Wnt/β-catenin and Bmp signals control distinct sets oftranscription factors in cardiac progenitor cellsAlexandra Klausa, Marion Müllera, Herbert Schulzb, Yumiko Sagac, James F. Martind, and Walter Birchmeiera,1

aDepartment of Cancer Research and bDepartment of Cardiovascular Research, Max Delbrueck Center for Molecular Medicine, 13125 Berlin, Germany;cDivision of Mammalian Development, National Institute of Genetics, 1111 Yata, Mishima Shizuoka 411-8540, Japan; and dDepartment of MolecularPhysiology and Biophysics, Baylor College of Medicine, Houston, TX 77030

Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved May 22, 2012 (received for review December 24, 2011)

Progenitor cells of the first and second heart fields depend on car-diac-specific transcription factors for their differentiation. Usingconditional mutagenesis of mouse embryos, we define the hierar-chy of signaling events that controls the expression of cardiac-specific transcription factors during differentiation of cardiacprogenitors at embryonic day 9.0. Wnt/β-catenin and Bmp actdownstream of Notch/RBPJ at this developmental stage. MutationofAxin2, the negative regulator of canonical Wnt signaling, enhan-ces Wnt and Bmp4 signals and suffices to rescue the arrest of car-diac differentiation caused by loss of RBPJ. Using FACS enrichmentof cardiac progenitors in RBPJ and RBPJ/Axin2 mutants, embryocultures in the presence of the Bmp inhibitor Noggin, and by cross-ing a Bmp4 mutation into the RBPJ/Axin2 mutant background, weshow that Wnt and Bmp4 signaling activate specific and nonover-lapping cardiac-specific genes in the cardiac progenitors: Nkx2-5,Isl1 and Baf60c are controlled by Wnt/β-catenin, and Gata4, SRF,and Mef2c are controlled by Bmp signaling. Our study contributesto the understanding of the regulatory hierarchies of cardiac pro-genitor differentiation and outflow tract development and hasimplications for understanding and modeling heart development.

cardiogenesis | MesP1-cre | mesoderm patterning | canonical Wnt |progenitor differentiation in culture

Distinct cell types, such as the mesodermal progenitors of thefirst and second heart fields (FHF and SHF) as well as car-

diac neural crest cells, contribute to the development of thevertebrate heart. The FHF lineage generates the left ventricle,whereas the SHF forms the right ventricle and contributes tooutflow tract and atria. SHF and cardiac neural crest cells co-operate during the development of outflow tract vessels andvalves (1–3). Mesodermal cells of the anterior postgastrulatingmouse embryo and FHF and SHF progenitors express the tran-scription factor MesP1, which is essential for heart induction (4).FHF and SHF progenitors can be distinguished by their level ofproliferation and differentiation. Cells of the FHF begin to dif-ferentiate by expressing Gata4, Baf60c, and Nkx2-5 in the cardiaccrescent and cardiac tube. Cells of the SHF express Isl1, whichmarks undifferentiated cells (5). The development of the outflowtract involves Isl1-expressing cardiac progenitors from the ante-rior SHF, endocardial cells, and cardiac neural crest cells (6).Mesodermal progenitor cells pass through several developmen-

tal stages before they become fully differentiated cardiomyocytes.During a first phase, mesodermal progenitors express a corecomplex of essential transcription factors and chromatin remod-elers—Gata4, Brg1, and Baf60c—which induce transcription fac-tors such as Nkx2-5, Tbx5/20, and Isl1 that define cells as cardiacprogenitors (5, 7). During predifferentiation of cardiac progenitorsinto cardioblasts, these transcription factors induce the expressionofMef2c andHand1/2 and also early cardiac muscle-specific genes,e.g., the genes that encode cardiac actin and myosin light chain 2a(8–10). In the differentiation phase, down-regulation of Isl1 andinteraction of Mef2c/Hand1/2 with the core complex induce theexpression of cardiac muscle-specific genes for structural proteinssuch as troponin T2 and myosin heavy chains (11, 12).How the Notch, Wnt, and Bmp developmental signaling path-

ways control heart development and the heart-specific transcrip-tion factors has been studied. Notch signals repress differentiation

by inhibiting Mef2c expression during early cardiogenesis inXenopus and in embryonic stem cells and promote differentiationin both myocardium and endocardium during later developmentalstages (6). The Notch intracellular domain translocates to thenucleus to form a transcriptional complex with the DNA-bindingprotein RBPJ and the coactivator Mastermind-like to activatetarget genes (6). Studies in mouse, chick, frog, and fish demon-strate that high levels of Bmp activity are necessary for the ex-pression of Nkx2-5 and Gata4 and for myocardial differentiation(5, 9, 13, 14). Canonical Wnt signals are essential for proliferationof Isl1-expressing SHF progenitors and also promote Nkx2-5 ex-pression and subsequent cardiac differentiation by down-regu-lating HDAC1 (5, 13, 15). In the presence of canonical Wntligands, β-catenin is stabilized and translocates to the nucleus,where it interacts with Lef/Tcf transcription factors to activatetarget genes (16–18). Axin proteins are negative regulators andcontrol β-catenin degradation. Axin2 is a target gene of canonicalWnt signals, and its expression is a useful marker to define cellsexposed to Wnt (19–22). Axin2-null mice are viable and have noobvious defects in cardiac development. However, increased ca-nonical Wnt signaling is found in homozygous Axin2−/− cells aftertransplantation into wild-type tissues and embryos (21, 22). Def-icits in skull formation in Axin2-mutant mice appear to be causedby tissue-specific up-regulation of canonical Wnt signals (20).

