Endocardial Hippo signaling regulates myocardial growth and cardiogenesis

Abstract

The Hippo signaling pathway has been implicated in control of cell and organ size, proliferation, and endothelial-mesenchymal transformation. This pathway impacts upon two partially redundant transcription cofactors, Yap and Taz, that interact with other factors, including members of the Tead family, to affect expression of downstream genes. Yap and Taz have been shown to regulate, in a cell-autonomous manner, myocardial proliferation, myocardial hypertrophy, regenerative potential, and overall size of the heart. Here, we show that Yap and Taz also play an instructive, non-cell-autonomous role in the endocardium of the developing heart to regulate myocardial growth through release of the paracrine factor, neuregulin. Without endocardial Yap and Taz, myocardial growth is impaired causing early post-natal lethality. Thus, the Hippo signaling pathway regulates cell size via both cell-autonomous and non-cell-autonomous mechanisms. Furthermore, these data suggest that Hippo may regulate organ size via a sensing and paracrine function in endothelial cells.

Figure 1. Mutant embryos display loss of both Yap and Taz in endocardial cells, but not other vascular endothelial cells

(A,B) Transverse sections of hearts from E13.5 (A) control (Yap^flox/+; Taz^flox/+; Nfatc1^IRES-Cre/+) and (B) mutant (Yap^flox/flox; Taz^flox/flox; Nfatc1^IRES-Cre/+) embryos stained for Yap and eNOS (endothelial marker) with nuclear counter-stain (Hoechst). Throughout, boxed areas are shown at higher magnification in images to the right. White arrowheads indicate cells co-expressing Yap and eNOS. Yellow arrowheads denote eNOS-positive/Yap-negative cells. Transverse sections of hearts from E13.5 control (C) and mutant (D) embryos stained for Taz and eNOS with nuclear counter-stain (Hoechst). White arrowheads indicate cells co-expressing Taz and eNOS. Yellow arrowheads denote eNOS-positive/Taz-negative cells. RV=right ventricle, LV=left ventricle, Scale bars: 100 μm. (E) Bright field and corresponding fluorescence image of whole-mount E13.5 control mouse embryo on the background of the R26^td-Tomato reporter (Nfatc1^IRES-Cre/+; Taz^flox/+;Yap^flox/+;R26^td-Tomato) shows specificity of Cre activity to the heart. Cross sections of hearts from control (F) (Nfatc1^IRES-Cre/+; Taz^flox/+;Yap^flox/+;R26^td-Tomato) and mutant (G) (Nfatc1^IRES-Cre/+; Taz^flox/flox;Yap^flox/flox;R26^td-Tomato) E13.5 mouse embryos stained with Pecam1 and RFP antibodies. RFP expression is restricted to endocardial/endothelial cells and the mesenchyme of the endocardial cushions derived from endocardium. Scale bars: 100 μm, inset: 10 μm.

Figure 2. Loss of endocardial Yap and Taz results in thin myocardium

(A) Control (Nfatc1^IRES-Cre/+; Taz ^flox/+;Yap ^flox/+) E13.5 embryo imaged in bright field. (B–D) H&E stained transverse sections from the heart of a control E13.5 embryo. Blue lines indicate representative region used in ventricular wall thickness calculations. (E) Mutant (Nfatc1^IRES-Cre/+; Taz ^flox/flox;Yap ^flox/flox) E13.5 embryo imaged in bright field. (F–H) H&E stained transverse sections from the heart of a mutant E13.5 embryo. Blue lines indicate representative region used in ventricular wall thickness calculations. (I,J) Quantification of RV wall thickness (I) or LV wall thickness (J) in control and mutant embryos. Data represent the mean ± standard deviation. Statistics were completed using a Student’s t-test, *** p<0.001. RA=right atrium, LA=left atrium, RV=right ventricle, LV=left ventricle, Scale bars: 100 μm.

