James W. Fristrom

Talin Is Essential for Integrin Function in Drosophila

We show that the Drosophila gene rhea, isolated because its wing blister phenotype is typical of mutants affecting integrin function, encodes talin. Embryos deficient in talin have very similar phenotypes to integrin (betaPS) null embryos, including failure in germ band retraction and muscle detachment. We demonstrate that talin is not required for the presence of integrins on the cell surface or their localization at muscle termini. However, talin is required for formation of focal adhesion-like clusters of integrins on the basal surface of imaginal disc epithelia and junctional plaques between muscle and tendon cells. These results indicate that talin is essential for integrin function and acts by stably linking clusters of ECM-linked integrins to the cytoskeleton.

Gene within a gene: Nested Drosophila genes encode unrelated proteins on opposite DNA strands

A pupal cuticle protein gene has been found within an intron of a Drosophila gene that encodes three purine pathway enzymatic activities. The intronic gene is encoded on the DNA strand opposite the purine pathway gene and is itself interrupted by an intron. Whereas the purine pathway gene is active throughout development, the intronic cuticle protein gene is expressed primarily over a 3 hr period in the abdominal epidermal cells of prepupae that secrete the pupal cuticle. Therefore, a housekeeping gene and a developmentally regulated gene function in a nested arrangement.

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.

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

Abstract Imaginal discs are induced by β-ecdysone to evaginate and undergo imaginal differentiation in completely defined culture medium (Robbs). 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.

The recovery and preliminary characterization of X chromosome mutants affecting imaginal discs ofDrosophila melanogaster

A selection method especially designed for isolation of X-linked lethals in Drosophila having defective imaginal discs has generated 26 mutants with high larval viability, but which terminate development in late larval and prepupal stages. The mutants are tentatively classified into four categories, according to the morphological appearance of their imaginal discs in third-instar larvae, and are further characterized with reference to autonomy/nonautonomy and to capacity for in vitro evagination of discs. The four categories are as follows: 1. discs degenerate = extreme reduction of disc tissue (8 mutants). Of 7 mutants tested, all appear to be autonomous. 2. discs small = reduction in size of disc tissue (7 mutants). Of 6 mutants tested, 5 are nonautonomous. However, discs from 4 of the nonautonomous mutants fail to evaginate in vitro . 3. discs large = hypertrophy of disc tissue (1 mutant). The mutant is phenotypically similar to an allele of lethal giant larvae (l(2)gl 4 ) . 4. discs normal = normal appearance of disc tissue (10 mutants). Of 9 mutants tested, 5 are nonautonomous. Discs from one mutant evaginate abnormally in vitro and in vivo .

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

Abstract 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 (Laws compound, C 17 and C 18 Cecropia hormones, farnesol). It was found that disks do not metabolize α - or β -ecdysone. A concentration of β -ecdysone of 0·1 μ g/ml or of α -ecdysone of 20 μ g/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 C 18 Cecropia juvenile hormone was rapidly metabolized by disks. Large quantities of the hormone ( ca. 100 μ g/ml) were needed to inhibit evagination in vitro . Thus, unlike the assay of ecdysones, the assay of juvenile hormones on disks is insensitive and complicated by metabolic conversions of the hormones. Juvenile hormones were also tested for their ability to inhibit the normal rotation of male external genitalia.

20-Hydroxyecdysone is required for, and negatively regulates, transcription of Drosophila pupal cuticle protein genes.

Transcripts of ecdysone-dependent genes (EDGs) accumulate in isolated imaginal discs with 8 hr after exposure to a pulse of the steroid hormone 20-hydroxyecdysone (20-HE; 1 microgram/ml for 6 hr) but not in discs cultured in the continuous presence or absence of the hormone. Sequence analyses show that two of the EDGs are members of gene families encoding insect cuticle proteins. We conclude that a third EDG encodes a cuticle protein because the conceptual glycine-rich protein contains sequence motifs similar to those found in insect egg shell proteins and vertebrate cytokeratins and because expression of this gene is limited to tissues that deposit the pupal cuticle. Nuclear run-on assays show that the hormone-dependent expression of each of these EDGs is due to transcriptional regulation. Readdition of hormone to imaginal discs actively synthesizing the EDG messages causes rapid repression of EDG transcription. Thus, 20-HE acts as both a positive and a negative regulator of EDG transcription. Sequences in the promoter regions of two of the EDGs are similar to an ecdysone response element and may play a role in negative regulation.

The cuticle proteins of Drosophila melanogaster: Stage specificity

Abstract Five stage-specific cuticles are produced during the development of Drosophila . Urea-soluble proteins were extracted from each developmental stage and compared by gel electrophoresis. Proteins from first and second instar cuticle are identical except for minor differences in two proteins. Each subsequent stage, third instar, pupa, and adult, has a unique set of cuticle proteins. Qualitative changes within stages are seen in proteins from third instar and adult cuticle. Third instar cuticle proteins can be divided into “early” [proteins 2a, 3, 4, 5, 7, and 8] and “late” [proteins 2 and 1] groups. Adult cuticle proteins change in relative amounts during pharate adult development and change mobility at eclosion. The lower abdominal pupal cuticle lacks a protein found in the pupal cuticle covering the head and thorax. Cuticle proteins from each stage are immunologically related. Nonetheless, electrophoretic variants of three larval proteins do not affect any major changes in the electrophoretic mobility of proteins from other stages. We propose that each stage (except first and second instar) has proteins encoded by discrete genes.

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.

Prepupal larval mosaics in Drosophila melanogaster

THE metamorphosis of insects provides a model system for the study of hormone action. In Drosophila, metamorphosis is initiated by the formation of a puparium with a rigid cuticle that becomes tanned early in the prepupal period. Subsequently many larval tissues degenerate and the imaginal tissues develop to produce the adult insect. These processes are initiated in Drosophila by the steroid moulting hormone, β-ecdysone1. We are interested in the mechanisms by which β-ecdysone elicits adult development. The recovery of mutants which cannot respond to the hormone would facilitate investigation into the nature of the action of β-ecdysone. We have isolated systematically a series of late larval and prepupal X-linked lethals (ref. 2 and unpublished results of I.K. et al.), about 15% of which are non-pupariating (npr) lethals in which metamorphosis is not initiated. We have investigated whether the npr condition is a result of the autonomous inability of the mutant target tissues to respond normally to β-ecdysone or rather is a stage-specific failure in the production of the hormone. Our results indicate that in most npr lethals the tissue cannot respond to the hormone. Futhermore, we estimate that there are 100–200 such lethals in the genome of D. melanogaster.