Effects of alpha-ecdysone, beta-ecdysone and inokosterone on the in vitro evagination of Drosophila leg discs and the subsequent differentiation of imaginal integumentary structures

The morphogenetic activity of three hormonal substances—α-ecdysone, β-ecdysone, and inokosterone—has been studied in vitro on isolated imaginal leg discs of third-instar larvae of Drosophila melanogaster.In the presence of α-ecdysone (0.3–3 μg/ml) and also of the phytohormone inokosterone (0.3–3 μg/ml), the discs underwent metamorphosis, as characterized by complete evagination (in less than 24 hr), secretion, and shedding (48 hr after explanation) of the pupal cuticle, secretion, and structural differentiation of the imaginal cuticle, namely pigmentation and formation of claws, bristles, and hairs (during days 3–6).In the presence of β-ecdysone (10, 6, 3, 0.3, 0.03, 0.003 μg/ml), evagination was always abnormal and incomplete. With all concentrations but the lowest, the partially everted legs had a swollen appearance and, at all concentrations, the subsequent development was inhibited. No imaginal differentiation occurred at any of the concentrations tested.Larval fat body or larval epidermis added to the isolated discs had no influence on their response to either α-ecdysone or β-ecdysone.Changing the osmotic pressure of the β-ecdysone containing medium likewise did not alter the noxious effect of β-ecdysone.Discs cultured first in the presence of β-ecdysone (for 24 hr), then transferred to fresh medium containing α-ecdysone were unable to undergo normal development. The inhibitory effect of β-ecdysone thus appears to be irreversible.Discs cultured first in the presence of α-ecdysone (for 24, 48 or 72 hr), then transferred to β-ecdysone containing medium, were unable to continue their normal differentiation. Further development was blocked within a few hours after the transfer.Results are discussed in view of results obtained with other in vitro and in vivo cultivation techniques. In conclusion, isolated leg discs of Drosophila are unable to respond physiologically to exogenous β-ecdysone. Only α-ecdysone and inokosterone will induce complete and normal metamorphosis in leg discs cultured in vitro.     

Hormonal control of growth and differentiation of insect tissues cultured in vitro

Summary This paper reviews the effects of insect hormones on lepidopteran imaginal discs cultured in vitro.β-ecdysone stimulated both evagination and cuticle deposition of wing discs ofPlodia interpunctella (Hübner). However, evagination required a shorter exposure to ecdysone than did cuticle deposition. Cuticle deposition was obtained under the following conditions: (a) a 24-hr pulse ofβ-ecdysone (0.5–5.0μg/ml); (b) continuous treatment with 0.2μg/mlβ-ecdysone; or (c) continuous treatment with 0.5 to 50.0μg/mlβ-ecdysone in medium conditioned with larval fat body. Investigations of some biochemical effects of ecdysone showed that RNA and protein synthesis was required for evagination and cuticle deposition. In particular, studies with actinomycin D and cycloheximide (at nontoxic levels) showed that RNA and protein synthesis during the ecdysone-dependent period was essential for subsequent development. These findings support the hypothesis that stimulation of macromolecular synthesis is fundamental to the action of ecdysone on imaginal discs. The influence of beta-ecdysone on chitin synthesis was also examined.β-ecdysone stimulated uptake and incorporation of tritiated-glucosamine by culturedP. interpunctella wing discs. Addition of hexosamines to the culture medium had no influence on ecdysone-induced cuticle deposition, but inhibition of glucose-uptake by cytochalasin B prevented the formation of cuticle. The action of ecdysone on particular enzymes in the chitin pathway remains to be elucidated. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Effect of cytochalasin B on the evagination in vitro of leg imaginal discs

Cytochalasin B (1 μg/ml) completely inhibited the evagination of isolated leg imaginal discs cultured in vitro in a synthetic medium (ME) containing α-ecdysone (3 μg/ml). In discs precultured for 6 hr in medium ME without the drug, then transferred to cytochalasin B-containing medium, continuation of evagination was stopped immediately. The inhibition of evagination was completely reversible, provided pretreatment with cytochalasin B did not exceed 8 hr. Results are discussed in view of what is known on the effect of cytochalasin B on other developmental systems. Findings are compatible with the primary action of the drug being an alteration of cell surface properties, thus bringing to light the importance of these properties in the course of normal imaginal disc evagination.     

