DOI: 10.1126/science.1199010
, 458 (2011);
332 Science , et al.
Todd Heallen Proliferation and Heart Size Hippo Pathway Inhibits Wnt Signaling to Restrain Cardiomyocyte
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waters at these latitudes,which is supported by the extremely high d 18O values for sirenian tooth enamel sampled from these locations.Combined results of AGCM simulations that span the Eocene and temperature-independent d 18O sw values from early to middle Eocene si-renian tooth enamel indicate an identifiable latitudinal gradient in d 18O sw and an associated enhanced tropical hydrolog
ic 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 d 18O sw 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 d 18O sw sug-gest that,despite falling atmospheric p CO 2and widespread cooling (1,4),the atmospheric cir-culation and hydrologic cycle of the Eocene green-house world were broadly stable.Furthermore,the tropical d 18O sw 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 d 18O sw 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 d 18O data have previously indicated.
References and Notes
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Nature 445,639(2007).
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Acknowledgments:We thank S.Bajpai,D.P.Domning,
S.Sorbi,and the museums and institutions that made fossil specimens available for this study (a list of all institutions is provided in the SOM);P.Haselhorst and S.L.Peek for assistance with sample preparation;the University of Wyoming Stable Isotope Facility for stable isotope analyses;and E.Speelman.Funding for travel and analyses was provided by the American
Chemical Society Petroleum Research Fund (M.T.C.)and the NSF (M.T.C.).Computation was supported by Virginia Tech Advanced Research Computing and the Department of Geosciences.We also thank two anonymous reviewers for critical and thoughtful comments that improved this manuscript.
Supporting Online Material
/cgi/content/full/332/6028/455/DC1Materials and Methods Figs.S1to S3Table S1References
2December 2010;accepted 4March 201110.1126/science.1201182
Hippo Pathway Inhibits Wnt Signaling to Restrain Cardiomyocyte Proliferation and Heart Size
Todd Heallen,1Min Zhang,1Jun Wang,1Margarita Bonilla-Claudio,1Ela Klysik,1Randy L.Johnson,2James F.Martin 1*
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 -cat
enin on Sox2and Snai2genes.These data uncover a nuclear interaction between Hippo and Wnt signaling that restricts cardiomyocyte proliferation and controls heart size.I
n 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 Ste20family
kinases Mst1and 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,Lats1and Lats2,which
are homologous to Drosophila W arts .Lats kinases complex with Mob to phos-phorylate Y ap 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 transcriptionally inactive.
To determine whether the Hippo signaling path-way functions in the determination of mammalian heart size,we inactivated the single mammalian Salv ortholog in the mouse heart by using a Salv conditional null allele and the Nkx2.5cre allele that directs cardiac cre activity (5–7).Yap phos-phorylation at Ser 127(pY AP)was examined in Nkx2.5cre :Salv f/f (Salv CKO )mutants via Western blot and immunofluorescence (IF)(fig.S1).IF of embryonic day 9.5(E9.5)sagittal sections re-vealed pYap signal in both second heart field
1
Institute of Biosciences and Technology,Texas A&M System Health Science Center,2121West Holcombe Boulevard,Houston,TX 77030,USA.2Department of Biochemistry and Molecular Biology,M.D.Anderson Cancer Center,Houston,TX 77030,USA.
*To whom correspondence should be addressed.E-mail:jmartin@ibt.tamhsc.edu
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(SHF)progenitors and outflow tract and ventric-ular cardiomyocytes (fig.S1,A to D).Salv CKO hearts had reduced pY AP 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 ar-rangement of chambers and valves were unaffected (Fig.1,C and D),consistent with preserved pattern-ing observed in Hippo mutant imaginal discs (4).Hearts of some Salv CKO mutant animals had a ventricular septal defect (VSD),indicating that Hip-po 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,E14.5and earlier stages.
Salv CKO mutant hearts had expansion of trabecular and subcompact ventricular myo-cardial layers,thickened ventricular walls,and enlarged ventricular chambers without a change in myocardial cell size (Fig.1,E and F,and fig.S2).Lats2and Mst1/2CKO E11.5mutant hearts had similar myocardial expansion phenotypes (fig.S3).
