The long term goal of this proposal is to understand the biology of negative regulators of branching during kidney organogenesis. The collecting system of the kidney arises from iterative branching of an embryonic structure known as the ureteric bud (UB). Branching morphogenesis determines nephron number, which is believed by some to be an important factor in the development of hypertension and the progression of renal injury. Investigators in the field of renal development have generally tended to focus on stimulators of branching, rather than those that might act as negative regulators or """"""""stop signals."""""""" Here we argue that there is no reason, a priori, that the study of these negative regulators should be of secondary importance, since it is the balance of positive and neqative factors that ultimately determines the deqree of arborization of the developing kidney's collecting system (as well as nephron number). We also arque that studies of total RNA levels during branching morphogenesis are of limited value without similar information on spatiotemporal expression at tips, branch points and stalks for understanding how the arborization pattern of the developing tree becomes established. Our lab has devised a number of in vitro model systems for analyzing branching morphogenesis of the ureteric bud. These include the isolated UB culture model and cell culture-based models (UB and IMCD) of branching morphogenesis. We have amassed a considerable amount of preliminary data that demonstrates the robustness of these model systems and how they can be employed, together and separately, to understand regulatory pathways involved in branching morphogenesis, tn particular, we have shown that the branching pattern is negatively modulated by a number of soluble growth factors, including members of the TGF-beta superfamily, and an as yet unidentified activity elaborated by the conditioned medium of a metanephric mesenchyme cell line. In this proposal, we aim to: a) utilize the aforementioned model systems to better understand how negative modulators of branching exert their inhibitory effects by examining spatiotemporal expression patterns on tips, branch points and stalks under conditions of: 1) branching, 2) inhibition of branching AND proliferation, and 3) inhibition of branching WITHOUT major effects on proliferationofollowed by functional perturbation experiments (SA1: cell and organ culture, in vitro functional assays, immunocytochemistry, arrays, laser microdissection, knockout animals); and b) purify an apparently novel activity that inhibits branching of the cultured isolated UB (SA2: column chromatography, immunoblotting, microsequencing). In the preliminary data, we provide examples of our expertise in all the techniques required to successfully complete the aims of this project.
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