Coup-tf dependent mechanisms of ventricular and hemangioblast specification

描述(由申请人提供)：先天性心血管畸形是最常见的先天性出生缺陷之一，几乎发生在1%的人口中。然而，我们并不了解这些缺陷中大多数潜在的分子本质。为了开发有效的体外干细胞和再生疗法，旨在预防出生缺陷和治疗成人心血管疾病，对体内心血管发育的机制有一个准确的了解是至关重要的。因此，我们实验室的长期目标是了解脊椎动物发育过程中心血管规范的机制。这项资助的具体目的是阐明鸡卵清蛋白上游促进转录因子(COUP-TFS)通过斑马鱼限制脑室规格和促进血管母细胞规格的机制。在人类中，COUP-TFS是正常心脏发育所必需的。在小鼠身上的研究表明，Coup-TF2是心血管正常早期发育所必需的，但在这些早期缺陷中COUP-TF2的作用机制尚不清楚。我们的初步结果表明，斑马鱼Coup-Tf1a存在心血管缺陷，这让人想起哺乳动物的Coup-TF2。此外，我们的初步结果支持这一假设，即斑马鱼Coup-tf1a具有限制心室细胞规范和促进血管母细胞规范的明确要求。在具体目标1中，我们将使用细胞移植和谱系追踪实验来确定哪些细胞需要Coup-tf1a来限制心室细胞的规格。我们还将确定FGF8和Coup-tf1a在限制心肌细胞特性中的遗传关系。之前还没有人认识到COUP-TFS在脊椎动物血管母细胞规范中的作用。在特定目标2中，我们将使用上位性和细胞移植分析来确定COUP-TF1a与已知的血管母细胞规范调节因子的关系，以及哪些细胞需要COUP-TF1a来满足血管母细胞规范。目前尚不清楚COUP-TF1a在心室细胞和血管母细胞中的下游因子。在具体目标3中，我们将使用功能丧失方法来确定是否需要候选基因来调节Coup-tf1a下游的这些不同的过程。总之，这些研究将提高我们对发育过程中正常心血管规范事件的理解，为未来旨在修复儿童先天性心血管缺陷和成人受伤或患病心血管组织的治疗提供基础。

DESCRIPTION (provided by applicant): Congenital cardiovascular malformations are among the most common congenital birth defects, occurring in almost 1% of the population. However, we do not understand the underlying molecular nature of the majority of these defects. In order to develop effective in vitro stem cell and regenerative therapies aimed at preventing birth defects and healing cardiovascular diseases in adults, it is critical to have a precise understanding of the mechanisms directing cardiovascular development in vivo. Therefore, the long-term goal of our lab is to understand the mechanisms of cardiovascular specification during vertebrate development. The specific aims of this grant are to elucidate the mechanisms by which chicken ovalbumin upstream promoting-transcription factors (Coup-tfs) are required to restrict ventricular specification and promote hemangioblast specification using zebrafish. In humans, Coup-tfs are required for normal heart development. Studies in mice have demonstrated that Coup-tf2 is required for proper early cardiovascular development, but the mechanisms underlying Coup-tf2 function in these early defects are not understood. Our preliminary results suggest that zebrafish Coup-tf1a has cardiovascular defects reminiscent of mammalian Coup-tf2. Moreover, our preliminary results support the hypothesis that zebrafish Coup-tf1a has distinct requirements restricting ventricular cell specification and promoting hemangioblast specification. In Specific Aim 1, we will use cell transplantation and lineage tracing experiments to determine which cells required Coup-tf1a to restrict ventricular cell specification. We will also determine the genetic relationship of FGF8 and Coup-tf1a in restricting ventricular cell specification. A role for Coup-tfs in vertebrate hemangioblast specification has not been previously recognized. In Specific Aim 2, we will use epistasis and cell transplantation analysis to determine the relationship of Coup-tf1a to known regulators of hemangioblast specification and what cells require Coup-tf1a for hemangioblast specification. Factors downstream of Coup-tf1a in ventricular cell and hemangioblast specification are not known. In Specific Aim 3, we will use loss-of-function methods to determine if candidate genes are required for regulating these distinct processes downstream of Coup-tf1a. Altogether, these studies will improve our understanding of normal cardiovascular specification events during development, providing the basis for future therapies aimed at healing congenital cardiovascular defects in children and injured or diseased cardiovascular tissues in adults.