ResultsWnt Gain-of-Function Mutation Axin2−/− Rescues Defects of SHFMorphogenesis in Conditional RBPJ/Notch Mice. Interference withNotch signaling arrests cardiac development (6, 23–25). In ac-cordance, MesP1-cre–induced mutation of RBPJlox/lox (hereaf-ter, “RBPJ mutant”) reduced the size and compactness of theheart, as revealed by analyses of H&E-stained sections at embry-onic day (E) 9.75 (Fig. 1 A and B and Table S1). The right ven-tricles were strongly affected, and the expression of thepredifferentiation factor Hand2 was lost (Fig. 1 C and D). In situhybridizations showed a pronounced down-regulation of Gata4 inthe heart and of Isl1 in the splanchnic mesoderm and outflow tractmyocardium at E9.25 (Fig. 1 E and F and Fig. S1 A and B). Ab-lation of RBPJ (Fig. S1 C–D′) strongly attenuated canonical Wntsignaling, as seen by the reduced expression of the Wnt targetgenes Axin2 and Lef1, particularly in the rudimentary anteriorSHF and SHF derivatives at E8.5–9.25 (Fig. S1 E–L and TableS2). Moreover, a major reduction in Lef1 and Isl1 coexpressionoccurred in the splanchnic mesoderm (Fig. S1 M–P). Axin2 ex-pression also was visualized in mice that carried a Wnt reporter,the heterozygous Axin2LacZ allele (19, 21). LacZ was expressed insplanchnic mesoderm and SHF derivatives in control mice (13)

Author contributions: A.K. and W.B. designed research; A.K. and M.M. performedresearch; A.K., Y.S., and J.F.M. contributed new reagents/analytic tools; A.K. and H.S.analyzed data; and A.K. and W.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The data reported in this paper have been deposited in the Gene Ex-pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE36804).1To whom correspondence should be addressed. E-mail: wbirch@mdc-berlin.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1121236109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1121236109 PNAS | July 3, 2012 | vol. 109 | no. 27 | 10921–10926

DEV

ELOPM

ENTA

LBIOLO

GY

Dow

nloa

ded

by g

uest

on

Janu

ary

3, 2

021

but was reduced at these sites in the RBPJ mutants (Fig. 1 G andH). These data indicate cross-talk between Notch/RBPJ and ca-nonical Wnt signaling, operating particularly in the SHF, in earlyheart morphogenesis.We confirmed by genetic means that Notch/RBPJ and Wnt/

β-catenin interact during SHF morphogenesis, and we generatedcompound MesP1-cre;RBPJlox/lox;Axin2−/− mutants (hereafter“RBPJ/Axin2 double mutants”). Remarkably, the Axin2−/− mu-tation substantially but not completely rescued the heart phe-notypes at E9.25 in RBPJ mutants (Fig. 1 I–L and Table S1). Inparticular, the size of the right ventricles and outflow tracts andalso the expression of Nkx2-5 were normalized (Fig. 1 J and K).Elimination of Axin2 alone did not produce heart hyperplasia(Fig. 1L). Pronounced activation of canonical Wnt signaling byβ-galactosidase expression occurred in splanchnic mesoderm andventricular walls of RBPJ/Axin2LacZ/LacZ mutants, as analyzed

by immunohistology of Cre+ cells (Fig. S2 A–B′′). Canonical Wntsignals previously had been shown to be essential for pro-liferation of Isl1-expressing SHF progenitors (5, 13). However,proliferation in Isl1-expressing cells was little affected in RBPJsingle mutants and RBPJ/Axin2+/− mutants (Fig. S2 C and D).However, in RBPJ/Axin2+/− mutants, a pronounced rescue of thenumber of Isl1-expressing cells in the splanchnic mesoderm andof ventricular cardiomyocyte differentiation was observed, evi-denced by the restored expression of troponin T2 (Fig. 1 M–Tand Fig. S2E). Thus, coexpression of Nkx2-5 and troponin T2were not observed in RBPJ mutant hearts, whereas robust coex-pression was seen in RBPJ/Axin2 double mutants (Fig. 1 Q–T).We used gene-expression profiling of dissected cardiac tissue

(heart, including SHF) to study the mechanism of the rescue ofthe cardiac differentiation program in RBPJ/Axin2 mutants (Fig.1U). Compared with RBPJ single-mutant tissues, the expression of

OFT

RV

OFT

RV

Axi

n2-L

acZ

OFT

RVRVLV

RVLVH

/E

Isl1

Han

d2

DC

52.9E52.9E

E

HG

BA

MesP1-cre; RBPJ lox/loxControl MesP1-cre;

RBPJ lox/loxControl

Control MesP1-cre; RBPJ ;Axin2

lox/+-/-

MesP1-cre; RBPJ

lox/lox-/-

MesP1-cre; RBPJ ;Axin2lox/lox

Nkx

2-5 I

RV LV

OFT

L

RVLV

OFTOFT

LV

RV

J K

RV LV

OFT

Trop

T2/N

kx2-

5

E9.2

5

control Axin2 mutant RBPJ mutant RBPJ/Axin2 mutant

BMPWNTHEART

Rel

ativ

e ex

pres

sion

(A

U)V

Q

FGFGFG

FG

RV

LV RRV LV

S

RV

LVTRV

LV

Isl1

/cre

M N O P

M’ N’ O’ P’

