Notch Signaling in Mouse Arterial-Venous Specification

描述(申请人提供)：动脉和静脉(AV)分支的形成必须精确协调，以产生对血管功能至关重要的适当的房室回路。房室协调的机制，特别是成对的、平行的动脉和静脉之间的协调机制，人们知之甚少。我们的长期目标是阐明哺乳动物房室回路的遗传程序。在之前的资助时期，我们报道了发育中的背主动脉(DA)和大静脉(CV)的管腔大小是同步的。Noch信号控制动脉的规格和动脉和静脉内皮细胞(ECs)在各自血管中的分配，从而平衡发育中的DA和CV的大小。我们还在小鼠身上获得了初步数据，表明DA和CV的形成并不像之前认为的那样，是由预先确定的动脉和静脉内皮细胞启动的。相反，我们的工作提出了一个新的哺乳动物平行房室对形态发生的循序渐进模型：原始的未指定动脉在静脉之前组装；随后在两个血管中经历一段混合的房室身份；最后，混合的内皮细胞被分离到具有协调大小的统一指定的血管中。这项资助的具体目的是测试这一新的范例，并确定在小鼠平行的房室对的形态发生中由房室信号介导的细胞机制。我们的战略是采取跨学科的方法，包括尖端小鼠遗传学、细胞谱系命运图和成像技术。我们最近建造了一种定制的双光子激发荧光显微镜，能够对活着的小鼠组织中1000 5米深的血管系统进行成像，实现了对以前无法接触到的血管结构的前所未有的分辨率。目的1研究血管内皮细胞生长因子(VEGF)介导的细胞分化在原始DA(PDA)和CV(PCV)中形成异质性动静脉内皮细胞的机制。目的2研究细胞分离作为一种机制将PDA中静脉命定的内皮细胞分选到PCV。目的3确定Notch信号在平行动、静脉配对发育中的作用。目的4确定成人平行动静脉对声学指标中内皮细胞Notch1和COUP-TFII的需求量。这项研究的成功完成将从概念上促进我们对平行房室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。对AV规范的分子机制的基础知识将启发研究疾病背景下的血管再生和静脉移植工程的新方法。将双光子高分辨率成像与先进的小鼠胚胎细胞谱系追踪相结合，将是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，并阐明这些过程潜在的分子机制。未来，对Notch在哺乳动物出生前和出生后血管功能的基本了解将指导对其在病理条件下的血管再生功能的研究，如心脏病发作、中风和其他缺血性疾病。最终，我们对Notch通路的了解可能会导致识别新的药物靶点和心血管疾病的治疗干预措施。

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.