Endocardial Pathways Regulated by Tbx20

描述（由申请人提供）：先天性心脏畸形通常包括流出道（OFT）和间隔形态发生的扰动。在人类中，TBX 20的突变与瓣膜发生、隔膜形成和心肌病的缺陷有关。OFT形态发生和心脏分隔需要多个细胞系之间的串扰，包括咽内胚层，神经嵴，第二心脏领域，心肌，内皮细胞和垫间充质。Tbx 20的整体消融或敲低已证明Tbx 20在心脏发生的多个方面中的关键作用。尽管这些研究深入了解了Tbx 20的生物学作用，但它们没有回答关于Tbx 20在早期心脏发育中的组织特异性需求的关键问题。为了研究Tbx 20在内皮细胞中的潜在作用，我们利用Tie 2Cre在小鼠发育早期消融Tbx 20。Tie 2Cre; Tbx 20突变体在E14左右死亡，伴有心脏缺陷，包括DORV、发育不全垫和房室（AV）间隔缺损。Tie 2Cre的内皮细胞; Tbx 20突变体表现出参与OFT形态发生、垫重塑和心脏分隔所需途径的许多基因的表达降低。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）。我们通过全基因组染色质免疫沉淀（ChIP）在胚胎心脏中鉴定了这些基因中的几个作为Tbx 20的直接靶基因。这些目标中的两个，Wnt 5a和Wnt 11，通过非经典Wnt途径发挥作用，以调节心脏发生方面所需的途径，这些途径在Tie 2Cre; Tbx 20突变体中受到干扰，包括OFT形态发生和心脏分隔。我们已经确定了Tbx 20结合的Wnt 11增强子，其驱动发育中小鼠心脏内膜内报告基因的表达。根据上述内容，我们的假设是，Tbx 20在内皮细胞中通过调节EMT、ECM重塑和心肌PCP所需的基因在OFT和垫形态发生中起重要作用。为了了解Tbx 20调节内皮细胞靶基因表达的机制，我们提出了一个综合平台，以深度和广度的范围询问Tbx 20在内皮细胞中的作用。我们将研究内分泌TBX 20在垫和OFT形态发生中的表型作用，同时揭示内分泌TBX 20调控的遗传途径。我们将确定信号通路和转录因子作为辅助因子与TBX 20调节EMT，ECM重塑和PCP途径。我们将评估信号通路的功能意义及其与TBX 20在内皮细胞中的交叉。该提案的总体目标是确定Tbx 20在内皮细胞中的关键靶点，这可能是患者表型的基础，通过了解Tbx 20如何与其他因素一起调节这些通路以及其干扰先天性心脏病的功能后果来获得机制见解。

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.