Regulation of cardiovascular transcription

描述（由申请人提供）：先天性心脏缺陷影响了近1%的活产婴儿，是美国婴儿死亡的最常见原因。最严重的先天性心脏缺陷形式是那些涉及流出道和室间隔，而瓣膜异常是最常见的先天性心脏缺陷形式。提出的研究的长期目标是确定控制心血管发育的分子机制和途径，并强调这些先天性心脏异常。具体来说，该应用的重点是鉴定第二心野（SHF）及其在OFT和RV中的衍生物在发展过程中发挥作用的转录途径和机制。lim同源域转录因子Isl1和MADS结构域转录因子MEF2C是SHF发展的重要调控因子。最近的研究表明，Mef2c基因是SHF中Isl1的直接转录靶点，在AHF中形成了转录途径的基础。然而，尽管Isl1在SHF谱系中具有早期的重要作用，但SHF中Isl1基因的上游转录调节因子仍未明确。同样，MEF2C的下游靶点尚不清楚，尽管MEF2C是心脏发育所必需的，并且其功能在SHF中特别需要用于适当的OFT对齐和心内膜垫重构。因此，该提案将验证以下假设：SHF中需要MEF2C来控制SHF来源的细胞数量或向OFT心内膜缓冲层迁移，MEF2C是心内膜缓冲层中Nfatc1的直接转录激活剂。这是很重要的，因为转录因子NFATc1是心内膜缓冲重塑和心脏瓣膜成熟所必需的。该研究还验证了一种假说，即SHF中Isl1基因的表达是通过一种新型模块化增强子调控的，该增强子将上游信号整合到前外侧中胚层中心血管祖细胞的直接表达中。提出了两个具体目标。具体目标之一将通过检查MEF2C对shf来源的细胞向心脏迁移的需求，以及使用条件敲除方法在小鼠中进行细胞增殖和存活的需求，确定MEF2C在OFT对齐和心内膜缓冲发育中的需求。Specific Aim 1还将通过使用转基因小鼠方法，通过Nfatc1心内膜特异性增强子中保守的、一致的MEF2位点，确定Nfatc1是否是流出道心内膜垫中MEF2C的直接转录靶点。Specific Aim 2将通过定义一种新型SHF特异性Isl1转录增强子的上游调控子，确定SHF中Isl1转录的调控子。目标是将Isl1置于转录和信号通路中，这将为控制心脏发育和Isl1+祖细胞自我更新和分化的早期事件提供重要信息。

DESCRIPTION (provided by applicant): Congenital cardiac defects affect nearly 1% of all live births and are the most common cause of infant mortality in the United States. Among the most severe forms of congenital heart defects are those involved in outflow tract and interventricular septation, whereas valve anomalies are the most common form of congenital heart defect. The long-term goal of the proposed studies is to define the molecular mechanisms and pathways that control cardiovascular development and underscore these congenital cardiac anomalies. Specifically, this application is focused on the identification of transcriptional pathways and mechanisms that function during the development of the second heart field (SHF) and its derivatives in the OFT and RV. The LIM-homeodomain transcription factor Isl1 and the MADS domain transcription factor MEF2C are essential regulators of SHF development. Recent work has established that the Mef2c gene is a direct transcriptional target of Isl1 in the SHF, forming the basis of a transcriptional pathway in the AHF. However, the upstream transcriptional regulators of the Isl1 gene in the SHF remain undefined in spite of the early, essential role for Isl1 in that lineage. Similarly, the downstream targets of MEF2C are not known, even though Mef2c is required for heart development, and its function is required specifically in the SHF for proper OFT alignment and endocardial cushion remodeling. This proposal will therefore test the hypothesis that MEF2C is required in the SHF to control cell number or migration of SHF-derived cells into the OFT endocardial cushions and that MEF2C is a direct transcriptional activator of Nfatc1 in the endocardial cushions. This is significant since the transcription factor NFATc1 is required for endocardial cushion remodeling and heart valve maturation. This proposal will also test the hypothesis that the expression of the Isl1 gene in the SHF is regulated via a novel modular enhancer that integrates upstream signals to direct expression in cardiovascular progenitors in the anterior lateral mesoderm. Two specific aims are proposed. Specific aim one will determine the requirement of MEF2C in OFT alignment and endocardial cushion development by examining the requirement of Mef2c for migration of SHF-derived cells into the heart and for cell proliferation and survival using a conditional knockout approach in mice. Specific Aim 1 will also determine if Nfatc1 is a direct transcriptional target of MEF2C in the outflow tract endocardial cushions via a conserved, consensus MEF2 site present in the Nfatc1 endocardial- specific enhancer using a transgenic mouse approach. Specific Aim 2 will identify regulators of Isl1 transcription in the SHF by defining upstream regulators of a novel SHF-specific Isl1 transcriptional enhancer. The goal is to place Isl1 into a transcriptional and signaling pathway, which will provide essential information about the early events controlling heart development and Isl1+ progenitor self-renewal and differentiation. PUBLIC HEALTH RELEVANCE: Heart defects are the most common class of birth defect in the United States, affecting nearly 1% of all newborns. Among the most serious of these defects are the cyanotic lesions, which affect septation of the ventricles and outflow tracts and allow oxygenated and deoxygenated blood to mix. The proposed studies will contribute to the understanding of the molecular pathways and the biochemical and genetic mechanisms controlling heart development. Defining these pathways is essential to determine how to reactivate the genetic programs controlling the development of heart tissue for the purpose of regeneration and repair, tissue engineering, directed differentiation of induced pluripotent cells, and diagnosis and intervention in cardiovascular birth defects.