Roux's Arch Dev Biol (1983) 192:~64-170 F{oux's Archives of Developmental Biology ,~, Springer-Verlag 1983

The Role of the Peripodial Membrane in the Morphogenesis of the Eye-Antennal Disc of Drosophila melanogaster Martin John Milner, Alison Jane Bleasby, and Andrew Pyott Department of Zoology, The University, St. Andrews, Fife KYt6 9TS, Great Britain

Summary. The early morphogenesis of the eye-antennal disc of Drosophila in response to 20-hydroxy ecdysone involves the curling of the eye anlagen dorsally over the antenna. During this process, the area of the peripodial membrane is substantially reduced. The peripodial membrane is taut at this stage, and if it is cut the curling of the disc cannot continue, and the eye anlagen returns to its original position within one minute of the operation. In contrast, cutting the columnar epithelium between the eye and antennal anlagen does not disrupt curling, but actually facilitates it. During curling, the cells of the peripodial membrane appear healthy, and exhibit basal extensions. We suggest that the curling of the eye is mediated by the conversion of cuboidal peripodial membrane cells into pseudostratified columnar epithelium at the edges of the peripodial mem- brane. Subsequently, cells of the peripodial membrane secrete first a pupal cuticle, and then an imaginal cuticle.

Key words: Drosophila - Imaginal disc - Morphogenesis - Tissue culture

Introduction

Imaginal discs consist of a hollow sac composed of a single layer of cells, enclosing a cavity, the lumen. On one side, the cells are organised into a thick, columnar epithelium, which is folded. The other side consists of a thinner, un- folded monolayer of squamous or cuboidal cells, called the peripodial membrane.

Early sudies of disc ultrastructure and morphogenesis concluded that the peripodial membrane did not differenti- ate to form imaginal cuticular structures, but degenerated and/or was removed by phagocytes during the pupal stage (Poodry and Schneiderman 1970; Ursprung 1972). The only function attributed to the peripodial membrane by these and earlier authors would appear to be that of maintaining structural continuity between the columnar epithelium of the disc and the larval epidermis (Poodry and Schneiderman 1970, Fig. 31).

Some recent investigations have suggested a more active role for the peripodial membrane. Spray and Oldenhave (t974) constructed a fate map for the wing disc of Calli- phora, and concluded that both the thick peripheral and thin central parts of the peripodial membrane contribute to the differentiation of imaginal structures. The peripodial membrane of the Drosophila wing disc may also contribute to imaginal differentiation (Bryant 1975). The morphogene-

sis and differentiation of the labial disc of Drosophila has been examined by Kumar et al. (1979) who concluded that the distal part of the peripodial membrane contributes to the formation of the proboscis, whereas the proximal part has a temporary morphogenetic function, but eventually disappears.

The majority of these studies have examined the ever- sion and differentiation of leg and wing imaginal discs, and so we considered it worthwhile to investigate the role played by the peripodial membrane in the metamorphosis of the eye-antennal disc. This disc possesses a large area of peripo- dial membrane, and the morphogenetic movements it un- dergoes during the prepupal and pupal stages differ mark- edly from the wing disc. As the antenna starts to evaginate, the eye tissue moves upwards to fold over it forming a vesicle (Gottschewski 1960; Sprey 1970), and this sequence of events occurs in vitro under the influence of the moulting hormone (Schneider 1964; 1966; Milner and Haynie 1979; Milner 1980). We have examined the early morphogenesis of eye-antennal discs cultured in vitro using histological and microscopical techniques. We use the term morphogen- esis to refer to the changes in the gross morphology of discs which occur in situ during the prepupal period under the influence of the hormone 20-hydroxy ecdysone.

Materials and Methods

The procedures used for the sterile culture of larvae from a wild-type (Oregon) stock of Drosophila melanogaster, and for the dissection and culture of their imaginal discs have been previously described (Milner and Sang 1974, with modifications in Edwards et al. 1978 and Milner 1980). The culture medium used was Shields and Sang's medium 3 (Shields and Sang 1977), supplemented with 2% foetal bovine serum, and with 0.1 gg/ml of 20-hydroxy ecdysone added. Cultures were maintained at 25 ~ C.

