My laboratory is interested in how patterns are formed in developing organisms. In particular, we are interested in how cellular communication contributes to developmental decisions. We previously discovered that the decapentaplegic gene of Drosophila is a member of the secreted transforming growth factor-beta (TGF-beta) family and is therefore involved in cellular communication. Recently, a number of proteins have been identified which complex with, and alter the activities of TGF-beta members. One candidate gene from Drosophila, the tolloid gene, encodes a protein which augments the activity of decapentaplegic. This proposal is aimed at determining the developmental role and the mechanism by which tolloid contributes to the development of Drosophila, focusing on its interaction with the decapentaplegic gene. Both these genes are involved in establishing dorsal-ventral cell fates in the dorsal ectoderm. Several lines of genetic and molecular evidence link the activity of tolloid and decapentaplegic. In particular, the two vertebrate homologs of these genes, bone morphogenetic protein-1 (BMP1) and bone morphogenetic protein-4 (BMP4) copurify with each other, suggesting their interactions are at the protein level. Recent molecular data indicates that the tolloid gene is duplicated in tandem in the 96A region of the Drosophila chromosome. The tolloid related-1 (tldr-1) gene lies between a P element, where our molecular walk began, and tolloid, which is about 30 kb from the P element. Both these genes contain a protease domain, EGF repeats and the repeats found in the blood complement genes, Clr and Cls. Presently, there are only a few genes implicated in pattern formation which encode proteases, and this study should provide general insights into how they regulate cell growth. To study the tolloid-like genes in Drosophila, we plan to: 1) characterize their expression in embryos and larval tissues in detail, 2) generate mutations in the tldr-1 gene to aid in determining its role during development, 3) dissect structure-function aspects of the tolloid gene by sequencing mutant alleles and generating in vitro mutations for reintroduction into the fly, 4) express tolloid and decapentaplegic in cell lines to biochemically characterize their interaction by immunoprecipitation and to characterize the protease activity of the tolloid protein. Since the expression pattern of tolloid and tldr-1 includes larval and pupal stages, they may interact with decapentaplegic at later times of development. We already have a putative point mutation in the tldr-1 gene. These experiments will provide insight into the action and mechanism of this interesting growth factor in Drosophila and contribute to a general understanding of the role of proteases in pattern formation and cellular communication in animals.
