Relative activities of alpha-ecdysone and beta-ecdysone for the differentiation in vitro of Drosophila melanogaster imaginal discs

Both α- and β-ecdysone cause explanted Drosophila prothoracic leg and wing discs to evert and differentiate when added to culture media. But the amount of α-ecdysone needed is about 100 times greater than the amount of β-ecdysone. As well as the expected complement of exoskeletal structures, the differentiated discs may show muscular movement. Low doses of both hormones cause partial eversion, without differentiation: Intermediate quantities cause partial eversion and differentiation.     

Patterns of protein synthesis in imaginal discs of Drosophila melanogaster.

Patterns of polypeptide synthesis in wing, leg and eye-antenna imaginal discs and in whole larvae of wild-type and and mutant Drosophila melanogaster have been examined using two-dimensional polyacrylamide gel electrophoresis and autoradiography. After 2 hr of labeling with 35S during the third larval instar, the synthesis of more than 318 polypeptides has been detected in imaginal discs. Of these, 268 are present in similar amounts in all three disc types. The remaining polypeptides detected in the three imaginal disc types fall into two categories: those unique to a particular disc type, and those specific for a particular pair of disc types. These results are discussed in relation to the spectrum of gene expression in imaginal discs.     

Transdetermination of Drosophila imaginal discs cultured in vitro

Imaginal discs of Drosophila melanogaster undergo transdetermination when cultured in vivo in the abdominal cavity of adult female hosts. We report here that leg discs cultured in vitro, in a recently developed system, also undergo transdetermination. Whether cultured in vivo or in vitro, leg discs produce a similar range of specific transdetermined structures. Moreover, in comparison to discs cultured in vivo, the discs cultured in vitro exhibit a similar correlation between the amount of growth and the total frequency of transdetermination.     

Ecdysone-stimulated RNA synthesis in imaginal discs of Drosophila melanogaster

Cytoplasmic RNA from imaginal discs of Drosophila melanogaster, labeled by uridine incorporation in organ culture, has been assayed by hybridization to cytological preparations of polytene chromosomes. RNA labeled during the early stages (first four hours) of ecdysone stimulation was compared to RNA labeled in the absence of the hormone. For the poly(A)-containing fraction (oligo-dT bound), several loci hybridize only RNA labeled in the presence of ecdysone; one locus hybridizes only control RNA. The majority of hybridizing loci are unaffected by the hormone. Of the loci hybridizing RNA not bound to oligo-dT, several appear specific for the ecdysone-treated sample, though most are labeled more heavily with this RNA than with the control. None of the ecdysone-sensitive loci visualized by in situ hybridization are the sites of salivary gland puffs induced by ecdysone on the same time scale.     

The in vitro synthesis and characteristics of ribosomal RNA in imaginal discs of Drosophila melanogaster

Summary Late third instar imaginal discs of Drosophila melanogaster cultured in vitro in Robb's tissue culture medium synthesize 38S, 28S and 18S ribosomal RNAs which are qualitatively indistinguishable from their in vivo synthesized counterparts (Fig. 1). As found in other insect systems, the 38S molecule appears to be the precursor for both the 28S and 18S rRNAs (Figs. 2, 3 and 4). The 28S rRNA and a portion of the 38S pre-rRNA shift in sedimentation value upon exposure to heat or dimethylsulfoxide (Figs. 5 and 8). Studies of the thermal denaturations of these molecules (Figs. 6, 7 and 9) indicate the existence of a single class of 28S rRNA, but three classes of 38S pre-rRNAs. The addition of -ecdysone to the in vitro culture medium stimulates the net amount of rRNA synthesized, increases the rate of processing of the 38S precursor and increases the relative amount of 18S material produced (Figs. 10 and 12).This work was supported in part by grants from the National Science Foundation (GB-8176) and from the Atomic Energy Commission (AT-04-3-34).Predoctoral Trainees, PHS Training Grant No. 2-Tl-GM367 from Research Training Grants Branch, National Institute of General Medical Sciences.1 For purposes of simplification we shall refer to the rRNA molecules of D. melanogaster as being 38S, 30S, 28S and 18S; however, it should be noted that these values are approximate (see Hastings and Kirby, 1966; Greenberg, 1969; Tartof and Perry, 1970).     

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.     

Metamorphosis of imaginal discs of Drosophila melanogaster

Summary Imaginal discs ofDrosophila melanogaster larvae, 24–53 hrs after oviposition, were transplanted into mature immobile larval hosts. The transplants did not respond to the hormonal stimuli of metamorphosis, but instead completed their larval development. When reinjected into mature larval hosts, they now differentiated the full set of their presumptive imaginal structures. The process of acquiring competence for metamorphosis appears to be independent of the hormonal conditions.Supported by a credit of the Swiss National Foundation granted to Prof. Dr. E. Hadorn. I thank Dr. R. Nöthiger for his valuable criticism during this investigation.     

