9630621 Vize This proposal aims to investigate the genetic interactions required for pronephric specification and patterning. Xenopus orthologs of a number of genes previously demonstrated to play essential, but poorly defined, roles in development of the mammalian adult kidney have been isolated. These genes include frog orthologs of the Wilms' tumor suppressor gene WT1, pax2, wnt4, c-met, HGF, N-myc and lim~1. A novel Xenopus pax gene which is expressed at high levels very early in kidney development has also been identified. Vertebrates form at least two different forms of kidney during their life span, normally an embryonic kidney of low or moderate complexity, and an adult kidney of much greater complexity. Once the more complex kidney is functional, the embryonic kidney either degenerates or becomes integrated into the reproductive system. The pronephros is a simple embryonic kidney used by the embryos and larvae of the lower vertebrates to excrete wastes and control water balance. The structure of one entire pronephros resembles a much larger version of a single nephron from an adult kidney. A pair of these large nephrons sustain the embryo until the adult kidney is functional, which in amphibians is around the time of metamorphosis. In comparison, the human adult kidney requires around one million nephrons to perform all of its required functions. The pronephros has a number of advantages as an experimental model for studying organogenesis of the kidney. The simple organization and large size of the organ make it easy to visualize, and as it undergoes a stereotypical pattern of morphogenesis, subtle changes in its development are easy to detect. Molecular markers are available for each of the different components of the pronephros. The pronephros is fully formed in under three days in a Xenopusembryo, so the effects of experimental interventions can be rapidly analyzed. This early development also means that this organ is induced and patterned in the period of develo pment that is easily manipulated in Xenopus by either microdissection or microinjection. In this proposal the mechanism by which ectopic Xenopus WT1 disrupts kidney development will be determined by analyzing the effect of this gene product on cell proliferation, apoptosis, and the expression of other pronephric genes. The second part of the proposal will examine the role of each of the genes listed above in pronephric development. All of these experiments will be performed by microinjecting either in vitro synthesized mRNA encoding the gene product, or expression plasmids into early blastulae, that will then be allowed to develop to tadpole stages. The effects of aberrant gene expression will be analyzed by examining the effect on the expression of other pronephric genes, and by studying the structure of the differentiated pronephros with immunohistochemistry and confocal microscopy. These experiments will use a simple model system to determine how these genes interact to pattern and form a nephron. The results will have direct relevance not only to organogenesis of other types of kidney, but also to kidney disease, as a number of these genes are involved in the development of Wilms' tumor, a pediatric tumor of the urogenital system.
