Role of Plcg1 in Vegf signaling

描述(申请人提供)：在正常和疾病状态下控制血管形成的信号是相似的，并且在脊椎动物的进化过程中一直是保守的。这使得能够使用遗传上易驯化的动物模型，如斑马鱼，来剖析和分析这些信号。我们发现，在斑马鱼的动脉形成和动脉内皮细胞分化过程中，血管内皮生长因子(VEGF)是特异的。由于血管内皮生长因子已成为人类疾病血管治疗的靶点，更好地了解血管内皮生长因子驱动动脉发育的机制具有重要的临床意义。我们的工作证明了磷脂酶C-伽马-1(PLCG1)在血管内皮生长因子介导的动脉发育中的作用，并表明其他信号也聚集在PLCG1上以驱动这一过程。在这项提案中，我们将首先通过体内结构/功能分析确定PLCG1中与动脉发育所需的结构域，从而确定在动脉发育过程中与PLCG1相互作用的分子。基于这些结果，我们将识别与PLCG1相互作用的分子，并确定它们在动脉发育过程中的功能。我们还将确定在动脉发育过程中影响血管内皮生长因子和PLCG1信号的新斑马鱼突变体。为此，我们将使用转基因斑马鱼进行基因筛查，这些斑马鱼带有荧光标记的血管，可以容易地识别突变表型。最后，我们将描述节段性动脉突变的表型，并确定与这些表型相关的候选基因。这些研究将传统生化技术与通过使用斑马鱼系统可获得的强大遗传工具相结合，将提供一种全面的方法，以更好地了解血管内皮生长因子和PLCG1如何管理动脉发育。由于这些信号机制在进化上是保守的，我们在这项建议中识别的分子代表了临床操作病理性血管形成的可能靶点。

DESCRIPTION (provided by applicant): The signals that govern blood vessel formation in both normal and disease states are similar and have been conserved throughout vertebrate evolution. This enables the use of a genetically tractable animal model, such as the zebrafish, to dissect and analyze these signals. We have found that vascular endothelial growth factor (VEGF) is required specifically for the formation of arteries and the differentiation of arterial endothelial cells in zebrafish. Since VEGF has become a target for therapeutic manipulation of blood vessels in human diseases, it is clinically relevant to better understand the mechanism by which VEGF drives artery development. Our work demonstrates a role for phospholipase C gamma-1 (Plcg1) during VEGF-mediated artery development and suggests that other signals also converge on Plcg1 to drive this process. In this proposal, we will identify molecules that interact with Plcg1 during artery development by first identifying domains in Plcg1 that are required for artery development through in vivo structure/function analysis. Based on these results, we will identify molecules that interact with Plcg1 and determine their function during artery development. We will also identify new zebrafish mutants that affect VEGF and Plcg1 signaling during artery development. For this purpose we will perform a genetic screen using transgenic zebrafish with fluorescently labeled blood vessels that allow easy identification of mutant phenotypes. Finally, we will characterize segmental artery mutant phenotypes and identify candidate genes responsible for these phenotypes. These studies integrate traditional biochemical techniques with the powerful genetic tools available through the use of the zebrafish system and will provide a comprehensive approach to better understand how Vegf and Plcg1 govern artery development. Since these signaling mechanisms are evolutionary conserved, the molecules we identify in this proposal represent possible targets for clinical manipulation of pathological blood vessel formation.