mutant

Axin2

mutant

RBPJ/Axin

2

BMP

WNT

HEART

Gpr83SRFAc tc 1Me f2cTNCWnt2Wnt5aWnt9bDkk1Le f1LRP6Smad6Bmp4Msx2

NogginBmpRIa

Nkd2PCDH7U

mutant

RBPJ

Control

OFT

RV

F

SMA SRF Noggin Bmp7 Bmp4 Wnt5a Isl1 TropT2 Axin2

Fig. 1. The Axin2 mutation restores Wnt and Bmp signalingand substantially rescues the RBPJ phenotype in the heart.(A and B) H&E staining of transverse sections of hearts ofcontrol and RPPJ mutants at E9.75. Right ventricles (RV) aremarked by arrows. LV, left ventricle. (C and D) Whole-mount insitu hybridization for Hand2 in controls and RBPJ mutants atE9.75. (E and F) In situ hybridization on sections for Isl1 atE9.25. (G and H) Canonical Wnt activation as determined ontransverse sections of whole-mount in situ hybridizations forAxin2-LacZ in controls (Axin2LacZ/+) and RBPJ mutants (RBPJ/Axin2LacZ/+) at E9.25. Right ventricles (RV; see Inset magnifica-tions) and outflow tract myocardium (OFT) are marked byarrows and arrowheads, respectively, and splanchnic meso-derm is marked by asterisks. (I–L) Whole-mount in situ hy-bridization for Nkx2-5 in controls, RBPJ single-mutant, RBPJ/Axin2 double-mutant, and Axin2 single-mutant embryos atE9.25. (M–T) Immunofluorescence analysis for Isl1 (red) and cre(green) (M–P), and TropT2 (red) and Nkx2-5 (green) (Q–T) inindicated genotypes. See also magnifications of splanchnicmesoderm (M′–P′). FG, foregut. (U) Heatmap of gene-expressionarrays of RBPJ/Axin2 double mutants vs. controls and RBPJ andAxin2 single mutants. The restored expression of particulargenes is shown in orange for cardiac-specific genes, green forWnt genes, and magenta for Bmp-controlled genes. (V) qRT-PCRanalyses of mRNA expression at E9.25 in the heart region ofcontrols (gray bars), Axin2 single (blue bars), RBPJ single mutants(magenta bars), and RBPJ/Axin2 double mutants (blue/magenta-striped bars). Error bars represent SEM (n = 4). *P < 0.05, **P <0.01, ***P < 0.005. AU, arbitrary units.

10922 | www.pnas.org/cgi/doi/10.1073/pnas.1121236109 Klaus et al.

Dow

nloa

ded

by g

uest

on

Janu

ary

3, 2

021

many down-regulated genes was largely restored in RBPJ/Axin2double mutants (compare the third and second columns in Fig. 1Uand see Tables S1 and S3). Among these genes were those re-quired for cardiac differentiation, e.g., cardiac actin (Actc1), SRF,and Mef2c (identified in orange in Fig. 1U). Moreover, the ex-pression of >20 genes that encode components and targets ofcanonical Wnt signaling, e.g., Wnt2, Dkk1, and Lef1 (identified ingreen in Fig. 1U; see also Table S3, and for Wnt target genes seewww.stanford.edu/group/nusselab/cgi-bin/wnt/) were up-regulatedalso. Surprisingly, the expression of many genes encoding mem-bers of the Bmp pathway (Bmp4, BmpR1a) and Bmp target genes(Smad6, Msx2) (indentified in magenta in Fig. 1U; see also TableS3) was rescued also. We confirmed restoration of expression ofselected cardiac differentiation genes (SMA, SRF, and troponinT2) and of Wnt and Bmp target genes by quantitative RT-PCR(qRT-PCR) (Fig. 1V). Furthermore, Bmp4 but not Bmp7 expres-sion was restored (Fig. 1V; see also below). The majority of thesegenes were not or were only slightly affected in Axin2 singlemutants [Fig. 1U (compare first and fourth columns) and V]. Thus,our genetic data indicate that Wnt/β-catenin acts downstream ofRBPJ/Notch and controls Bmp signals in morphogenesis anddifferentiation of cardiac cells.

Wnt/β-Catenin and Bmp Signals Downstream of Notch/RBPJ AreCrucial for Different Stages of Cardiac Progenitor Differentiation.The minimal core complex, Gata4 and Baf60c, that inducescardiac progenitor specification was down-regulated in the RBPJmutants and was rescued by Axin2 ablation, as confirmed by in situhybridization (Fig. 2 A–D; see also below). The factors produced

during progenitor specification, Nkx2-5, Isl1, SRF, and Tbx20 weresimilarly down- and up-regulated (Figs. 1 I–L and V and Figs. 2 E–H and Table S2). The expression of factors that mark prediffer-entiation, Hand2 and Mef2c, also was down-regulated in RBPJmutants and was rescued by Axin2 ablation (Fig. 2 I–P). Elimi-nation of one allele of Axin2 sufficed to rescue, in part, the ex-pression of Mef2c (quantifications are given in Fig. 2P). In situhybridization also confirmed the reduced expression of Lef1,Wnt2, and the Bmp target gene Smad6 in SHF derivatives of RBPJmutants and their up-regulation in RBPJ/Axin2 double mutants(Fig. 2 Q–X and Fig. S2 F–I). The expression of the Notch/RBPJtarget gene Hey3 was not restored, but Hey1 and Hey2 were nor-malized by Axin2 ablation (Fig. S2 J–P and Table S3) (6). Theexpression of Tenascin C (Fig. S2 Q–T) and of other genes not yetdescribed as being involved in SHF morphogenesis [e.g., proto-cadherin (PCDH7) (Fig. S2 U–X) and G protein-coupled receptorGpr83, Nkd2, and others (identified in Table S3)] was normalized.Thus, Wnt/β-catenin and further signals regulated by Wnt arerequired for the expression of transcription factors that are es-sential for specification of cardiac progenitors as well as for dif-ferentiation, i.e., the induction of heart-specific structural proteins.

