Development of Drosophila Larval Sensory Organs: Spatiotemporal Pattern of Sensory Neurones, Peripheral Axonal Pathways and Sensilla Differentiation

Development 102, 869-886 (1988) 869 Printed in Great Britain © The Company of Biologists Limited 1988

Development of Drosophila larval sensory organs: spatiotemporal pattern of sensory neurones, peripheral axonal pathways and sensilla differentiation

VOLKER HARTENSTEIN*

Institut fuer Entwicklungsphysiologie der Umversilaet zu Koeln, Gyrhofstr. 17, 5000 Koeln 41, FRC

* Present address: Department of Biology, University of California, San Diego La Jolla, California 92093, USA

Summary

The sensilla of Drosophila larval thoracic and abdomi- of the sensillum cells resembles the development of nal segments appear in a constant temporal sequence larval epidermal cells in many aspects. Thus, the during stage 13/14 (9-5-11-5 h) of embryonic develop- sheath processes formed by sensillum accessory cells ment. Those sensilla innervated by more than one and the axons formed by sensory neurones develop dendrite (basiconical sensilla, chordotonal organs, from processes transiently formed by all cells. During some of the trichoid sensilla and campaniform sensilla) the phase of cuticle secretion, apical portions of the appear earlier than sensilla innervated by a single presumptive accessory cells are modified to form the dendrite (majority of trichoid sensilla and campani- cuticular apparatus responsible for receiving the sen- form sensilla). Furthermore, a dorsoventrally directed sory stimuli. Finally, two sets of subepidermally gradient underlies the sequence in which sensilla of a located cells which differ with respect to their mor- given type appear. Sensory axons are emitted in the phology and, probably, their origin envelop somata same sequence. Thus, axons of the polyinnervated and axons of the sensory neurones. sensilla appear first. Together with a distinct set of efferent axons they form 'pioneer tracts' of the two Key words: sensory organs, Drosophila, morphogenesis, fascicles of the segmental nerves. Cytodifferentiation axonogenesis.

Introduction of sensillum this neurone belongs to (Ghysen, 1980; Ghysen et al. 1983). Sensillum quality, on the other Insect sensilla described as 'Kleinorgane' (Henke, hand, seems to be determined by factors such as the 1953) or organules (Lawrence, 1966) are composed of small sets of highly specialized cells, each with pre- time during development when the sensilla are gener- cisely defined morphological and functional charac- ated, their position within a segment and the identity teristics. Sensilla possess one or more bipolar of this segment (Murphey et al. 1980; Shankland & neurones and a set of accessory cells which forms Bentley, 1983; Dambly-Chaudiere & Ghysen, 1986). sheaths around the neurone(s) and produce the Thus, developmental mechanisms controlling the pat- stimulus-receiving cuticular apparatus. The pattern of terning, differentiation and axonal projection of the distribution, architecture and axonal projection of sensilla seem to be closely interrelated. Further sensilla is highly invariant among different individuals analyses directed at unravelling the control of sensil- of one species. Therefore, sensilla are a favourable lum development require a detailed knowledge of the system to analyse the determinants of cell patterning, temporal and spatial frame in which these different cell differentiation and axonal projection during phenotypic traits of the sensilla unfold. neural development. Experimental studies have Since the application of genetic and molecular shown that one of the determinants of the axonal methods is well advanced in Drosophila melanogas- projection pattern of a sensory neurone is the quality ter, this animal represents one of the favourite objects 870 V. Hartenstein to study developmental mechanisms. Though its propylene oxide and then for 5-10 h in unpolymerized anatomy and general development is known in con- Epon. They were oriented sagittally or horizontally and siderable detail (Hertweck, 1931; Poulson, 1950; placed at 60°C for 12 h to permit polymerization of the Bownes, 1975; Kankel et al. 1980; Campos-Ortega & Epon. Blocks were sectioned with a LKB Ultrotome III. Hartenstein, 1985; Dambly-Chaudiere & Ghysen, For reconstruction of sense organs, the lateral body wall of 12 saggitally oriented specimens of different stages was 1986), our knowledge of the pattern in which the sectioned in almost complete series of ultrathin sections sensory organs are assembled and the morphogenetic (5-10 nm). For the analysis of developing peripheral events shaping their development is scarce. In the nerves, ultrathin sections were cut at intervals of 5 ^m from present study, labelling of peripheral neurones in five horizontally oriented specimens. Sections were whole-mount preparations of embryos and recon- mounted on formvar-coated slot grids and treated with structing the architecture of thoracic and abdominal uranyl acetate and lead citrate (Reynolds, 1963). Sections sensilla at sequential embryonic stages (from serial were inspected and photographed with a Siemens Elmiskop EM sections) were combined to investigate sensilla 101. For reconstruction of sensilla, photographs of sections development in the larva of Drosophila melanogaster. of representative sensilla (lateral basiconical sensillum of Special attention was attributed to (i) the time se- thoracic segments; lateral pentascolopidial chordotonal quence in which sensory neurones appear and their organ, lateral trichoid sensilla and lateral campaniform axons grow out, (ii) the projection pattern of a subset sensillum of abdominal segments) at approximately 1 fim intervals were taken. From the prints, contours and ultra- of early appearing axons which form 'pioneer tracts' structural details of sensilla cells were drawn on translucent for the peripheral nerves and (iii) the movements and plastic sheets. By superimposing the drawings of sequential differentiative events performed by the developing sections three-dimensional reconstructions of the sensilla sensillum cells. were obtained.

