian cluster of A8, which project long, slender dendrites into the tracheal opening, where they extend parallel to the Filzk6rper, en- sheathed by special cells. No scolopales, nor any kind of cuticular specialisations, excepting the crown of hairs sur- rounding the posterior spiracles, are associated with these sensory receptors, which we have tentatively called spiracu- lar sensory organ (sso). No ventral neurons are present in either A8 or Ag, except for a single median, unpaired soma located immediately rostral of the anal plate, whose axon

reaches the CNS via the nerve of the abdominal segment A9. No specialisation has yet been detected in the larval cuticle which could be assigned to this neuron.

In a previous account on the PNS of the Drosophila larval sensilla in different segments were homologised according to various morphological criteria (Campos-Ortega and Hartenstein 1985a). However, we did not attempt to include gnathal and procephalic sensilla in the homology because, in the wild-type embryo, profound morphogenetic movements affect both the procephalon and gnathocepha- lon, which makes the task of establishing homologies very difficult. Hyperplasia of the CNS in the neurogenic mutants described in the present report largely prevents the gnathal and procephalic territories from undergoing their major

Fig. 3A-D. Photographs illustrating anti- HRP-labelled whole mounts of 16 h embryos of Drosophila wild-type (A), and homozygous for weak (mare c2"4) (B), intermediate (bib I~176 (C), and strong (neu ~F65) (D) alleles. Lateral view. Ventral epidermis of mare c2"4 (B) is defective at locations marked by large arrowheads. In bib ID~ (C) a considerable portion of the ventrolateral epidermis is missing along the entire length of the embryo. In neu Ir65 (D) ventrolateral epidermis is entirely absent and only a narrow segmented strand of dorsal epidermis (small arrowheads) is left. Note occurrence of differentiated sensilla in wt, mare c2"4, and bib ID~ but not in neu IF65. In all mutants, ventral shifts of gnathal segments and head involution is incomplete (note position of epiphysis and hypophysis, compare with Fig. 1). Moreover, in neu Iv65 germ-band shortening has not taken place. Abbreviations: amx: antenno-maxillary complex; dg: dorsal group of sensilla; epi: epiphysis; hypo : hypophysis; lg: lateral group of sensilla; ve: ventral cord; vg: ventral group of sensilla. Bar = 100 gm

morphogenetic dislocation, namely ventral shift (Technau and Campos-Ortega 1985) and involution. Therefore, gnathal and procephalic territories retain the position and shape of their primordia, which are in fact organised in a similar way to those of thoracic and abdominal segments (Fig. 4). Correspondingly, in the present account gnathal and procephalic sensilla have been tentatively homologised to thoracic and abdominal sensilla on the basis of their topological relationships. We propose that the labial com-

plex corresponds to the dorsal group of sensilla, the hypophysis to the lateral group, and that the terminal-ventral organ and the dorsal organ correspond to both the lateral and dorsal group (Fig. 1). No ventral (sternal) sensilla seem to occur in gnathal segments.

Segmental nerves are composed of two fascicles. The anterior fascicle (ajO contains axons of the lateral and dorsal sensilla; the posterior fascicle (pJ) carries axons from the ventral sensilla. The anterior fascicle emerges from the yen-

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Fig. 4. Photograph and diagram showing rostral part of anti-HRP-labelled 16-h, homozygous mam c24 embryo to illustrate the location of gnathal and procephalic sensilla in the absence of head involution. The primordia of the gnathal se~nents (MD/MX: mandible and maxilla; LB: labium) retain their original dorsal position. This permits one to recognize that the corresponding complex sensilla (to/vo: terminal organ and ventral organ; lbo: labial organ; hy: hypophysis) can be homologous to the lateral and dorsal groups of sensilla of thoracic and abdominal segments. Note that the somata belonging to the dorsal organ fuse with the hyperplasic CNS (hatched region in diagram). Other abbreviations: dbd: dorsal black dot; dch 3: dorsal triscolopidial chordotonal organ; lbd: lateral black dot; mg: midgut; PL: procephalic lobe; TI-2: prothorax and mesothorax. Bar = 50 gm

tral cord by two roots, one from the homotopic neuromere and the other from the neuromere anteriorly adjacent to the homotopic one (Fig. 1); the posterior fascicle has only one root, emerging from the homotopic neuromere.