RNA synthesis in imaginal disks of Galleria mellonella: Effects of alpha- and beta-ecdysone and fat body in vitro

The effects of alpha-ecdysone (α-E), beta-ecdysone (β-E), and larval fat body on morphogenesis and total RNA synthesis in wing imaginal disks of Galleria mellonella were studied. Both ecdysones induce morphogenesis of disks in vitro. Alpha-ecdysone and β-E (0·3–3·0 μg/ml) stimulate RNA synthesis 30 and 60 per cent above control levels, respectively. While less α-E (0·3 μg/ml) is required to increase RNA synthesis than to induce morphogenesis, the reverse is true for β-E. Morphogenesis (i.e. tracheole migration and evagination) can proceed in the presence of concentrations of β-E (0·03 μg/ml) that are subthreshold for the induction of RNA synthesis (0·3 μg/ml). We conclude, therefore, that the increase in total RNA (presumbly ribosomal) is unrelated to and not a prerequisite for tracheole migration or evagination. If morphogenically active preconditioned medium (i.e. medium in which α-E and fat body have been incubated for 48 hr and the fat body then removed) is added to disk cultures, RNA synthesis is not stimulated. Apparently, increases in total RNA caused by both ecdysones may not be necessary for early in vitro disk development. The independent nature of some ecdysone-induced events and implications of our conclusions are discussed.     

The appearance of dopa decarboxylase activity in imaginal discs of Sarcophaga bullata, undergoing development in vitro

During the postembryonic development of Sarcophaga bullata, two large peaks of dopa decarboxylase activity were observed. These were associated with the sclerotization (hardening) of the puparium and the adult cuticle, respectively. A small peak of activity 5.5–6.5 days after pupariation was possibly associated with the sclerotization of the prothoracic spiracles.A premature increase in enzyme activity was observed in young, third-instar larvae injected with 20 μg of β-ecdysone. However, the advantage of studying the effect of the hormone on enzyme activity in vitro led to an attempt to induce² dopa decarboxylase in cultured wing discs.In the presence of β-ecdysone, wing discs underwent evagination and a substantial increase in dopa decarboxylase activity was observed in these discs. The enzyme activity began to appear after the rupture of the peripodial membrane and reached a maximum about the time disc evagination ceased. We suggest that this enzyme activity was responsible for the slight sclerotization of a fine cuticle secreted by the discs. The cultured imaginal discs underwent changes that are very similar to those which occur in intact animals. Therefore, this system appears promising for further studies on the role in differentiation of the hormonal control of enzyme activity.     

The secretion and metabolism of alpha-ecdysone by cockroach (Leucophaea maderae) tissues in vitro

Hemolymph β-ecdysone levels are high (～1.6 μg/ml) in late last instar cockroach ($\text{Leucophaea}$$\text{maderae}$) nymphs; the level of α-ecdysone (～0.1 μg/ml) is evidently subphysiological. Cultured leg regenerates, target organs of ecdysone, are capable of slowly converting α- to β-ecdysone. Cultured prothoracic glands secrete α-ecdysone, which was identified by complete mass spectrometry. These results are consistent with the view that α-ecdysone, secreted by the prothoracic gland, functions as a prohormone which is converted into the active moulting hormone, β-ecdysone, in other tissues.     

Metamorphic shortening of interganglionic connectives of Galleria mellonella (Lepidoptera) in vitro: stimulation by ecdysone analogues

In the absence of other organ systems, β-ecdysone (0·05 to 0·10 μg/ml culture medium) stimulates the shortening of interganglionic connectives of Galleria mellonella that occurs during metamorphosis. There is a direct relations-ship between the amount of β-ecdysone in the medium and the fraction of the sample shortening to at least half the initial length. β-Ecdysone is ～ 140 × more active than α-ecdysone in eliciting the response. When β-ecdysone and other ecdysone analogues are assayed on this system at uniform dosages (10 μg/ml tissue culture medium), the order of effectiveness (percentage sample shortening to at least half the initial length) is: cyasterone > ponasterone-A = β-ecdysone > inokosterone > α-ecdysone. 22-Iso-α-ecdysone is ineffective in stimulating shortening.     

