Molecular and cellular mechanisms of vascular patterning by PlexinD1 signaling

描述（由申请人提供）：早期胚胎血管形成具有很大的解剖再现性，以确保体内平衡和存活。人们对脉管系统是如何形成其固有结构的了解甚少。为了揭示血管模式的遗传和细胞基础，我们研究了斑马鱼节段血管（Se）的发育。这些血管模式简单，由少数内皮细胞组成，其行为很容易观察到，可以用遗传学、化学和胚胎学工具进行研究。我们已经证明，内皮特异性PlexinD1 （PlxnD1）受体感知的旁分泌排斥信号素（Sema）信号塑造了Se血管的解剖结构。Sema-PlxnD1信号传导过程中内皮细胞内发生哪些分子事件？细胞内PlxnD1功能的机制基础和所涉及的分子参与者尚不清楚。PlxnD1具有细胞质结构域，与轴突Plxns中具有排斥活性的结构域相似，并且具有PlxnD1特异性序列特征。PlxnD1的血管模式活动是否需要这些基序尚不清楚。因此，在Specific Aim 1中，我们将询问plxnd1介导的硒血管模式是否需要这些结构域，并定义它们的细胞和生化作用。接下来，我们将描述gipc1， PlxnD1信号的第一个候选调制器/效应器。gipc1在发育中的血管中特异性转录，它编码一种与PlxnD1细胞质尾部物理相关的蛋白质，其活性对硒血管的形成至关重要。因此，在Specific Aim 2中，我们将确定GIPC1在plxnd1介导的硒血管模式中的分子和细胞作用。为此，我们将定义介导其与PlxnD1物理关联的GIPC1结构域，询问这种相互作用对于PlxnD1介导的硒血管模式是否必要，定义GIPC1是否促进或拮抗PlxnD1信号传导，并分析GIPC1水平降低或升高的动物的硒血管模式表型。最后，在Specific Aim 3中，我们将重点放在通往灭亡之路（rtp）上，这是一种具有Se血管缺陷的突变体，类似于plxnD1 null的突变体。我们将从克隆rtp开始，探索其与PlxnD1信号的关系。然后，我们将确定依赖于rtp的内皮细胞行为。我们的研究有望为健康和疾病中形成血管树的遗传程序和细胞行为提供关键见解。公共卫生相关性：培养系统的可复制解剖组织对体内平衡和生存至关重要。PlexinD1是一种内皮特异性受体，在血管形态形成中起关键作用。在本研究中，我们以斑马鱼为研究对象，探讨了PlexinD1信号在血管形成过程中的分子和细胞机制。

DESCRIPTION (provided by applicant): Early embryonic blood vessels form with great anatomical reproducibility to ensure homeostasis and survival. How the vasculature acquires its stereotypical architecture is poorly understood. To uncover the genetic and cellular basis of vascular patterning we study the development of the zebrafish Segmental (Se) vessels. These vessels have a simple pattern, are made of few endothelial cells whose behaviors are easily observed and can be studied with genetic, chemical and embryological tools. We have shown that paracrine, repellent Semaphorin (Sema) cues sensed by the endothelial-specific PlexinD1 (PlxnD1) receptor shape the Se vessel anatomy. What molecular events occur inside the endothelial cell during Sema-PlxnD1 signaling? The mechanistic basis of intracellular PlxnD1 function and the molecular players involved are unknown. PlxnD1 has cytosolic domains similar to those essential in axonal Plxns for repulsive activity and also PlxnD1- specific sequence features. Whether these motifs are required for PlxnD1's vascular patterning activity remains unexplored. Thus, in Specific Aim 1 we will ask if these domains are required for PlxnD1-mediated Se vessel patterning and define their cellular and biochemical roles. Next, we will characterize gipc1, the first identified candidate modulator/effector of PlxnD1 signaling. gipc1 is specifically transcribed in the developing vessels, it encodes a protein that physically associates with the PlxnD1 cytosolic tail and its activity is essential for Se vessel patterning. Thus, in Specific Aim 2 we will determine the molecular and cellular roles of GIPC1 for PlxnD1-mediated Se vessel patterning. To do this we will define the GIPC1 domains that mediate its physical association with PlxnD1, ask if this interaction is necessary for PlxnD1-mediated Se vessel patterning, define if GIPC1 promotes or antagonizes PlxnD1 signaling and analyze the Se vessel patterning phenotypes of animals with reduced or increased levels of gipc1. Finally, in Specific Aim 3 we focus on road to perdition (rtp), a mutant with Se vessel defects similar to those of plxnD1 nulls. We will start by cloning rtp to explore its relationship to PlxnD1 signaling. Then, we will determine the endothelial cell behaviors that are rtp-dependent. Our studies promise to offer key insights into the genetic programs and cellular behaviors that shape the vascular tree in both health and disease. PUBLIC HEALTH RELEVANCE: The reproducible anatomical organization of the vaculature is essential for homeostasis and survival. PlexinD1 is an endothelial specific receptor that plays a key role in shaping the vascular pattern. In this proposal we address the molecular and cellular mechanism of vascular patterning by PlexinD1 signaling using zebrafish.