Cuticle secretion in Drosophila wing imaginal discs in vitro: parameters of exposure to 20-hydroxy ecdysone

We have cultured Drosophila wing imaginal discs in vitro under a variety of hormonal conditions in order to determine whether cuticle secretion is enhanced by a withdrawal of 20-hydroxy ecdysone at one of two points in development, corresponding to the drop in hormone titer during the prepupal period, and to the fall in hormone levels during the later stages if imaginal differentiation. We found that these treatments did not enhance either pupal or adult cuticle secretion.     

The pupal cuticle of Drosophila: biphasic synthesis of pupal cuticle proteins in vivo and in vitro in response to 20-hydroxyecdysone

We investigated the synthesis and localization of Drosophila pupal cuticle proteins by immunochemical techniques using both a complex antiserum and monoclonal antibodies. A set of low molecular weight (15,000-25,000) pupal cuticle proteins are synthesized by the imaginal disk epithelium before pupation. After pupation, synthesis of the low molecular weight proteins ceases and a set of unrelated high molecular weight proteins (40,000-82,000) are synthesized and incorporated into the pupal cuticle. Ultrastructural changes in the cuticle deposited before and after pupation correlate with the switch in cuticle protein synthesis. A similar biphasic accumulation of low and high molecular weight pupal cuticle proteins is also seen in imaginal discs cultured in vitro. The low molecular weight pupal cuticle proteins accumulate in response to a pulse of the insect steroid hormone 20-hydroxyecdysone and begin to appear 6 h after the withdrawal of the hormone from the culture medium. The high molecular weight pupal cuticle proteins accumulate later in culture; a second pulse of hormone appears to be necessary for the accumulation of two of these proteins.     

In vitro differentiation ofPieris brassicae imaginal wing discs: Effects and metabolism of ecdysone and ecdysterone

Summary Imaginal wing discs ofPieris brassicae can be cultured in vitro for extended periods. Their ultrastructural development was investigated after culture in the presence of various concentrations of ecdysone and ecdysterone. When ecdysone or low concentrations of ecdysterone (2×10^–7 M) were added to culture media, larval discs secreted a pupal cuticle and they subsequently differentiated scales; prepupal discs completed their imaginal development. With a higher concentration of ecdysterone (2×10^–6 M), all discs produced abundant but fragmentary cuticular material.Prepupal discs were able to metabolize both hormones in vitro. Ecdysterone was mainly converted into a polar compound detectable after a short period of incubation. Ecdysone was transformed, at a slower rate, forming a polar compound and 26-hydroxyecdysone but no ecdysterone.     

Effects of a protein synthesis inhibitor on the hormonally mediated regression and death of motoneurons in the tobacco hornworm,Manduca sexta

The larval–pupal transformation of Manduca sexta is accompanied by the loss of the abdominal prolegs. The proleg muscles degenerate, the dendritic arbors of proleg motoneurons regress, and a subset of the proleg motoneurons dies. The regression and death of proleg motoneurons are triggered by the prepupal peak of ecdysteroids in the hemolymph. To investigate the possible involvement of protein synthesis in these events, we gave insects repeated injections of the protein synthesis inhibitor, cycloheximide (CHX), during the prepupal peak. Examination of insects 3–5 days following CHX treatment showed that CHX inhibited the death of proleg motoneurons and the production of pupal cuticle in a dose-dependent fashion. When insects were allowed to survive for 10 days after the final CHX injection, motoneuron death and pupal cuticle production sometimes occurred belatedly, apparently in response to the ecdysteroid rise that normally triggers adult development. CHX treatments that inhibited motoneuron death were less effective in inhibiting dendritic regression in the same neurons. In another set of experiments, abdomens were isolated from the ecdysteroid-secreting glands prior to the prepupal peak, and infused with 20-hydroxyecdysone (20-HE). Single injections of CHX delivered just prior to the start of the 20-HE infusion inhibited motoneuron death and pupal cuticle production, but in the range of doses tested, did not prevent dendritic regression. Our findings suggest that protein synthesis is a required step in the steroid-mediated death of proleg motoneurons, and that dendritic regression is less susceptible to inhibition by CHX than is motoneuron death. © 1993 John Wiley & Sons, Inc.     

Development of the prepupal Verson's gland of the tobacco hornworm, Manduca sexta, and its hormonal control

The segmentally arranged Verson's glands are epidermal derivatives comprised of three cells: the duct, saccule, and secretory cells. The development of these glands was followed through the 5th instar and larval-pupal transition of Manduca sexta. The glands are relatively small during the feeding stage, begin to grow at wandering, and undergo about a 50-fold increase in size during the prepupal period. The increase in size is due mainly to the hypertrophy of the secretory cell which synthesizes a heterogeneous set of proteinaceous secretory products. Three prominent 11 to 12 kiloDalton (kD) polypeptides are made by the pharate fifth larval gland, whereas the pupal gland produces polypeptides ranging from 14 to 75 kD with a major complex at 30 to 34 kD. The secretory product is poured out onto the surface of the new cuticle at the time of ecdysis and contains all of the major proteins detected in extracts of the whole gland. The accumulation of secretory products by the gland occurs during the prepupal peak of ecdysteroid and is blocked if this rise is prevented by abdominal isolation. Infusion of 30 micrograms 20-hydroxyecdysone (20-HE) into such isolated abdomens caused synthesis of the pupal products. Treatment with the juvenile hormone mimic, methoprene, during the fifth instar showed that the commitment of the glands to produce the pupal proteins is independent of and occurs before the overlying epidermis becomes committed to make pupal cuticle.     