Materials and methods Results A series of staged embryos of wild-type Drosophila melanogaster (Oregon R) was stained with antibody against Time sequence of development of the thoracic and horseradish peroxidase (HRP) and the mouse monoclonal abdominal sensilla antibody 22C10. Both antibodies show an affinity for the membranes of immature neural cells (Jan & Jan, 1982; The distribution and external morphology of the Canal & Ferrus, 1986). A second series of staged embryos various types of Drosophila larval sensory organs has was processed for transmission electron microscopy. been investigated in a number of studies (Hertweck, For precise staging, freshly laid eggs were dechorionated, 1931; Kankel et al. 1980; Singh & Singh, 1984; mounted on Scotch tape, covered with halofiuorocarbon oil Campos-Ortega & Hartenstein, 1985; Dambly-Chau- (Voltalef 3S) and left at 25°C. For each specimen, the time diere & Ghysen, 1986). In the thoracic and abdominal when the germ band had retracted to 50 % egg length was segments, four classes of sensory organs are present: recorded. This stage, which is easily identified in the living hairs (sensilla trichoidea), papillae (sensilla campani- embryo, lasts for less than lOmin; it was taken as time Oa.r. formia), 'Koelbchen' (Hertweck, 1931; classified as (after 50 % retraction). Time 0 a.r., at 25°C, corresponds to sunken sensilla basiconica in Campos-Ortega & Har- 8h40min-8h50min after egg fertilization. Embryos were fixed and devitellinized according to the methods of tenstein, 1985) and chordotonal organs. These sen- Zalokar & Erk (1977) and Mitchison & Sedat (1983) at the silla are assembled into distinct patterns different for desired intervals (0—4ha.r. at increments of 20 min; 5ha.r.; the prothoracic, meso- and metathoracic, first 6ha.r.; first instar larva). through seventh abdominal, and terminal abdominal Antibody staining followed the protocol described in a segments (Campos-Ortega & Hartenstein, 1985; previous publication (Hartenstein & Campos-Ortega, Dambly-Chaudiere & Ghysen, 1986; see Fig. 1A). 1986). For electron microscopy, 25 % glutaraldehyde mixed Presumptive sensory neurones first develop an with heptane was used as a fixative. After devitellinization, antigenity towards the antibodies anti-HRP and embryos were washed in 015M-cacodylate buffer (pH6-9) 22C10 at the beginning of dendrite formation (Figs and postfixed for 30 min in a 1:1 mixture of 2-5 % glutaral- IB, 2). Shortly thereafter, axons grow out of the dehyde and 2% osmium tetroxide, then for 2 h in 2% somata opposite the dendrites and contact one of the osmium tetroxide in 015M-cacodylate buffer. Larvae had to be cut into halves in order to allow sufficient fixation. tracts pioneering the peripheral nerves. Within each Specimens were washed several times in distilled water and segment, a dorsoventral and a rostrocaudal temporal dehydrated in graded ethanol and propylene oxide (ethanol gradient characterizes the sequence in which sensilla 70%, 90%, 95%, 5min each; 100% 15min; propylene of equal morphological type appear. Homologous oxide (15 min). Postfixation, washing and dehydration was sensory neurones of different segments, on the other carried out in ice to prevent precipitation of osmium. hand, appear at the same time (Fig. 2). Sensilla Embryos were left overnight in a 1:1 mixture of Epon and appear in two waves: chordotonal organs, basiconical Development of Drosophila larval sensory organs 871

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Fig. 1. Distribution and development of the Drosophila larval sensilla. (A) Schematic depiction of the sense organs and nerves of the fully developed embryo. Homologies between sensilla of different segments as proposed by Campos- Ortega & Hartenstein (1985) are indicated by corresponding symbols at the same vertical level. Shading indicates topological groups (ventral group, shading; lateral group and dorsal group, blank). The neuropile is vertically hatched and the commissures are shown cross hatched. Vertical dotted lines mark borders of gnathal segments; the dotted circle encloses sensilla belonging to the antennomaxillary complex. Sensilla are symbolized as follows: small circles, campaniform sensilla; large circles with central dots, basiconical sensilla; large open circles, various types of head sensilla; circles with triangle, trichoid sensilla; arrowheads, chordotonal organs; arrows, sensilla of the posterior spiracle. (B) The sequence of appearance of the Drosophila larval sensilla in synoptic view. The horizontal depicts time; numbers at the top give stages (upper panel; after Campos-Ortega & Hartenstein, 1985) and minutes after 50% germ band retraction (at 25°C). A sensillum is represented by a bar; the left end of the bar indicates the time when the sensillum starts expressing the neural-specific antigen detected by anti-HRP and 22C10. Left end of shaded part of a bar marks the time when the axon of the corresponding sensillum has reached a pioneer tract leading towards the CNS. Interrupted lines at the beginning of bars give estimated variability in time of appearance of the corresponding sensilla. Sensilla with several neurones appearing at different times are indicated by bars which taper towards their left end; the tip marks the time when the first neurone appears, full width of the bar gives the time when all neurones are present. Abbreviations: A1-A9, abdominal segments; af, anterior fascicle of segmental nerve; amx, antennomaxillary complex; esc, caudal sensory cone; dbd, dorsal basiconical sensillum; dcl-3, dorsal campaniform sensilla 1-3; dch3, dorsal (triscolopidial) chordotonal organ; dcsc, dorsocaudal sensory cone; dhl-2, dorsal trichoid sensilla 1-2; disc, dorsolateral sensory cone; dmsc, dorsomedial sensory cone; do, dorsal organ; dpo, dorsal pharyngeal organ; epi, epiphysis; hy, hypophysis; ko, Keilin's organ; LB, labium; Ibd, lateral basiconical sensillum; Ibo, labial complex; Ibr, labrum; lcl-2, lateral campaniform sensilla 1-2; lchl/3/5, lateral (mono-/tri-/pentascolopidial) chordotonal organ; lhl-2, lateral trichoid sensilla 1-2; MD, mandible; MX, maxilla; pchl, paryngeal chordotonal organ; pf, posterior fascicle of segmental nerve; PR, procephalic nervous system; sso, spiracle sense organ; sto, stemmatal organ; 77-3, thoracic segments; to, terminal organ; vas, ventral anal sensillum; vbd, ventral basiconical sensillum; vcl-5, ventral campaniform sensilla 1-5; vchl, ventral (monoscolopidial) chordotonal organ, vo, ventral organ.

