Molecular and cellular mechanisms of vascular patterning by PlexinD1 signaling

描述（由申请人提供）：早期胚胎血管的形成具有很好的解剖学再现性，以确保体内平衡和存活。血管系统如何获得其定型的架构是知之甚少。为了揭示血管模式的遗传和细胞基础，我们研究了斑马鱼分节（Se）血管的发育。这些血管具有简单的模式，由少数内皮细胞组成，其行为易于观察，并且可以用遗传学，化学和胚胎学工具进行研究。我们已经表明，旁分泌，排斥脑信号蛋白（Sema）的线索感觉内皮特异性丛蛋白D1（PlxnD 1）受体形状的硒血管解剖。在Sema-PlxnD 1信号传导过程中，内皮细胞内发生了什么分子事件？细胞内PlxnD 1功能的机制基础和参与的分子球员是未知的。PlxnD 1的胞质结构域相似的轴突Plxns的排斥活动，也PlxnD 1特异性的序列特征。PlxnD 1的血管图案化活性是否需要这些基序仍然未被探索。因此，在具体目标1中，我们将询问这些结构域是否是PlxnD 1介导的Se血管图案化所需的，并定义其细胞和生化作用。接下来，我们将表征gipc 1，这是第一个确定的PlxnD 1信号转导的候选调节剂/效应子。gipc 1在发育中的血管中特异性转录，它编码与PlxnD 1胞质尾部物理相关的蛋白质，并且其活性对于Se血管图案化是必需的。因此，在具体目标2中，我们将确定PlxnD 1介导的硒血管图案化的GIPC 1的分子和细胞作用。要做到这一点，我们将定义的GIPC 1域介导的物理协会与PlxnD 1，问如果这种相互作用是必要的PlxnD 1介导的硒血管图案，定义如果GIPC 1促进或拮抗PlxnD 1信号传导和分析硒血管图案表型的动物减少或增加的gipc 1水平。最后，在具体目标3中，我们关注的是毁灭之路（road to perdition，简称RDD），这是一种具有类似于plxnD 1空值的Se血管缺陷的突变体。我们将从克隆pxD开始，以探索其与PlxnD 1信号传导的关系。然后，我们将确定内皮细胞的行为是依赖于rtp。我们的研究有望为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见解。公共卫生相关性：血管的可再生解剖组织对于体内平衡和生存至关重要。丛蛋白D1是一种内皮特异性受体，在形成血管模式中起关键作用。在这个建议中，我们解决了血管图案的分子和细胞机制PlexinD 1信号使用斑马鱼。

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.