Discs were transferred from the culture medium by pipette for fixing in 2.5% glutaraldehyde solution buffered with 0.02 M phosphate buffer (pH 7.6). Post-fixation was done in buffered (pH 7.6) I % osmium tetroxide solution and discs were rinsed in buffer before dehydration (Tucker 1967). Fixed material was embedded in araldite epoxy resin and sections were cut on an LKB ultrotome III. Thick sec- tions (1 gm) for light microscopy were stained with 1% methylene blue in 1% borax. Thin sections (50-60 nm) were stained with uranyl acetate and lead citrate (Reynolds 1963) for observation on a Philips 301 electron microscope.

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Results

The columnar epithelium of the antennal region of the mature disc is thrown into folds representing the future antennal segments, while that of the eye is unfolded but convex in shape, fitting the curved surface of the brain lobe (Fig. 1 a, b). The cells are pseudostratified, nuclei of adjacent cells being located at different levels in order to increase packing efficiency (Milner and Pyott, unpublished observations, see Ready et al. 1976 Fig. 10). This columnar epithelium is covered by the peripodial membrane, the cells of which are monolayered and cuboidal in appearance. It is of relatively uniform thickness (4-8 gm) in the region of the eye (Fig. 1 c) and of similar thickness in the medial region of the antenna (Fig. I d). The peripodial membrane overlying the lateral region of the antenna is thicker (Fig. I d).

When single eye-antennal discs are cultured in vitro with 0.1 lag/ml of 20-hydroxy ecdysone, they undergo a sequence of morphogenetic events identical to that observed in situ (Robertson 1936; Gottschewski 1960; Milner and Bleasby, unpublished observations). After 4-6 h of culture at 25 ~ C, the eye begins to curl dorsally (Fig. 2 a, b), a process which results in the formation of a vesicle in which the eye anlagen is curled over the everted antenna (Fig. 2c, d). During this process, the peripodial membrane may be seen to be tightly stretched between the anterior of the antenna and the distal region of the eye (Fig. 2 a). Serial sections showed that the surface area occupied by the peripodial membrane after curling had occurred (Fig. 2 d) was approximately one third of that found in late third instar discs (Fig. 1 b, c, d). Peripo- dial membrane was defined as tissue thinner than 10 gm. During curling, the cells of the peripodial membrane ap- peared healthy, and no evidence of necrosis was obtained. Cells at this stage were cuboidal, and basal cell extensions were evident (Fig. 3 a). The basement lamina was in all cases perfectly straight, and this contrasts with the situation in discs taken from wandering stage (late third instar) larvae where the basement lamina of the peripodial membrane cells was found to be wavy (Fig. 3 b).

There would appear to be two possible mechanisms for curling the eye over the antenna. Firstly, the reduction in surface area of the peripodial membrane during curling could actively pull the eye anlagen dorsally over the anten- na. Secondly, the active mechanism could reside in the col- umnar epithelium. Changes in cell shape and/or cell rear- rangement might actively initiate the dorsal movement of the eye, the reduction in the surface area of the peripodial membrane being concomitant with, but not causing, this morphogenetic event.

In order to distinguish between these two possibilities, eye-antennal discs at the stage of curling shown in Figs. 2 a and b were dissected from white or lightly tanned prepupae. A sharp tungsten needle was inserted into the lumen through a small tear so that it traversed the disc from the lateral to the medial side, at the level of the junction of eye and antennal anlagen. A single cut was then made across the disc, by means of a scissors action with a second needle. The cut was either through the peripodial membrane (single arrow, Fig. 2b, n = 29) or through the columnar epi- thelium at the eye-antennal junction (double arrow, Fig. 2 b, n = 37). It was thus possible to assess the effect of damaging separately each of the two sites which might be responsible for curling.

