Here we show that Hippo signaling components are

Here, we show that Hippo signaling components are expressed during epicardium formation. To determine the significance of Yap and Taz in the developing epicardium, we generated epicardium-specific Yap/Taz double-knockout mice. Genetic deletion of Yap and Taz using Sema3d mice leads to embryonic lethality between E11.5 and E12.5 due to cardiac defects. Furthermore, the inducible genetic deletion of Yap and Taz using Wt1 mice reveals impaired coronary vasculature development. Pharmacological and genetic experiments suggest that the impaired coronary vasculature development observed in Yap/Taz mutants is due to defects in epicardial cell proliferation, EMT, and fate determination. We provide further evidence that Yap/Taz control epicardial cell proliferation, EMT, and fate determination, in part by regulating Tbx18 and Wt1 expression.
Results
Discussion Recent studies have implicated Hippo signaling in cardiac development and regeneration. However, a role for Hippo signaling in coronary vasculature formation has not been explored. In the present study, we have demonstrated that Hippo signaling components are not only strongly expressed in the epicardium but also required for coronary vasculature development and remodeling and for expression of some epicardial-derived growth factors, including Fgf9, Raldh2, and Wnt5a. Hippo signaling mediators Yap and Taz regulate epicardial EMT and epicardial cell proliferation and differentiation into coronary endothelial cells. We provide evidence that Yap and Taz control epicardial cell behavior, in part by regulating Tbx18 and Wt1 expression. Both Tbx18 and Wt1 promoters are more strongly activated by Yap than by Taz, and our genetic data support a dominant role for Yap compared to Taz during coronary vasculature formation. Proepicardial GSK1904529A migrate and adhere to the embryonic myocardial surface and spread over the myocardium enveloping the entire heart. The molecular mechanisms that regulate adhesion and migration of the proepicardial cells as they migrate over the myocardium appear to be intact in Sema3d:Yap:Taz hearts. The formation of epicardium is similarly initially normal in other mouse models engineered with other mutated epicardial genes (Greulich et al., 2012, von Gise et al., 2011, Wu et al., 2013). The formation of the epicardium requires extensive proliferation and migration of proepicardial cells. Consistent with the role of Yap and Taz as regulators of proliferation in other systems, a significant decrease in the proliferation rate of Sema3d:Yap:Taz epicardial cells was observed when compared to controls. Proliferation of epicardial cells is required for EMT as epicardial cells undergoing cell-cycle arrest fail to invade the myocardium (Wu et al., 2010). Reduced proliferation of epicardial cells seen in Wt1:Yap:Taz mice may therefore contribute to deficient EMT and subsequent migration of epicardial derivatives into underlying myocardium. Paracrine signals from the epicardium are required for myocardial expansion. The observed decrease in Fgf9, Raldh2, and Wnt5a expression suggests that signaling between the epicardium and myocardium is affected in Sema3d:Yap:Taz embryos, which likely contributes to the myocardial defects. Hippo signaling has been recently implicated in cell fate determination in multiple organs (Imajo et al., 2015, Yimlamai et al., 2014). However, the role of Hippo signaling components in epicardial cell fate determination has not been explored. Recently, we demonstrated that Sema3d/Scx-positive proepicardial cells are competent to differentiate into coronary endothelial cells in vivo and in vitro (Katz et al., 2012). We were unable to definitively determine if cell fate determination is affected in Sema3d:Yap:Taz embryos due to early embryonic lethality. We observed decreased endothelial, smooth muscle, and fibroblast cells as a percentage of total epicardial derivatives in mutant hearts, but embryonic lethality precluded us from determining if these deficiencies resulted from delayed differentiation or a failure of fate specification.