Pattern formation in the imaginal discs of a temperature-sensitive cell-lethal mutant of Drosophila melanogaster

To explore the effects of cell death on pattern formation in the developing imaginal discs of Drosophila melanogaster, I have isolated a number of cell-autonomous temperature-sensitive lethal mutants. Sex-linked temperature-sensitive lethals were screened for cell-autonomy by scoring the survival of lethal-bearing clones in genetic mosaics. The mutant with the strongest effect on clone viability gave rise to a high frequency of structural deficiencies and duplications in the derivatives of the eye-antennal discs, when subjected to pulse-treatments at the nonpermissive temperature during the late second and third instars. The patterns produced were nonrandom, with some structures showing a tendency to become deficient, and others a tendency to duplicate. Duplicated structures were only found in heads in which other structures were missing. Genetic tests identified the lethal as a point mutation at the suppressor-of-forked locus. Recombination, and complementation tests with a small duplication of this region showed that a second mutational lesion is in all probability not involved in the generation of abnormal patterns in the imaginal discs. It is therefore proposed that the cell-lethal action of the mutant is sufficient to account for phenotypic effects described. According to this hypothesis, cell death primarily causes deficiencies, and duplications occur as a response of the discs to injury. In agreement with this, it was found that in gynandromorphs, pattern duplications can be found in wild-type tissue in the presence of lethal tissue in the same disc. Thus, a cell-autonomous lethal may affect the process of pattern formation in a nonautonomous way.     

Pattern specification in imaginal discs ofDrosophila melanogaster

Summary l(1)su(f)^mad-ts (mad) is a new temperature-sensitive (ts) lethal mutant ofDrosophila melanogaster which produces duplicated legs after temperature pulse treatment during larval development. The ts-lethality was studied in temperature experiments and genetic mosaics. Temperature pulses given during two distinct TSPs of larval development result in two different types of leg pattern duplication. Total differ from partial duplications with respect to the affected leg compartments and the orientation of the planes of symmetry which are perpendicular to the dorso-ventral and the proximo-distal leg axes in total and partial duplications, respectively. Genetic mosaic studies indicate (i) disc autonomy of leg pattern duplication, (ii) clonal separation of the anlagen of the two pattern copies, and (iii) clonal restriction along the antero-posterior compartment border in the two pattern copies of totally duplicated legs.The results suggest thatmad leg pattern duplication is caused by a change in positional information rather than by cell death and subsequent regeneration. Our data are compatible with the assumption that during normal development the leg disc cells acquire information about their position within the disc with respect to the different leg axes independently and at different times.     

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.     

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.     

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.     

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.     

Localization of the Antennapedia protein in Drosophila embryos and imaginal discs

[This corrects the article on p. 3327 in vol. 5.].     

Localization of the Antennapedia protein in Drosophila embryos and imaginal discs

Antibodies have been raised against a fusion protein containing the 3' region of the coding sequence of the Antennapedia (Antp) gene fused to β-galactosidase. The distribution of the protein on whole mount embryos and imaginal discs of third instar larvae was examined by immunofluorescence. In young embryos, expression of the Antp protein was limited to the thoracic segments in the epidermis, whereas it was found in all neuromeres of head, thorax and abdomen. At the end of embryogenesis, the Antp protein mainly accumulated in the ventral nervous system in certain parts of the thoracic neuromeres, from posterior T1 to anterior T3, with a gap in posterior T2. Comparison of Antp protein distribution in nervous systems from wild-type and Df P9 embryos, lacking the genes of the Bithorax-complex (BX-C), revealed a pattern of expression which indicated that the BX-C represses Antp in the posterior segments with the exception of the last abdominal neuromeres (A8-9) which are regulated independently. The protein pattern in nervous systems from Sex combs reduced(Scr^xF9) mutant embryos was indistinguishable from that found in wild-type embryos; thus, neurogenic expression of Antp in T1 and the more anterior segments does not appear to be under the control of Scr⁺. All imaginal discs derived from the three thoracic segments express Antp protein. The distribution was distinct in each disc; strongest expression was observed in the proximal parts of the discs. In the leg discs the protein distribution seemed to be compartmentally restricted, whereas in the wing disc this was not the case. Antp protein was not detected in the eye-antennal disc. In embryos, as well as in imaginal discs, the protein is localized in the nucleus.     

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.     

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.     

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.     

Transplanted wing and leg imaginal discs in Drosophila melanogaster demonstrate interactions between epidermis and myoblasts in muscle formation

 Adult muscle development in Drosophila is intimately associated with the development of the nervous system and epidermis. During metamorphosis, myoblasts from the wing imaginal disc reach target sites on the developing pupal epidermis and begin the formation of multinucleate myofibres of the dorsal thorax. The paths taken by pupal myoblasts could be specified by the nervous system and/or the epidermis. Using genetically marked donor pupal wing and leg discs transplanted onto pupal hosts, we have generated animals that have ectopic wings or legs and have examined the formation of adult muscle types. We show that thoracic myoblasts migrate over both host and donor epidermis when the transplant site on the host is thoracic. However, when the transplant site is on the abdomen, thoracic myoblasts do not migrate over abdominal epidermis. Our results show that the epidermis plays an important role in determining the migration pattern of myoblasts. Since muscles are multinucleate cells that form by the fusion of myoblasts, one way in which their molecular characteristics could be achieved is by some myoblasts acting as ”founders”. These myoblasts could influence the pattern of gene expression of those nuclei that fuse with them. We have examined, again using disc transplant experiments, if myoblasts on discs have the capacity to express fibre-specific genes as distinct from this property being conferred by other extra-discs myoblasts. Our results demonstrate that disc-associated myoblasts can indeed fuse with each other to express fibre-specific genes. We synthesize the results presented here with those from earlier experiments to suggest a mechanism for muscle patterning in the adult thorax. Received: 8 January 1995 / Accepted in revised form: 22 January 1996     

Embryonic origin of the imaginal discs of the head of Drosophila melanogaster

The embryonic development of the primordia of the Drosophila head was studied by using an enhancer trap line expressed in these structures from embryonic stage 13 onward. Particular attention was given to the question of how the adult head primordia relate to the larval head segments. The clypeo-labral bud to the stage 13 embryo is located at a lateral position in the labrum adjacent to the labral sensory complex (epiphysis). Both clypeo-labral bud and sensory complex are located anterior to the engrailed-expression domain of the labrum. Throughout late embryogenesis and the larval period, the clypeo-labral bud forms integral part of the epithelium lining the roof of the atrium. The labial disc originates from the lateral labial segment adjacent to the labial sensory complex (hypophysis). It partially overlaps with the labial en-domain. After head involution, the labial disc forms a small pocket in the ventro-lateral wall of the atrium. The eye-antenna disc develops from a relatively large territory occupying the dorso-posterior part of the procephalic lobe, as well as parts of the dorsal gnathal segments. Cells in this territory are greatly reduced in number by cell death during stages 12–14. After head involution, the presumptive eye-antenna disc occupies a position in the lateral-posterior part of the dorsal pouch. Evagination of this tissue occurs during the first hours after hatching. In the embryo, no en-expression is present in the presumptive eye-antenna disc. en-expression starts in three separate regions in the third instar larva.