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由不同的信号通路调节提供了宝贵的见解。然而，调控发育中胚胎内皮细胞系最初规范的细胞和分子机制在很大程度上是未知的。为了描绘内皮细胞系的发育起源，我们先前在斑马鱼原肠胚的最腹侧区域制作了一张激光辅助单细胞分辨率命运图，提供了血管母细胞的第一个详细的分布模式，血管母细胞是假设的内皮和造血系的共同前体。此外，在我们之前的研究中，我们发现血管母细胞只产生内皮细胞谱系的一部分，而大多数起源于内皮细胞特异的前体细胞，这表明内皮细胞谱系的异质性发育起源。我们最近的观察发现，无血管突变胚胎在发育后期从最初的内皮细胞缺失中恢复过来，进一步支持了这一观点，并表明内皮细胞谱系的祖先由空间和时间上不同的亚群组成。在这个提案中，我们计划使用多方面的方法来描绘内皮祖细胞的异质性，并阐明不同亚群的内皮祖细胞如何对Wnt信号做出不同的反应，根据我们的初步数据，Wnt似乎是内皮细胞谱系指定的关键调节器。为了实现这些目标，我们设计了三个具体目标。首先，我们将扩展我们的单细胞分辨率命运图分析，以测试原肠胚的其他区域是否存在血管母细胞。这种方法还将确定具有内皮潜能的前体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力的内皮前体细胞亚群。在第二个目标中，我们计划确定内皮细胞在无血管突变胚胎血管恢复中的细胞来源。对之前分离的两个无血管斑马鱼突变体的分析将确定以一种在时间上独特的方式产生内皮血统的祖细胞亚群。最后，我们将通过使用能够操纵Wnt活动时间和地点的转基因品系来检查内皮祖细胞亚群是否对Wnt信号做出不同的反应。了解内皮细胞系的发育异质性将为内皮细胞系在发育过程中如何建立提供宝贵的见解。此外，拟议的研究将加强我们对多能祖细胞在发育过程中如何产生不同类型的细胞的了解。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，这些祖细胞能够改善影响人类循环系统的临床条件。

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.