James W. Fristrom - US grants

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.

Cuticle protein genes are comprised of 2 or 3 multigene families that are differentially expressed in response to the steroid hormone, 20-hydroxyecdysone (20HOE) during Drosophila development. Genes for larval cuticle proteins are expressed in the larval epidermis. Genes for pupal cuticle proteins, related to but distinct from the larval ones, are expressed in imaginal discs in prepupae. Expression of the pupas genes is induced in discs by 20HOE in vitro. Genomic clones containing a cluster of four 3rd instar cuticle protein genes have been recovered and characterized (Snyder et al., 1981) (35). Variants affecting the expression of these genes have been recovered. We propose to clone the genes for the pupal cuticle proteins and produce polyclonal and monoclonal antibody against these proteins. These reagents will be used to describe the molecular details of the onset of synthesis, in response to 20HOE, of cuticle proteins and their mRNAs in imaginal discs in vitro. We will systematically recover variants affecting the expression of larval and pupal cuticle genes. Both "cis"-acting and "distant regulators" of gene expression will be recovered and characterized genetically and at the DNA level. Pupal cuticle gene variants will be analyzed in discs cultured in vitro to determine the molecular nature of the alteration in expression using the cloned genes and the antibody to monitor synthesis. New information about the regulation of gene expression during development should result from these studies.

The Broad-Complex (Br-C) is a Drosophila regulatory gene. Its transcription is controlled by a steroid hormone (20-hydroxyecdysone). The Br-C encodes a set of four DNA-binding Zn finger proteins (Z1, Z2, Z3, Z4) that participate in the hierarchial transcriptional regulation essential for metamorphosis. The Br-C is one of at least such genes that govern metamorphosis. Different Zn finger proteins are produced by the Br-C through differential splicing of Zn finger coding exons to a common "core." Our current results demonstrate that different Br-C Zn finger proteins have different roles in metamorphosis, but they may also be functionally related in a redundant, cooperative and/or competitive manner. Other evidence suggests that the locus may regulate itself both positively and negatively. Additionally, some proteins are expressed in some tissues but not in others, e.g. Z2 and Z4 are transcribed in imaginal disc but not in salivary gland. Finally, the timing of expression of the different transcripts within a given tissue differs. The studies we propose are intended to illuminate the roles this gene and its component parts have in the propagation of a complex hormonally regulated developmental process. Accordingly, we propose to define the functional relationships between Br-C proteins through mutant analyses and induced expression of different Zn finger proteins. A most important objective is to investigate possible autoregulation and cross-regulation at the locus. These studies will be facilitated by the systematic in situ mutagenic dissection of the Br-C to produce mutants that on the one hand, are true null mutations for each Zn finger domain, or, on the other hand, can express only one Zn finger protein. These studies will help to describe the general nature of hormone control of gene expression and development.