A comparison of protein synthesis in imaginal wing discs of wild-type and vestigial of Drosophila melanogaster

In vitro incorporation of labelled amino acids into TCA insoluble protein in the imaginal wing discs of vg mutant and wild-type Drosophila melanogaster is compared and correlated with the extent of cellular degeneration during development. (1) With 96–100-hr-old larvae, wing discs isolated from vg mutant incorporate radiolabelled amino acids at a higher rate than wild-type discs, although cellular degeneration is more extensive in vg discs than wild-type discs. This may reflect the larger size of the vg discs since they have more protein than the wild-type discs. (2) The vg discs of 105–110-hr-old larvae have a lower rate of incorporation than the wild-type disc, although both types of discs have the same protein content. This may be due to the presence of more degenerating cells in the vg discs. (3) The rate of incorporation in vg discs of 115–118-hr-old larvae is lower than that of wild-type discs; the vg discs have less protein but more degenerative cells than the wild-type discs. The rates of protein degradation are similar in both discs. Electrophoretic comparison reveals a general reduction in protein synthesis in vg discs as compared with wild-type discs.     

Patterns of protein synthesis in the imaginal discs of Drosophila melanogaster: a comparison between different discs and stages

High-resolution two dimensional gel electrophoresis has been used to study the patterns of protein synthesis in imaginal discs of Drosophila melanogaster. In this paper we first compare the patterns of protein synthesis in wing, haltere, leg 1, leg 2, leg 3 and eye antenna imaginal discs of late third instar larvae. We have detected only quantitative changes: differences in 17 proteins among the different imaginal discs. In addition, we have analysed the variations in pattern of proteins in the wing disc of the last larval stage and early pupae as well as in wing discs cultured in vivo for 6 days. Variations in these patterns affect more than 20% of the proteins and involve both qualitative and quantitative changes. Some of the changes may correspond to protein phosphorylation. Correlations of these changes between discs and through development are also discussed. Correspondence to: F. Santaren     

Extracellular peptidases of imaginal discs of Drosophila melanogaster

The imaginal discs of Drosophila melanogaster give rise to the adult epidermis during metamorphosis. During this developmental period several peptidase genes are expressed in disc cells, but there is a paucity of biochemical information regarding substrate specificity. We have used peptides and peptidyl 7-amino-4-methylcoumarin (AMC) substrates to detect several peptidases either positioned on the surface of wing discs or secreted by the imaginal cells. Using [Leu(5)]enkephalin as a substrate, a captopril sensitive dipeptidyl carboxypeptidase (angiotensin I-converting enzyme) and an amastatin-sensitive aminopeptidase were detected as prominent activities associated with intact discs. The formation of [Leu(5)]enkephalin-derived Phe was attributed to the concerted action of the D. melanogaster angiotensin I-converting enzyme (Ance) and a dipeptidase. The disc Ance also showed endopeptidic activity towards locust tachykinin-1 (LomTK-I) by cleaving the Gly-Val peptide bond, but this enzyme was not the sole endopeptidase activity associated with discs. Complete inhibition of the endopeptidic hydrolysis of the LomTK-1 by a disc homogenate required a combination of captopril and the neprilysin inhibitor, phosphoramidon, providing biochemical evidence for a neprilysin-like peptidase, in addition to Ance, in imaginal discs of D. melanogaster. Peptidyl AMC substrates for furin, prohormone convertase and tryptase provided evidence for trypsin-like serine endopeptidases in addition to the metalloendopeptidases. We conclude that imaginal discs are endowed with a variety of peptidases from different families that together are capable of hydrolyzing a broad range of peptides and proteins. Some of these peptidases might be responsible for the metabolic activation/inactivation of signaling peptides, as well as being involved in the production of dipeptides and free amino acids required for protein synthesis and osmotic balance during adult morphogenesis.     

Pyridine nucleotide metabolism in imaginal discs of Drosophila melanogaster

The pyridine nucleotide metabolism of imaginal discs of Drosophila melanogaster has been studied in vitro by incubating discs with labeled nicotinic acid in the presence and absence of ecdysterone. The major labeled compounds found within the discs are NAD, NADP, and nicotinic acid. There is preferential uptake of nicotinamide over nicotinic acid, although the Priess-Handler pathway is used exclusively. The presence of ecdysterone produces a small increase in the NADP/NAD ratio, and an increase in NAD synthesis, probably to compensate for increased NAD turnover.Supported by Grant GB 43569 from the National Science Foundation.     