A

Fig. 1. a Dorsal aspect of a left eye-antennal disc from a larva shortly before metamorphosis. The eye (E) and antennal (AN) anlagen are marked, and the anterior-posterior (A and P) and medial-lateral (M and L) axes of the larva are indicated, b Longitu- dinal section through eye-antennal disc. c Transverse section through disc in the region of the eye. d Transverse section through antennal region of the disc. The peripodial membrane is thicker in the lateral region of the antenna (single arrow) than in the medial region (double arrow). PM= peripodial membrane. Bar represents t00 gm

Both operations were followed within seconds by an alteration in the shape of the curling disc, indicating that the tissue was under tension at this stage of morphogenesis. When the peripodial membrane was cut, the curling eye anlagen rapidly reverted to its original position. This re- sulted in a substantial gap opening between the cut edges of the peripodial membrane within 5 min of the operation (Fig. 4a). After 3 h of culture at 25 ~ C, the gape of the wound had increased, and the margins of the previously thin, uniform sheet of peripodial membrane cells had become columnar (Fig. 4b). In one region, folding of this tissue had also occurred (Fig. 4c). After one day of culture, ventral movement of the margins of the eye had completed the exposure of its apical surface to the culture medium, and eversion of the antenna had been completed.

A single cut through the columnar epithelium at the junction of the eye and antennal discs produced a different result. The eye anlagen moved dorsally immediately after

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a b

d

Fig. 2. a Eye-antennal disc during the curling stage of morphogenesis. Note taut peripodial membrane (arrow). b Longitudinal section through curling disc. Single arrow and double arrows indicate the position of cuts through the peripodial membrane and columnar epithelium respectively, the results of which are illustrated in Fig. 4. e Brightfield micrograph and d longitudinal section through an eye-antennal disc at the completion of the curling stage. The eye anlagen is folded over the everted antenna, and the peripodial membrane is considerably reduced in area (cf. Fig. lb). P M = peripodial membrane; E~ eye; A N = antenna. Bar represents 100 gm

the cut, and a small gap opened at the site of the wound (Fig. 4d). Little change in the relative positions of the eye and antennal anlagen was apparent after 3 h, and the wound had not opened further (Fig. 4e). Conversion of the marginal regions of the peripodial membrane into folded, columnar epithelium was again apparent (Fig. 4f). At this stage, the disc was very similar in appearance to an unoperated disc after curling had been completed (Fig. 2d). After one day of culture, the position of the eye anlagen was in most cases unchanged, the continuing eva- gination of the antenna increasing the gape of the wound to allow it exit from the lumen of the disc.

The possibility that the peripodial membrane ceils might persist and secrete cuticle was examined by serially section- ing single eye-antennal discs cultured for one or three days after cutting the columnar epithelium at the eye antennal junction. The cut allowed the antenna to evert, and served as a marker for the correct orientation of sections, as tissue connecting the eye and antennal anlagen after such an oper- ation must be derived from the peripodial membrane. Fol- lowing this operation, the peripodial membrane retained

the morphology shown in Fig. 4f, a fact which assisted its identification in sectioned material. A region of columnar cells was visible adjacent to the eye, and the central parts remained cuboidal and monolayered, simply folding in to- wards the disc lumen.

Cells derived from the peripodial membrane secreted first a pupal cuticle (Fig. 5a and b), and subsequently an imaginal cuticle bearing trichomes and bristles (Fig. 5c). This occurred both in folded columnar epithelium derived from the peripodial membrane, and from peripodial mem- brane cells which had retained their original cuboidal, monolayered appearance. Thus, cells of the peripodial membrane persist in development and contribute to im- aginal differentiation. A much thicker layer of imaginal cuticle was produced after seven days of culture, but as this became thicker, and as the underIying ceils degenerated, it became more difficult to recognise specific areas of the disc in sections.