General aspects o f the phenotype o f neurogenic mutations

All mutants included in this study exhibited hyperplasic CNS and PNS (ref. to Figs. 2 B, 3 and 4). Depending on the allele considered the peripheral effect ranged from a slight increase in the number of some types of sensilla (weak phenotype), over an intermediate phenotype, in which al- most all types of sensilla were present 2- to 4-fold, to a strong phenotype in which the major part of the lateral and dorsal ectodermal derivatives had developed into large clusters of ill-differentiated, anti-HRP binding cells. The extent of the epidermal sheath and, consequently, differentiated cuticle of the mutants was inversely correlated to the degree of CNS and PNS hyperplasia. Thus, in the strong phenotype, the epidermal sheath was restricted to a narrow dorsomedial region flanked by anti-HRP binding cells, whereas in the weak and intermediate phenotypes the mutant effect led to an increase in fairly well developed sensilla, generally integrated in well-differentiated epidermis. These observations explain why so little epidermis, and cuticle, succeeds in developing in embryos homozygous for extreme neurogenic mutant alleles (Lehmann et al. 1983) although the dorsal epidermal primordium is apparently normal during early stages of development (discussed in Jimenez and Campos-Ortega 1982). An important aspect of the weak and intermediate phenotypes is that, nothwithstanding the distortions brought about by neural hyperplasia, several different features of sensory organs, e.g. type, relative posi-

tion to each other and segment-specific distribution, were maintained in the neurogenic mutants (Fig. 2 B). This suggests that genes acting at determining segmental identity, as well as the pattern of spatial distribution of sensilla and nerves in the PNS, are independent of the function of neurogenic loci, thus able to exert their function in the absence, or dysfunction, of the latter genes. In strong phenotypes, however, the spatial distribution of sensilla was grossly altered, most probably due to the absence of naked epidermis and cuticle, and in the more extreme phenotype there were hardly any differentiated sensilla left (Fig. 3 D). In embryos homozygous for these alleles, it is impossible to determine to what extent segmental identitiy and intrasegmental pat- terns of neural elements are maintained.

Quantitative differences

Little variability was found among individuals homozygous for any given allele with respect to the degree of PNS hyperplasia, although topological differences became evident between the different regions (Tables 1 and 2). By counting sensilla in the mutants we could calculate incremental factors for each individual type in the various alleles with weak and intermediate phenotype, i.e. those with fairly well differentiated, or identifiable sensilla, with the following results. Firstly, the number of homologous sensilla was found to increase homogeneously in the different segments of a given individual; for instance, all pentascolopidial chordotonal organs, or any other type, were affected to the same extent in A I - 7 irrespective of the degree of CNS hyperplasia (see Table 2). Thus, with respect to the lateral and dorsal clusters of the PNS, there is no evidence for differences in the expressiveness of the neurogenic genes along the an-

Table 1. The effects of neurogenic mutations on CNS and PNS development"

Locus Al le le Effect Effect on PNS Cuticle on CNS defect % VD Differ- Increment

entiation

N 2 16.8_+1.9 + 2.0_+0.15 + 55ell 51.3• - e + + +

amx 1 43.0+2.2 +_ 2.0_+0.39 + /+ +/ + + +

roam c2.4 g; 19.8___2.3 + 1.3_+0.32 + /2199 43.0-+ 3.63 - e

bib ID05 24.4_+3.8 + 2.1 +0.3 + +

neu KE2 g + 1.3 • 0.43 + IF65 46.4_+4.0 -- e + + +

Dl 6B37 g + 1.5_+0.67 + (18 ~ C) 9P39 48.3_ 6.8 - e + + +

E(spl) R14.8 g -- 1.4-t-0.40 + B251 48.9_+ 3.8 - e + + +

Abbreviations: e: large increase in cell number, though no quantifi- cation possible due to the lack of differentiated sensilla; g: major part of individuals exhibiting isolated gaps within the ventral cuticle through which parts of the CNS of variable width protrude