Axin2Mutation Restores Expression of Crucial Transcription Factors inFACS-Sorted Cardiac Progenitors of RBPJ Mutants. Our analysesshow that Axin2 ablation rescues cardiac development of RBPJmutants during the formation of cardiac progenitor cells. Toconfirm the stage and cell type precisely, we used FACS to iso-late cardiac progenitors. A reporter line that expresses EYFP ina cre-inducible manner was crossed into controls and mutants

Control MesP1-cre; RBPJ

lox/lox-/-

MesP1-cre; RBPJ ;Axin2lox/lox

MesP1-cre; RBPJ ;Axin2

lox/+-/-

Han

d2

GF HE

Tbx2

0B

af60

c

X

Smad

6 W

Rel

ativ

e ex

pres

sion

(AU

)

VU

KJ LI

A CB D

RBPJ/Axin2 mutantRBPJ mutantAxin2 mutant tnatum2nixA/JPBRlortnoc +/-

Lef1

T

Mef

2c

NM PO

Rel

ativ

e ex

pres

sion

(AU

)R

elat

ive

expr

essi

on (A

U)

R SQ

-/- -/-Smad6

Lef1

Mef2c

Fig. 2. Canonical Wnt and Bmp signaling downstream ofNotch/RPBJ are crucial for the different stages of cardiac dif-ferentiation at E9.25. (A–L) In situ hybridization on transversesections of hearts for Baf60c, Tbx20, and Hand2 in indicatedgenotypes at E9.25. (M–X) Whole-mount in situ hybridizationsof embryos or on transverse sections of hearts forMef2c, (M, N,O), Lef1 (Q, R, S), and Smad6 (U, V, W) (outflow tract/rightventricles are marked by arrows) and corresponding qRT-PCRanalyses (P, T, X) in controls, RBPJ single mutants, RBPJ/Axin2+/−,RBPJ/Axin2 double mutants, and Axin2 single mutants. Errorbars represent SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.005.AU, arbitrary units.

Klaus et al. PNAS | July 3, 2012 | vol. 109 | no. 27 | 10923

DEV

ELOPM

ENTA

LBIOLO

GY

Dow

nloa

ded

by g

uest

on

Janu

ary

3, 2

021

that carry MesP1-cre (26). Reporter expression was observed inMesP1-derived mesodermal cardiac progenitor cells that expressIsl1, in particular in the splanchnic mesoderm and distal outflowtracts, and in cells of the left and right ventricles (arrowheads inFig. 3A; merged yellow fluorescence marks coexpression of Isl1and GFP). Two distinct EYFP+ cell populations from the car-diac tissue (heart, including SHF) were isolated: small-diameterP1 cells (outlined in orange in Fig. 3B) and large-diameter P2cells (outlined in blue in Fig. 3B). The identity of the sorted cellsobtained from control hearts was verified by analyses of Isl1 andαMHC expression. RT-PCR showed that P1 cells strongly ex-pressed the progenitor-specific factors Isl1, Tbx20, and SRF,suggesting that they represent cardiac progenitors (orange barsin Fig. 3C). In contrast, P2 cells expressed reduced levels of Isl1,Tbx20, and SRF but high levels of MLC2a and αMHC typical for

differentiated cardiac cells (blue bars in Fig. 3C), suggesting thatP2 cells are cardioblasts and/or cardiomyocytes. Immunofluo-rescence showed that isolated P1 progenitors expressed Nkx2-5,whereas P2 cells had high levels of cardiac troponin (Fig. 3 D andE). When isolated P1 and P2 cells were cultured for 7 d, P1 cellsunderwent cardiac differentiation in the presence of Activin Aand Bmp4 (27), as analyzed by immunofluorescence for Nkx2-5and troponinT2 (Fig. S3 A–B′). At the end of the culture period,cardiac troponin in P1 and P2 cells was equal. Thus, P1 cells areindeed cardiac progenitors that retain the capacity to differen-tiate. P1 progenitor cells strongly expressed Notch and Wntpathway components and target genes, in particular Jag2 andNotch4 and Wnt2, Axin2, and Lef1, but these genes were seen toa lesser extent in P2 cells (Fig. S3C). These genes were expressedin cardiac tissue of RBPJ/Axin2 double mutants but not in RBPJmutants (Table S3). Other Notch pathway components, i.e., Dll4,Jag1, and Notch1/2, were highly expressed in the differentiatedP2 cardiac cells (Fig. S3C). We used P1 and P2 cell populationsof RBPJ single-mutant and RBPJ/Axin2+/− mutant hearts to de-fine potential differences in their response to the mutations.Remarkably, loss of RBPJ in P1 and P2 cells abolished Nkx2-5,Isl1, Tbx20, and Mef2c expression (yellow bars in Fig. 3F andlight blue bars in Fig. 3G). However, loss of Axin2 (one allelesufficed) substantially normalized the expression of Nkx2-5, Isl1,Tbx20, and Mef2c in P1 and P2 cells of RBPJ mutants (stripedbars in Fig. 3 F and G). Furthermore, Bmp4 expression was re-stored in RBPJ/Axin2+/− mutant P1 cells (Fig. 3 F and G Right;see also below and Fig. S4 B–D). Thus, Axin2 ablation restoredthe expression of the crucial transcription factors Nkx2-5, Isl1,Tbx20, and Mef2c and the expression of Bmp4 in RBPJ mutantcardiac cells, indicating that the sequential regulation of cardiac-specific transcription factors mediated by RBPJ, Wnt, and Bmpsignals occurs in mesoderm-derived cardiac progenitors as wellas in the early differentiated cardiac cells.

Nkx2-5, Isl1, and Baf60c are Wnt/β-Catenin Target Genes, and Gata4and SRF Are Bmp4 Targets in Cardiac Progenitors. We next definedthe heart-specific transcription factors controlled by canonicalWnt and/or Bmp signaling at this developmental stage. WholeE8.75 embryos were cultured in the presence or absenceof Noggin, a soluble Bmp inhibitor (Fig. 4A) (14). Analysis ofmRNA from dissected cardiac tissue (heart, including SHF) ofcontrol embryos treated with Noggin revealed decreased ex-pression of all cardiac-specific transcription factors and also re-duced Lef1, indicating that Noggin also affected Wnt signaling(Fig. S4A). However, analysis of RBPJ/Axin2+/− mutants, inwhich Wnt signaling is up-regulated, showed that Noggin treat-ment strongly reduced the expression of Gata4, SRF, Mef2c, andMsx2 (Fig. 4A′), indicating that Gata4, SRF, and Mef2c arecontrolled by Bmp signaling in the presence of Wnt signaling. Incontrast, Nkx2-5, Baf60c, and Isl1 expression was not affectedsignificantly by Noggin (Fig. 4 A′), showing that Nkx2-5, Baf60c,and Isl1 are controlled by Wnt but not by Bmp signals.We also investigated the expression of various Bmp ligands in

