The molecular basis of directional migration of endothelial cells and the patterning of the developing vasculature during kidney organogenesis are poorly understood. We showed that VEGF is a chemoattractant for endothelial cells and directs the migration of endothelial cells towards developing nephrons. Other guidance cues are necessarily involved to modulate vascular growth. The nature of these guidance cues is unknown. Two VEGF co-receptors, neuropilins 1 and 2 are also receptors for semaphorins 3A and 3F. Semaphorins are guidance proteins that induce axon chemorepulsion. Sema 3A decreases endothelial cell migration by competing with VEGF for neuropilin binding. We showed that sema 3A and 3F are expressed during renal morphogenesis and localize to renal epithelial cells in a complementary fashion to their receptors located in endothelial cells, suggesting that semaphorins are important for vascular patterning. The objectives of this proposal are to elucidate the mechanisms of endothelial cells directional migration leading to the spatial organization of the developing vasculature and the function of semaphorins 3A and 3F during kidney morphogenesis. We hypothesize that sema 3A and 3F produced by renal epithe!ial cells generate chemorepulsive cues that modulate VEGF's endothelial cell chemoattraction by creating boundaries to endothelial cell migration. The combinatorial possibilities of ligand binding for sema 3 A, sema 3F and VEGF to their shared receptors may result in """"""""paths"""""""" for vessel formation. We also hypothesize that the scatter factor-like properties of semaphorins may be responsible, at least in part, for branching morphogenesis of renal epithelia. To test our hypotheses: 1) we will study the mechanims of sema 3A and sema 3F guidance cues for endothelial cell migration by live cell microscopy using co-culture, migration assays, and cell-specific overexpression of sema 3A and 3F in transgenic mice. 2) We will examine the function of semaphorins 3A and 3F in branching morphogenesis and tubulogenesis using tubular epithelial cells, organ cultures and transgenic mice. 3) We will examine the role of FAK and the downstream signaling mechanisms involved in semaphorin-mediated guidance and morphogenetic cues. This proposal should provide novel and important information regarding semaphorin-induced directional migration and advance our knowledge of the molecular mechanisms governing vascular spatial organization. Understanding the molecular basis of guidance cues for cell migration should enable us to generate new strategies for diagnosis and treatment of congenital renal abnormalities and to develop organogenesis in vitro.
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