" Hyperplasia of the CNS is indicated as a percentage of dorsoventral perimeter (% VD) occupied by the ventral cord (n: 8 ani- mals) Degree of differentiation of the PNS has been classified as well differentiated (+), partly undifferentiated (_+, most sensilla still recognisable), and undifferentiated (-) . Classification of cuticle defect in weak phenotype (+), intermediate phenotype (+ +), and strong phenotype (+ + +) follows the criteria described in Lehmann et al. (1983)

tern-posterior axis, although such differences exist concerning the effects of these genes on central neurogenesis (see Lehmann et al. 1983). Secondly, significant differences became evident concerning the effects of a given allele on the various sensilla of a given segment (Table 2). Ventral sensilla (re 1-vbd in T1-T3, vc l - r e 5 in A1-A7) only develop in alleles with weak phenotype (e.g. mam c24, Nz), where a sufficient number of ventral ectodermal cells escapes neuralisation of the ventral neurogenic region. However, in these alleles most ventral sensilla are either rather undifferentiated or they show the same incremental factor as the lateral sensilla. The number of lateral sensilla (lc 1-1ch3 in T1, lc 1-1bd in T2-T3, and lh 2-leh 1 in AI-A7, dlsc and dcsc in A8 and A9, respectively) and of the ventralmost sensilla of the dorsal group (deh 3, dc I in T1-T3, dh 1 in AI-A7) of embryos homozygous for a given allele, was generally increased by the same factor. In homozygotes for the weak allele mare C24 hyperplasia preferentially affected the chordotonal organs and the basiconical sensilla, the remaining types being in most cases wild-type in number. In all of the alleles studied, the dorsalmost sensilla (dh 1- dh 2 in T1-T3, dc 1-de 2 in A1-A7, dmsc and sso in A8) were much less affected.

Qualitative differences

All neurogenic mutants investigated so far share the ability to increase the number of central and peripheral neural

cells and, in principle, all affect the same regions of the ectoderm, though to a variable extent (Figs. 2, 4). Compar- ing mutations in the different neurogenic loci, however, qualitative differences become evident which mainly concern the degree of sensilla differentiation. As stated before, sensilla differentiation was disturbed in all alleles with extreme phenotype, e.g. N 55~11, amx 1, mare u199, neu IF6s, DI 9P39 and E(spl) n251. Alleles with intermediate or weak phenotype, on the other hand, could be assigned to either of two gene groups. In the first group, comprising mam c2"4 and bib zn~ all sensilla were fully differentiated, i.e. all specialisations of the sensory neuron and supporting cells, typi- cal of the wild-type embryo, were also present in the mutants' sensilla. The other group comprised amx I, N 2, neu K~2, Dl 6B37 and T(3 ; 4)E(spl)R, 14"8, which exhibited sensilla with altered morphology, irrespective of a weak or strong increment in their number. In these cases orienta- tion of dendrites and their sheaths, as well as relative positions of neighbouring sensilla, were abnormal. Dendritic sheaths appeared either irregularly enlarged or were small, reminiscent of the morphology of wild-type immature ones (Campos-Ortega and Hartenstein 1985a; Hartenstein, unpublished work). In general, not all sensilla of a given individual homozygous for one of the alleles under discussion were equally affected, but they exhibited abnormalities to a variable degree. These observations suggest that the affected gene functions are necessary not only for the development of a normal complement of sensilla, but also to allow normal differentiation.

