Endothelial lineage specification and differentiation in vertebrate embryos

描述（由申请人提供）：细胞培养研究的最新进展为内皮细胞谱系如何被多种信号通路调节提供了宝贵的见解。然而，在发育中的胚胎中调节内皮细胞谱系初始规格的细胞和分子机制在很大程度上是未知的。为了描述内皮细胞谱系的发育起源，我们之前在斑马鱼原肠胚最腹侧区域生成了激光辅助单细胞分辨率命运图，该图提供了成血管细胞的第一个详细分布模式，这是内皮细胞和造血细胞谱系的假设共同前体。此外，在我们之前的研究中，我们发现成血管细胞只产生内皮谱系的一个子集，而大多数来源于内皮特异性祖细胞，这表明内皮谱系的发育起源是不均匀的。我们最近观察到，无血管突变胚胎在发育后期从最初缺乏内皮细胞中恢复过来，这进一步支持了这一观点，并表明内皮谱系的祖细胞由空间和时间上不同的亚群组成。在这一建议中，我们计划采用多方面的方法来描述内皮祖细胞的异质性，并阐明内皮祖细胞不同亚群对Wnt信号的不同反应，根据我们的初步数据，Wnt信号似乎是内皮谱系规范的关键调节剂。为实现这些目标，制定了三个具体目标。首先，我们将扩展我们的单细胞分辨率命运图分析，以测试成血管细胞是否存在于原肠管的其他区域。这种方法还将确定具有内皮潜能的祖细胞在胚胎范围内的分布模式，并将使我们能够识别具有不同发育潜能的内皮祖细胞亚群。在第二个目标中，我们计划确定参与无血管突变胚胎血管恢复的内皮细胞的细胞起源。对两个先前分离的无血管斑马鱼突变体的分析将确定以一种暂时独特的方式产生内皮谱系的祖细胞亚群。最后，我们将通过使用能够操纵Wnt活性时间和地点的转基因系来研究内皮祖细胞亚群对Wnt信号的反应是否不同。了解内皮谱系的发育异质性将为内皮谱系在发育过程中如何建立提供宝贵的见解。此外，提出的研究将增强我们对发育过程中多能祖细胞如何产生不同细胞类型的认识。此外，所提出的研究可能为多能祖细胞的治疗应用提供必要的基础，这些祖细胞具有改善影响人类循环系统的临床状况的能力。公共卫生相关性。本研究旨在了解内皮细胞是如何从祖细胞发育而来的。从拟议的研究中获得的信息将增强我们目前对多能祖细胞（例如干细胞）向特定细胞类型分化的调节机制的了解。此外，它还将有助于我们了解许多血管疾病的病因，并设计一种更好的方法来利用多能祖细胞进行治疗。

DESCRIPTION (provided by applicant): Recent advances in cell culture studies provide invaluable insights on how the endothelial lineage is modulated by diverse signaling pathways. However, the cellular and molecular mechanisms that regulate the initial specification of endothelial lineage within developing embryos are largely unknown. To delineate the developmental origin of the endothelial lineage, we previously generated a laser assisted single-cell resolution fate map in the most ventral region of zebrafish gastrula that provided the first detailed distribution pattern of the hemangioblast, a hypothetical common precursor for both endothelial and hematopoietic lineages. Additionally, in our previous study, we found that hemangioblasts only produce a subset of the endothelial lineage, while the majority originates from endothelial specific progenitors, indicating the heterogeneous developmental origin of endothelial lineage. Our recent observation that avascular mutant embryos recover from their initial lack of endothelial cells later in development, further supports this idea, and suggests that the progenitors of the endothelial lineage consist of spatially and temporally distinct subpopulations. In this proposal, by using a multifaceted approach, we plan to delineate the heterogeneity of endothelial progenitors, and elucidate how distinct subpopulations of endothelial progenitors respond differently to Wnt signaling, which, based on our preliminary data, appears to be a key modulator of endothelial lineage specification. Three specific aims are designed to achieve these goals. First, we will expand our single-cell resolution fate map analyses to test whether hemangioblasts exist in other areas of the gastrula. This approach will also determine the embryo-wide distribution patterns of progenitors with endothelial potential, and will allow us to identify subpopulations of endothelial progenitors with distinct developmental potentials. In the second aim, we plan to define the cellular origin of endothelial cells involved in the vascular recovery of avascular mutant embryos. The analyses on two of previously isolated avascular zebrafish mutants will identify subpopulations of progenitors that generate endothelial lineages in a temporally distinctive manner. Lastly, we will examine whether subpopulations of endothelial progenitors respond differently to Wnt signaling by using transgenic lines that are capable of manipulating the time and place of Wnt activity. Understanding the developmental heterogeneity of the endothelial lineage will provide invaluable insights on how endothelial lineage is established during development. In addition, the proposed research will enhance our knowledge on how distinct cell types emerge from pluripotent progenitors during development. Furthermore, the proposed research may provide essential groundwork for the therapeutic application of pluripotent progenitors with the ability to ameliorate clinical conditions affecting the circulatory system in humans. PUBLIC HEALTH RELEVANCE. The proposed research aims to understand how endothelial cells emerge during development from progenitors. Information acquired from the proposed research will enhance our current knowledge on the mechanisms that regulate the differentiation of pluripotent progenitors (for example, stem cells) into specific cell types. Furthermore, it will also help us to understand the etiology of many vascular diseases and design a better way of using pluripotent progenitors for the therapeutic purposes.