Fig. 4. Yap interacts with b-catenin. (A) Quantification of pHH3 indices
in control, salv CKO, and Nkx2.5cre; Salv f/f; b-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 de-
sign. IgG, immunoglobulin G. (E) Luciferase reporter assays with co-
DNA binding partners potentiates transcriptional
The Yap and b-catenin interaction on genes
such as Snai2 and Sox2 uncovers a nuclear
mechanism for antagonistic control of cardiomyocyte growth by Hippo and canonical Wnt
signaling. Our model is supported by genetic
suppression of Hippo-enlarged hearts by reduced
b-catenin. Hippo signaling inhibits a pro-growth
Wnt/b-catenin-Yap interaction in differentiating
cardiomyocytes as the heart transitions from
rapid progenitor cell growth to more measured
growth in the maturing heart.
Although our p Yap data indicate that there is
some Hippo activity in E9.5 SHF cardiac progenitors, cell proliferation in Hippo mutant SHF
was unchanged from control. This may reflect
Salv-independent Hippo activity and perhaps overlapping functions with other Hippo pathway kinases (i.e., Lats1). Our data support the previous
observation that Hippo signaling was low in E10.5
myocardium (14). Although Salv CKO
cardiomyocyte cell size was unchanged, this question requires
further study under stressed conditions (15).
Hippo regulates growth and progenitor genes
like Sox2, Snai2, Ccdn1, Cdc20, and l-Myc in
cardiomyocytes. In livers overexpressing Yap
and Hippo loss-of-function mutants, expression
of c-Myc and Ccdn1 is up-regulated, suggesting
shared mechanisms between liver and heart (4).
Although apoptosis inhibitors Birc2 and Birc5
were up-regulated in Salv CKO mutant hearts,
apoptosis was unchanged.
Important recent work uncovered a repressive
cytoplasmic interaction between p Taz and Dvl in
kidney (16). Our findings, uncovering a nuclear
interaction between Wnt and Hippo, suggest a
two-tiered mechanism by which Hippo negatively modulates Wnt signaling in multiple contexts
Wnt signaling has distinct functions in the
two cardiac progenitor fields (13). Nonetheless,
we find no proliferation difference between first
heart field–derived left ventricle and SHF-derived
right ventricle in Salv CKO hearts, indicating
shared organ size-control mechanisms for the
two myocardial lineages. Also Wnt signaling is
low in myocardium even though Wnt ligands
are expressed (17). Our findings suggest that, in
Hippo-low cardiac progenitors, progrowth genes
regulated by Hippo and Wnt are actively transcribed. In cardiomyocytes, Hippo signaling restricts Yap from the nucleus, resulting in diminution
of Hippo-Wnt–regulated genes.
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Acknowledgments: We thank J. Epstein, S. Sasakura,
T. Gridley, M. Sudol, F. Long, and B. Amendt for reagents.
Supported by NIH grants (J.F.M. and R.L.J.), T32
DE15355-04 (M.B.C.), R01HD052785 and
R01HD060579 (R.L.J.), AHA09PRE2150024 (J. W.),
and AHA10POST4140029 (T.H.). Microarray data
(accession number GSE27259) can be accessed at the
Gene Expression Omnibus (GEO) repository ( www.ncbi.
Supporting Online Material
Materials and Methods
Figs. S1 to S9
Tables S1 and S2
13 October 2010; accepted 14 February 2011