Dianne Fristrom

The mechanism of evagination of imaginal discs of Drosophila melanogaster: I. General considerations

Abstract Morphological and ultrastructural observations of evaginating discs are presented along with studies of the biochemical and cellular effects of inhibitors of evagination, particularly cytochalasin B (CB). The ultrastructure of the evaginating disc is described with special reference to elements which might be important in evagination, specifically microfilaments and microtubules. There is circumstantial evidence that CB sensitive microfilaments distributed along the basal cell surface may play a role in evagination. No effects of CB were found on β-ecdysone-stimulated synthetic activities. A model for the evagination of appendages from imaginal discs which requires both cell flattening and cell rearrangement is presented. Some implications of the model for mosaic and cell lineage studies are discussed.

Cellular degeneration in the production of some mutant phenotypes in Drosophila melanogaster

SummaryA number of mutants of Drosophila melanogaster are characterized by the absence of structures present in the wild type. Imaginal discs from the wing mutants vestigial, apterous-Xasta, Beadex and cut and from the eye mutants Bar, eyeless and lozenge were examined by light and electron microscopy. In all these mutants, with the exception of lozenge, clear evidence of degeneration was found. The onset and duration of degeneration and the number and distribution of dying cells were specific characteristics of each mutant. In most cases the degenerate areas of the disc could be correlated with the missing parts of the adult wing or eye. In contrast, in wild type wing and eye discs and in wing discs from the mutant miniature, which has a wing reduced in size but fully formed, extensive cell death was not observed.The ultrastructural features of the degenerating areas weresimilar in all the mutants studied. Conspicuous aspects of the cytolytic process included condensation and fragmentation of the dying cells followed by phagocytosis of the cell fragments by neighboring disc cells.The results indicate that localized cell death during development is a widespread occurrence among Drosophila mutants which exhibit structural deficiències.

The mechanism of evagination of imaginal discs of Drosophila melanogaster. III. Evidence for cell rearrangement.

The shape and arrangement of cells in leg discs of Drosophila melanogaster at different stages of evagination were examined by scanning electron microscopy. The observations indicate that the change in shape of the disc during evagination is largely a result of cell rearrangement. This process involves small movements of many cells within the disc epithelium while close associations between neighboring cells are maintained.

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.

The ultrastructure of the differentiating pupal leg ofDrosophila melanogaster

SummaryThe ultrastructure of the developing pupal leg ofDrosophila melanogaster is investigated at various stages from white prepupa to pharate adult. All the different cell types found in the leg at each stage examined are traced developmentally. This includes general epithelium which secretes both a pupal and adult cuticle and specialized epithelial structures such as apodemes, bristles and other sense organs. Muscle, nerve and tracheal tissue develop within the leg cavity. Particular attention was paid to the development of muscle tissue which was shown to differentiate from the adepithelial cells of the larval disc. The development of theDrosophila leg is compared with that ofCalliphora (van Ruiten and Sprey, 1974) especially with respect to the different stages at which new structures appear in the two species.

Effects of ecdysone and juvenile hormone on DNA metabolism of imaginal disks of Drosophila melanogaster

Abstract Imaginal disks of Drosophila melanogaster isolated en masse and incubated in Robbs tissue culture medium incorporate 3 H-thymidine into nuclear DNA. Both α- and β-ecdysone stimulate the rate of 3 H-thymidine incorporation into disk DNA. Concentrations of ecdysone that induce complete evagination of disks in vitro cause the initiation of DNA synthesis in some disk cells. Juvenile hormone has no effect on DNA synthesis in control disks. However, juvenile hormone blocks the ecdysone stimulation of DNA synthesis. It is proposed that juvenile hormone and ecdysone act in a balanced fashion to regulate DNA synthesis in imaginal disks.