Hormonal control of growth and differentiation of insect tissues cultured in vitro.

This paper reviews the effects of insect hormones on lepidopteran imaginal discs cultured in vitro. Beta-ecdysone showed that RNA and protein synthesis was required for evagination and cuticle deposition. In particular, studies with actinomycin D and cycloheximide (at nontoxic levels) showed that RNA and protein synthesis during the ecdysone-dependent period was essential for subsequent development. These findings support the hypothesis that stimulation of macromolecular synthesis is fundamental to the action of ecdysone on imaginal discs. The influence of beta-ecdysone on chitin synthesis was also examined. Beta-ecdysone stimulated uptake and incorporation of tritiated-glucosamine by cultured P.interpunctella wing discs. Addition of hexosamines to the culture medium had no influence on ecdysone-induced cuticle deposition, but inhibition of glucose-uptake by cytochalasin B prevented the formation of cuticle. The action of ecdysone on particular enzymes in the chitin pathway remains to be elucidated.timulated both evatination and cuticle deposition of wing discs of Plodia interpunctella(Hubner). However, evagination required a shorter exposure to ecdysone than did cuticle deposition. Cuticle deposition was obtained under the following conditions: (a) a 24-hour pulse of beta-ecdysone (0.5-5.0 mug/ml); (b) continuous treatment with 0.2 mug/ml beta-ecdysone; or (c) continuous treatment with 0.5 to 50.0 mug/ml beta-ecdysone in medium conditioned with larval fat body...     

Hormonal requirements for the larval-pupal transformation of the epidermis of Manduca sexta in vitro

In the tobacco hornworm, Manduca sexta, metamorphosis occurs in response to two releases of ecdysone that occur 2 days apart. Epidermis was explanted from feeding final-instar larvae before the first release of ecdysone and was cultured in Grace's medium. When exposed to 1 μg/ml of β-ecdysone for 24 hr and then to hormone-free medium for 24 hr, followed by 5 μg/ml of β-ecdysone for 4 days, the epidermis produced tanned pupal cuticle in vitro. During the first 24 hr of exposure to β-ecdysone, the epidermis first changed its cellular commitment to that for pupal cuticle formation (ET₅₀ = 14 hr), then later (by 22 hr) it became committed to tan that cuticle. Then, for most of the pupal cuticle to be tanned, at least a 12-hr period of culture in hormone-free medium was required before the cuticle synthesis was initiated. Consequently, some events prerequisite to sclerotization of pupal cuticle not only occur during the ecdysone-induced change in commitment but also during the ecdysone-free period. When the tissue was preincubated in 3 μg/ml of juvenile hormone (JH I or a mimic epoxygeranylsesamole) for 3 hr and then exposed to both ecdysone and juvenile hormone for 24 hr, it subsequently formed larval cuticle. The optimal conditions for this larval cuticle formation were exposure to 5 μg/ml of β-ecdysone in the presence of 3 μg/ml of epoxygeranylsesamole for 48 hr. When the epidermis was cultured in Grace's medium for 3 days and then exposed to 5 μg/ml of β-ecdysone for 4 days, 70% of the pieces formed pupal cuticle. By contrast, if both ecdysone and JH were added, 77% formed larval cuticle. Therefore, the change from larval to pupal commitment of the epidermal cells requires not only the absence of JH, but also exposure to ecdysone.     

Effects of beta-ecdysone and juvenile hormone on the Na+/K+-dependent ATPase in imaginal disks of Drosophila melanogaster.