Temporal relationships between central and peripheral neuralisation

The allele D16B37 has been found to express a temperature- sensitive phenotype (Lehmann et al. 1983). In homozygotes developing at 18 ~ C hyperplasia of the CNS is weak, whereas when reared at 29 ~ C the embryos exhibit a strong phenotype; intermediate phenotypes can be obtained when keep- ing the embryos at temperatures between these two. Using temperature shift experiments, the restrictive period could be identified as the period of neuroblast segregation (Leh- mann eta1. 1983). After similar temperature shifts, we found that the restrictive period for the effect of the muta- tion D16~37 on PNS development is approximately the inter- val between maximal extension and shortening of the germ band (stages 11 and 12, Campos-Ortega and Hartenstein 1985). Moreover, it was possible to separate chronologically the mutant effects on CNS and on PNS by shifting the developing embryos from the restrictive to the permissive temperature, or vice versa, between neuroblast segregation (stage 9) and maximally extended germ band (stage 11).

The thoracic and abdominal PNS in the anti-HRP-labelled D16~37embryo reared at 18 ~ C is normal; this applies both to the amount of peripheral neural cells and the differentiation of sensilla elements (Fig. 5). In thoracic and abdominal levels the CNS exhibited normal and moderately hyperplasic regions, the latter located near the embryonic ventral midline. No differences could be found when com- paring the sensilla of segments with hyperplasic CNS to the sensilla in segments with normal CNS. The procephalic CNS, however, was strongly affected by neural hyperplasia in all individuals. After raising the temperature to 29~ between neuroblast segregation and maximally extended germ band, the fully grown embryos exhibited relatively normal CNS, although accompanied by moderate hyper-

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Fig. 5A-C. Photographs (A-C) to show lateral views of whole mounts of anti-HRP labelled 16-h embryos homozygous for the temperature- sensitive allele Dl ~B37 raised at different temperatures (diagram at top right shows temperatures at which embryos shown in A-C, were raised; for shading of the abscissa, refer to time scale in Fig. 7). The embryo in C was raised at 18 ~ C throughout embryogenesis and exhibits weak CNS hyperplasia (gaps of the ventral epidermis indicated by large arrowheads) and a wild-type-like PNS (see normal configuration of lateral pentascolopidial chordotonal organ, Ich 5, shown in C'). The embryo in A was raised at 29 ~ C and both CNS and PNS are strongly hyperplasic. Finally, the embryo in B was raised at 18 ~ C up to 6 h and shifted to 29 ~ C, where it completed development. This embryo exhibits slight CNS hyperplasia, comparable with that shown in C, whilst development of the PNS, proceeding well after 6 h, is disturbed. B' is a larger magnification of the region indicated in B showing high number and incomplete differentiation of elements belonging to the lateral pentascolopidial chordotonaI organ (Ich 5). dg: dorsal group of sensilla; epi: epiphysis; lg: lateral group of sensilla; rag: midgut

plasia and defective differentiat ion of the PNS. The number of an t i -HRP binding per ipheral cells was clearly increased for all sensilla. In many individuals the number of differen- t iated sensilla, especially those o f the lateral group, was also increased by a factor of about two. Dendr i tes and scolopales were irregularly or ientated and deformed. Lack of differentiat ion was most p ronounced for lateral ehordo- tonal organs which, on the average, were decreased in number. A strong per ipheral phenotype, as found in embryos reared at 2 9 ~ th roughout development, could be observed in no individual.

Shif ts-down from 29~ to 18~ in the embryo with extended germ band resulted in intermediate CNS hyperplasia (ventral cuticle entirely absent) and slight hyperplasia , or even wild-type development, of the PNS.

Discassion

The most conspicuous effect of loss of function from any of the neurogenic loci is the misrout ing into neurogenesis o f all cells of the ventral neurogenic region, as opposed to only one-fourth of these cells in the wild-type. Conse-

quently, mutant embryos develop a huge CNS and lack a substantial part of the epidermis (Poulson 1937; Lehmann et al. 1983; Hartenstein and Campos-Ortega 1984; see review in Campos-Ortega 1985). Dietrich and Campos-Or- tega (1984) described additional effects of neurogenic mutations upon development of imaginal epidermis and sensory organs, e.g. macro- and microchaetae as well as compound eye, the intensity of which was found to depend on the allele considered. Here, we present the results of observations indicating that loss of function at the neurogenic loci inflicts on the embryonic epidermis the same developmental defect as on the imaginal epidermis, namely larval sensilla development was found to be affected in different ways, depending on the allele used. All of these phenotypic traits permit the generalisation that the function of the neurogenic genes is required whenever a decision between epidermal and neural (either central or peripheral) cell lineages is made. In the following, we discuss the three main conclu- sions of the present observations.

