Hippo pathway inhibits Wnt signaling to restrain cardiomyocyte proliferation and heart size

Abstract

Genetic regulation of mammalian heart size is poorly understood. Hippo signaling represents an organ-size control pathway in Drosophila, where it also inhibits cell proliferation and promotes apoptosis in imaginal discs. To determine whether Hippo signaling controls mammalian heart size, we inactivated Hippo pathway components in the developing mouse heart. Hippo-deficient embryos had overgrown hearts with elevated cardiomyocyte proliferation. Gene expression profiling and chromatin immunoprecipitation revealed that Hippo signaling negatively regulates a subset of Wnt target genes. Genetic interaction studies indicated that β-catenin heterozygosity suppressed the Hippo cardiomyocyte overgrowth phenotype. Furthermore, the Hippo effector Yap interacts with β-catenin on Sox2 and Snai2 genes. These data uncover a nuclear interaction between Hippo and Wnt signaling that restricts cardiomyocyte proliferation and controls heart size.

Fig. 1

Salvador mutant cardiomegaly. (A to D) Control [(A) and (C)] and Salv CKO [(B) and (D)] P2 neonate hearts. ra indicates right atrium; la, left atrium; rv, right ventricle; lv, left ventricle. Hearts in (A) and (B) were sectioned and stained with hematoxylin and eosin (H&E), as shown in (C) and (D). Arrow, ventricular septum defect. (E and F) H&E-stained control (E) and Salv CKO (F) hearts. High magnification of (E) and (F) are shown in the right-hand images; subcompact, sc; trabecular, tr myocardium. Control genotype is Nkx2.5^cre; Salv^f/+.

Fig. 2

Cardiomyocyte proliferation in Salvador mutant ventricles. E12.5 control (top) and Salv CKO (middle) coronal sections of left ventricles: stain with TO-PRO-3, blue; α-pHH3, red; and α-MF20, green. Arrows, pHH3/MF20-positive cells. (Bottom) pHH3-positive cardiomyocytes quantification. Control genotype is Nkx2.5^cre; Salv^f/+.

Fig. 3

Salvador deletion potentiates canonical Wnt signaling. Heat map (A) and qRT-PCR validation (B) showing relative transcript levels of Wnt/β-catenin target genes. Values were determined as a mean of three samples ± SD with glyceraldehyde-3-phosphate dehydrogenase control. (C) qRT-PCR of Wnt/β-catenin target genes in β-catenin CKO hearts. (D and E) IF images with quantification (F) of E12.5 heart sections: stain with TO-PRO-3, blue, and α-β-catenin, green. Nuclear β-catenin identified by β-catenin/TO-PRO-3 signal overlap (arrows). Control genotype is Nkx2.5^cre; Salv^f/+.

Fig. 4

Yap interacts with β-catenin. (A) Quantification of pHH3 indices in control, salv CKO, and Nkx2.5^cre; Salv^f/f; β-catenin ^F/+ E12.5 hearts. (B) qRT-PCR with indicated genes. (C) Immunoprecipitation/Western with indicated antibodies. IB, immunoblot. (D) ChIP and sequential ChIP with indicated antibodies. Control ChIP sites proximal to Sox2 and Snai2 loci were tested (asterisks). Refer to fig. S7D for ChIP assay design. IgG, immunoglobulin G. (E) Luciferase reporter assays with co-transfected factors.