Endocardial Pathways Regulated by Tbx20

描述(申请人提供)：先天性心脏畸形通常包括流出道扰动(OFT)和间隔形态发生。在人类中，TBX20的突变与瓣膜形成、间隔形成和心肌病的缺陷有关。常见的形态发生和心脏间隔需要多种细胞系之间的串扰，包括咽内胚层、神经脊、第二心区、心肌、心内膜和垫状间充质。Tbx20的整体消融或敲除已经证明了Tbx20在心脏发生的多个方面发挥着关键作用。虽然这些研究深入了解了Tbx20的生物学作用，但它们没有回答关于Tbx20在心脏早期发育中的组织特异性需求的关键问题。为了研究Tbx20在心内膜中的潜在作用，我们在小鼠发育早期使用Tie2Cre来消融Tbx20。Tie2Cre；Tbx20突变体在胚胎14岁左右死于心脏缺陷，包括DORV、发育不良的垫子和房室间隔缺陷。Tie2Cre；Tbx20突变体的心内膜细胞表现出一些涉及OFT形态发生、垫层重塑和心脏间隔所需途径的基因表达减少。这些途径包括上皮-间充质转化(EMT)、细胞外基质(ECM)重塑和平面细胞极性(PCP)。我们在胚胎心脏中通过全基因组染色质免疫沉淀(CHIP)鉴定了其中几个基因是Tbx20的直接靶点。其中两个靶点，Wnt5a和Wnt11，通过非规范的Wnt途径来调节Tie2Cre；Tbx20突变体中受干扰的心脏发生所需的通路；Tbx20突变，包括OFT形态发生和心脏间隔。我们已经确定了一个与Tbx20结合的Wnt11增强子，它可以驱动一个报告基因在发育中的小鼠心脏内膜中的表达。综上所述，我们的假设是心内膜中的Tbx20通过调节EMT、ECM重塑和心肌PCP所需的基因，在OFT和缓冲形态发生中发挥重要作用。为了了解Tbx20调节心内膜靶基因表达的机制，我们提出了一个综合平台，从深度和广度两个方面探讨Tbx20在心内膜中的作用。我们将研究心内膜TBX20在垫层和OFT形态发生中的表型作用，同时揭示心内膜中TBX20调控的遗传途径。我们将确定信号通路和转录因子作为与TBX20共同调控EMT、ECM重塑和PCP通路的因子。我们将评估信号通路的功能意义以及它们与心内膜中的TBX20的交叉点。这项建议的总体目标是确定心内膜中Tbx20的关键靶点，这些靶点可能是患者表型的基础，通过了解Tbx20如何与其他因素一起调节这些途径，以及它们对先天性心脏病的干扰的功能后果，获得机制上的洞察。

DESCRIPTION (provided by applicant): Congenital cardiac malformations often include perturbations in outflow tract (OFT) and septal morphogenesis. In humans, mutations in TBX20 are associated with defects in valvulogenesis, septum formation and cardiomyopathy. OFT morphogenesis and cardiac septation require crosstalk between multiple cell lineages, including pharyngeal endoderm, neural crest, second heart field, myocardium, endocardium, and cushion mesenchyme. Global ablation or knockdown of Tbx20 has demonstrated a key role for Tbx20 in multiple aspects of cardiogenesis. Although these studies give insight into biological roles of Tbx20 they leave unanswered the critically important question as to tissue specific requirements for Tbx20 in early heart development. To investigate a potential role for Tbx20 in endocardium, we have utilized Tie2Cre to ablate Tbx20 during early mouse development. Tie2Cre;Tbx20 mutants die around E14 with cardiac defects including DORV, hypoplastic cushions and atrioventricular (AV) septal defects. Endocardial cells of Tie2Cre;Tbx20 mutants demonstrated decreased expression of a number of genes involved in pathways required for OFT morphogenesis, cushion remodeling, and cardiac septation. These pathways include epithelial-mesenchymal transition (EMT), extracellular matrix (ECM) remodeling, and planar cell polarity (PCP). We identified several of these genes as direct targets of Tbx20 by genome-wide chromatin immunoprecipitation (ChIP) in embryonic heart. Two of these targets, Wnt5a and Wnt11, act through a non-canonical Wnt pathway to regulate pathways required for aspects of cardiogenesis which are perturbed in Tie2Cre;Tbx20 mutants, including OFT morphogenesis and cardiac septation. We have identified a Wnt11 enhancer bound by Tbx20 which drives expression of a reporter gene within endocardium of developing mouse heart. From the foregoing, our hypothesis is that Tbx20 in endocardium plays an essential role in OFT and cushion morphogenesis by regulating genes required for EMT, ECM remodeling, and myocardial PCP. To understand mechanisms by which Tbx20 regulates expression of target genes in endocardium, we propose an integrative platform that interrogates the roles of Tbx20 in endocardium with both depth and breadth of scope. We will investigate the phenotypical role of endocardial TBX20 in cushion and OFT morhpogenesis, while uncovering genetic pathways regulated by TBX20 in endocardium. We will identify signaling pathways and transcription factors that serve as co-factors with TBX20 to regulate EMT, ECM remodeling and PCP pathways. We will assess the functional significance of signaling pathways and their intersection with TBX20 in the endocardium. The overall aim of this proposal is to identify key targets of Tbx20 in endocardium which may underlie patient phenotypes, to gain mechanistic insight by understanding how Tbx20 works with other factors to regulate these pathways, and functional consequences of their perturbations for congenital heart disease.