the mutant hearts. Bmp2, Bmp4, and Bmp7 expression was lost inRBPJ single mutants. Bmp4 but not Bmp2 and Bmp7 expressionwas restored in the heart of RBPJ/Axin2 double mutants (Fig. S4B–O; see also Fig. 1V). It should be noted that in Bmp4 mutantsBmp7 was expressed in the entire heart, suggesting that Bmp7might substitute for Bmp4 loss. To assess the role of the restoredBmp4 expression in the RBPJ/Axin2 double mutants and to cor-roborate the above assignment of signal-dependent expression ofcardiac transcription factors, we introduced a conditional Bmp4mutation (28) into the RBPJ/Axin2 mutant background (MesP1-cre;RBPJlox/lox;Bmp4lox/lox;Axin2+/−; hereafter “triple mutants”).Bmp signaling indeed was lost in themutants, andBmp4 expressionas well as pSmad1/5/8-Isl1 costaining were abrogated in the SHF ofthe triple mutants at E9.25 (Fig. S4 P–T′ and Table S1). However,the quantity of Isl1-expressing cells was unchanged, demonstratingthat the low pSmad1/5/8 expression was not caused by a loss of SHFcells (Fig. S4U). It should be noted that Smad1/5/8 phosphorylationwas detectable in the ventricular walls of the triple mutants, in-dicating that these differentiated cells receive signals provided by

Rel

ativ

e ex

pres

sion

(AU

)R

elat

ive

expr

essi

on (A

U)

RBPJ/Axin2 mutant (P2)RBPJ mutant (P2)control (P2)

+/-

RBPJ/Axin2 mutant (P1)RBPJ mutant (P1)control (P1)

+/-

GFP

Nkx

2-5

Trop

T2

P2

P1

Rel

ativ

e ex

pres

sion

(AU

)

control P1 cellscontrol P2 cells progenitors

cardioblasts/cardiomyocytes

G

F

C

BA

E

DMesP1-cre; EYFP lox/+MesP1-cre; EYFP lox/+

LV

RV P1 progenitors

P2 cells

YFP+

cell s

ize

Fig. 3. Wnt/β-catenin activation by Axin2 ablation restores cardiac commit-ment and Bmp signaling in SHF progenitor cells at E9.25. (A) Immunofluo-rescence analysis of GFP (green) and Isl1 (red) on a transverse section of controlembryos (arrowheads mark merged yellow fluorescence in MesP1-derived SHFcells). (B) FACS of YFP+ cells of MesP1-cre; EYFPlox/+ embryos at E9.25 showingtwo populations of YFP+ cells: P1 progenitors (outlined in orange) and P2 cells(outlined in blue). (C) Relative mRNA expression levels in the YFP+ populations(P1, orange; P2, blue) of controls show that P1 cells express higher levels ofcardiac progenitor genes, and P2 cells show a marked increase in muscle-specific genes. (D and E) Immunofluorescence analysis of GFP (blue), Nkx2-5(green), and troponin T2 (red) in P1 cells (D) and P2 cells (E). (F and G) qRT-PCRanalysis of mRNA in the cardiac progenitor population (P1 cells) (F) and in P2cells (G) of controls (MesP1-cre;EYFPlox/+; RBPJlox/+;Axin2+/−), RBPJ singlemutants (MesP1-cre;EYFPlox/+; RBPJlox/lox), and double mutants (MesP1-cre;EYFPlox/+; RBPJlox/lox;Axin2+/−). Error bars represent SEM (n = 3–6). *P < 0.05,**P < 0.01, ***P < 0.005. AU, arbitrary units.

10924 | www.pnas.org/cgi/doi/10.1073/pnas.1121236109 Klaus et al.

Dow

nloa

ded

by g

uest

on

Janu

ary

3, 2

021

a Bmp ligand other than Bmp4. In accordance with the embryoculture in the presence of Noggin, FACS-sorted P1 progenitors ofthe triple-mutant embryos displayed strongly reduced Gata4, SRF,and Mef2c expression (Fig. 4B Upper Left; the Bmp target geneMsx2 served as a control). In contrast, the additional ablation ofBmp4 had no significant effect on the expression of Nkx2-5, Isl1,and Baf60c, as shown by comparison of triple-mutant and double-mutant P1 cells (Fig. 4B Left and Fig. S5A). In differentiated P2cells, the expression ofGata4, SRF, andMef2c was unchanged (Fig.4B Right and Fig. S5B). The ventricular wall contained differenti-ated pSmad1/5/8+ cardiac cells, which are a probable source of P2cells (Fig. S4 P–T). In situ hybridization confirmed that the ex-pression of Gata4, Mef2c, and Hand2 was down-regulated in theSHF at E9.25, but the expression of Isl1,Nkx2-5, and Tbx20was notaffected (Fig. 4C–L, Fig. S5C–R, and Table S2). The change in theexpression of the cardiac transcription factors was accompanied atE9.5 by a marked arrest of differentiation in the distal outflow tractof the triple mutants, a possible consequence of the loss of Hand2and Mef2c in the anterior SHF (Fig. 4 M–P; bars mark the area ofcells lacking troponin T2 in the distal outflow tract).In summary, we used genetics and embryo culture to show that

Wnt signaling controls the expression of Nkx2-5, Baf60c, andIsl1, whereas Bmp signaling regulates Gata4, SRF, and Mef2c.Wnt and Bmp signaling act downstream of Notch in cardiacprogenitors. We also unraveled a regulatory hierarchy in outflowtract development and showed that the Bmp- and Wnt-de-pendent expression of Hand2 and Mef2c in the anterior SHFresulted in arrested differentiation.