We investigated cardiomyocyte proliferation by double immunostaining with phosphorylated Ser 10histone 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).
SHF cardiac progenitors that contribute to right ventricle had normal cellular proliferation levels in Sal
v CKO ,Lats2CKO ,and Mst1/2CKO mutants,indicating that Salv CKO mutant cardio-megaly was unlikely to be due to elevated cardiac progenitor number (fig.S5).
Microarray revealed that known canonical Wnt target genes were up-regulated in Salv CKO embryos (Fig.3A).This Wnt-Hippo signature in-cluded Sox2,which has been implicated in cardiac repair and cell reprogramming (8,9);Snai2/Slug ,a tumorigenesis factor having roles in epithelial-mesenchymal transition and cell growth (10);and the anti-apoptosis gene Birc5/Survivin (11).
Reverse transcription quantitative polymerase chain reaction (qRT-PCR)validated up-regulated expression of Sox2and Snai2,as well as cdc20,l-Myc ,Birc2,and Birc5,in Salv CKO embryonic hearts (Fig.3B).With the exception of l-Myc ,reduced expression was observed in Mef2c cre ;b -catenin f/f mutants (Fig.3C).Other b -catenin –regulated genes,such as Fgf10and Isl1,were un-changed in Salv CKO hearts,indicating incom-plete overlap between Yap targets and b -catenin targets (fig.S6).In situ hybridization revealed Sox2and Snai2up-regulation in Salv CKO E12.5hearts (fig.S6).
E12.5hearts were immunostained with b -catenin –specific antibodies to assay the nuclear
b -catenin index,a readout for cells receiving a Wnt signal.Whereas b -catenin localized to plasma membrane junctions and cytosol of control myo-cardium,Salv CKO mutants had a fourfold in-crease i
n nuclear b -catenin staining (Fig.3,D to F).These findings indicate that canonical Wnt sig-naling in cardiomyocytes is derepressed upon Salv deletion and support the notion that Hippo sig-naling inhibits Wnt/b -catenin to regulate heart size.To obtain geneti
c evidence that Hippo signal-ing negatively regulates Wnt/b -catenin –dependent cardiac growth,we crosse
d Salv CKO mic
e to a b -catenin conditional null allele to generate Nkx2.5cre ;Salv f/
f ;b cat f/+(Salv/b cat f/+CKO )em-bryos that are Hippo-deficient with reduced b -catenin dosage.
Myocardial thickness of Salv/b cat f/+CKO hearts was significantly reduced by comparison to Salv CKO (figs.S7,B and C,and S2).V entricular proliferation rates,as well as subcompact and trabecular myocardial thickness,in Salv/b cat f/+CKO ;mutants resemble those of control (Fig.4A and figs.S2and S7,A and C).qRT -PCR in-dicated that Sox2,Snai2,Birc2,and Cdc20ex-pression levels reverted to control or were lower in hearts with reduced b -catenin dosage (Fig.4B).Suppression of the Hippo myocardial over-growth phenotype by reduced b -catenin indicates that Wnt signaling is required for up-regulated car-diomyocyte proliferation and cardiomegaly in Hippo mutants.
To investigate whether Yap and b -catenin are in the same molecular complex,we immunopre-cipitated protein extracts from E14.5hearts with Yap and pY AP-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)]P2neona
te 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 ;Salv f/+.
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only with nuclear,nonphosphorylated Yap,in-dicating that the Yap/b -catenin molecular inter-action is nuclear (Fig.4C).
Because Yap associates with DNA-binding Tead/Tef transcription factors whereas b -catenin binds to Lef/Tcf factors (12,13),we examined surrounding genomic sequence of genes within the Wnt-Hippo expression signature for Tead/Tef and Lef/Tcf binding sites.For Sox2and Snai2,candidate Yap/Tead binding sites were identified both upstream and downstream of open reading frames (fig.S7D).Conserved Tcf/Lef binding elements (CTTTG)were in close proximity to Sox2and Snai2downstream candidate Yap/Tead sites (fig.S7D).