sensilla and trichoid/campaniform sensilla innervated Development of the thoracic and abdominal by two dendrites (dhl, vc5) appear earlier than the peripheral nerves single innervated trichoid/campaniform sensilla of Peripheral axons form two fascicles within each corresponding dorsoventral level (Figs 1-3). hemisegment of the Drosophila embryo (Campos- 872 V. Hartenstein

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Fig. 2. Pattern of sensory neurones of Drosophila embryos stained with the neural specific antibody 22C10 at sequential developmental stages. A-D show whole mounts of embryos in lateral view (A, stage 13/70min a.r.; B, stage 13/90 min a.r.; C, stage 14/120 min a.r.; D, stage 15/160 min a.r.;). E-G show the anterior parts of embryos in dorsal view (E, stage 14/120 min a.r.; F, stage 15/160 min a.r.; G, stage 16/300min a.r.). For E and F, two planes of focus are depicted (top, dorsal plane; bottom, ventral plane). Abbreviations of head peripheral nerves; bo, Bolwig's nerve (axons of stemmatal organ); Ibr, labral nerve; nan, antennal nerve; nl, labial nerve. For abbreviations of sensilla see Fig. 1. Bar, 50 fim. Development of Drosophila larval sensory organs 873

Ortega & Hartenstein, 1985; see Figs 1A, 3). The segmental tracheal ramus. In the early stage-14 em- anterior fascicle receives axons of the lateral and bryo (2-2-5 h a.r.) efferent and afferent pioneer dorsal group of sensilla, respectively, and delivers axons meet and fasciculate. efferent axons to the dorsal musculature. The pos- At around the same stage, axons of the lateral terior fascicle receives the sensory axons of the basiconical sensilla (Ibd in the thoracic segments T2 ventral sensilla, including the lateral monoscolopidial and T3), the lateral pentascolopidial chordotonal chordotonal organ (Ichl in abdominal segments organs {Ich5, A1-A7), and the thoracic dorsal trisco- A1-A7; Ghysen et al. 1986). It supplies motor axons lopidial chordotonal organs (dch3) join the pioneer to the pleural transversal muscles and the ventral tract. The neurones of the later developing trichoid musculature. and campaniform sensilla emit their axons in the late All sensory neurones start axonal outgrowth ap- stage-14 embryo (2-7-3-5 h a.r.). All of these sensilla proximately 30min after the beginning of the ex- are located within a distance of less than lOjum from pression of the antigens recognized by anti-HRP and the trajectory of the pioneer tract of the anterior 22C10 (Figs IB, 3). Thus, axonal outgrowth in the fascicle. Their axons thus reach the pioneer tract various sensilla follows the sequence of appearance of immediately after outgrowth. Efferent branches ap- these sensilla. Axons of the basiconical sensilla, pear in the late stage-14 embryo. By stage 16 (6-7 h chordotonal organs and doubly innervated trichoid/ a.r.), all efferent fibres have reached their muscles of campaniform sensilla appear first. In concert with destination. some early extending efferent fibres, all of these axons form 'pioneer tracts' with which the later Posterior fascicle outgrowing sensory and motor axons will fasciculate. The posterior fascicle, also known as 'segmental Except for the axons of the thoracic chordotonal nerve' (Canal & Ferrus, 1986) arises somewhat later organ dch3 and prothoracic Ich3 all axons follow a than the anterior fascicle (Fig. 3). Similar to the straight, ventrally directed course. The neurones of anterior fascicle, it is pioneered by an efferent and an the dch3 have a reversed polarity; this is to say their afferent component (vbd in the thoracic segments, dendrites point ventrally and their axons grow out vc5/vcl/vc2 in the abdominal segments). The effer- dorsally (Fig. 2B). The axons then make a sharp turn ent axons are emitted in the late stage-13 embryo to grow ventrally, thereby fasciculating with the (l-5-2ha.r.) by two or three central neurones axons of the dhl. The axons of the prothoracic Ich3 located within the homotopic neuromere. Extending extend caudally, thereby crossing the T1/T2 bound- laterally, the axons leave the CNS and penetrate the ary (Fig. 2C). Apposed to the wall of the trachea, myoblasts of the ventral musculature. About 1 h later these axons are 'guided' into T2 where they fascicu- axons of the vbd and vc5 appear. Axons of the late with the pioneer tract of the anterior fascicle of thoracic vbd, after only 10-20/xm of ventrally di- that segment. rected growth, meet the dorsally extending efferent pioneer axons. The abdominal vc5 axons first meet Anterior fascicle the somewhat later appearing vcl/vc2 cells, whose The anterior fascicle within each segment is pio- axons in turn are the first to make contact with the neered by two efferent axons and two sensory axons efferent pioneer axons. The first axons to join the originating from the dorsal hair dhl (Fig. 3; see also pioneer tract are those of vchl and Ichl. Axons of the Campos-Ortega & Hartenstein, 1985; Ghysen et al. remaining trichoid and campaniform sensilla follow 1986). The efferent axons grow out in the late stage- between 3 and 3-5 h a.r. (late stage 14). Around 6-7 h 12 embryo (0-30 min a.r.) from a pair of central a.r. (stage 16), all of the efferent fibres travelling with neurones. Growing posteriorly, these axons are the the posterior fascicle have also reached their muscles first components of the presumptive connective of destination. (Canal & Ferrus, 1986). Upon reaching the posteriorly adjacent neuromere, the axons bend laterally Development of the peripheral nervous system of the and leave the CNS. The axons pioneer the anterior larval head root of the anterior fascicle of the segmental nerve, The head sensilla, most of which are assembled into also known as the 'intersegmental nerve' (Campos- large sensory complexes, have been described in a Ortega & Hartenstein, 1985; Canal & Ferrus, 1986). number of studies (Hertweck, 1931; Chu-Wang & The two axons penetrate the mass of myoblasts of the Axtell, I972a,b; Singh & Singh, 1984; Campos- ventral musculature which, by this stage, laterally Ortega & Hartenstein, 1985; Juergens et al. 1986). flank the CNS; they then grow along the wall of the These (predominantly chemosensory) complexes transversal segmental ramus of the trachea which comprise the hypophysis and labial complex, both extends along the segmental boundary. Sensory derived from the labial segment; the antennomaxill- axons of the dhl grow ventrally along the wall of the ary complex, derived from the maxillar segment 874 V. Hartenstein

