Molecular and cellular mechanisms in cardiac outflow tract formation and defects

项目摘要 心脏流出道(OFT)缺陷的患病率估计为每1000名活产儿中有1-2名。这个 22q11.2缺失综合征或22q11.2DS是心脏OFT缺陷最常见的遗传原因之一。一个 总共有60%患有22q11.2ds的患者患有从轻微到严重的先天性心脏病，包括 二尖瓣主动脉瓣(BAV)、孤立性室间隔缺损(VSD)至法洛四联症(TOF)或持续性 动脉干(PTA)。这些临床发现提示，遗传修饰物可能会影响表型表达。 。在……里面 本项目中，我们建议使用Lgdel/+小鼠模型来了解神经脊之间的关系 细胞(NCC)和邻近心内膜细胞(ECCs)在心脏OFT的形成和重塑中的作用。 NCCs来源的间充质细胞(MCs)和ECCs来源的间质细胞(MCs)分别位于OFT远端和近端。 在心脏发育过程中形成明显的OFT MC边界。正确部署来自两个谱系的MC 确保主-肺-室间隔和半月瓣的正确位置和形成以分离 心脏出口进入体循环和肺循环。NCC在OFT缺陷中的作用一直很好 然而，关于22q11.2DS的研究，ECCs在OFT畸形中的作用尚未被研究。 我们已经开始通过研究Lgdel/+鼠标来填补这个知识空白，Lgdel/+鼠标是通过删除一个鼠标而产生的 包含26个蛋白质编码基因(22q11.2DS基因)的人类22q11.2小鼠共线区域的拷贝。 我们发现了一系列OFT缺陷，范围从孤立的VSD到TOF。结构缺陷的前面是 心内膜向间充质转化过程中OFT-MC边界被破坏，Edn1表达增加 在EMT后OFT重塑过程中，NOTCH1信号和CTGF表达减少。 通过单细胞rna测序(scrna-seq)，我们确定edn1是一个独特的基因程序的一部分。 接受EMT的ECC的一个子集。基于这些发现，我们提出了一个总体假设：22q11.2ds 基因通过调节ECCs的功能和细胞与细胞之间的通讯来控制OFT的发展 来自ECC和NCC的MCs，通过与OFT形成所必需的基因相互作用而产生。我们将对此进行测试 三个具体目标的假设。目的1将确定22q11.2DS基因是否通过 调节EMT基因程序，如果Edn1作用于22q11.2DS基因下游调节这一过程。 目标2将确定22q11.2DS基因是否也通过细胞-细胞相互作用调节OFT重塑 OFT MC边界的模式网络，以及CTGF是否作为EMT后OFT所需的中枢基因发挥作用 重塑，22q11.2DS基因下游。目标3将定义Notch1单倍体不足是否可以 增强22q11.2DS OFT缺陷。在这项研究完成后，我们预计将有新的发现 由重要的综合征和非综合征CHD基因调控的遗传、分子和细胞串扰 对小鼠OFT形态发生的影响。这些信息将提供对心脏发育的更深层次的了解 OFT缺陷的生物学和致病机制，在先天性心脏病中具有更广泛的意义 疾病。

Project Summary Cardiac outflow tract (OFT) defects have an estimated prevalence of 1-2 in 1,000 live births. The 22q11.2 deletion syndrome or 22q11.2DS is one of the most frequent genetic causes of cardiac OFT defects. A total of 60% of patients with 22q11.2DS have congenital heart disease that ranges from mild to severe including bicuspid aortic valve (BAV), isolated ventricular septal defects (VSDs) to tetralogy of Fallot (TOF) or persistent truncus arteriosus (PTA). These clinical findings suggest genetic modifiers may affect phenotypic expression . In this project, we propose to use the Lgdel/+ mouse model to understand the relationship between neural crest cells (NCCs) and adjacent endocardial cells (ECCs) in forming and remodeling of the cardiac OFT. Mesenchymal cells (MCs) derived from NCCs and ECCs occupy the distal and proximal OFT, respectively, and form a distinct OFT MC boundary during heart development. Proper deployment of MCs from the two lineages ensures correct position and formation of aorto-pulmonary-ventricular septum and semilunar valves to separate the heart outlet into the systemic and pulmonary circulation. The function of NCCs in OFT defects has been well studied with respect to 22q11.2DS, however, the role of ECCs in OFT malformations has not been investigated. We have begun to fill this knowledge gap by studying the Lgdel/+ mouse, which was generated by deleting one copy of the mouse syntenic region of human 22q11.2 containing 26 protein-coding genes (22q11.2DS genes). We found a spectrum of OFT defects ranging from isolated VSD to TOF. The structural defects are preceded by a disrupted OFT MC boundary, increased expression of Edn1 during endocardial-to-mesenchymal transformation (EMT), and decreased NOTCH1 signaling and Ctgf expression during post-EMT OFT remodeling. By single cell RNA sequencing (scRNA-seq), we identified Edn1 as part of a unique gene program operating in a subset of ECCs undergoing EMT. Based on these findings, we propose an overall hypothesis that 22q11.2DS genes control OFT development by regulating the function of ECCs and the cell-cell communications between MCs from ECC and NCC origins, via interacting with genes essential for OFT formation. We will test this hypothesis in three specific aims. Aim 1 will determine whether the 22q11.2DS genes regulate EMT through modulating the EMT gene program, and if Edn1 acts downstream of 22q11.2DS genes to regulate the process. Aim 2 will ascertain whether 22q11.2DS genes also regulate OFT remodeling through a cell-cell interaction network that patterns the OFT MC boundary, and if Ctgf functions as a hub gene required for the post-EMT OFT remodeling, downstream of 22q11.2DS genes. Aim 3 will define whether Notch1 haploinsufficiency can potentiate the 22q11.2DS OFT defects. At the completion of this study, we expect discoveries that will establish genetic, molecular, and cell crosstalk regulated by important syndromic and non-syndromic CHD genes essential for mouse OFT morphogenesis. The information will provide deeper understanding of heart developmental biology and inform the disease mechanism of OFT defects, with a broader implication in congenital heart disease.