Distribution of cuticular protein mRNAs in silk moth integument and imaginal discs

The distributions of mRNAs for two cuticular proteins of Hyalophora cecropia were examined with RT-PCR and in situ hybridization. For major regions of larval and pupal cuticle, there was a strong correspondence between the type of cuticle and the predominant cuticular protein message found. Epidermal cells underlying soft cuticle had mRNA for HCCP12, with a RR-1 consensus attributed to soft cuticle, while the epidermal cells associated with hard cuticle had predominantly mRNA for HCCP66, a protein with the RR-2 consensus attributed to hard cuticle. Both messages were found in all areas of the pupal fore- and hind-wings, with modest area-specific difference in concentration being much less than differences in the relative abundance of these cuticular proteins.

mRNA for HCCP12 was present in imaginal discs of feeding larvae of H cecropia. Data from Bombyx mori available at SilkBase (http://www.ab.a.u-tokyo.ac.jp/silkbase/) revealed that imaginal discs from feeding larvae had abundant mRNA for RR-1 cuticular proteins, representing six distinct gene products. Only discs from spinning larvae had mRNAs that coded for RR-2 proteins arising from 10 distinct genes. Thus, lepidopteran wing imaginal discs can no longer be regarded as inactive in larval cuticle production.     

Commencement of pupal commitment in late penultimate instar and its hormonal control in wing imaginal discs of the silkworm, Bombyx mori

Pupal commitment of the wing imaginal disc of the silkworm, Bombyx mori, is completed shortly after the final (fifth) larval ecdysis. Pupal commitment was induced by in vitro culture with 20-hydroxyecdysone (20E). Shortly after the head capsule slippage (HCS) that occurs approximately 24 h before the final larval ecdysis, the discs become competent to respond to 20E, indicating that the process of pupal commitment begins in the late penultimate (fourth) instar. The simultaneous presence of methoprene (JHA) with 20E suppressed the pupal commitment at 4 ng/ml for the discs at 12 h after HCS and at 240 ng/ml for the discs at the ecdysis. Thus, the discs rapidly lose their sensitivity to JH at the end of the fourth instar. Day 0 fourth wing discs were not pupally committed by 20E when freshly dissected discs were exposed to 20E. By contrast, exposure to 20E after a pre-culture in a hormone free medium induced the pupal commitment. In those discs, the effective JHA concentration to suppress the 20E effects was 0.1 ng/ml. The present data suggest that pupal commitment proceeds through two stages from a reversible state that begins at around HCS to an irreversible state early in the fifth instar. The loss of sensitivity to JH is the primary impetus to begin the process and 20E is the factor that drives the discs to enter the reversible state.     

A screen for genes expressed in Drosophila imaginal discs

The development of Drosophila imaginal discs serves as a model system to understand how genes determine the shape and size of an organ. The identification of genes involved in this process is an important step towards this goal. Here we describe a P-element based enhancer trap screen for genes expressed in the larval imaginal discs. Our aim was to establish a large collection of enhancer trap lines each showing expression of Gal4 in imaginal discs. To this end, we improved the well established P-element vector pGawB in order to obtain higher in vivo transposition frequencies. In addition we chose an F1-screening approach using UAS-GFP as a reporter gene. This system permits the efficient screening of larval and pupal stages of living animals and the detection of imaginal gene expression patterns through the transparent cuticle. The procedure has been optimized for high-throughput. 2'000 P-element insertions have been established which exhibit expression in imaginal discs.     

The role of nutrition in creation of the eye imaginal disc and initiation of metamorphosis in Manduca sexta

With the exception of the wing imaginal discs, the imaginal discs of Manduca sexta are not formed until early in the final larval instar. An early step in the development of these late-forming imaginal discs from the imaginal primordia appears to be an irreversible commitment to form pupal cuticle at the next molt. Similar to pupal commitment in other tissues at later stages, activation of broad expression is correlated with pupal commitment in the adult eye primordia. Feeding is required during the final larval instar for activation of broad expression in the eye primordia, and dietary sugar is the specific nutritional cue required. Dietary protein is also necessary during this time to initiate the proliferative program and growth of the eye imaginal disc. Although the hemolymph titer of juvenile hormone normally decreases to low levels early in the final larval instar, eye disc development begins even if the juvenile hormone titer is artificially maintained at high levels. Instead, creation of the late-forming imaginal discs in Manduca appears to be controlled by unidentified endocrine factors whose activation is regulated by the nutritional state of the animal.     