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Fig. 3. Camera-lucida drawings taken from whole mounts of Drosophila embryos stained with the neural specific antibody 22C10. The sensory neurones and their axons of one thoracic (upper row) and abdominal (lower row) hemisegment at sequential developmental stages are shown in lateral view (A, stage 13/70mina.r.; B, stage 14/120mina.r.; C, stage 15/l60mina.r.; D, stage 15/180mina.r.; E, stage 16/300mina.r.). Segmental boundaries are indicated by fine dotted lines; large dots outline the CNS. Neurones of the ventral group are shown with dark shading, those of the lateral group light shading; dorsal group neurones are blank. Stippling marks cells bodies of the efferent pioneer neurones. For symbols and abbreviations of sensilla innervated by the neurones see Fig. 1. Numbers to the left give percent of perimeter (ventral midline, 0%; dorsal midline, 100%). Bar, 30 jim.

(terminal organ), mandibular segment (ventral or- from the rudimentary intercalary segment; the epi- gan), and antennal segment (dorsal organ); the hypophysis and dorsal pharyngeal organ; both derived pharyngeal organ ('organ X' of Hertweck), derived from the preantennal segment; the stemmatal organ. Development of Drosophila larval sensory organs 875

Fig. 4. Morphogenesis of a basiconical sensillum (A-D; A, stage 14/100min a.r.; B, stage 15/180min a.r.; C, stage 16/300 min a. r.; D, first instar larva) and a trichoid sensillum (E-H; E, stage 15/160 min a.r.; F, stage 15/l80mina.r.; G, stage 16/300 min a.r.; H, first instar larva). Drawings depict three-dimensional reconstructions of the sensilla at different stages in lateral view; they were obtained from superimposing the profiles of sensilla cells on EM pictures taken at different levels. Hidden contours of neurones and dendrite-related sheaths are indicated by dashed lines; other hidden lines (contours of cell bodies of accessory cells) were omitted for sake of clarity. Bar, 5^m. During phase 1 (A for basiconical sensillum; E for trichoid sensillum) the presumptive sensory neurones (sc, dark shading) penetrate the thecogen cell (th, light shading). A macula adherens (arrowheads) surrounds the dendrites just beneath their apical tips. From two basal bodies (black dots) located at this position, outgrowth of the outer dendritic segments starts. The outer segments reach the apical surface of the thecogen cell at phase II (C). The trichogen cell (tr) and tormogen cell (to) wrap mesaxon-like processes around the thecogen cell. At the beginning of this process, only a narrow apical fringe of the thecogen cell is entirely enclosed. Gradually, the sheath is completed at more basal levels. In phase II (B,C for basiconical sensillum; F,G for trichoid sensillum) regularly spaced microvilli appear at the apical surface of the trichogen and tormogen cell. Cuticle first covers the apices of the microvilli only; then a continuous sheath of increasing thickness is formed. During cuticle secretion the trichogen cell produces the hair shaft which, in the basiconical sensilla, is invaded by two dendrites. The thecogen cell secretes the dendritic sheath (hatched) which appears in the apical part of the cleft between dendrites and thecogen cell. During phase III (D for basiconical sensillum; H for trichoid sensillum) the soma sheath cell (ss) encloses the neural somata by an extremely thin, electron- dense process. A perineural cell of unknown origin (stippled) wraps around the axons. The basal lamina appears at the basal surface of the epidermal layer. No sheath enclosing the somata of the sensory neurones is formed in the trichoid and campaniform sensilla. 876 V. Hartenstein derived from the antennal segment. This sensory that the distances between most sensory neurones of organ, which has been described for the Drosophila the head and the positions where their axons will larva recently (Steller et al. 1987) consists of a circular enter the CNS are very short. Elongation of the array of 12-15 bipolar neurones which are located in peripheral tracts is secondarily effected by the dislo- the dorsal sac and project their axons in close cation of the head sensilla during head involution. proximity to the antennal nerve (innervating the Only the axons of the hypophysis and the labial antennal part of the antennomaxillary complex). The complex (which will form the labial nerve) and those axons terminate superficially in a ventrocaudal region of the epiphysis and dorsal pharyngeal organ (which of the supraoesophageal ganglion which corresponds will form the labral nerve) have to travel for longer to the primordium of the optic lobe (Hofbauer, 1979). distances before reaching the CNS (Fig. 2E,F). They With respect to its location and axonal projection, appear in a cascade-like manner: neurones of the this sensory complex corresponds to the stemmatal hypophysis and epiphysis emit axons at a stage when organ described for the larva of Musca (Bolwig, 1946) these sensory complexes are located at the rostral tip and other dipterans. of the embryo. Extending caudally, these axons meet The time sequence in which the sensilla of the and fasciculate with the axons of the labial complex larval head appear is shown in Figs 1 and 2E-G. It and dorsal pharyngeal organ, respectively, which lie should be noted that all sensilla are strongly dislo- midway towards the CNS. cated during head involution. Axonal outgrowth occurs at a stage similar to that described for the Ultrastructure of the thoracic and abdominal sensilla axons of the thoracic and abdominal sensilla. By that The Drosophila larval sensory organs comprise three stage, involution of the head has not yet occurred so types of external sensilla (trichoid, campaniform,

