Molecular and cellular mechanisms in cardiac outflow tract formation and defects

项目摘要 心脏流出道（OFT）缺陷的估计患病率为1-2/1，000活产。的 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 共有60%的22q11.2DS患者患有轻度至重度的先天性心脏病，包括 二叶式主动脉瓣（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）。这些临床研究结果表明遗传修饰剂可能影响表型表达 .在 本项目中，我们建议使用Lgdel/+小鼠模型来了解神经嵴与 细胞（NCC）和相邻的内皮细胞（ECCs）在心脏OFT的形成和重塑中的作用。 来源于NCC和ECC的间充质细胞（MC）分别占据远端和近端OFT，并且 在心脏发育过程中形成明显的OFT MC边界。适当部署来自两个血统的MC 确保右肺心室间隔和半月瓣的正确位置和形成， 进入体循环和肺循环的心脏出口。NCC在OFT缺陷中的作用一直很好 然而，关于22q11.2DS的研究，尚未研究ECC在OFT畸形中的作用。 我们已经开始通过研究Lgdel/+小鼠来填补这一知识空白， 人22q11.2的小鼠同线区的拷贝，含有26个蛋白质编码基因（22q11.2DS基因）。 我们发现了一系列的OFT缺陷，从孤立的VSD到TOF。结构性缺陷之前， OFT MC边界被破坏，心内膜-间质细胞转化过程中Edn 1表达增加， 在EMT后OFT重构期间，N 0 TCH 1信号传导和Ctgf表达降低。 通过单细胞RNA测序（scRNA-seq），我们鉴定了Edn 1作为一个独特的基因程序的一部分， 接受EMT的ECC子集。基于这些发现，我们提出了一个总体假设，即22q11.2DS 基因通过调节内皮细胞的功能和细胞间的通讯来控制OFT的发育。 来自ECC和NCC起源的MC，通过与OFT形成所必需的基因相互作用。我们将测试这个 三个具体目标。目的1将确定22q11.2DS基因是否通过调节EMT 调节EMT基因程序，如果Edn 1作用于22q11.2DS基因下游以调节该过程。 目的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 如果Ctgf作为后EMT OFT所需的枢纽基因发挥作用， 重塑，22q11.2DS基因下游。目标3将定义Notch 1单倍不足是否可以 强化22q11.2DS OFT缺陷。在这项研究完成后，我们希望发现， 由重要的综合征性和非综合征性CHD基因调控的遗传、分子和细胞串扰 用于小鼠OFT形态发生。这些信息将有助于更深入地了解心脏发育 生物学和告知OFT缺陷的疾病机制，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.