The evagination of imaginal disks of Drosophila melanogaster is induced in vitro by β-ecdysone and inhibited by juvenile hormone. The possibility that these hormones act by changing intracellular Na⁺ and K⁺ levels was investigated by studying their effects on the sodium-potassium dependent adenosinetriphosphatase (NaK ATPase), an enzyme with a major rôle in regulating Na⁺ and K⁺ levels in cells. We find that β-ecdysone has no effect on this enzyme and can induce evagination even when intracellular Na⁺ concentrations are increased 2 to 3 fold by ouabain. Juvenile hormone stimulates the enzyme, but still acts to inhibit evagination when NaK ATPase activity is inhibited by ouabain. We conclude that the actions of β-ecdysone and juvenile hormone on imaginal disk evagination do not directly involve the NaK ATPase or require specific changes in Na⁺ and K⁺ concentrations.     

Pupal cuticle formation by Manduca sexta epidermis in vitro: Patterns of ecdysone sensitivity

During the larval-pupal transformation, various regions of the epidermis of Manduca sexta larvae have previously been found to require different lengths of exposure to the prothoracic glands in order to form pupal cuticle. To distinguish between requirements for differing threshold concentrations of ecdysone and those for differing durations of exposure to ecdysone, wandering stage larval epidermis was cultured in Grace's medium. When most of the thick larval cuticle was removed, the epidermis responded to concentrations of β-ecdysone of 1.0 μ/ml or greater for 4 days by forming cysts which later formed tanned pupal cuticle. No fat body or protein supplement was required. When the larval integument was explanted intact, similar requirements for cuticle formation and for tanning were found. All regions of the fifth abdominal segment required similar concentrations of β-ecdysone (0.4–0.6 μg/ml) for 4 days for 50% to form pupal cuticle, but gin trap epidermis required the least exposure to a threshold concentration of ecdysone (1.5 days in 0.9 μg/ml). The anterior dorsal intersegmental region required about 0.5 day longer, followed by the posterior intersegmental and the dorsal intrasegmental regions. Thus, the duration of exposure seemed more important. About 1 day longer of exposure to ecdysone was required for subsequent tanning of the new cuticle than for cuticle formation, yet tanning of the cuticle did not occur with prolonged exposure to ecdysone.     

Selective activation of RNA polymerase I in fat body nuclei of Sarcophaga peregrina larvae by beta-ecdysone.

RNA synthesis in fat body nuclei of Sarcophaga peregrina larvae was temporarily activated after injection of β-ecdysone: increased synthesis was detectable 2 hr after injecting the hormone and lasted for at least 2 hr. This increased RNA synthesis was insensitive to α-amanitin and was observed in KCl-free reaction mixture, indicating that β-ecdysone activated RNA polymerase I but not RNA polymerase II. No activation was observed when protein synthesis was inhibited by cycloheximide, suggesting that protein synthesis was essential for the activation of the nuclei.     

Nascent glycoproteins with a chitin-like carbohydrate component in Drosophila melanogaster imaginal discs

The incorporation of [1-³H]d-glucosamine in Drosophila melanogaster imaginal discs revealed the synthesis of glycoproteins represented by a family of subfractions with roughly the same molecular mass of about 80,000 and discrete isoelectric point values in the range of 5.0 to 6.5 pH units. The incorporation of [1-³H]d-glucosamine was not inhibited by tunicamycin, an inhibitor of N-glycosylation. This family of glycoproteins is relatively protease-resistant but can be digested by high concentrations of pronase E (100 μg/ml) or pepsin (1 mg/ml). The carbohydrate component of these glycoproteins is sensitive to chitinase. The properties of the glycoproteins in imaginal discs are similar to those of chitinase sensitive glycoproteins found in established cell cultures of D. melanogaster [Kramerov et al., Insect Biochem. 16, 417–432 (1986)]. Incorporation of [1-³H]d-glucosamine into the family of glycoproteins decreases as the imaginal discs undergo evagination induced by 20-hydroxyecdysone.     

Non-linear relationship between oxygen saturation and proton release, and equivalence of the Bohr and Haldane coefficients in human hemoglobin.

The effects of cordycepin on RNA metabolism in plants are studied at various drug concentrations (5–200 μg/ml) as a function of time. It is shown that high concentrations totally inhibit RNA synthesis. At lower concentrations rRNA and tRNA syntheses are selectively severely depressed. The drug appears to have no direct effect on in vivo protein synthesis at any of the studied doses. Thus high concentrations of the drug can be used to get informations concerning mRNA stability by measuring in vivo protein synthesis: some plant mRNA are stable for more than 7 hrs.     