Fig. 5. Morphogenesis of a pentascolopidial chordotonal organ (A, stage 14/100mina.r.; B, stage 15/180mina.r.; C, stage 16/300 min a.r.; D, first instar larva). Drawings were designed as described for Fig. 4. Bar, 5/jm. During phase I (A,B) the development of the neurones (sc, darkly shaded) and scolopale cells (sco, lightly shaded) closely resembles the development of the corresponding cells in the external sensilla (neurones, thecogen cells). The elongating dendrites do not, however, grow beyond the apical surface of the scolopale cells. The scolopale cells penetrate the cap cells (cc) which in turn apically insert at an epidermal cell. Membrane contacts between dendrites, scolopale cells and cap cells are strengthened by prominent adherent desmosomes. During phase II (C) the scolopale cells secrete extracellular dendritic sheaths (hatched) and form intracellular scolopales (black). These extend from the dendritic sheath (apically) to the level of the basal bodies (black dots) basally. During phase III (D) processes of the soma sheath cell (ss) enclose the sensory neural somata and their basally elongating 'anchoring processes'. Bar, 5/«n. Development of Drosophila larval sensory organs 877

Fig. 6. Longitudinal (A,C,E) and transverse (B,D) sections of a basiconical sensillum of a first instar larva. A shows the cell bodies of two sensory neurones (sc), the soma sheath cell (ss), and the tormogen cell (to). For one of the neurones, the outer dendritic segment (a"), inner segment (d), basal bodies (bb) and rootlet fibres (rf) are shown. The sheath processes of trichogen cell (tr) and thecogen cell (th) reach the apical surface of the sensory neurones; there they border the electron-dense sheath formed by the soma sheath cell. Basally, a perineural cell (gc), enclosing a sensory axon (ax) is depicted. B shows the outer dendritic segments enclosed within an extracellular sheath (ds). In C-E, the basiconical sensory process is illustrated. It is filled by two dendrites (D); only the thicker and less electron-dense of these dendrites (dl) reaches the tip of the sensory process (E). The third dendrite (d3) ends below the hair base (C). Bars, 3/im (A); (B-E). 878 V. Hartenstein

Fig. 7. Lxingitudinal (A,C,D,G) and transverse sections (E,F) of a trichoid sensillum of a stage-16 Drosophila embryo (300mina.r.) and longitudinal section of a first instar larval trichoid sensillum (B). A shows the hair-forming process of the trichogen cell (pi) covered with a thin cuticle layer (CM). A single dendrite (d') ends at the hair base; the extracellular sheath is continuous with the cuticle. In G the transition between inner (d) and outer (d') dendritic segment with the basal bodies (bb) and rootlet fibres (rf) are pictured. C shows the cell bodies of the sensory neurone (sc) and the thecogen cell (th). E illustrates the dendrite, its sheaths and the adherent desmosomes between dendrite and thecogen cell (arrowhead). F shows the sensillum vc5 which is innervated by two dendrites. The microtubule-filled tubular body (tb) at the end of the outer dendritic segment is depicted in B. Bar, 3^m. basiconical) and the the subepidermally located chor- Chordotonal organs are built of a variable number dotonal organs. The dendrites innervating the exter- of units called scolopidia. Each scolopidium com- nal sensilla are ensheathed by three accessory cells. prises distinctive types of cells which have been The inner one of these (thecogen cell or neurilemma tentatively identified as homologous to the different cell) secretes an extracellular sheath around the tip of cells of the external sensilla (Schmidt, 1969). An the dendrite(s) (Steinbrecht & Mueller, 1976). A individual scolopidium is innervated by one or more sensory process of variable shape and length is bipolar sensory neurone(s). Dendrites are enclosed produced by the middle accessory cell (trichogen within the scolopale cell, a homologue of the theco- cell). The outer accessory cell (tormogen cell) forms a gen cell of an external sensillum. Like the thecogen circular socket or excavation at the base of the cell the scolopale cell secretes an extracellular sheath sensory process. An additional, subepidermally around the dendritic tip. Additionally, it forms the located cell surrounds the soma of the sensory scolopale, an intracellularly located framework of neurone. A cell similar in structure and position has densely packed microtubules surrounded by an elec- been described as glial or perineural cell for a variety tron-dense material. Apically, the scolopale cell is of sensilla (Zacharuk, 1985). I prefer the term 'soma connected to the elongated cap cell which anchors the sheath cell' to emphasize the fact that this type of cell, scolopidium in the epidermis. The cap cell might be as opposed to the perineural cells surrounding the homologous to either the trichogen or tormogen cell peripheral axons, seems to originate from the epider- of an external sensillum. The soma of the sensory mally derived) sensilla precursor cells. neurone is enclosed within a soma sheath cell. Development of Drosophila larval sensory organs 879

