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提供了关于内皮细胞谱系如何通过不同信号传导途径调节的宝贵见解。然而，在发育胚胎内调节内皮细胞谱系的初始特化的细胞和分子机制在很大程度上是未知的。为了描绘内皮细胞谱系的发育起源，我们先前在斑马鱼原肠胚的最腹侧区域中产生了激光辅助的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，在我们以前的研究中，我们发现成血管细胞只产生内皮细胞谱系的一个子集，而大多数来源于内皮特异性祖细胞，表明内皮细胞谱系的异质性发育起源。我们最近的观察，无血管突变胚胎恢复从他们最初缺乏内皮细胞在后来的发展，进一步支持了这一观点，并表明，内皮细胞系的祖细胞组成的空间和时间上不同的亚群。在这个提议中，通过使用多方面的方法，我们计划描绘内皮祖细胞的异质性，并阐明不同的内皮祖细胞亚群如何对Wnt信号做出不同的反应，根据我们的初步数据，Wnt信号似乎是内皮谱系特异性的关键调节剂。为实现这些目标，制定了三个具体目标。首先，我们将扩大我们的单细胞分辨率命运图分析，以测试是否成血管细胞存在于原肠胚的其他领域。这种方法还将确定具有内皮潜能的祖细胞的全胚胎分布模式，并使我们能够鉴定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。对先前分离的两个无血管斑马鱼突变体的分析将鉴定以时间上独特的方式产生内皮谱系的祖细胞亚群。最后，我们将研究是否内皮祖细胞亚群不同的Wnt信号通过使用转基因株系，能够操纵的时间和地点的Wnt活性。了解内皮细胞谱系的发育异质性将为内皮细胞谱系在发育过程中如何建立提供宝贵的见解。此外，拟议的研究将增强我们对不同细胞类型如何在发育过程中从多能祖细胞中出现的知识。此外，拟议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。这项研究旨在了解内皮细胞在祖细胞发育过程中是如何出现的。从拟议研究中获得的信息将增强我们目前对调节多能祖细胞（例如干细胞）分化为特定细胞类型的机制的了解。此外，它也将帮助我们了解许多血管疾病的病因，并设计一个更好的方法，利用多能祖细胞的治疗目的。

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.