To interrogate the molecular basis for

To interrogate the molecular basis for the myocardial phenotype in mutant hearts, we performed quantitative real-time PCR (qRT-PCR) expression analysis of genes encoding critical endocardium-derived molecules including Nrg1 and Efnb2. Nrg1 expression was reduced in E13.5 mutant hearts as compared to controls, while Efnb2 expression levels did not notably differ (Fig. 3A). We also measured the expression of other endocardium-enriched genes, Vegfr2, Nfatc1 and Pecam1, and found these transcripts to be present at similar levels in control and mutant hearts (Fig. 3A). These observations are consistent with a specific Yap/Taz-dependent regulation of Nrg1 in endocardium. Consistent with the qRT-PCR results, antibody staining revealed reduced Nrg1 expression in the Pecam1-positive endocardial cells of Yap/Taz mutants compared to control hearts (Fig. 3B-G). Consistent with reduced endocardial Nrg1 ligand expression, we also noted reduced phosphorylation of the Nrg1 co-receptor, ErbB2 (phospho-ErbB2, p-ErbB2), on adjacent cardiac troponin T (cTnT)-positive myocardial cells in the mutants compared to controls (Fig. 3H-M). Collectively, these data are consistent with the Hippo effector molecules Yap and Taz acting in endocardium upstream of Nrg1/ErbB2 signaling in the developing heart. Given the reduced levels of Nrg1 expression observed in the Yap/Taz mutants, we sought to examine the possibility that Nrg1 expression requires Yap and Taz. To do so, we examined murine embryonic fibroblasts (MEFs) isolated from Yap;Taz embryos. Treatment with an adenovirus-expressing Cre recombinase (AAV1-Cre) significantly reduced Yap and Taz expression in these cells (Supplemental Fig. 3A). We observed a reduction of Nrg1 expression in response to Cre-mediated deletion of Yap and Taz similar to that found in mutant mouse hearts (Supplemental Fig. 3A). Transient overexpression of Yap and Taz, individually or in combination, in the AAV1-Cre-treated Yap;Taz MEFs rescued Nrg1 expression (Supplemental Fig. 3B), confirming the Yap/Taz-dependent expression of Nrg1. Further, we asked whether Yap binds Nrg1 regulatory elements in endothelial cells. We used published chromatin profiles of human umbilical vein endothelial cells (HUVECs) to identify relevant Nrg1 regulatory elements (Ernst et al., 2011) that control the most highly expressed Nrg1 transcript (Fig. 4A). We identified two well conserved putative TEAD KN-62 (one in the promoter and one in a strong enhancer) and demonstrated Yap occupancy in HUVECs by chromatin immunoprecipitation followed by qPCR (Fig. 4B). These chromatin regions showed Yap enrichment of 2.5- to 4-fold over IgG controls. As expected, Yap was not enriched at either of two nearby control genomic regions (NC1, NC2) upstream of these putative TEAD binding sites, or at another more distant region (DC1) on the same chromosome (Fig. 4B and see methods for genome coordinates). This suggests that Yap/Taz may directly regulate Nrg1 expression in endothelial cells. To determine whether Nrg1 acts as the primary molecular effector underlying the Yap/Taz mutant heart phenotype, we cultured explanted E12.5 embryonic hearts in serum-free medium supplemented with insulin/transferrin/selenium formulation (Sigma) with or without recombinant Nrg1. Following 24 h of culture, with or without Nrg1, both control and mutant hearts were beating, suggesting myocardial viability. Vehicle-treated mutant hearts, however, had thin compact myocardium compared with control hearts, which is consistent with our in vivo analysis (Fig. 5A,C). Nrg1-treated mutant hearts had a significantly thickened compact myocardial wall compared to vehicle-treated mutant hearts (Fig. 5C,D) and had enlarged to a degree similar to Nrg1-treated control hearts (Fig. 5). We assessed phenotypic rescue of the thin compact myocardium by comparing the number of cells in the compact myocardium of vehicle- and Nrg1-treated control and mutant hearts. Upon treatment with exogenous Nrg1, the thin compact myocardium of mutant hearts was rescued and the number of cells in this region increased to similar levels as control hearts (Fig. 5E). This is consistent with the reported role of Nrg1 as a growth factor essential for ventricular trabeculation (Meyer and Birchmeier, 1995, Rentschler et al., 2002). Since Nrg1 has also been shown to affect the cell size of myocardial cells (Zhao et al., 1998), we assessed the size of the compact myocardial cells by staining heart sections with wheat germ agglutinin to demarcate the cell boundary, and measuring the distance between nearby nuclei. Nrg1-treated control and mutant hearts had larger compact myocardial cells compared to vehicle-treated control and mutant hearts (Fig. 6). Thus, exogenous Nrg1 treatment can rescue the thin myocardium of mutant hearts by increasing myocardial cell number as well as cell size. Taken together, these data suggest that endocardial Yap and Taz are required for proper Nrg1 expression and for normal myocardial development.