Basiconical sensilla poreless hair of 5-6/irn length and l-l-5jum diam- The Drosophila larval basiconical sensilla (Fig. 6) eter. The hair base is sunken into an excavation of the closely resemble sensilla of the same morphological cuticle. Basiconical sensilla are innervated by three quality that have been electrophysiologically defined dendrites. Two of these reach into the lumen of the as combined thermo- and hygroreceptors in other hair shaft; the third one terminates beneath the hair insects (Altner et al. 1981). They possess a stout, base.

Fig. 8. Oblique sections of a lateral pentascolopidial chordotonal organ of a first instar larva. A depicts the basal 'anchoring processes' of the sensory neurones iyp). They are enclosed within cylindrical processes of the soma sheath cells (55) which basally insert at an epidermal cell (epi). Apically, soma sheath cells border at the scolopale cells (sco; the border is indicated by arrowheads in B). The sheath enclosing the sensor)' neurones is incomplete; part of it is formed by a perineural cell which also wraps the sensory axons (outside the level of section). The rootlet apparatus (r) forms a cylinder which ends within the ventral 'anchoring processes' of a sensory neurone. C and D show oblique sections of four scolopidia at the level of the outer dendritic segment (d'\ left one in D) and the inner dendritic segment (d), respectively. The scolopale forms six longitudinal rods (sr) which apically fuse into a massive cylinder contiguous with the (extracellular) dendritic sheath. Bar, 3/im. 880 V. Hartenstein Development of Drosophila larval sensory organs 881

