Novel Regulators of Epithelial-to-Mesenchymal Transformation in Cardiogenesis are Identified Through Next-Generation Sequencing

Abstract

Epithelial-to-Mesenchymal Transformation (EMT) is an important process in development, and occurs during key steps in both valvular and coronary vessel development. This dissertation uses transcriptional profiling strategies to identify novel regulators of EMT during cardiogenesis. An early step in valvulogenesis occurs when endocardial cells overlaying the cushions of the atrioventricular canal (AVC) and outflow tract (OFT) undergo EMT to yield valvular interstitial cells. We developed an unbiased strategy to identify genes important in endocardial EMT using a spatial transcriptional profile. RNA-seq analysis of gene expression between AVC, OFT, and ventricles (VEN) isolated from chick (HH18) and mouse (E11.0) embryos at comparable stages of development was performed. EMT occurs in the AVC and OFT cushions but not the VEN at this time. We identified genes >2-fold enriched in chick and mouse cushions compared to VEN. Gene ontology and Gene Regulatory Network (GRN) analysis of cushion-enriched genes were consistent with cells undergoing EMT. Further analysis accurately identified and validated previously unrecognized novel candidate genes (Meis2, Id1, Hapln1, Foxp2) and the NF-κB pathway as regulators of endocardial cell EMT in vitro. Epicardial EMT is a critical step in coronary vessel formation which is dysregulated in mice lacking TGFβR3. To elucidate the role of TGFβR3 in EMT we developed a strategy to identify genes downstream of TGFβR3 in cultured epicardial cells. Tgfbr3+/+ and Tgfbr3-/- immortalized epicardial cells were incubated with vehicle or ligands known to promote TGFβR3-dependent invasion (TGFβ1, TGFβ2, BMP2) and harvested for RNA-seq analysis. GRN analysis of genes >2-fold differentially expressed between Tgfbr3+/+ and Tgfbr3-/- cells in each ligand incubation group revealed dysregulated NF-ĸB signaling. TGFβ2 or BMP2 incubation stimulated NF-ĸB activity in Tgfbr3+/+ but not Tgfbr3-/- epicardial cells. Inhibiting NF-ĸB signaling reduced TGFβ2- or BMP2-promoted invasion of Tgfbr3+/+ cell, further supporting a role for NF-ĸB signaling in promoting invasion downstream of TGFβR3. The genes and signaling pathways identified through our analysis yield the first comprehensive list of candidate genes whose expression is dependent on TGFβR3 signaling. These transcriptional profiling strategies identified and validated novel regulators of endocardial and epicardial EMT.