Myocardial growth and maturation is

Myocardial growth and maturation is regulated in part by signals from the underlying endocardium (Brutsaert, 2003, de la Pompa and Epstein, 2012, Rentschler et al., 2010). More generally, endothelial MK1775 play an increasingly appreciated role in instructing organogenesis by providing inductive signals and by secreting growth factors and cell surface ligands in addition to their important role in blood vessel formation, and thus in delivering oxygen and nutrients to developing tissues. In endodermal tissues, endothelial cells play an instructive function in the formation of liver and pancreas (Matsumoto et al., 2001, Yoshitomi and Zaret, 2004). In the developing heart, endocardial cells secrete neuregulin 1 (Nrg1) and express the cell surface ligand ephrin B2 (Efnb2), both of which signal to neighboring myocardial cells to regulate growth and maturation of the myocardium. Notch signaling between endocardium and myocardium is also required for myocardial growth and trabeculation (Gassmann et al., 1995, Lee et al., 1995, Meyer and Birchmeier, 1995). Active Notch signaling in the endocardium up-regulates Efnb2, which subsequently enhances Nrg1 expression (Grego-Bessa et al., 2007). Nrg1 is a ligand for the erb-b2 receptor tyrosine kinases 2 and 4 (ErbB2 and ErbB4) on the adjacent myocardium and engagement of ligand stimulates ErbB2-ErbB4 dimerization and activation of ErbB2 tyrosine kinase activity to promote differentiation of trabecular myocytes (Grego-Bessa et al., 2007). In this report, we show that Hippo signaling in endocardium regulates myocardial growth during embryonic development. In the absence of endocardial Yap and Taz, Nrg1 expression is deficient and myocardial ErbB2/4 signaling is impaired. This culminates in thin myocardial walls and early post-natal lethality. Interestingly, activation of Notch in endocardium is intact, suggesting that both Notch and Hippo converge on Nrg1 expression. These results indicate that the Hippo pathway can regulate cell, and possibly organ size via both cell autonomous and non-cell autonomous mechanisms.
Results Yap and Taz are each expressed in the developing heart by both myocardial cells and eNOS-expressing endocardial cells at embryonic day 13.5 (E13.5) (Fig. 1A,C). We used the Nfatc1 allele (Wu et al., 2012) to delete Yap and Taz in endocardial cells, but not other extra-cardiac vascular endothelial cells, to produce Yap°°; Taz°; Nfatc1 embryos (hereinafter referred to as mutants). We show by immunohistochemistry that endocardial Yap and Taz are efficiently deleted, while myocardial Yap and Taz expression remain intact (Fig. 1B,D). By examining E13.5 embryos that harbor a Td/tomato Cre-reporter allele (recognized by an RFP antibody), we confirmed that Nfatc1 activity was restricted to the heart in both control and mutant embryos (Fig. 1E-G). Endothelial cells outside of the heart were not labeled. At this stage of development, endocardial cells also exhibit active Notch signaling as evidenced by co-expression of the cleaved Notch intracellular domain (NICD, Supplemental Fig. 1). Genetic deletion of Yap and Taz from endocardial cells produced mutant embryos that appear grossly normal at E13.5 (Fig. 2A,E), and mutant animals were present at normal Mendelian ratios at that time point; however, mutants die between postnatal day P1 and P7 (Table 1). In the mutants, we observe abnormal intervetricular septae, suggestive of serpiginous ventricular septation defects; no overt valve mesenchyme or coronary vasculature defects were appreciated. Histological sections of hearts from E13.5 mutant embryos also reveal thin right and left ventricular walls as compared to controls (Fig. 2B-D,F-H). The thickness of the ventricular myocardium in E13.5 mutant hearts was significantly decreased in both the right and left ventricles compared with controls (Fig. 2I,J), and this thin ventricular myocardial phenotype remained evident at perinatal time points (E18.5 and P0) (Supplemental Fig. 2).