The first conlcusion refers to topological aspects of gene expression. A comprehensive fate map of the Drosophila blastoderm has recently been published (Hartenstein et al. 1985; Hartenstein and Campos-Ortega 1985; Technau and Campos-Ortega 1985), according to which the anlage of the germ-band ectoderm comprises two subdivisions, the ventral neurogenic region and the dorsal epidermal anlage. We find, however, that the reactivity of epidermal cells to the loss of genetic activity from so-called neurogeuic loci varies among the different regions of the ectoderm and, consequently, the dorsal epidermal anlage should be further subdivided into a lateral, a dorsolateral and a dorsal region, each with different neurogenic abilities. Therefore, with respect to neurogenesis the embryonic ectoderm of the germ band consists of four longitudinal subdivisions: the ventral neurogenic region, and the lateral, dorsolateral and dorsal subdivisions of the dorsal epidermal anlage (Fig. 6).

The ventral neurogenic region contains progenitors of the CNS, progenitors of 0-45% of the VD perimeter of the larval integument, and progenitors of all sensilla of the ventral group; additionally the labial segment contains the anlage of the salivary glands. The function of the neurogenic loci is necessary at two consecutive phases of development of the ventral neurogenic region, during segregation of CNS neuroblasts and during segregation of sensilla precursors. This function is formally similar in both instances and consists of preventing a majority of anlage cells from becoming either neuroblasts or sensilla progenitors. In strong alleles of Dl or E(spl) , or after completely removing the genetic function from the loci of N, mare and neu, all cells of the ventral neurogehic region, including presump-

t i v e progenitor cells of sensory organs, acquire the neurogenic fate. Thus, no cell is left in those mutants for the development of ventral sensilla. The lateral subdivision of the dorsal epidermal anlage contains the progenitors of a lateral strip of the larval integument (45%-80% VD), which includes the precursors of the lateral group of sensilla and a ventral subset of the dorsal group. In neurogenic alleles expressing a strong phenotype, the entire complement of cells in this region is transformed into sensilla cells (see below) and no cuticle differentiates. The dorsolateral subdivision of the dorsal epidermal anlage contains the epidermal progenitors of a narrow strip of the larval integument (80%-90% VD extend) and the progenitors of the dorsalmost sensilla. Dorsolateral sensory organs behave differ-

T2/3 A 1-7 - 1 0 0 %

0 dc l - 8 0

Ich~ -60

~il~ ~ Ich~

" - - ~ - " Ih2 | vc5 .40

e~ vc4

O vc3 @~' vch' - 2 0

�9 vc2

Fig. 6. Correspondence of sensilla to subdivisions of the ectoderm, shown on a scheme of a transverse section through an embryo at the blastoderm stage (arrows point to the midline). Each ectodermal subdivision is characterised by a distinct neurogenic ability. The ventral neurogenic region (vNR) yields central neuroblasts and sensilla of the ventral group (darkly shaded symbols). The lateral portion (intermediate hatching) of the dorsal epidermal anlage (dEpi) contains the anlagen of the lateral group of sensilla (lightly shaded symbols) and the ventralmost part of the dorsal group (white symbols). This territory may undergo complete differentiation into peripheral neural tissue in the absence of the genetic function of any of the neurogenic loci. In contrast, the neighbouring dorsolateral portion (widely hatched) of the dorsal epidermal anlage, to which the dorsalmost sensilla of the dorsal group belong, seems to resist complete transformation into peripheral neural tisue. Fi- nally, the dorsal part of the dorsal epidermal anlage (white) does not give rise to any sensilla and retains its epidermal fate even in the complete absence of the genetic function of neurogenic loci. Other abbreviations: as: anlage of amnioserosa; ms: anlage of mesoderm. For other abbreviations and symbols, see legend to Fig. 1. Scale to the right indicates percentage of embryonic perimeter (0% : ventral midline; 100% : dorsal midline

