Analysis of the Drosophila Gene Rhomboid

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Bier 9603801 The goal of this research proposal "Analysis of the Drosophila gene rhomboid" is to understand how the Drosophila rhomboid (rho) gene amplifies signaling through the EGF-Receptor (EGF-R) pathway. rhomboid encodes an integral membrane protein (Rho) which is concentrated in unique plaque-like structures at the apical cell surface. The products of the rho and Star genes are likely to promote EGF-R signaling since mutations in rho, Star, Egf-r, and a gene encoding an EGF/TGF-a type of growth factor (spitz) lead to extremely similar embryonic phenotypes. rho is expressed in a spatially restricted pattern corresponding to cells requiring hyperactivity of the EGF-R pathway, while genes encoding EGF-R, Star, and the Spitz (Spi) ligand are ubiquitously expressed. This localized expression of rho is critical for limiting augmented EGF-R signaling to appropriate cells. The following are specific research goals for this proposal: I. Dr. Bier will determine whether Rho interacts physically with other proteins by generating epitope tagged versions of the Rho protein and using characterized antibodies against the epitope tags to immune-precipitate active tagged Rho proteins along with other potential interacting proteins such as Star or EGF-R. He will also determine whether Rho and Star proteins co-localize in plaques. II. He will identify functionally important regions of the Rho protein by generating a limited set of targeted rho mutants using in-vitro mutagenesis and random in vivo mutagenesis to revert the dominant ectopic vein phenotype of a fly stock carrying a mix-expressed rho transgene. He will sequence recovered rho mutants to determine whether they cluster in potential functional sub-domains of Rho and whether mutant Rho proteins can form plaques. III. He will compare the genetic requirements for rho, Star, and spi during wing vein development and mix-express rho clonally using the flip-out technique to further investigate the locally non-autonomous action Of rho. He also will us e mosaic analysis to determine the role of spi in wing development.