Tbx20调控心脏瓣膜生长发育的信号通路研究

Cardiac valves are highly organized, yet delicate, structures that ensure unidirectional blood flow through the cardiac chambers and large vessels. Proper formation of the cardiac valves is crucial for normal heart function. Disturbed development of the cardiac valves leads to aberrant heart formation and function, accounting for a significant portion (~30%) of CHDs in humans. T-box genes encode transcription factors that play vital roles for the development of various organs. Mutations in T-box genes are associated with human diseases with birth defects. Tbx20 is an ancient T-box family gene with orthologues present in species from Drosophila to human. In mice, Tbx20 is dynamically expressed in the endocardial cushion and valves throughout heart formation. We recently found that genetic deletion of Tbx20 in the endocardium led to shortened valve leaflets with less proliferative cushion endocardial cells and regurgitant blood flow, and expression of Wnt/β-catenin pathway genes was disturbed in the cushion endocardium at mid-to-late gestation of mouse heart development. Our long-term objectives are to apply state-of-the-art genetic and biochemical approaches to study the biology of Tbx20, in order to fully understand how this important transcription factor orchestrates various signals to coordinate cardiac valve formation, and how Tbx20-mediated transcriptional disorders lead to congenital valve defects in humans. Towards these goals, our specific aims are: 1) To define the role of Wnt/β-catenin pathway in Tbx20 signaling cascades of cardiac valve development. We will delete β-catenin (Ctnnb1) in the cushion endocardium by crossing β-catenin-flox (Ctnnb1f/f) mice to Nfatc1 enhancer Cre mice (Nfatc1EnCre/+), to determine the role of Wnt/β-catenin pathway in cardiac valve development at mid-late gestation of mouse heart development; 2) To characterize the transcriptional network of Tbx20 during cardiac valve formation. We will perform ChIP-Seq assay with mouse valvular tissues, to identify genome-wide targets of Tbx20 during cardiac valve formation; 3) We will further sequence TBX20 coding regions from ~600 congenital heart disease patients with malformed valves, to investigate if mutated TBX20 cause human congenital valvular disease. Results of these studies will provide a comprehensive overview of Tbx20 with respect to cardiac valve development. They may also provide novel insights into the etiology of human congenital heart diseases (CHDs) with valvulogenesis defects from perturbations of TBX20 and its target genes.

心脏瓣膜是心脏内不可缺少的重要且精巧的结构，瓣膜发育缺陷约占先天性心脏病的1/3，发病机制不明。本课题组研究发现，Tbx20能够在心内膜发育中调节Wnt/β-catenin一系列基因正常表达，且Tbx20基因突变会导致心脏瓣膜发育异常，由此推测Tbx20是通过调控Wnt/β-catenin信号通路来维持心脏瓣膜正常发育。本研究拟完成：1.通过构建心内膜β-catenin缺失小鼠模型，确定妊娠中后期Wnt/β-catenin通路在Tbx20信号传导链中对心脏瓣膜延伸发育的作用;2.通过ChIP-Seq实验，确定Tbx20在心瓣膜发育中的下游靶基因及其DNA调控序列，阐明Tbx20在心瓣膜发育过程中的转录调控网络;3.对患有累及瓣膜的先天性心脏病患儿血液和瓣膜组织进行Tbx20基因突变分析。研究结果将阐明Tbx20调控心瓣膜发育的信号网络以及TBX20突变后导致先天性瓣膜心脏病的分子病理基础。

心瓣膜形成是一个复杂的动态过程，涉及到一系列重要发育环节，包括谱系决定、细胞增殖、分化和迁移。TBX20在小鼠的心内膜和心瓣膜中高度表达。小鼠心内膜的TBX20对心瓣膜的延伸生长至关重要，心内膜的TBX20缺失会降低其瓣膜心内膜细胞的增殖率，并致瓣叶生长缓慢。然而，课题组对临床收集的103例瓣膜相关的先心病患儿外周血进行基因检测，并未发现高致病性的TBX20突变，但在42例瓣膜病变相关的法洛四联症（tetralogy of Fallot，TOF）患儿心脏组织中，发现TBX20表达上调。TBX20被公认以剂量敏感的方式调节心脏发育，是增殖和分化遗传网络的关键组成部分，由此推测，TBX20部分区域DNA甲基化修饰异常亦可能是导致心脏瓣膜发育异常原因之一。本项目的主要研究内容包括：1.研究TBX20基因在TOF发展过程中的表观遗传调控机制。以TOF患儿右室流出道及肺动脉瓣组织样本为研究对象，以法医尸检非心源性死亡心脏相应部位组织为对照，首先发现TOF患儿心肌组织中TBX20启动子M1区（-945bp至-635bp）甲基化水平较正常对照组明显降低，呈低甲基化状态；然后利用相关分子生物学技术对TBX20启动子M1区进行甲基化功能验证，显示该区域具有增强转录活性的作用，说明TOF患儿TBX20基因启动子M1区的低甲基化状态导致TBX20转录水平的增高；最后证实与心脏发育相关的重要转录因子Sp1能增强TBX20启动子M1区转录活性，从而上调TBX20的表达，阐明了TOF患儿心肌组织中TBX20基因启动子区异常甲基化引起TBX20异常表达的转录调控机制。2.对临床收集的瓣膜病变患儿的外周血进行基因测序，筛选包括TBX20基因在内的有意义的基因突变。对17例瓣膜病变相关的房室间隔缺损患儿进行全外显子序列分析并经一代测序验证，筛选得到SETD1B和SCAF1候选基因，开始进行细胞功能研究。3.利用谱系示踪小鼠，对心外膜细胞进行命运定位，确定心脏瓣膜组织中心外膜来源的细胞类型及其分布，为心脏瓣膜发育分子机制研究提供新的思路。本课题从心脏瓣膜缺陷的临床问题出发，结合基础研究，从DNA甲基化角度诠释了TBX20对心脏瓣膜发育的可能调控机制，为阐明心脏瓣膜发育缺陷的发病机制提供理论依据。