DiscussionHere we used mouse genetics, FACS enrichment of cardiacprogenitors, and embryo culture with pharmacological inhibitorsto unravel the regulatory hierarchies of Wnt/β-catenin, Bmp, andNotch signaling during cardiac progenitor differentiation andoutflow tract development. The study may have implications forunderstanding and modeling heart development.We show that cardiac defects of a conditional RBPJ mutation

at E9 could be rescued substantially by ablating the negativeWnt regulator Axin2, indicating that Wnt/β-catenin is a centraldownstream effector of Notch signaling in cardiac progenitor dif-ferentiation and outflow tract morphogenesis (Fig. S6). Moreover,the expression of Bmp4 and Bmp-controlled genes was restored insuch double mutants, suggesting that Wnt/β-catenin acts upstream

of Bmp signaling in this developmental context. The rescue byAxin2 was virtually complete with respect to the restoration ofcanonical Wnt signaling in the heart, as assessed by the expressionof the endogenous Axin2 promoter (Axin2LacZ) and of Wnt targetgenes such as Lef1 or by the expression of cardiac-specific genes inisolated cardiac progenitors. However, cardiac morphogenesis wasattenuated, indicating that, in addition to Wnt signaling, Notchsignaling may control other essential target genes, e.g., Bmp2 andBmp7 (shown in this study) or Fgf (5, 23). Outflow tract morpho-genesis is known to depend on signals provided by cardiac pro-genitors from the anterior SHF as well as endocardial, neural crest,and endodermal cells (5, 6, 29). In RBPJ/Axin2 double mutants,diffusible morphogens (Wnt ligands and Bmp4, here shown bygene profiling and by in situ hybridization) may affect gene ex-pression and cell death in MesP1− cells, such as neural crest orendodermal cells. Thus the up-regulated expression of suchmorphogens in double mutants also might contribute indirectly tothe rescued development of nonmesoderm-derived cells (29). Inprevious analyses of mice that carried a conditional Jagged1 mu-tation or that expressed a dominant-negativeMaml usingMef2c-crealso exhibit SHF morphogenesis affects and reduced Bmp and Fgfsignaling (23). In contrast, in studies using Isl1-cre, elevated Wnt/β-catenin signaling has been observed after conditional mutation ofNotch1 but not RBPJ (24, 25). The difference in phenotypes mightbe caused by the use of distinct cre lines to target cardiac cells or byeffects that are not mediated by canonical Notch signaling.The question arises as to how the two developmental signaling

systems, Wnt/β-catenin and Bmp, control the expression of thetranscription factors required for cardiac differentiation. Ourresults suggest a model in which Wnt/β-catenin regulates thecrucial transcription factors Baf60c, Nkx2-5, and Isl1 at an earlystage of progenitor formation (Fig. S6). At this stage Wnt/β-cat-enin also regulates Bmp4 signaling, which in turn activates Gata4and SRF. Together, these two sets of effectors activate the tran-scription factors Mef2c and Hand2, thus allowing cardioblast for-mation. To corroborate the nature of genes that are regulated byeither Wnt/β-catenin or Bmp signaling, we used embryo culturein the presence of the Bmp inhibitor Noggin and introduced aconditional Bmp4 mutation into RBPJ/Axin2 mice. Both experi-ments confirmed the distinct regulation of the two sets of non-overlapping transcription factors by Wnt/β-catenin and Bmpsignaling (Fig. S6). Analyses of Bmp4mutants indicate that Bmp4signals control the expression ofGata4 and SRF; however, residual

1lsI4ataG Baf60cNkx2-5Lef1Msx2Mef2cSRF

A’A

E9.25

E8.75

RBPJ/Axin2 RBPJ/Axin2 (+nog)+/-

+/-

+/-

RBPJ/Axin

2 /

Bmp4 mutan

t

RBPJ/Axin

2

mutant

Control +/-+/-

RBPJ/Axin

2 /

Bmp4 mutan

t

RBPJ/Axin

2

mutant

Control +/-

B

Gata4

Baf60c

GAPDH

Isl1

SRFMef2c

Msx2

Nkx2-5

Rel

ativ

e ex

pres

sion

(AU

)

12hembryoculture±Noggin

Control+/-

MesP1-cre;RBPJ ;Bmp4 ;Axin2

lox/lox

lox/lox

+/-

MesP1-cre;Bmp4 ;Axin2

lox/lox

+/-

MesP1-cre;RBPJ ;Axin2

lox/loxMesP1-cre;RBPJ lox/lox

MesP1-cre;RBPJ ;Bmp4 ;Axin2

lox/lox

lox/lox

E9.5

Control+/- +/- +/-

MesP1-cre;Bmp4 ;Axin2

lox/loxMesP1-cre;RBPJ ;Axin2

lox/lox

Nkx

2-5/

Trop

T2 POM N

Gat

a4Is

l1FEC D

KJH I

E9.25

LV

RV

OFT

G

L

Fig. 4. Wnt/β-catenin and Bmpsignaling target different setsof cardiac-specific transcriptionfactors to maintain differen-tiation of cardiac progenitorswithin the distal outflow tract.(A) Scheme of mouse embryoculture. Culture for 12 h in theabsence or presence of Nogginwas from E8.75–9.25. (A′) qRT-PCR analysis of mRNA levels ofthe indicated genes of RBPJ/Axin2+/− double mutants in theabsence (plain green bars) andpresence of Noggin (+Noggin;striped green bars). A 2.5- tothreefold decrease of Gata4,SRF, and Mef2c expression wasobserved by treatment withNoggin. Error bars representSEM (n = 4). *P < 0.05, **P <0.01, ***P < 0.005. AU, arbitraryunits. (B) Relative expression ofindicated mRNAs in FACS P1progenitor cells and P2 cells ofcontrols, RBPJ/Axin2+/− double mutants, and RBPJ/Bmp4/Axin2+/− triple mutants. (C–L) In situ hybridization on transverse sections of hearts for Gata4 (C–G)and Isl1 (H–L) in indicated genotypes at E9.25 (shown are magnifications of the SHF as indicated in the scheme at left). (M–P) Immunofluorescence analysisin control (M ), RBPJ/Axin2+/−(Ν), RBPJ/Bmp4/Axin2+/− triple-mutant (O), and Bmp4/Axin2+/− mutant (P) embryos for TropT2 (red) and Nkx2-5 (green) atE9.5. Bars mark the area of undifferentiated cells in the distal outflow tracts.

