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Science 332 (6028): 458-461

Copyright © 2011 by the American Association for the Advancement of Science

Hippo Pathway Inhibits Wnt Signaling to Restrain Cardiomyocyte Proliferation and Heart Size

Todd Heallen1, Min Zhang1, Jun Wang1, Margarita Bonilla-Claudio1, Ela Klysik1, Randy L. Johnson2, and James F. Martin1,*

1 Institute of Biosciences and Technology, Texas A&M System Health Science Center, 2121 West Holcombe Boulevard, Houston, TX 77030, USA.
2 Department of Biochemistry and Molecular Biology, M. D. Anderson Cancer Center, Houston, TX 77030, USA.

Figure 1
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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 Nkx2.5cre; Salvf/+.


Figure 2
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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 Nkx2.5cre; Salvf/+.


Figure 3
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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 Nkx2.5cre; Salvf/+.


Figure 4
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Fig. 4. Yap interacts with β-catenin. (A) Quantification of pHH3 indices in control, salv CKO, and Nkx2.5cre; Salvf/f; β-cateninF/+ 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 cotransfected factors.


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