Chromatin immunoprecipitation (ChIP)re-vealed that Yap and b -catenin were recruited to Sox2and Snai2downstream regions but not upstream sites (Fig.4D).Sequential ChIP re-vealed that Yap and b -catenin concurrently oc-cupied Sox2and Snai2,suggesting that Yap and b -catenin are contained within a common reg-ulatory complex on Sox2and Snai2(Fig.4D).Transfection experiments indicated that Yap and b -catenin transfection along with their DNA binding co-factors induced luciferase expression from both Sox2and Snai2reporter plasmids.
Luciferase reporter activity was significantly reduced by preventing Yap or b -catenin recruit-ment to t
he reporter either individually or con-currently (Fig.4E).These findings support the model that Yap and b -catenin recruitment to Sox2and Snai2chromatin through their respective
Fig.2.Cardiomyocyte proliferation in Salvador mutant ventricles.E12.5control (top )and Salv CKO (middle )coronal sections of left ventricles:stain with TO-PRO-3,blue;a -pHH3,red;and a -MF20,green.Arrows,pHH3/MF20-positive cells.(Bottom )pHH3-positive cardiomyocytes quantification.Control genotype is Nkx2.5cre ;Salv f/+
.
Fig.3.Salvador deletion potentiates canonical Wnt signaling.Heat map (A )and qRT-PCR validation (B )showing relative transcript levels of Wnt/b -catenin target genes.Values were determined as a mean of three samples T SD with glyceraldehyde-3-phosphate dehydrogenase control.(C )qRT-PCR of Wnt/b -catenin target genes in b -catenin CKO hearts.(D and E )IF images with quantification (F )of E12.5heart sections:stain with TO-PRO-3,blue,and a -b -catenin,green.Nuclear b -catenin identified by b -catenin/TO-PRO-3signal overlap (arrows).Control genotype is Nkx2.5cre ;Salv f/+.
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DNA binding partners potentiates transcriptional activity.
The Yap and b -catenin interaction on genes such as Snai2and Sox2uncovers a nuclear mechanism for antagonistic control of cardio-myocyte 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 pYap data indicate that there is some Hippo activity in E9.5SHF cardiac pro-genitors,cell proliferation in Hippo mutant SHF was unchanged from control.This may reflect Salv -independent Hippo activity and perhaps over-lapping functions with other Hippo pathway ki-nases (i.e.,Lats1).Our data support the previous observation that Hippo signaling was low in E10.5myocardium (14).Although Salv CKO cardiomyo-cyte 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 Ccdn1is up-regulated,suggesting shared mechanisms between liver and heart (4).
Although apoptosis inhibitors Birc2and Birc5were up-regulated in Salv CKO mutant hearts,apoptosis was unchanged.
Important recent work uncovered a repressive cytoplasmic interaction between pTaz and Dvl in kidney (16).Our findings,uncovering a nuclear interaction between Wnt and Hippo,suggest a two-tiered mechanism by which Hippo negative-ly modulates Wnt signaling in multiple contexts (fig.S8).
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 tran-scribed.In cardiomyocytes,Hippo signaling re-stricts Y ap 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.),T32DE15355-04(M.B.C.),R01HD052785and
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 (bi.v/geo/).
Supporting Online Material
/cgi/content/full/332/6028/458/DC1Materials and Methods Figs.S1to S9Tables S1and S2References
13October 2010;accepted 14February 2011
10.1126/science.1199010
Fig.4.Yap interacts with b -catenin.(A )Quantification of pHH3indices in control,salv CKO ,and Nkx2.5
cre ;Salv f/f ;b -catenin F/+E12.5hearts.(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 Sox2and Snai2loci were tested (asterisks).Refer to fig.S7D for ChIP assay de-sign.IgG,immunoglobulin G.(E )Luciferase reporter assays with co-transfected factors.
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