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.     

Time studies of ecdysone-action on in vitro apolysis of Chilo suppressalis integument

The relationship between induction of in vitro apolysis and the duration of hormone treatment, and the effects of metabolic inhibitors on the ecdysone-induced apolysis were investigated in the cultured integument taken from the rice stem borer larva, Chilo suppressalis. When fragments of integument were subjected to 0.3 μg/ml β-ecdysone for more than 5 hr and then transferred to hormone free medium, they were induced to apolyse one day after treatment. If the fragments of integument were treated with hormone for 1 to 4 hr at first and then treated with hormone for 2 to 5 hr again after a 5 day interval in hormone free medium, almost all the fragments were induced to apolyse one day after treatment. This result suggests that the action of β-ecdysone on the cultured integument is accumulative. If the fragments of integument were cultured in the medium containing actinomycin-D and then transferred to medium containing β-ecdysone, a strong inhibitory effect on the apolysis of the integument was observed. Similarly, an inhibitory effect appeared when fragments of integument were treated first with hormone and then with puromycin. These results show that the m-RNA synthesis necessary for apolysis was completed within 6 hr after hormone treatment. However, the protein synthesis required for apolysis was not. The relationship of the results obtained from these in vitro experiments to the mode of action of ecdysone is discussed.     

Mitotic responses of the anterior pericardial wall of Biomphalaria glabrata (Mollusca) subjected to challenge

Using light microscope autoradiography and electron microscopy we studied the effect of juvenile hormone III (JHIII) and β-ecdysone insect molting hormone (β-ecd) on the replication of Tipula iridescent virus (TIV) in suspension cultured cells of Estigmene acrea. JHIII at a concentration of 87.5 μg/ml completely inhibited viral DNA synthesis, but upon removal of JHIII, [³H]thymidine was incorporated into the cytoplasm as detected by autoradiography and virions in developmental stages from the same cell samples were-readily seen by electron microscopy. β-ecd at a concentration of 17.5 μg/ml, unlike JHIII, permitted viral DNA synthesis in the presence of the hormone although at a reduced level when compared to TIV-infected cells. But the presence of β-ecd seemed to prevent capsid formation, although islands similar in fine structure to those of viroplastic centers were seen by electron microscopy. Once β-ecd was removed from the medium, TIV-inoculated cells appeared to synthesize new virions in a normal pattern. Both hormones inhibited host cell DNA synthesis in noninfected cells.     

The assay of ecdysones and juvenile hormones on Drosophila imaginal disks in vitro

Evagination of mass-isolated imaginal disks of Drosophila melanogaster is induced in vitro by ecdysones (α- and β-ecdysone, inokosterone, cyasterone, rubrosterone) and inhibited by juvenile hormones (Law's compound, C₁₇ and C₁₈ Cecropia hormones, farnesol). It was found that disks do not metabolize α- or β-ecdysone. A concentration of β-ecdysone of 0·1 μg/ml or of α-ecdysone of 20 μg/ml was needed to induce complete evagination. Thus, β-ecdysone is about two hundred times more active than α-ecdysone. It is suggested that β-ecdysone is responsible for in situ moulting hormone activity in Drosophila.     

The time during which beta-ecdysone is required for the differentiation in vitro and in situ of wing imaginal discs of Drosophila melanogaster.

The time during which β-ecdysone is required for the apolysis and imaginal differentiation of wing discs of Drosophila both in vitro and in situ has been examined, and it is concluded that β-ecdysone is required as a sustained stimulus rather than as a trigger for differentiation. These results are compared with the requirement for β-ecdysone for the puffing of salivary gland polytene chromosomes during the prepupal stage (Richards, G. P., 1976, Develop. Biol.48, 191–195). It is suggested that imaginal discs and larval salivary glands require different exposures to β-ecdysone to fulfill their developmental commitments and that the drop in β-ecdysone titer during the early prepupal stage, which is necessary for the subsequent puffing of the polytene chromosomes, plays little or no part in imaginal disc differentiation.