We wished to determine whether the integrity of the peripodial membrane was required for 20-hydroxy ecdy- sone induced morphogenesis in other imaginal discs. The

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: ii!!!:!!i/i !ii i i �84 ii i !!!iii/ i i i iiiiiii! iii i!/ * Fig. 3. a Electron micrograph showing peripodial membrane cells from a disc in the process of curling, after 6 h of culture in vitro with 0.1 gg/ml of 20-hydroxy ecdysone. The cells are healthy in appearance, and possess basal processes (arrow heads), x 8,000. b The peripodial membrane from a wandering-stage third instar eye-antennal disc exhibits a wavy basement lamina (BL). x 8,000

peripodial membrane was dissected from ten first leg discs and ten wing discs, all of which subsequently underwent normal evagination. Thus, a requirement for an intact peri- podial membrane for the early stages of morphogenesis is not common to all imaginal discs.

Discussion

If the peripodial membrane remained unchanged during the curling of the eye over the antenna, it would be expected to "bag out" during the folding process, as the distance between the anterior of the antenna and the posterior of the eye is decreased. Such a phenomenon is not observed.

On the contrary, the area of the peripodial membrane has been found to decrease substantially during curling. A pos- sible mechanism for this contraction could be cell death in the peripodial membrane, as suggested by Poodry and Schneiderman (1970) to account for the elimination of the peripodial membrane during leg disc morphogenesis. If ne- crosis were responsible for the reduction in surface area, extensive cell death and basal cell extrusion should be ap- parent in the peripodial membrane during the curling process, and this is not found (Fig. 2b, d; Fig. 3a).

We wished to determine whether the motive force for the dorsal curling of the eye resides in the peripodial mem- brane, or in the underlying columnar epithelium. Cutting

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a

b e

C

Fig. 4. a A curling stage eye-antennal disc, 5 rain after a cut had been made through the peripodial membrane. The eye has moved ventrally, and a large gap has opened between the edges of the wound in the peripodial membrane, indicated by arrowheads, b Longitudinal section of (a), after 3 h of culture with 20-hydroxy ecdysone. The gape of the wound has increased (arrowheads), and the margins of the peripodial membrane have thickened, c As (b), sectioned at a different level. Here, the margin of the peripodial membrane can be seen to have become columnar and folded, while cells adjacent to the cut remain cuboidat. Ommatidial differentiation is apparent in the eye. d A curling stage eye-antennal disc, 5 min after a cut had been made through the columnar epithelium at the junction of the eye and antennal anlagen. The eye remains curled dorsally over the antenna. The wound, which has not opened substantially, is marked with an arrowhead, e Longitudinal section of (d), alter 3 h of culture. The relative positions of the antenna and eye anlagen remain unchanged, and the wound has not gaped (arrowhead). The peripodial membrane has become columnar and folded in the region adjacent to the eye. f Shows an enlargement of this region. PM=peripodial membrane; E=eye; AN=antenna. Bar represents 50 gm

the peripodial membrane during curling results in ventral movement of the eye as it returns to its original position, whereas cutting the underlying colmlanar epithelium allows the eye to accelerate its rate of dorsal curling. This strongly suggests that the reduction in the area of the peripodial membrane actively pulls the eye over the antenna.

Locke and Huie (1981) have investigated the decrease in area of Calpodes larval epidermis at the time of pupation. The application of 20-hydroxy ecdysone causes these epi- dermal cells to extend numerous basal cell processes, which subsequently contract at the same time as the decrease in epidermal cell area. Locke and Huie (1981) and Locke

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Fig. 5a--c, Cuticle secretion by eye-antennal discs, cut through the columnar epithelium at the eye-antenna[ junction, and cultured for one or three days with 1 gg/ml of 20-hydroxy ecdysone before fixation, a After one day of culture. Peripodial membrane derivatives can be seen to join eye and antennal anlagen, and are highly folded in this plane of section. The site of the cut through the epithelium is marked with an arrowhead. Bar represents 50 ktm. b Enlargement of (a) to show pupal cuticle secretion by peripodial membrane derivatives. Peripheral regions of columnar epithelium (X) and a fold of cuboidal cells from the more central region of the peripodial membrane (Y) are indicated. Bar represents 50 I-tm. c After three days of culture, cuboidal cells from the central region of the peripodial membrane have shed their pupal cuticle and secreted an imaginal cuticle. The folded peripodial membrane is similar in appearance to that seen in (lo). Bar represents 25 p-m. P M = peripodial membrane; E = eye; A N - antenna; PC = pupal cuticle; IC = imaginal cuticle bearing bristles and trichomes