Trichoid and campaniform sensilla (Fig. 7) epidermis. A similar, basal neuronal 'anchoring' The trichoid and campaniform sensilla morphologi- process has so far not been described for any other cally correspond to the sensilla of this denomination chordotonal organ investigated. However, as pointed described as mechanoreceptors in numerous species out by Moulins (1976), the basal anchorage of a (for review see Altner, 1977; Zacharuk, 1985; Keil & chordotonal organ has rarely been studied ultrastruc- Steinbrecht, 1986). These organs possess a slender turally in great detail. Neuronal 'anchoring' pro- hair or a dome, respectively. Hairs measure 5-8^m cesses, which might exist in other species as well, in length and 0-5-0-7 fim in diameter. Domes are of might have been overlooked or misinterpreted. the same diameter. With the exception of the abdominal dorsal hair dhl and the ventral campaniform Morphogenesis of the thoracic and abdominal sensillum vc5, both of which are innervated by two sensilla neurones, all trichoid and campaniform sensilla pos- The sensillum cells develop from ectodermal precur- sess one neurone each. The dendrite terminates sor cells during stage 11 and 12 of embryonic develop- beneath the base of the hair or dome. Unlike the ment (Hartenstein & Campos-Ortega, 1985). A fixed dendrites of the basiconical sensilla, it possesses an spatial relationship exists between the sensilla cells oval tubular body at its distal tip. A complete sheath throughout development. In the external sensilla, around the sensory neurone is lacking; however, a presumptive neurones and soma sheath cells are subepidermally located cell which might represent an subepidermally located, whilst presumptive theco- undifferentiated homologue of the soma sheath cell gen, trichogen and tormogen cells lie within the of a basiconical sensillum has been observed in most epidermal layer (Figs 4A,E; 9). In the chordotonal trichoid and campaniform sensilla. organs, all presumptive sensilla cells are subepidermally located (Figs 5A, 10). The neurone, scolopale Chordotonal organs (Fig. 8) cell and cap cell of each of the scolopidia are arranged The Drosophila larval chordotonal organs in struc- in a vertical column; the soma sheath cell lies ture and position correspond to those chordotonal medially and somewhat basally to the neurone. Thus, organs which, in other dipteran species, have been the primordia of chordotonal organs and external identified as stretch receptors (Moulins, 1976; Finn- sensilla differ with respect to the spatial arrangement layson, 1976). The scolopidia are innervated by one of the presumptive accessory cells right from the neurone each. Opposite the dendrite, the neurone beginning of sensillum morphogenesis. gives rise to the axon and a further, more ventrally Sensillum morphogenesis comprises a series of directed, process. Enclosed within an extension of the events most of which are similar to those events soma sheath cell, this process also inserts in the characteristic of the development of normal epidermal cells. Three phases can be distinguished (I) Fig. 9. EM photographs of transverse sections of a outgrowth of processes (Figs 4A,E; 5A, B; IOC): in basiconical sensillum of a stage-14 Drosophila embryo the stage-13 embryo (after 60mina.r.) all epidermal (A-D, 180mina.r.; E-F, lOOmina.r.). A-D were taken cells form horizontal, sleeve-like processes which, to from the same sensillum. They show dendrites (d) and a variable extent, wrap their neighbouring cells. their sheath (th, thecogen cell; tr, trichogen cell; to, During this phase, presumptive thecogen, trichogen, tormogen cell) at different levels (A, apical; D, basal). E tormogen, scolopale and cap cells start forming a and F picture cell bodies of the sensory neurones (sc) and permanent sheath around the elongating dendrites; the thecogen cell. Apically, the sheath cells possess microvilli which have started cuticle secretion (A). The the horizontal processes of normal (i.e. nonsensil- tormogen cell forms a cavity: outgrowth of the sensory lum) epidermal cells are lost during further develop- process formed by the trichogen cell starts from the ment. The cell bodies of the accessory cells are fundus of this cavity (A). Dendrites have not yet reached dislocated basally to reach a level distinctly below the the apical surface of the thecogen cell (compare B and neighbouring epidermal cells. At their basal surface C). C shows the three dendrites near the transition zone most, if not all, epidermal cells extend short, filo- between inner and outer segment; the outer segment (d1) podia-like extensions which, in the presumptive sen- by this stage is very short and possesses no extracellular sory neurones, develop into centripetally elongating sheath. Longitudinally oriented microtubules appear in axons. In other cells, the basal processes are with- the elongating outer dendritic segments and the sheath- drawn prior to formation of the basal lamina (stage forming processes of the accessory cells. The sheath 16). An event that exclusively occurs in the presump- processes formed by the trichogen cell and tormogen cell surround the thecogen cell only apically (A,B). Further tive sensory neurones is dendrite formation. Sensory symbols and abbreviations: large arrowheads indicate neurones apically penetrate into the overlying inner maculae adherentes or adherent desmosomes, sheath cells (thecogen cells in the external sensilla, respectively; small arrowheads point at septate scolopale cells in the chordotonal organs), thereby desmosomes; rf, rootlet fibres. Bars, forming the later inner dendritic segments. From the 882 V. Hartenstein Development of Drosophila larval sensory organs 883 two basal bodies (presumably derivatives of the pair the first wave (chordotonal organs, basiconical sen- of centrioles which occurs at the apical pole of any silla, doubly innervated trichoid and campaniform epidermal cell), the outgrowth of the outer dendritic sensilla), are distributed at regular distances, in such segments is initiated. (II) Cuticle formation (Figs a way that each of the topographically defined groups 4B,C,F,G; 5C): cuticle secreting microvilli appear at of sensilla (dorsal, lateral, ventral) is represented by the apical surface of the epidermal cells. Trichogen one or two sensilla. During the second wave, trichoid and tormogen cells produce the sensory processes and and campaniform sensilla possessing only one sensory sockets, respectively, and lay down the cuticle layer neurone are generated. A dorsoventral gradient has around these structures. Inner sheath cells secrete the also been reported for the proliferation pattern of extracellular dendritic sheaths (which are composed ectodermal cells (Hartenstein & Campos-Ortega, of a cuticle-like material) and, in the chordotonal 1985). Ectodermal cells proliferate in three mitotic organs, the intracellular scolopales. (Ill) Formation waves that occur in a 3h interval following gastru- of basal sheaths (Figs 4D,H; 5D); soma sheath cells lation. During each of these waves, mitosis starts at and perineural cells envelop sensory neural somata the dorsal margin of the ectoderm. Sweeping ven- and axons, respectively. This process is completed trally, the mitotic front reaches the ventral midline only for the basiconical sensilla; the somata of the approximately 1 h later. other sensilla of a first instar larva are only partially The evidence presented here suggests that (i) the ensheathed or lack a sheath altogether. mechanisms that control the pattern of proliferation within the ectoderm and the temporal sequence in which sensilla are generated might be related. Thus, a Discussion common positional cue might determine when an ectodermal cell initiates mitosis and when it is 'ready' Sequence of generation, distribution and axonal to segregate as a sensillum precursor cell. It has been projection of the sensilla shown in different insect species that the sequence of The sensilla of the Drosophila larval thoracic and generation of sensilla reflects the sequence in which abdominal segments appear in two dorsal-to-ventral the precursor cells of the corresponding body part waves across the epidermal primordium. Sensilla of divide (Edwards & Chen, 1979; Shankland & Bent- ley, 1983). Fig. 10. EM photographs of sections taken at different planes of the lateral body wall of a stage-13 Drosophila (ii) The temporal pattern underlying the gener- embryo (lOOmina.r.). The oblique section (A) and ation of sensilla might be involved in determining the tangential section (B) show details of a lateral spatial pattern in which they are distributed. The pentascolopidial chordotonal organ. Sensory neurone mechanisms controlling the distribution of sensilla in (sc), scolopale cell (sco), and cap cell (cc) of each the insect integument are poorly understood. In those scolopidium are arranged in a vertical column (B). models developed to explain the sensillum pattern in Homologous cells of the neighbouring scolopidia lie in the insect integument (Wigglesworth, 1940; Stern, horizontal rows (A). Basal bodies (bb) are present at the 1954; Lawrence, 1970; Richelle & Ghysen, 1979; tips of dendrites. Outgrowth of the basally directed Meinhardt, 1982), not much attention has been paid rootlet fibres has started. Outer dendritic segments are to the sequence in which sensilla are generated as a not yet present. Soma sheath cells, which also form a possible determinant of their spatial distribution. horizontal row medially to the neurones, are out of the Meinhardt (1982) speculated that the growth of a level of the sections. C shows a horizontal section at the level of the lateral pentascolopidial organ (Ich5). The positional field during cell proliferation might yield a pioneer tract of the anterior fascicle (large arrow) lies in- dilution of the suppressor emitted by the already between the segmental branch of the trachea (str) and existing sensilla, a process which could then result in myoblasts of the ventral musculature (vm). The insert D the appearance of new sensilla. In the Drosophila shows the growth cones constituting the pioneer tract of larva, as in larvae of other dipteran species (Finnlay- the anterior fascicle at higher magnification. The growth son, 1976), most thoracic and abdominal sensilla are cones are filled with microtubules and mitochondria and arranged in a transverse line in the middle of each emit filopodia. Further symbols and abbreviations: epi, segment. Thus, the position in the longitudinal axis of epidermal cell; pet, pleural transversal muscles; sf, a segment is almost identical for all sensilla. What segmental furrow; vm, ventral muscles. Arrowheads remains to be determined then is the position of the indicate adherent desmosomes in-between dendrite, sensilla in the transverse axis. It might be speculated scolopale cell and cap cell. Small arrows point at horizontal, sleave-like processes formed by sensory that the distance between sensilla of a given type is neurones (A). Black triangles delineate similar processes, linked to the sequence in which these sensilla appear. as well as some basal, filopodia-like processes, which are In addition, the regularly spaced sensilla of the first transiently formed by epidermal cells at this stage (C). wave may be involved in determining the spatial Bars, 3/im (A,B); 5fim (C); 2/im (D). pattern of the sensilla of the second wave. 884 V. Hartenstein

