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，几乎占人口的1%。然而，我们并不了解大多数这些缺陷的潜在分子性质。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，精确了解体内指导心血管发育的机制至关重要。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。该资助的具体目的是阐明鸡卵白蛋白上游促进转录因子（Coup-tfs）限制斑马鱼心室特异性和促进成血管细胞特异性的机制。在人类中，Coup-tfs是正常心脏发育所必需的。小鼠研究表明，Coup-tf2是早期心血管发育所必需的，但Coup-tf2在这些早期缺陷中的作用机制尚不清楚。我们的初步结果表明，斑马鱼Coup-tf1a具有与哺乳动物Coup-tf2相似的心血管缺陷。此外，我们的初步结果支持斑马鱼Coup-tf1a具有限制心室细胞特化和促进成血管细胞特化的不同需求的假设。在Specific Aim 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来限制心室细胞的分化。我们还将确定FGF8和Coup-tf1a在限制心室细胞分化方面的遗传关系。Coup-tfs在脊椎动物成血管细胞分化中的作用以前没有被认识到。在Specific Aim 2中，我们将使用上位性和细胞移植分析来确定Coup-tf1a与已知的成血管细胞规范调节因子的关系，以及哪些细胞需要Coup-tf1a来实现成血管细胞规范。Coup-tf1a在心室细胞和成血管细胞分化中的下游因子尚不清楚。在Specific Aim 3中，我们将使用功能丧失方法来确定候选基因是否需要调节Coup-tf1a下游的这些不同过程。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，为未来治疗儿童先天性心血管缺陷和成人心血管损伤或病变组织提供基础。

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.