Application of Small Organic Molecules Reveals Cooperative TGFβ and BMP Regulation of Mesothelial Cell Behaviors

Abstract

Epicardial development is a process during which epithelial sheet movement, single cell migration, and differentiation are coordinated to generate coronary arteries. Signaling cascades regulate the concurrent and complex nature of these three events. Through simple and highly reproducible assays, we identified small organic molecules that impact signaling pathways regulating these epicardial behaviors. Subsequent biochemical analyses confirmed the specificity of these reagents and revealed novel targets for the widely used dorsomorphin (DM) and LDN-193189 molecules. Using these newly characterized reagents, we show the broad regulation of epicardial cell differentiation, sheet movement, and single cell migration by Transforming Growth Factor β (TGFβ). With the DM analogue DMH1, a highly specific Bone Morphogenetic Protein (BMP) inhibitor, we demonstrate the cooperative yet exclusive role for BMP signaling in regulation of sheet migration. The action of DMH1 reveals that small organic molecules (SOM) can intervene on a single epicardial behavior while leaving other concurrent behaviors intact. All SOM data were confirmed by reciprocal experiments using growth factor addition and/or application of established non-SOM inhibitors. These compounds can be applied to cell lines or native proepicardial tissue. Taken together, these data establish the efficacy of chemical intervention for analysis of epicardial behaviors and provide novel reagents for analysis of epicardial development and repair.