(iii) The sequence in which sensory neurones send for the development of the sensillum cells are (tran- out their axons might be involved in the control of the siently) expressed by epidermal cells as well. Sensil- establishment of neural connections. Recent studies lum development thus might be envisaged as a have led to the suggestion that one of the factors process in which the series of differentiative steps allowing sensory axons to establish their appropriate shaping epidermal cells in general is modified by the peripheral pathways might be the temporal sequence action of intrinsic (genetic) and extrinsic factors. It in which sensory neurones are distributed and send might be speculated that some principal steps in out their axons. Peripheral tracts are generally pio- sensillum morphogenesis (for example, subepidermal neered by sets of early differentiating neurones which position, formation of dendrites and axons, or lack of start axonal outgrowth in a cascade-like manner. A cuticle secretion in the presumptive sensory given pioneer neurone might serve as a 'guide-post' neurones) are controlled by a relatively small number for the incoming axon of the distally adjacent of intrinsic factors. Other, more generally occurring, neurone, whilst its own axon pioneers the interval phenotypic traits (for example, the dendrite-enclos- towards the next proximal neurone (Ho & Goodman, ing processes of the accessory cells), might be modi- 1982; Keshishian & Bentley, 1983a,6; Shankland & fied in sensillum development as the result of cell-cell Bentley, 1983; Murray et al. 1984). The distance interaction. between neighbouring pioneer neurones is so small The rare experimental studies related to sensilla that the proper polarity of axonal outgrowth, in development suggest that cell-cell interactions in- concert with the exploratory activity of the filopodia deed play a role in organizing the morphogenesis of of the axonal growth cones, might be sufficient for an sensillum accessory cells. Thus, eliminating the pre- axon to find its way towards the next proximal sumptive neurones innervating the wing bristles of pioneer neurone. The problem of establishing the Galleriu mellonella prevented formation of the bris- appropriate peripheral pathways then would be the tles, although the trichogen and tormogen cells were problem of generating the 'guide post' neurones at not primarily affected by the experimental procedure the right positions and timing the outgrowth of their (Clever, 1961). In Drosophila, the characterization of axons in the right sequence. mutants affecting sensilla morphology yields a deeper insight into when and how genetic factors are in- In the Drosophila embryo, the sequence in which volved in sensilla morphogenesis. Lees & Wadd- sensory neurones appear and start axonogenesis is ington (1942) described a number of mutations most correlated with the temporal pattern of proliferation of which, as judged by light microscopic observation of the ectoderm in the early postgastrula embryo. It of the adult bristle phenotype, primarily affect the seems possible, therefore, that some of the mechan- division pattern of the sensillum precursor cells isms controlling complex developmental phenomena, and/or the position of the sensillum cells. Thus, some such as the patterning of sensory organs and the early occurring alterations in sensillum development guidance of sensory axons, might be the same mech- seem to have far reaching epigenetic consequences. It anisms designed for controlling general events of is hoped that a more subtle descriptive and exper- early embryogenesis, such as the proliferative activity imental analysis of the mutations affecting sensillum within the germ layers. morphology many of which are most probably found among the embryonic lethals, might improve our understanding of the mechanisms controlling sensil- Sensillum morphogenesis lum development. The development of external sensilla has been investigated electron microscopically for a variety of I thank Jose Campos-Ortega and James Posakony for species (Ernst, 1972; Gnatzy & Schmidt, 1972; Sanes critical reading of the manuscript. This work was supported & Hildebrand, 1976; Gnatzy, 1978; Keil, 1978; Han- by the Deutsche Forschungsgemeinschaft Grant Ca60/7. I sen & Hansen-Delkeskamp, 1983; Kuhbandner, thank Dr Seymour Benzer for kindly providing the anti- 1984; DeKramer & van der Molen, 1984). The body 22C10. development of chordotonal organs has been described light microscopically (Child, 1894; Schoen, References 1911; Jaegers-Roehr, 1968; electron microscopic studies only exist for late developmental stages ALTNER, H. (1977). Insektensensillen: Bau und (Schmidt, 1968; van Ruiten and Sprey, 1974). Funktionsprinzipien. Verh. Dtsch. Zool. Ges. 70, 139-153. The present study confirms and extends these ALTNER, H., ROUTIL, C. & LOFTUS, R. (1981). The investigations. It further reveals the strong simi- structure of bimodal chemo- thermo-, and larities in the development of sensilla cells and hygroreceptive sensilla on the antenna of Locusta epidermal cells: most morphogenetic events typical migratoria. Cell. Tiss. Res. 215, 289-308. Development of Drosophila larval sensory organs 885

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