The hormonal coordination of cuticulin deposition and morphogenesis in Drosophila imaginal discs in vivo and in vitro

Cuticulin is the first layer of the insect cuticle to be deposited and is laid down as a continuous inelastic sheet over the apical surface of cuticle-secreting cells. During metamorphosis in Drosophila melanogaster, imaginal discs deposit the cuticulin layer of the pupal cuticle between 3 and 7 hr after puparium formation. This is a period of rapid morphogenesis involving cell shape changes and cell rearrangements. We have examined cuticulin deposition in vivo and in vitro with a view to understanding the coordination of cuticulin deposition with morphogenesis. We find that the optimum hormonal regimen (of the steroid hormone, 20-hydroxyecdysone) for the completion of both morphogenesis and cuticulin deposition in vitro parallels the changes in hormone titer observed in vivo. We also find that cuticulin is deposited last over cell boundaries, thereby allowing cell rearrangements to occur as cuticulin is laid down. We have identified in vitro conditions under which cuticulin deposition is completed precociously, inhibiting further morphogenesis. Cytochalasin B and colchicine do not inhibit cuticulin deposition and we therefore conclude that an intact cytoskeleton is not necessary for secretion of this extracellular structure. Finally, we present a preliminary protocol for the partial purification of cuticulin synthesized in vitro by mass isolated discs.

Genes expressed during imaginal disc morphogenesis: IMP-E1, a gene associated with epithelial cell rearrangement☆

A unique set of genes that encodes hormone-inducible transcripts found on membrane-bound polysomes is expressed during ecdysone-dependent morphogenesis of Drosophila imaginal discs cultured in vitro. The pattern of expression of one of these genes, IMP-E1, was analyzed in tissues from late third instar larvae and white prepupae by hybridization of asymmetric RNA probes to tissue sections. The IMP-E1 transcript was detected in all anterior and posterior imaginal discs except the ommatidial region of the imaginal eye disc. Within the imaginal leg disc, the IMP-E1 transcript is expressed more abundantly in the proximal than in the distal portions of the epithelium. The distribution of transcripts is consistent with the hypothesis that the IMP-E1 gene product is involved in the cell rearrangements associated with morphogenesis of the disc epithelium. The IMP-E1 transcript is also expressed at pupariation in glial cell layers that ensheath the brain. This glial cell transcript is the same size (7.5 kb) and polarity as the imaginal disc transcript, and is also transcribed in response to 20-hydroxyecdysone. Similarities between the morphogenetic changes in imaginal disc and glial cell layers during metamorphosis are discussed.

The mechanism of evagination of imaginal discs of Drosophila melanogaster: V. Evagination of disc fragments☆

Experiments designed to test the hypothesis that imaginal disc evagination may be accomplished by a continuous circumferential constriction of the basally located microfilaments in disc cells [Fristrom, D., and Fristrom, J. W. (1975). Develop. Biol. 43, 1–23] are presented. Discs were cut in various planes so as to interrupt the hypothesized lines of constriction and the resulting fragments tested for the ability to evaginate in vitro. Fragments from each type of cut were able to undergo evagination without resealing of the would surfaces. Furthermore, sections of evaginated cut fragments showed no evidence for a general contraction of the basal cell surface. We conclude that the ability to evaginate is an intrinsic property of each fragment independent of its attachment to the rest of the disc.

Genes expressed during imaginal discs morphogenesis: IMP-L2, a gene expressed during imaginal disc and imaginal histoblast morphogenesis.

We describe our analysis of IMP-L2, one of a set of six ecdysone-inducible genes in imaginal discs of Drosophila whose transcripts are associated with membrane-bound polysomes. The spatial and temporal patterns of expression of the IMP-L2 transcript were analyzed. This transcript is first expressed in imaginal discs in areas that are precursors of head and thoracic epithelium, particularly the peripodial epithelia. It is later expressed in the imaginal histoblasts, precursors of the adult abdomen. The appearance of IMP-L2 transcript in each of these tissues precedes the spreading and fusion of the separate imaginal anlagen to form the continuous adult epidermis.