Klaus et al. PNAS | July 3, 2012 | vol. 109 | no. 27 | 10925

DEV

ELOPM

ENTA

LBIOLO

GY

Dow

nloa

ded

by g

uest

on

Janu

ary

3, 2

021

Bmp7 expression observed in such mutants cannot take over therole of Bmp4. Apparently, Notch signaling regulates Bmp2 andBmp7 independently of Wnt, because the expression of Bmp2 andBmp7 is not rescued in RBPJ/Axin2 mice, whereas Bmp4 expres-sion is rescued in aWnt-dependent manner. InRBPJ/Axin2 doublemutants, the initiation of Hand2 and Mef2c expression is rescued,and this rescue depends on Bmp4, as assessed by the analysis ofRBPJ/Axin2/Bmp4 triple mutants. In Bmp4 single mutants, how-ever, Mef2c and Hand2 are not misexpressed. These observationsmight reflect the fact that Bmp2 and Bmp7 are expressed in Bmp4mutants, whereas all these Bmp ligands are silenced in RBPJ/Axin2/Bmp4 mutants. We find that Wnt signaling promotes Nkx2-5, Isl1, and Baf60c. That Wnt controls Nkx2-5 has been notedpreviously, and it has been suggested that this control is mediatedby a down-regulation of HDAC1 (15). Several effects of Bmpsignaling in cardiomyocyte differentiation have been describedpreviously High Bmp activity is required for Gata4 expression andfor the initiation of Nkx2-5 expression at E8, but our data indicatethat at E9 themaintenance of Nkx2-5 no longer is Bmp dependent(9, 13, 14). Furthermore, miRNA-dependent fine-tuning of Isl1also is regulated by Bmp signals (12).Several laboratories have identified cardiac progenitor and

stem cells by using various protocols for sorting and culture (27).We isolated MesP1-derived EYFP+ cardiac progenitor cells ofthe dissected tissue (heart, including SHF) by FACS to in-vestigate gene expression in cells that carried the cre-mediatedRBPJ mutation. Cardiac progenitors (P1 cells) from control miceexpressed Nkx2-5 and produced troponin T2 after 1 wk of cul-ture in differentiation medium (27). This result demonstratesthat P1 cells indeed represent cardiac progenitors that retain thecapacity to differentiate into cardiomyocytes. P1 cells exhibitedlow Wnt and Bmp4 signaling in the absence of RBPJ, as assessedby the analysis of the respective target genes, whereas Wnt andBmp4 signaling levels were high in controls and in RBPJ/Axin2double mutants. The expression of Wnt- and Bmp4-dependentheart-specific transcription factors also was restored in P1 cells.The restoration did depend on Bmp4, because the expressionof Mef2c and Hand2 was down-regulated in RBPJ/Axin2/Bmp4triple-mutant P1 cells. Thus, the sequential activation of Notch,Wnt, and Bmp signaling is required during specification anddifferentiation of cardiac progenitor cells. In the future, the

culture of isolated cardiac progenitors will be useful for bio-chemical investigations to define the mechanisms by whichNotch, Bmp, and Wnt cooperate to control the production of theessential core transcription complexes of the embryonic heart.

Materials and MethodsA detailed description of materials and methods used in this study is given inSI Materials and Methods. Methods are briefly explained below.

The differentmouse strains, RBPJlox, Axin2LacZ, Bmp4lox, MesP1-cre, and cre-inducible EYFPlox [Gt(ROSA)loxP-STOP-loxP-EYFP] and the conditions for breedingmice andgenotypingprimers havebeen described previously (4, 19, 26, 28, 30).Mutant embryos were identified by PCR using amnion or yolk sac tissue.

Whole E8.75–9.25 embryos were cultured in RPMI1640 (Lonza), 6.7% (vol/vol) FBS (Invitrogen), 10% (vol/vol) rat serum (Sigma), 0.3% D-glucose-mono-hydrate (Merck), 1.5% (vol/vol) penicillin-streptomycin (Invitrogen), and 16mMHEPES (Sigma) in the absence or presence of recombinant Noggin (350 ng/μL)(R&D Systems), as adapted from methods described previously (31).

Embryos were fixed and used for whole-mount in situ hybridizations orfixed, embedded, and sectioned for immunofluorescence, in situ hybridiza-tion, or H&E staining.

For microarray analyses, total RNA of E9.25 hearts, including SHF fromthree or four independent controls, RBPJ single-mutant embryos, and RBPJ/Axin2 double-mutant embryos were isolated using TRIzol (Invitrogen) andthe RNeasy Mini cleanup kit (Qiagen). Total RNA (500 ng) was labeled andhybridized on the MouseRef-8 v2 array (Illumina) as specified by the man-ufacturer and data were analyzed (accession number GSE36804).

For FACS, cardiac tissue (including SHF) was collected at E9.25, trypsinizedfor 5 min at 37 °C, and centrifuged at 150 × g for 3 min at 4 °C. Pellets weresuspended in 500 μL DMEM (Invitrogen), washed once with PBS, and passedthrough a cell-strainer cap (35μm) (BD Biosciences). Yellow fluorescent cellswere sorted using FACS Aria (BD Biosciences), and apoptotic cells were ex-cluded by eliminating 7-AAD+ cells (BD Biosciences).

Cells isolated by FACS were used for RNA isolation and subsequent qRT-PCR analysis or were cultured for immunofluorescence analysis.