The cell biology of Drosophila wing metamorphosis in vitro

Summary We have examined the metamorphosis of the wing imaginal disc of Drosophila during culture in vitro in the continuous presence of 20-hydroxy ecdysone (0.1 g/ ml). We find that the sequence of cellular changes in the wing blade during culture closely match those occurring in situ, involving two periods at which the dorsal and ventral surfaces are joined only by cell processes containing trans-alar microtubule arrays. Good pupal and imaginal cuticle secretion is found in this system.     

Changes in translatable mRNAs during the larval-pupal transformation of the epidermis of the tobacco hornworm

At the initiation of metamorphosis when exposed to ecdysteroid in the absence of juvenile hormone (JH), the lepidopteran epidermis changes its commitment from one for larval differentiation to one for pupal differentiation. Changes in mRNA populations during this change both in vivo and in vitro were followed by a one-dimensional SDS-gel electrophoretic analysis of translation products made in a mRNA-dependent rabbit reticulocyte lysate system. The larval epidermal cell was found to lose its translatable mRNAs for larval cuticular proteins and the larval-specific pigment insecticyanin during the change in commitment; these never reappeared. For Class I cuticular proteins and for insecticyanin, this loss occurred during the exposure to ecdysteroid, each with a differing time course. By contrast, Class II cuticular mRNAs first increased during this time, then also disappeared by the time the cells were pupally committed. In vitro these mRNAs appeared in only trace amounts in response to 20-hydroxyecdysone (20-HE). The pupally committed cell (late in the wandering stage) contained mRNAs for three low-molecular-weight proteins which were precipitable with the pupal cuticular antiserum. The remainder of the pupal cuticular mRNAs were not translatable until the third day after wandering, a time when pupal cuticle is being deposited in response to a molting surge of ecdysteroid. The pupally committed cell also had at least one new noncuticular mRNA which coded for a 34K protein and which was absent from both larval and pupal epidermal cells making cuticle. Since its appearance in response to 20-HE in vitro is repressed by JH, it is called a pupal commitment-specific protein. Thus, during the change of commitment 20-HE inactivates larval-specific genes irreversibly in a sequential cascade of events. The activation of most pupal-specific genes then requires a subsequent exposure to more ecdysteroid.     

The formation of the pupal cuticle by Drosophila imaginal discs in vitro

Mass-isolated imaginal discs of Drosophila melanogaster form a chitin-containing pupal procuticle In vitro. Optimal procuticle deposition occurs when the discs are incubated for 4–6 hr with 0.5–1.0 μg/ml of 20-hydroxyecdysone and then with less than 0.05 μg/ml of 20-hydroxyecdysone. The formation of the chitin-containing procuticle is demonstrated using three independent assays: with fluorescene-conjugated cuticle proteins that bind to chitin; by electron microscopy; by incorporation of [³H]glucosamine into a chitin fraction. Synthesis and deposition of pupal cuticle proteins are also demonstrated. Incorporation of [³H]glucosamine into chitin is sensitive to inhibitors of protein, RNA and chitin synthesis, but has little sensitivity to inhibitors of DNA synthesis, and dolichol-dependent glycosylation.     

Synthesis of the major adult cuticle proteins of Drosophila melanogaster during hypoderm differentiation

Differentiating imaginal hypodermal cells of Drosophila melanogaster form adult cuticle during the second half of the pupal stage (about 40 to 93 hr postpupariation). A group of proteins with molecular weights of 23,000, 20,000, and 14,000 is identified as putative major wing cuticle proteins with the following biological properties: These proteins are abundant components of cuticle and are major synthetic products of cuticle-secreting hypodermal cells. They are leucine-rich and methionine-free and are the most prominent proteins of this type synthesized by wing hypoderm at 65 hr, during the period of procuticle formation. Electron microscopic autoradiography shows that leucine-rich, methionine-free proteins specifically localize to the apical cell surface and newly secreted cuticle of 65-hr wing cells. This strongly suggests the export of these proteins to the cuticle. Lastly, these proteins undergo a reduction in extractability just after eclosion, during the period of cuticle protein crosslinking (sclerotization). The synthesis of these major hypoderm proteins is temporally regulated in development. In wing cells, the 14-kDa proteins are synthesized first, from 53 to 78 hr, and the 20- and 23-kDa proteins are synthesized from 63 to 93 hr. The pattern of synthesis for these proteins is similar in abdominal cells but delayed by 6 to 10 hr. Two-dimensional gel electrophoresis shows that each of the 23-, 20-, and 14-kDa size classes contains at least two component polypeptides. Patterns of protein synthesis in cells of the imaginal hypodermis are regulated in a precise temporal sequence during the production of adult cuticle. Their study yields a useful system for the analysis of molecular events in gene control and cell differentiation.