(SHF) progenitors and outflow tract and ventricular cardiomyocytes (fig. S1, A to D). Salv CKO
hearts had reduced p YAP but no change in total
Yap, revealing that Hippo signaling is active in
the developing heart and that Salv deletion
reduces cardiac Hippo activity (fig. S1, E and F).
Salv CKO mutants survived development, but
most mutants expired postnatally with obvious heart
enlargement or cardiomegaly (Fig. 1, A and B, and
table S1). Histological examination of Salv CKO
mutant hearts revealed that, although organ size is
affected, arterioventricular connections and arrangement of chambers and valves were unaffected
(Fig. 1, C and D), consistent with preserved patterning observed in Hippo mutant imaginal discs (4).
Hearts of some Salv CKO mutant animals had a
ventricular septal defect (VSD), indicating that Hippo signaling regulates ventricular septation (Fig.
1D). Because VSD can cause heart failure, we
confined further analysis of Salv CKO mutants to
stages before ventricular septation is completed,
E14.5 and earlier stages.
Salv CKO mutant hearts had expansion of
trabecular and subcompact ventricular myocardial layers, thickened ventricular walls, and
enlarged ventricular chambers without a change
in myocardial cell size (Fig. 1, E and F, and fig.
S2). Lats2 and Mst1/2 CKO E11.5 mutant hearts
had similar myocardial expansion phenotypes
We investigated cardiomyocyte proliferation
by double immunostaining with phosphorylated
Ser10 histone H3 (pHH3) antibodies to detect
mitotic cells and sarcomeric myosin (a-MF20)
(Fig. 2, top and middle). Less than 1% of control
ventricular cardiomyocytes were pHH3-positive,
whereas about 4.5% of Salv CKO mutant cardio-
myocytes were pHH3-positive, indicating exces-
sive cardiac proliferation in cardiomyocytes
(Fig. 2, bottom). Cardiomyocyte proliferation was
elevated in both left and right Salv CKO ventricles.
Cell counting indicated elevated ventricular car-
diomyocte number in Salv CKO mutants but no
changes in ventricular smooth muscle (a-SMA)
or cardiac fibroblasts (a-DDR2) (fig. S4).
b-catenin index, a readout for cells receiving a
Wnt signal. Whereas b-catenin localized to plasma
membrane junctions and cytosol of control myocardium, Salv CKO mutants had a fourfold increase in nuclear b-catenin staining (Fig. 3, D to F).
These findings indicate that canonical Wnt signaling in cardiomyocytes is derepressed upon Salv
deletion and support the notion that Hippo signaling inhibits Wnt/b-catenin to regulate heart size.
To obtain genetic evidence that Hippo signaling negatively regulates Wnt/ b-catenin–dependent
cardiac growth, we crossed Salv CKO mice to
a b-catenin conditional null allele to generate
Nkx2.5cre; Salv f/f;bcat f/+ (Salv/bcat f/+ CKO) embryos that are Hippo-deficient with reduced
Myocardial thickness of Salv/bcatf/+ CKO hearts
was significantly reduced by comparison to Salv
CKO (figs. S7, B and C, and S2). Ventricular
proliferation rates, as well as subcompact and
trabecular myocardial thickness, in Salv/bcatf/+
CKO; mutants resemble those of control (Fig.
4A and figs. S2 and S7, A and C). qRT-PCR indicated that Sox2, Snai2, Birc2, and Cdc20
expression levels reverted to control or were lower
in hearts with reduced b-catenin dosage (Fig.
4B). Suppression of the Hippo myocardial overgrowth phenotype by reduced b-catenin indicates
that Wnt signaling is required for up-regulated cardiomyocyte proliferation and cardiomegaly in
To investigate whether Yap and b-catenin are
in the same molecular complex, we immunopre-cipitated protein extracts from E14.5 hearts with
Yap and pYAP-specific antibodies (Fig. 4C).
Western blotting with antibodies against b-
catenin revealed that b-catenin forms a complex
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/+.