ently from those of the lateral subdivision in that they are considerably less affected in neurogenic mutants. Finally, the dorsal subdivision of the dorsal epidermal anlage contains cells which are not susceptible to giving rise to neuroblasts or to progenitor cells of sensilla, its cells invariably developing as epidermis. We have recently postulated mini- mal requirements in the genetic control of neuroblast segregation (Campos-Ortega and Hartenstein 1985b; Campos- Ortega 1985), one of these requirements being genes ep- istatic to the neurogenic genes and related to dorsoventral patterning in the embryo, which apparently define the ventral neurogenic region as the exclusive source of neuroblasts. The present observations on the PNS of neurogenic mutants confirm and extend earlier observations on a differential responsiveness of the ectodermal regions to neuralisation (Campos-Ortega 1983), perhaps in consequence of the same factors that decide about the limits of the neurogenic ectoderm itself. That is to say, the present results suggest that the neurogenic responsiveness of the epidermis

PNS CNS 16

Fig. 7. Diagram to illustrate sequential steps in the development of central (CNS) and peripheral (PNS) nervous systems, and the time at which function of the neurogenic loci is assumed to be exerted (large arrows). The scale to the left indicates time (0 : fertil- isation). The ventral neurogenc region (vNR) gives rise to central neuroblasts (dark shading), ventral sensilla (light shading) and ventral epidermis. Dark shading in time scale indicates neuroblast segregation (S) from the ectoderm; S/M means that after neuroblasts segregate (S) the remaining cells start dividing (M), and behave like those of the dorsal epidermal anlage (dEpi), i.e. all remaining cells undergo two equal mitoses (M). Neuroblasts proliferate by unequal divisions (P), and the progeny differentiates (D). Segrega- tion of peripheral neural precursors occurs well after neuroblast segregation (see light shading in time scale). These precursors, called sensillum mother cells, apparently yield by differential mitotic divisions (riM) both neural and supporting elements of sensilla. Differ- entiation (D) of these elements into mature sensilla follows. The effect of the neurogenic alleles on segregation of neuroblasts and sensilla mother cells (black arrows) is experimentally well estab- lished. The effect of these alleles on central and peripheral neural differentiation (open arrows) may be indirect, i.e. secondary to the increased amount of neural tissue. However, some of the loci may also directly influence neural differentiation. It is possible that the presumably differential mitosis of the sensilla mother cells are also under the control of the neurogenic loci (open arrow with question mark)

depends on the genetic identity of epidermal cells along the dorsoventral body axis.

With respect to the anteroposterior dimension, no differences were detected concerning the behaviour of sensilla in any of the four ectodermal subdivisions (Table 2), i.e. the ventral neurogenic region, and the lateral, dorsolateral and dorsal subdivisions of the dorsal epidermal anlage. The degree of hyperplasia, or structural modification, of the various types of sensory organs was of comparable intensity along the entire anteroposterior extent of each ectodermal subdivision, as shown by quantitative assessments of the phenotype of homozygotes for a given allele. Concerning the effects of neurogenic mutations on early central neu-

rogenesis, however, earlier observations had already pointed to differences in the responsiveness of the ectoderm along the anteroposterior dimension to the central neural- ising effect of these mutations (Lehmann et al. 1983), and the material studied for this report confirms this previous finding. We are unable to explain why the mutant alleles under discussion affect central neurogenesis differentially at head and truncal levels, whereas there does not seem to be any difference along the antero-posterior body length concerning the development of sensory organs.

