The Role of Endoglin in Hemangioblast Development

描述（由申请人提供）：该资助旨在了解多能细胞对造血和内皮细胞命运的谱系决定所涉及的事件。这一知识是基于胚胎干细胞治疗血液和内皮疾病的再生医学发展的基础。我们目前的模型假设造血和内皮谱系起源于中胚层的一个共同的双能祖细胞——血管母细胞。尽管存在针对成血管细胞的体外实验，但对于是什么使一些中胚层细胞采用这种命运而其他细胞不采用这种命运，以及成血管细胞后代如何在内皮或造血命运之间进行选择，我们知之甚少。在我们寻找成血管细胞调节分子的过程中，我们发现内皮素（engin），一种tgf - β超家族成员的辅助受体，在成血管细胞规范中起着关键作用。我们使用体外分化的胚胎干细胞进行的初步研究表明，在缺乏内啡肽的情况下，成血管细胞的频率显著降低。此外，我们发现内皮素在胚胎样体（EB）分化的第3天标记成血管细胞。迄今为止，唯一已知的标记成血管细胞的细胞表面抗原是Flk-1。我们的试点结果指出了内啡肽在成血管细胞规范中的作用，并提供了内啡肽在造血功能而不是内皮功能中所需要的证据。在这项研究中，我们建议剖析内啡肽调节成血管细胞规格的机制，并了解其在造血和内皮承诺中的作用。在Aim 1中，我们建议使用诱导基因表达来探索内啡肽在时间定义窗口中的功能，以评估其在促进成血管细胞规范和造血或内皮谱系选择中的作用。为了阐明内啡肽在成血管细胞发育中的作用机制，在Aim 2中，我们将从生物化学角度研究tgf - β信号相关成分的级联反应，包括Smads和上游受体，这些成分在从eng -/-分离的成血管细胞前体中高度富集的细胞组分中，以及从EBs第3天开始的内啡肽过表达和控制细胞中。这些结果将通过用适当的活化蛋白挽救eng /-表型来补充遗传。在Aim 3中，我们将通过研究内啡肽在体内的作用来验证我们的体外结果。我们假设在胚胎中观察到的贫血不是由于血流量减少而引起的继发性影响，而是红细胞生成缺陷的结果，这与成血管细胞发育不当有关。

DESCRIPTION (provided by applicant): This grant aims toward an understanding of the events involved in lineage determination as pluripotent cells commit to hematopoietic and endothelial fates. This knowledge is fundamental to the development of regenerative medicine based on embryonic stem (ES) cells for the treatment of diseases of hematologic and endothelial origin. Our current model postulates that the hematopoietic and endothelial lineages arise in mesoderm from a common bipotent progenitor - the hemangioblast. Although an in vitro assay exists for the hemangioblast, very little is known about what enables some mesodermal cells to adopt this fate while others do not, and how the hemangioblast progeny select between endothelial or hematopoietic fates. In our search for hemangioblast regulatory molecules, we have discovered that endoglin (eng), an ancillary receptor for several members of the TGF-beta superfamily, plays a critical role in hemangioblast specification. Our preliminary studies using ES cells differentiated in vitro demonstrate a profound reduction in hemangioblast frequency in the absence of endoglin. Furthermore, we show that endoglin marks the hemangioblast on day 3 of embryoid body (EB) differentiation. To date the only cell surface antigen known to mark the hemangioblast is Flk-1. Our pilot results point to a role for endoglin in hemangioblast specification as well as provide evidence that endoglin is required for hematopoietic commitment but not endothelial. In this study, we propose to dissect the mechanisms by which endoglin regulates hemangioblast specification as well as to understand its role in hematopoietic and endothelial commitment. In Aim 1, we propose to use inducible gene expression to probe the function of endoglin in temporally defined windows to evaluate its role in promoting hemangioblast specification, and hematopoietic or endothelial lineage selection. To elucidate the mechanism associated with endoglin's function in hemangioblast development, in Aim 2, we will investigate biochemically the cascade of components associated with TGF-beta signaling, including Smads and upstream receptors, in cell fractions highly enriched for hemangioblast precursors isolated from eng -/- as well as endoglin overexpressing and control cells from day 3 EBs. These results will be complemented genetically by rescuing the eng -/- phenotype with appropriate activated proteins. In Aim 3, we will validate our in vitro results by studying the role of endoglin in vivo. We hypothesize that the anemia observed in eng -/- embryos is not a secondary effect due to reduced blood flow but a result of defective erythropoiesis, which is associated with improper hemangioblast development.