The ureter develops out of the ureteric bud (UB), but most work on the UB to date has focused on branching morphogenesis as it relates to development of the kidney collecting ducts. Little is known about the genes specifying ureter formation out of the UB. This is an important question because, as we show, the developing ureter, though non-branching in vivo, has the capacity to branch in vitro in a manner similar to the portion of the UB destined to undergo branching morphogenesis as it develops into the kidney collecting ducts. Based on extensive preliminary data (Figs. 1-39), we hypothesize that two major steps are involved in ureter formation: 1) outgrowth of the UB from the Wolffian duct (a step in common with the kidney collecting ducts) (SA1); and 2) a branch-inhibitory influence on a subpopulation of UB cells that results in the formation of a non-branching ureter (SA2). The goal of this proposal is to employ in vitro assays, many of which have been developed by our group (e.g. isolated UB culture, isolated ureter culture, non-branching 1MCD tubulogenesis assay) together with a demonstrated ability to perform large scale gene expression (array) analysis on small quantities of DNA, to identify specific subsets of morphogenetic genes critical for: 1) outgrowth of the UB from the Wolffian duct (SA1) and 2) the formation of a non-branched ureter out of the UB (SA2). Those genes that are expressed in an appropriate spatiotemporal manner for a role in ureter development (immunocytochemistry, in situ hybridization) will be functionally perturbed in the in the appropriate vitro system (e.g. isolated Wolffian duct culture, isolated UB culture, isolated ureter culture, branching and non-branching IMCD tubulogenesis assay). This will be done using a variety of techniques (neutralizing antibodies, antisense/morpholinos/RNAi, Specific inhibitors or activators, transfections). We have published experience with nearly all these techniques, and considerable data is presented to indicate the feasibility of the approach and our ability to perform sophisticated computational analyses of large sets of genes such as those that will be obtained in the proposed array experiments. Together, these studies should lead to the identification of genes that are critical for UB formation and that determine the fate of cells in the UB destined to form the ureter. (This proposal is specifically responsive to Program Announcement PAR-02- 143).
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