Kidney vascularization: semaphorin-mediated mechanisms.

描述（申请人提供）：在肾脏器官发生过程中，内皮细胞定向迁移的分子基础和血管发育的模式尚不清楚。我们发现VEGF是内皮细胞的化学引诱剂，并指导内皮细胞向发育中的肾单位迁移。其他的引导信号也必然参与到血管生长的调节中。这些引导线索的性质是未知的。两种VEGF共受体neuropilins 1和2也是信号蛋白3A和3F的受体。信号蛋白是诱导轴突化学排斥的指导蛋白。Sema 3A通过与VEGF竞争neuropilin结合而减少内皮细胞迁移。我们发现信号素3A和3F在肾脏形态发生过程中表达，并以一种互补的方式定位于肾上皮细胞，这表明信号素对血管模式很重要。本研究的目的是阐明内皮细胞定向迁移导致血管发育的空间组织的机制，以及信号蛋白3A和3F在肾脏形态发生中的功能。我们假设sema 3A和3F是由肾上皮产生的。上皮细胞产生化学排斥信号，通过创建内皮细胞迁移的边界来调节VEGF对内皮细胞的化学吸引。sema 3a、sema 3F和VEGF与它们的共享受体结合的配体组合可能性可能导致血管形成的“路径”。我们还假设信号蛋白的散点因子样特性可能至少部分地导致肾上皮的分支形态发生。为了验证我们的假设：1)我们将通过活细胞显微镜研究sema 3A和sema 3F引导内皮细胞迁移的机制，使用共培养、迁移实验和sema 3A和3F在转基因小鼠中的细胞特异性过表达。2)我们将利用小管上皮细胞、器官培养和转基因小鼠研究信号蛋白3A和3F在分支形态发生和小管发生中的功能。3)我们将研究FAK在信号蛋白介导的引导和形态发生线索中的作用和下游信号机制。这一建议将为信号蛋白诱导的定向迁移提供新的重要信息，并促进我们对血管空间组织的分子机制的了解。了解细胞迁移的引导线索的分子基础将使我们能够为先天性肾脏异常的诊断和治疗提供新的策略，并在体外发展器官发生。

DESCRIPTION (provided by applicant): 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.