Polycystic kidney disease is one of the most common human genetic conditions, and causes the slow swelling of cysts within a subset of nephrons over the course of years, which eventually results in loss of nephritic function and end- stage renal disease. The genes most often mutated in this disease have been sequenced, but it is not clear how the normal proteins act to maintain the narrow tubular structure of the nephron, as well as other similar structures such as hepatic ducts. Several theories exist as to the root cause of polycystic kidney disease, but are difficult to prove, in part because there is not model extant where the initial stages of cystogenesis can be studied. The nematode Cae norhabditis elegans offers such a model. It possesses rudimentary renal tubules, the excretory canals, that can be observed in living organisms during embryogenesis. I have isolated and characterized mutants in 12 genes that cause a novel phenotype, designated Exc, in which the excretory canals form large cysts that are formally analogous to the polycystic nephrons of vertebrates. This application proposes a detailed molecular study of the function of four exc genes and their products. The genes have been chosen based on the variety of their phenotypes, and on genetic interactions with each other and with known cytoskeletal compounds. The four exc genes will be cloned by microinjection of wild-type DNA into mutant worms, and their cDNA sequences determined. The four genes will then be tagged by linkages to the gene encoding the auto-fluorescent protein GFP, and injection to form transgenic worms. By observation of the site and time of organismal development where fluorescent protein appears, They will deduce where and when these proteins normally function, at both the organismal and subcellular levels. Finally, these four exc genes will be tested via yeast two-hybrid assay for the ability to bind to each other and tpo known cytoskeletal components directly. These experiments will provide a detailed framework for understanding the range of cytoskeletal structures necessary for maintaining a narrow tubular structure, and how that structures can be deranged to form large cysts both in model organisms and in humans.