Figure 3. Endocardial deletion of Yap/Taz causes reduction of Nrg1 expression and Nrg1-ErbB signaling

(A) qRT-PCR of RNA isolated from E13.5 hearts for Nrg1, Efnb2, Vegfr2, Nfatc1 and Pecam1. Data depicted are the mean + standard error of the mean (SEM) and the statistics were completed using a Student’s t-test * p<0.05; n=3. (B–D) Transverse sections from control E13.5 embryo hearts stained for (B) Nrg1 or (C,D) Nrg1 and Pecam1. White arrowheads indicate Pecam1/Nrg1 co-positive endocardial cells. (E–G) Transverse sections from mutant E13.5 embryo hearts stained for (E) Nrg1 or (F,G) Nrg1 and Pecam1. Yellow arrowheads denote Pecam1-positive/Nrg1-negative endocardial cells. (H–J) Transverse sections from control E13.5 embryo hearts stained for (H) phosphorylated ErbB2 (p-ErbB2) or (I,J) p-ErbB2 co-stained with cardiac troponin T (cTnT) and DAPI nuclear counterstain. White arrowheads indicate p-ErbB2/cTnT co-positive myocardial cells. (K–M) Transverse sections from mutant E13.5 embryo hearts stained for (K) p-ErbB2 or (L,M) p-ErbB2 co-stained with cTnT and nuclear counterstain. Yellow arrowheads denote cTnT-positive/p-ErbB2-negative myocardial cells. LV=left ventricle, Scale bars: 100 μm, inset: 10 μm.

Figure 4. Yap occupies promoter and enhancer regions of NRG1

(A) Schematic diagram of the human NRG1 locus showing active promoter (red) and enhancer (orange) regulatory regions in HUVECs. including two putative TEAD binding sites (purple) within evolutionary conserved regions of NRG1 promoter and enhancer regions. (B) ChIP-qPCR signal for Yap occupancy and IgG control at putative TEAD binding sites (TEAD1 and TEAD2), nearby experimental control regions (NC1, NC2), or distal experimental control region (DC1). See methods for genomic coordinates of control regions. Yap occupancy expressed as percent enrichment of Yap ChIP signal normalized to input. Data depicted are the mean + standard error of the mean (SEM) and the statistics were completed using a Student’s t-test * p<0.05; n=3.

Figure 5. Exogenous Nrg1 rescues the thin myocardium phenotype of Yap/Taz mutant hearts

Transverse sections of control (Yap^flox/+; Taz^flox/+; Nfatc1^IRES-Cre/+) (A,B) and mutant (Yap^flox/flox; Taz^flox/flox; Nfatc1^IRES-Cre/+) (C,D) E12.5 cardiac explants cultured for 24 hours with either vehicle (A,C) or recombinant Nrg1 (B,D). Sections were stained for cTnT to mark myocardial cells and with nuclear counter-stain (Hoechst). Merged images were generated by combining respective green and blue channels using ImageJ software. Dotted white lines demarcate the compact myocardium. (E) Quantification of cell number in the compact myocardium in vehicle- or Nrg1-treated control and mutant hearts. Box and whiskers plot with median, minimum, and maximum indicated depict cell number per section. Statistics were completed using a One-way ANOVA, P < 0.01 (Tukey’s multiple comparisons, ***P < 0.001, *P < 0.05). Scale bars: 100 μm.

Figure 6. Exogenous Nrg1 increases the size of compact myocardial cells

(A) Representative cross section of an E12.5 cardiac explant that was stained for wheat germ agglutinin (WGA), cTnT, and nuclear counter-stain (Hoechst) (left) and rendered in 3D (right) for quantification of cell size. Nuclei within the cTnT-low compact myocardium are dark blue. (B–E) Three-dimensional renderings of cross sections of control (Yap^flox/+; Taz^flox/+; Nfatc1^IRES-Cre/+) (B,D) and mutant (Yap^flox/flox; Taz^flox/flox; Nfatc1^IRES-Cre/+) (C,E) E12.5 cardiac explants that were treated with vehicle or recombinant Nrg1. For each representative explant cross section, only the nuclei of the compact myocardial cells are shown (blue spheres) with and without WGA-marked cell outlines (red). (F) Quantification of cell size as measured by average distance (in μm) between nuclei of the compact myocardial cells for each treatment. Statistics were completed using a Student’s t-test; * p < 0.001; N > 180 for each condition.