Dependence of transdetermination frequency on the developmental stage of cultured imaginal discs of Drosophila melanogaster

Male foreleg tissue from prepupal stages of Drosophila melanogaster was tested for its capacity to grow when cultured in the adult fly hemocoel and for its capacity, after culture, to produce adult cuticular structures when differentiated in a metamorphosing larva. Evaginated, segmented leg tissue from 8-hr-old prepupae (at 25°C), still retained the capacity to grow well in culture. Growth was, however, restricted to cells of the proximal half of the leg. Tissue from 11- and 24-hr stages (pupal ecdysis at 11 hr) was not successfully cultured. Cultured proximal halves of 8 hr prepupal legs frequently differentiated not only proximal structures, but also distal structures, such as sex combs and claws, indicating regeneration of missing leg structures during the culture period. Transdetermination to wing tissue occurred only rarely (once in 90 implants) whereas third-instar leg tissue in culture transdetermined frequently (50% of the implants) to wing, even though growth of tissue of the two stages was equivalent. Identical results were obtained with third-instar foreleg discs evaginated in vitro with β-ecdysone. This is the first in vitro treatment reported to reduce transdetermination frequency, without affecting growth proportionately. These results indicate that cell proliferation in culture, while probably a necessary condition for transdetermination, is not a sufficient condition. The developmental stage of the cultured tissue strongly affects the frequency of transdetermination.     

The effect of ecdysterone and juvenile hormones on protein synthesis and development of imaginal wing discs ofDrosophila melanogaster

The effect of ecdysterone and juvenile hormone on protein synthesis and development of imaginal wing discs ofDrosophila melanogaster has been studied. It is found that juvenile hormone apparently does not inhibit the synthesis of the ecdysterone-inducible proteins, although wing disc development is inhibited to various extent by different juvenile hormones. It is suggested that the ecdysterone-inducible proteins are not involved directly in the initiation of wing disc evagination, it is possible that some of these proteins are involved in the maintenance of chromatin activities or they are involved in gene activation.     

The synthetic and minimal culture requirements for evagination of imaginal discs of Drosophila melanogaster in vitro

Imaginal discs are induced by β-ecdysone to evaginate and undergo imaginal differentiation in completely defined culture medium (Robb's). The minimal nutritional requirements for evagination are salts, glucose, and 6 or 7 amino acids. Concentrations of β-ecdysone which cause evagination also produce increases in RNA and protein synthesis. Inhibitors of RNA and protein synthesis and amino acid starvation block evagination. Inhibitors of DNA synthesis do not inhibit evagination. The effects of β-ecdysone are concentration dependent. To produce complete evagination, discs must be exposed to low concentrations (0.1 μg/ml) of β-ecdysone for a longer time than to high concentrations (10 μg/ml). However, high concentrations of hormone reduce the rate, and under some conditions, the degree of evagination.     

Chitin biosynthesis in imaginal discs cultured in vitro

Summary We have investigated the stimulation of cuticle production by imaginal discs ofPlodia interpunctella in tissue culture. We turned to biochemical methods to assess the quantitative effects of beta-ecdysone on chitin biosynthesis in wing discs incubated with 0.5 C of C¹⁴-glucosamine for the final 24 h of culture.We demonstrated that imaginal discs ofP. interpunctella respond to increasing concentrations of -ecdysone with increased synthesis. The threshold is between 0.01 and 0.1 g/ml of hormone (2×10^–8 M to 2×10^–7 M). These data represent the first demonstration of quantitative biosynthesis of chitin by a developing tissue in vitro in relation to varying amounts of hormone. Additionally, protein synthesis during the -ecdysone-dependent period was necessary for chitin synthesis. This system thus lends itself to a detailed investigation of the control of chitin biosynthesis.We wish to dedicate this paper to the memory of a fine colleague and friend, Dr. Andrzej Dutkowski     

Proteomic analysis of the wing imaginal discs of Drosophila melanogaster

We have combined high-resolution two-dimensional (2-D) gel electrophoresis and mass spectrometry with the aim of identifying proteins represented in the 2-D gel database of the wing imaginal discs of Drosophila melanogaster. First, we obtained a high-resolution 2-D gel pattern of [35S]methionine + [35S]cysteine-labeled polypeptides of Schneider cells, a permanent cell line of Drosophila embryonic origin, and compared it with the standard pattern of polypeptides of the wing imaginal disc. These studies reveal qualitative and quantitative differences between the two samples, but have more than 600 polypeptides in common. Second, we carried out preparative 2-D polyacrylamide gel electrophoresis using Schneider cells mixed with radioactively labeled wing imaginal discs in order to isolate some of the shared polypeptides and characterize them by matrix-assisted laser desorption/ionization-time of flight MALDI-TOF analysis. Using this strategy we identified 100 shared proteins represented in the database, and in each case confirmed their identity by MALDI-TOF/TOF analysis.