Notch Signaling in Arterial-Venous Specification

描述（由申请人提供）：动脉和静脉（AV）分支的形成必须精确协调，以产生血管功能所必需的适当的AV电路。房室配位的机制，特别是在成对的，平行的动脉和静脉之间的机制尚不清楚。我们的长期目标是阐明哺乳动物AV电路的遗传程序。在之前的资助期内，我们报道了发育中的背主动脉（DA）和主静脉（CV）的管腔大小是同步的。Notch信号控制动脉规格以及动脉和静脉内皮细胞（ECs）在各自血管中的分配，从而平衡DA和CV的大小。我们还获得了小鼠的初步数据，表明DA和CV的形成不是由预先确定的动脉和静脉ECs引发的，正如之前认为的那样。相反，我们的工作提出了一种新的哺乳动物平行AV对形态发生的阶梯模型：原始的未指定动脉在静脉之前组装；然后是两个血管中混合AV身份的阶段；最后，混合的ECs被分离到尺寸一致的容器中。这项资助的具体目的是测试这一新的模式，并定义由AV信号介导的细胞机制在小鼠平行AV对的形态发生。我们的策略是采取跨学科的方法，包括尖端的小鼠遗传学，细胞谱系命运绘图和成像技术。我们最近建立了一个定制的双光子激发荧光显微镜，能够成像1000米深的活体小鼠组织血管，实现前所未有的分辨率以前难以接近的血管结构。目的1：研究血管内皮生长因子（VEGF）介导的细胞分化作为原初DA （pDA）和CV （pCV）中异质动脉和静脉内皮细胞的机制。目的2：研究细胞分离作为一种将pDA中静脉宿命的ECs分选到pCV的机制。目的3：确定Notch信号在协调平行动脉和静脉对发育中的作用。目的4确定内皮Notch1和Coup-TFII在成人平行动静脉对AV规格中的要求。这项研究的成功完成将从概念上推进我们对平行AV对的形态发生和维持的认识，为动脉和静脉的起源提供证据。了解AV规范的分子机制将激发研究疾病环境下血管再生和静脉移植工程的新方法。双光子高分辨率成像与尖端细胞谱系追踪在活体小鼠胚胎中的结合将是哺乳动物血管发育领域的一项重大技术创新。

DESCRIPTION (provided by applicant): The formation of arterial and venous (AV) branches must be exquisitely coordinated to generate proper AV circuitry essential for vascular function. The mechanism of AV coordination particularly that between paired, parallel arteries and veins is poorly understood. Our long-term objective is to elucidate the genetic program of mammalian AV circuitry. In the previous funding period, we reported that luminal sizes of the developing dorsal aorta (DA) and cardinal vein (CV) are synchronized. Notch signaling controls arterial specification and the allocation of both arterial and venous endothelial cells (ECs) into their respective vessels, thereby balancing the sizes of the developing DA and CV. We have also obtained preliminary data in mice suggesting that DA and CV formation is not initiated by pre-determined arterial and venous ECs, as previously thought. Instead, our work suggests a new step-wise model of mammalian parallel AV pair morphogenesis: the primitive unspecified artery assembles prior to the vein; followed by a phase of mixed AV identities in both vessels; finally the mixed ECs are segregated into uniformly-specified vessels with coordinated sizes. The specific aims of this grant are designed to test this new paradigm and to define the cellular mechanisms mediated by AV signaling in the morphogenesis of parallel AV pairs in mice. Our strategy is to take a cross- disciplinary approach including cutting-edge mouse genetics, cell lineage fate mapping, and imaging technologies. We recently built a custom 2-photon excited fluorescence microscope that is capable of imaging vasculature 1000 5m deep in living mouse tissue, achieving unprecedented resolution of previously inaccessible vascular structures. Aim 1 Examine Vascular Endothelial Growth Factor (VEGF)-mediated cell differentiation as a mechanism underlying heterogeneous arterial- and venous- fated ECs in the primordial DA (pDA) and CV (pCV). Aim 2 Examine cell segregation as a mechanism to sort venous-fated ECs in the pDA to the pCV. Aim 3 Determine the role of Notch signaling in coordinating the development of parallel artery and vein pairs. Aim 4 Determine the requirement of endothelial Notch1 and Coup-TFII in AV specification of adult parallel artery and vein pairs. Successful completion of this study will conceptually advance our knowledge of the morphogenesis and maintenance of parallel AV pairs, providing evidence regarding the origins of arteries and veins. Basic knowledge of the molecular mechanism of AV specification will inspire novel approaches to study blood vessel regeneration and vein graft engineering in disease settings. The combination of 2-photon high-resolution imaging with cutting-edge cell lineage tracing in living mouse embryos will be a major technological innovation for the field of mammalian vascular development at large.