waters at these latitudes, which is supported by
the extremely high d18O values for sirenian
tooth enamel sampled from these locations.
Combined results of AGCM simulations that
span the Eocene and temperature-independent
d18Osw values from early to middle Eocene si-
renian tooth enamel indicate an identifiable
latitudinal gradient in d18Osw and an associated
enhanced tropical hydrologic cycle that is per-
sistent across the Eocene. These results support
not only the long-held belief that the greenhouse
climate of the early Paleogene (and greenhouse
climates in general) was characterized by an
enhanced, but balanced, subtropical hydrologic
cycle and wetter mid-high latitudes than are seen
under modern conditions (7–9), but also suggest
that the early Paleogene tropics had substantially
decreased evaporation and increased precipita-
tion that both contributed to much lower d18Osw
values than those that exist today. The persistence
of the enhanced hydrologic cycle across the Eo-
cene simulations and its match to the sirenian
enamel-derived latitudinal gradient in d18Osw sug-
gest that, despite falling atmospheric pCO2 and
widespread cooling (1, 4), the atmospheric cir-
culation and hydrologic cycle of the Eocene green-
house world were broadly stable. Furthermore,
the tropical d18Osw signature associated with such
hydrologic-cycle changes is up to 1.0‰ lower
than the expected, ice-volume–induced offset
of −1.0‰ between icehouse and greenhouse
conditions. If this effect on d18Osw and, thence,
the isotopic composition of foraminiferal tests is
not recognized and considered, systematic over-
estimation of Eocene tropical sea-surface temper-
atures by up to 4°C could result. Our results, thus,
suggest that the Eocene tropics were not only
wetter but may have been cooler than foraminif-
eral d18O data have previously indicated.
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Acknowledgments: We thank S. Bajpai, D. P. Domning,
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institutions is provided in the SOM); P. Haselhorst
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Chemical Society Petroleum Research Fund (M. T.C.) and
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Supporting Online Material
Materials and Methods
Figs. S1 to S3
2 December 2010; accepted 4 March 2011
Hippo Pathway Inhibits Wnt Signaling
to Restrain Cardiomyocyte
Proliferation and Heart Size
Todd Heallen,1 Min Zhang,1 Jun Wang,1 Margarita Bonilla-Claudio,1 Ela Klysik,1
Randy L. Johnson,2 James F. Martin1*
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 b-catenin heterozygosity suppressed the
Hippo cardiomyocyte overgrowth phenotype. Furthermore, the Hippo effector Yap interacts with
b-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.
In mammals, organ-extrinsic influences such as nutritional status, circulating growth factors, and hormones have a large impact on organ
size control (1). Several lines of evidence indicate
that there are also organ-intrinsic mechanisms to
modulate organ size (1, 2). Insight into genetic
pathways regulating organ size has come from
Drosophila, where two main organ-size control
pathways are bone morphogenetic protein (Bmp)
and Hippo signaling pathways (3, 4). Whether
these growth control mechanisms are broadly
conserved in mammalian organs remains unclear.
Organ size is important in cardiac develop-
ment, because the heart must be large enough to
generate a physiological cardiac output but not so
large as to block cardiac outflow, as in obstructive
cardiomyopathies. The mammalian core Hippo
signaling components include Ste20 family
kinases Mst1 and Mst2, which are homologous
to Drosophila Hippo. Mst kinases form an active
complex with WW repeat scaffolding protein
Salvador (Salv), also called WW45, that phos-
phorylates large tumor suppressor homolog (Lats)
kinase. Mammals have two Lats genes, Lats1
and Lats2, which are homologous to Drosophila
Warts. Lats kinases complex with Mob to phos-
phorylate Yap and Taz, two related transcriptional
coactivators. Upon phosphorylation, Yap and Taz,
the most downstream Hippo signaling compo-
nents, are excluded from the nucleus and are
1Institute of Biosciences and Technology, Texas A&M System
Health Science Center, 2121 West Holcombe Boulevard,
Houston, TX 77030, USA. 2Department of Biochemistry and
Molecular Biology, M. D. Anderson Cancer Center, Houston, TX
*To whom correspondence should be addressed. E-mail: