Molecular and cellular mechanisms of vascular patterning by PlexinD1 signaling

描述(申请人提供)：早期胚胎血管的形成具有极大的解剖学重复性，以确保动态平衡和存活。血管系统是如何获得其典型的结构的，人们对此知之甚少。为了揭示斑马鱼血管构型的遗传和细胞基础，我们研究了斑马鱼节段性血管的发育。这些血管有一个简单的模式，由很少的内皮细胞组成，这些内皮细胞的行为很容易观察到，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)受体感受到的旁分泌、排斥性信号素(SEMA)信号塑造了血管解剖。在Sema-PlxnD1信号转导过程中，内皮细胞内发生了哪些分子事件？细胞内PlxnD1功能的机制基础和参与其中的分子成员尚不清楚。PlxnD1具有类似于轴突Plxns中具有排斥活性的胞浆结构域，以及PlxnD1特有的序列特征。这些模体是否是PlxnD_1‘S血管构图活动所必需的，目前尚不清楚。因此，在特定的目标1中，我们将询问这些结构域是否是PlxnD1介导的Se血管模式所必需的，并确定它们的细胞和生化作用。接下来，我们将对gipc1进行鉴定，它是第一个确定的plxnD1信号的候选调节器/效应器。Gipc1是在发育中的血管中特异转录的，它编码一种与PlxnD1胞浆尾巴物理相关的蛋白质，它的活性对于Se血管模式是必不可少的。因此，在特定的目标2中，我们将确定GIPC1在PlxnD1介导的Se血管构型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Se血管构型是否必要，定义GIPC1是促进还是拮抗PlxnD1信号转导，并分析giPC1水平降低或增加的动物的Se血管构图表型。最后，在特定目标3中，我们重点关注死亡之路(RTP)，这是一种突变体，具有与plxnD1零相似的Se血管缺陷。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RTP的内皮细胞行为。我们的研究承诺为在健康和疾病中塑造血管树的遗传程序和细胞行为提供关键的见解。公共卫生相关性：血管的可复制解剖组织对于体内平衡和生存是必不可少的。PlexinD1是一种内皮特异性受体，在塑造血管模式中发挥关键作用。在这项建议中，我们研究了斑马鱼通过PlexinD1信号来形成血管图案的分子和细胞机制。

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.