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.
We propose to expand the ENCODE-led efforts to map genome-wide binding sites of transcription factors and chromatin markers to the mouse genome. Toward that, we will develop on efforts initiated in our labs to generate molecular reagents, through BAC recombineering, that allow for ChIP-seq of factors which lack specific antibodies. We will create transgenic mice harboring these BACs, allowing tissue-specific experiments in multiple tissues and organs in the mouse, including tissues that are absent in the ENCODE, due to their difficulty in obtaining, paucity, or poor ability to culture in vitro.
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