The imaginal discs of Drosophila are epithelial structures that are the embryonic precursors of adult cuticular structures, e.g. legs, wings, eyes. Mass-isolated imaginal discs undergo morphogenesis-evagination-to form appendages in vitro when incubated with the insect steroid hormone, 20-hydroxyecdysone. The hormone acts via the genome to elicit the developmental responses. Evagination itself results from short-distance movements of disc cells. We propose to identify the genes and their protein products in a major pathway leading to evagination. A master regulatory gene, located cytogenetically at 2B5, will be investigated through recovery of genomic clones and subsequent studies on the clones of binding of the receptor for 20-hydroxyecdysone to DNA acceptor sites, of transcriptional units expressed in larval and imaginal tissues, of changes caused by 2B5 mutants. Hormone-dependent transcripts from other loci will be identified using cDNA cloning. Because of the apparent involvement of membrane proteins in evagination, special efforts will be made to identify cDNA clones representing membrane proteins. Genes involved in evagination will be identified because of the absence of their transcripts in discs of 2B5 mutants that block evagination. Genes for membrane proteins and/or with hormone-dependent transcription will be localized cytogenetically by in situ hybridization. cDNA clones will be related to their protein products by comparing in vitro translation products to in vivo synthesized proteins. Membrane proteins will be mapped cytologically within discs using a combination of labelling and immunological techniques. Membrane glycoproteins whose glycosylation occurs via the dolichol-dependent pathway have been implicated in evagination and will be mapped cytologically and characterized structurally. These studies should provide insights into gene regulation during hormone-induced morphogenesis of an animal epithelium and provide an understanding of the functions of some hormone-dependent genes. The research also forms the basis for a long-term study on the genetics of membrane proteins. The basic knowledge gained will be relevant to the action of steroid hormones and to the normal and abnormal development of epithelia in humans.