ACKNOWLEDGMENTS. We thank Dr. C. Birchmeier (Max Delbrueck Centerfor Molecular Medicine) for helpful discussions and critical reading of themanuscript, Dr. T. Honjo (Kyoto University) for providing RBPJ floxed mice,and Dr. H. P. Rahn’s FACS facility at the Max Delbrueck Center for MolecularMedicine for advice.

1. Cai CL, et al. (2003) Isl1 identifies a cardiac progenitor population that proliferates priorto differentiation and contributes a majority of cells to the heart. Dev Cell 5:877–889.

2. Kelly RG, Brown NA, Buckingham ME (2001) The arterial pole of the mouse heartforms from Fgf10-expressing cells in pharyngeal mesoderm. Dev Cell 1:435–440.

3. Waldo KL, et al. (2001) Conotruncal myocardium arises from a secondary heart field.Development 128:3179–3188.

4. Saga Y, et al. (1999) MesP1 is expressed in the heart precursor cells and required forthe formation of a single heart tube. Development 126:3437–3447.

5. Vincent SD, Buckingham ME (2010) How to make a heart: The origin and regulationof cardiac progenitor cells. Curr Top Dev Biol 90:1–41.

6. MacGrogan D, Nus M, de la Pompa JL (2010) Notch signaling in cardiac developmentand disease. Curr Top Dev Biol 92:333–365.

7. Takeuchi JK, Bruneau BG (2009) Directed transdifferentiation of mouse mesoderm toheart tissue by defined factors. Nature 459:708–711.

8. Dodou E, Verzi MP, Anderson JP, Xu SM, Black BL (2004) Mef2c is a direct transcrip-tional target of ISL1 and GATA factors in the anterior heart field during mouse em-bryonic development. Development 131:3931–3942.

9. Evans SM, Yelon D, Conlon FL, Kirby ML (2010) Myocardial lineage development. CircRes 107:1428–1444.

10. Olson EN (2006) Gene regulatory networks in the evolution and development of theheart. Science 313:1922–1927.

11. Liang FS, Crabtree GR (2009) Developmental biology: The early heart remodelled.Nature 459:654–655.

12. Wang J, et al. (2010) Bmp signaling regulates myocardial differentiation from cardiacprogenitors through a MicroRNA-mediated mechanism. Dev Cell 19:903–912.

13. Klaus A, Saga Y, Taketo MM, Tzahor E, Birchmeier W (2007) Distinct roles of Wnt/beta-catenin and Bmp signaling during early cardiogenesis. Proc Natl Acad Sci USA104:18531–18536.

14. Schultheiss TM, Burch JB, Lassar AB (1997) A role for bone morphogenetic proteins inthe induction of cardiac myogenesis. Genes Dev 11:451–462.

15. Liu Z, et al. (2009) WNT signaling promotes Nkx2.5 expression and early cardiomyo-genesis via downregulation of Hdac1. Biochim Biophys Acta 1793:300–311.

16. Behrens J, et al. (1996) Functional interaction of beta-catenin with the transcriptionfactor LEF-1. Nature 382:638–642.

17. Molenaar M, et al. (1996) XTcf-3 transcription factor mediates beta-catenin-inducedaxis formation in Xenopus embryos. Cell 86:391–399.

18. Klaus A, Birchmeier W (2008) Wnt signalling and its impact on development andcancer. Nat Rev Cancer 8:387–398.

19. Lustig B, et al. (2002) Negative feedback loop of Wnt signaling through upregulationof conductin/axin2 in colorectal and liver tumors. Mol Cell Biol 22:1184–1193.

20. Yu HM, et al. (2005) The role of Axin2 in calvarial morphogenesis and craniosynos-tosis. Development 132:1995–2005.

21. Zeng YA, Nusse R (2010) Wnt proteins are self-renewal factors for mammary stemcells and promote their long-term expansion in culture. Cell Stem Cell 6:568–577.

22. Qian L, Mahaffey JP, Alcorn HL, Anderson KV (2011) Tissue-specific roles of Axin2 inthe inhibition and activation of Wnt signaling in the mouse embryo. Proc Natl AcadSci USA 108:8692–8697.

23. High FA, et al. (2009) Murine Jagged1/Notch signaling in the second heart fieldorchestrates Fgf8 expression and tissue-tissue interactions during outflow tractdevelopment. J Clin Invest 119:1986–1996.

24. Kwon C, et al. (2009) A regulatory pathway involving Notch1/beta-catenin/Isl1 de-termines cardiac progenitor cell fate. Nat Cell Biol 11:951–957.

25. Kwon C, et al. (2011) Notch post-translationally regulates β-catenin protein in stemand progenitor cells. Nat Cell Biol 13:1244–1251.

26. Srinivas S, et al. (2001) Cre reporter strains produced by targeted insertion of EYFPand ECFP into the ROSA26 locus. BMC Dev Biol 1:4.

27. Kattman SJ, et al. (2011) Stage-specific optimization of activin/nodal and BMP sig-naling promotes cardiac differentiation of mouse and human pluripotent stem celllines. Cell Stem Cell 8:228–240.

28. Liu W, et al. (2004) Bmp4 signaling is required for outflow-tract septation andbranchial-arch artery remodeling. Proc Natl Acad Sci USA 101:4489–4494.

29. Prall OW, et al. (2007) An Nkx2-5/Bmp2/Smad1 negative feedback loop controls heartprogenitor specification and proliferation. Cell 128:947–959.

30. Tanigaki K, et al. (2002) Notch-RBP-J signaling is involved in cell fate determination ofmarginal zone B cells. Nat Immunol 3:443–450.

31. Jones EA, et al. (2002) Dynamic in vivo imaging of postimplantation mammalianembryos using whole embryo culture. Genesis 34:228–235.

10926 | www.pnas.org/cgi/doi/10.1073/pnas.1121236109 Klaus et al.

Dow

nloa

ded

by g

uest

on

Janu

ary

3, 2

021