Notch Signaling in Mouse 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，以生成血管功能所必需的适当AV回路。房室协调的机制，特别是成对的、平行的动脉和静脉之间的房室协调机制还知之甚少。我们的长期目标是阐明哺乳动物AV电路的遗传程序。在上一个资助期，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信号传导控制动脉特化以及动脉和静脉内皮细胞（EC）向其各自血管中的分配，从而平衡发育中的DA和CV的大小。我们还在小鼠中获得了初步数据，表明DA和CV的形成并不像以前认为的那样由预先确定的动脉和静脉EC启动。相反，我们的工作提出了一个新的逐步模式的哺乳动物平行AV对形态发生：原始的未指定的动脉组装之前的静脉，其次是一个阶段的混合AV身份在两个血管，最后混合EC被隔离到统一指定的血管与协调的大小。这项资助的具体目的是测试这种新的范例，并确定在小鼠中平行AV对的形态发生中由AV信号介导的细胞机制。我们的策略是采取跨学科的方法，包括尖端的小鼠遗传学，细胞谱系命运映射和成像技术。我们最近建立了一个定制的2光子激发荧光显微镜，能够成像血管1000 5米深的活小鼠组织，实现前所未有的分辨率以前无法访问的血管结构。目的1研究血管内皮生长因子（VEGF）介导的原始DA（pDA）和CV（pCV）内皮细胞分化的机制。目的2检测细胞分离作为将pDA中静脉命运的EC分选到pCV中的机制。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Notch 1和Coup-TFII在成人平行动、静脉对AV特化中的需求。这项研究的成功完成将在概念上推进我们对平行AV对的形态发生和维持的认识，提供有关动脉和静脉起源的证据。AV特化的分子机制的基础知识将激发新的方法来研究疾病背景下的血管再生和静脉移植工程。双光子高分辨率成像与活小鼠胚胎中的尖端细胞谱系追踪相结合，将是整个哺乳动物血管发育领域的重大技术创新。 公共卫生相关性：我们的研究旨在揭示Notch信号通路在血管分化和维持中的作用，并阐明这些过程的潜在分子机制。在未来，这种对Notch在出生前和出生后哺乳动物血管功能的基本理解将指导研究其在病理条件下（如心脏病发作，中风和其他缺血性疾病）的血管再生功能。最终，我们对Notch通路的理解可能会导致心血管疾病的新药物靶点和治疗干预的确定。

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. PUBLIC HEALTH RELEVANCE: Our study aims to reveal the role of the Notch signaling pathway in vascular differentiation and maintenance and illuminate potential molecular mechanisms underlying these processes. In the future, this basic understanding of Notch in pre- and post-natal mammalian vascular function will guide investigations into its function in vessel regeneration under pathological conditions, such as heart attack, stroke, and other ischemic diseases. Ultimately, our understanding of the Notch pathway may lead to the identification of novel drug targets and therapeutic interventions for cardiovascular diseases.