(1981) suggest that the contraction of these processes causes an alteration in cell shape from cuboidal to columnar, in- creasing the packing efficiency of ceIIs and hence reducing the area of the epithelium. A similar mechanism may operate to reduce the surface area of the peripodial mem- brane, a possibility supported by the presence of basal ex- tensions in the peripodial membrane (Fig. 3). We suggest that alteration of cuboidal peripodial membrane cells into columnar epithelium begins at the edges, as cells from the central part of the peripodial membrane remain cuboidal even at an advanced stage of curling and during cuticle secretion. This process of alteration may well be slowed by the tension induced in the peripodial membrane by the curling process. Removal of this tension by destroying the integrity of the vesicle allows a speedy conversion of peripo- diaI membrane cells into columnar epithelium (Fig. 4). Pseudostratification and folding subsequently effect a fur- ther reduction in surface area.

Evagination of leg and wing discs was able to occur in vitro despite the removal of the peripodial membrane. This experiment does not demonstrate that the peripodial membrane plays no part in imaginal differentiation in these discs, nor that evagination in situ occurs via the rupture of the peripodial membrane. However, one may conclude that the cellular processes which result in the evagination of these discs do not require the presence of an intact peri- podial membrane, and differ in this respect from the early morphogenesis of the eye-antennal disc. The dorsal curling of the eye over the antenna is essential for the subsequent morphogenesis of the head, as it juxtaposes the appropriate regions of the two eye-antennal discs for fusion to occur. Cutting the peripodial membranes of a disc pair inhibits further morphogenesis, and fusion cannot take place (MiIner and BIeasby, unpunished observations).

The peripodial membrane may well contribute to the differentiation of imaginal cuticular structures in a number

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of systems (Sprey and Oldenhave 1974; K u m a r et al. 1979). Examinat ion of the Drosophila eye-antennal disc fate map suggests a similar contr ibut ion. Haynie (1975) notes that the anter ior- la teral por t ion of the antennal disc, containing the anlagen for the post-occipi ta l sensilla, must meet the poster ior-medial areas of the eye disc, which will produce the occipital bristles, as these two groups of structures lie close together on the insect head (see Bryant 1978, Figs. 2 and 5). Our observat ions indicate that this " m e e t i n g " is more apparen t than real, as the anlagen for these and other imaginal structures may well be located on the per ipodia l membrane, and therefore are not as widely separated as the fate map implies. This suggestion may resolve an appar- ent discrepancy between fate maps obta ined by Ouweneel (1970) and Haynie (1975), and the morphologica l da t a o f Ready et al. (1976). The fate maps show the facet-forming region occupying only the central par t of the eye anlagen, the per ipheral areas being concerned with the differentia- t ion of the ocelli and the bristles surrounding the eye. How- ever, morphologica l observat ions suggest that vir tually the whole of the columnar epithelium of the eye disc is involved in facet format ion (see Ready et al. 1976 Figs. 7, 10, 15). Fa te maps of this disc may be misleading, because investiga- tors have ignored the possibil i ty of imaginal cuticle secre- t ion by per ipodia l membrane derivatives.

Deak (1980) has suggested that per ipodia l membrane cells may possess posi t ional informat ion, and par t ic ipate in the regulat ion of f ragmented imaginal discs. Karpen and Schubiger (1981) repor t that the blastema of a regulat ing leg disc fragment acquires novel propert ies before wound healing is complete and suggest that interact ions between the disc epithelium and the per ipodia l membrane might be involved in init iat ing regulation. Thus, the per ipodia l mem- brane may be involved in all major aspects of imaginal disc biology - morphogenesis , imaginal differentiation, and pat tern regulation.

Acknowledgements. This work was supported by grants from the Science and Engineering Research Council and from the Royal Society. We wish to thank Dr, J.B. Tucker for a critical reading of the manuscript.

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Received December 12, 1982 Accepted in revised form February 14, 1983