The second conclusion of the present study is that the effects on CNS and PNS development can be separated from each other in time, as indicated by the results of temperature shift experiments with the temperature-sensitive allele D16~s7. Thus, although under the influence of the same system, segregation of neuroblasts and sensilla progenitor cells may well correspond to two at least temporally independent events. The meaning of this observation is not clear. For example, it suggests two different, consecutive phases of (transcriptional?) activity of the D1 + gene during embryogenesis, one immediately after gastrulation and the other at germ-band shortening. Another possibility would be that the genetic activity remains unchanged and the responsiveness of the cells to the gene product changes during development.

The third main conclusion of the present findings refers to a possible effect of the neurogenic loci on steps of sensory organ development other than commitment of mother cells. Sensory organ development may be roughly subdivided into three phases (Fig. 7).

1. The phase of early development: Progenitors of sensilla are among the cells of the larval epidermal anlage in the blastoderm and, from what we know about the prolifer- ative abilities of embryonic cells in Drosophila (Hartenstein and Campos-Ortega 1985), we assume that these cells divide as frequently as the remaining, epidermal progenitor cells, since all ectodermal cells which do not develop as neuroblasts undergo at least two divisions during the period of extended germ band. Thus, in addition to the immediate progenitors of the sensilla, each ectodermal progenitor cell gives rise to a number of epidermal cells as well.

2. The phase of commitment of sensilla mother cells: At shortening of the germ band some epidermal cells, corresponding to sensilla mother cells, are seen to divide further; the remaining epidermal cells are at this time mitotically quiescent. Thus we assume that epidermal cells become committed at around this time to develop into sensory organs.

3. The progeny of the cells committed as mother cells undergoes differentiation to form the mature sensilla.

Contrary to the segregation Of neuroblasts from the ventral neurogenic ectoderm, which occurs without intervening mitosis, larval and imaginal sensory neurons originate together with other non-neural cells after divisions of their progenitor cells. The current evidence (present results; Die- trich and Campos-Ortega 1984) indicates that the neurogenic loci participate in the commitment of sensilla mother cells within the array of epidermal cells. In particu- lar weak and intermediate phenotypes suggest that the function of the genes under discussion consists in supressing the production of sensilla mother cells, permitting the development of naked epidermis, whereas the other phases of sensilla development remain unaffected. We have seen that in embryos homozygous for weak alleles, as well as in ira-

aginal discs carrying clones of cells homozygous for weak alleles (Dietrich and Campos-Or tega 1984), the number of otherwise perfectly normal sensilla is increased at the expense of the epidermis, and that no other anomalies can be detected. However, the epidermis of homozygotes for all of the strong alleles exhibits undifferentiated sensilla, along with a low number o f fairly well differentiated ones. Two non-mutual ly exclusive interpretat ions can account for this observation. In the first one, not only the commitment of progeni tors of sensory organs versus epidermis is altered in these alleles, but also the commitment of the progeny of sensilla mother cells itself, yielding more sensory neurons and fewer support ing cells; consequently normal sensilla cannot develop due to lack of support ing cells. In the second interpretat ion, all cell types are present in normal numbers, bu t cytodifferentiat ion of sensory neurons and support ing cells is impaired. The second possibil i ty appears to be true in alleles in which an increased number of sensilla occurs, though some of them have clearly detectable differentiat ion defects. However, since an t i -HRP in the wild-type binds to both sensory neurons and tr ichogen cells, the undifferentiated an t i -HRP binding cells occurring in some alleles may in fact belong to either cell type. Therefore, the first possibil i ty cannot be excluded from our data. The genetic system that controls the decision between neural and epidermal lineages consists of several genes, interrelated in a complex way (Campos-Ortega et al. 1984; Vfissin et al. 1985). Thus, it might well be that some of the genetic functions are related to the commitment of the progeny of the sensilla mother cells, whereas others are necessary for cytodifferentiation. This question must remain open.

Acknowledgment. This research was supported by the Deutsche Forschungsgemeinschaft (Ca 60/7-1).

References

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Received August 19, 1985 Accepted in revised form November 28, 1985

The peripheral nervous system of mutants of early neurogenesis in Drosophila melanogaster

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