Wnt/Β-Catenin and Bmp Signals Control Distinct Sets of Transcription Factors in Cardiac Progenitor Cells

Wnt/β-catenin and Bmp signals control distinct sets of transcription factors in cardiac progenitor cells

Alexandra 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 Molecular Physiology 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- by inhibiting Mef2c expression during early cardiogenesis in diac-specific transcription factors for their differentiation. Using Xenopus and in embryonic stem cells and promote differentiation conditional mutagenesis of mouse embryos, we define the hierar- in both myocardium and endocardium during later developmental chy of signaling events that controls the expression of cardiac- stages (6). The Notch intracellular domain translocates to the specific transcription factors during differentiation of cardiac nucleus to form a transcriptional complex with the DNA-binding progenitors at embryonic day 9.0. Wnt/β-catenin and Bmp act protein RBPJ and the coactivator Mastermind-like to activate target genes (6). Studies in mouse, chick, frog, and fish demon- downstream of Notch/RBPJ at this developmental stage. Mutation strate that high levels of Bmp activity are necessary for the ex- of Axin2, the negative regulator of canonical Wnt signaling, enhan- fi pression of Nkx2-5 and Gata4 and for myocardial differentiation ces Wnt and Bmp4 signals and suf ces to rescue the arrest of car- (5, 9, 13, 14). Canonical Wnt signals are essential for proliferation RBPJ diac differentiation caused by loss of . Using FACS enrichment of Isl1-expressing SHF progenitors and also promote Nkx2-5 ex- of cardiac progenitors in RBPJ and RBPJ/Axin2 mutants, embryo pression and subsequent cardiac differentiation by down-regu- cultures in the presence of the Bmp inhibitor Noggin, and by cross- lating HDAC1 (5, 13, 15). In the presence of canonical Wnt ing a Bmp4 mutation into the RBPJ/Axin2 mutant background, we ligands, β-catenin is stabilized and translocates to the nucleus, show that Wnt and Bmp4 signaling activate specific and nonover- where it interacts with Lef/Tcf transcription factors to activate fi Nkx2-5 – lapping cardiac-speci c genes in the cardiac progenitors: , target genes (16 18). Axin proteins are negative regulators and BIOLOGY Isl1 and Baf60c are controlled by Wnt/β-catenin, and Gata4, SRF, control β-catenin degradation. Axin2 is a target gene of canonical DEVELOPMENTAL and Mef2c are controlled by Bmp signaling. Our study contributes Wnt signals, and its expression is a useful marker to define cells to the understanding of the regulatory hierarchies of cardiac pro- exposed to Wnt (19–22). Axin2-null mice are viable and have no fl obvious defects in cardiac development. However, increased ca- genitor differentiation and out ow tract development and has −/− implications for understanding and modeling heart development. nonical Wnt signaling is found in homozygous Axin2 cells after transplantation into wild-type tissues and embryos (21, 22). Def- icits in skull formation in Axin2-mutant mice appear to be caused cardiogenesis | MesP1-cre | mesoderm patterning | canonical Wnt | by tissue-specific up-regulation of canonical Wnt signals (20). progenitor differentiation in culture Results istinct cell types, such as the mesodermal progenitors of the Axin2−/− fi fi Wnt Gain-of-Function Mutation Rescues Defects of SHF D rst and second heart elds (FHF and SHF) as well as car- Morphogenesis in Conditional RBPJ/Notch Mice. Interference with diac neural crest cells, contribute to the development of the Notch signaling arrests cardiac development (6, 23–25). In ac- vertebrate heart. The FHF lineage generates the left ventricle, cordance, MesP1-cre–induced mutation of RBPJlox/lox (hereaf- whereas the SHF forms the right ventricle and contributes to “ ” fl ter, RBPJ mutant ) reduced the size and compactness of the out ow tract and atria. SHF and cardiac neural crest cells co- heart, as revealed by analyses of H&E-stained sections at embry- operate during the development of outflow tract vessels and – onic day (E) 9.75 (Fig. 1 A and B and Table S1). The right ven- valves (1 3). Mesodermal cells of the anterior postgastrulating tricles were strongly affected, and the expression of the mouse embryo and FHF and SHF progenitors express the tran- predifferentiation factor Hand2 was lost (Fig. 1 C and D). In situ scription factor MesP1, which is essential for heart induction (4). hybridizations showed a pronounced down-regulation of Gata4 in FHF and SHF progenitors can be distinguished by their level of the heart and of Isl1 in the splanchnic mesoderm and outflow tract proliferation and differentiation. Cells of the FHF begin to dif- myocardium at E9.25 (Fig. 1 E and F and Fig. S1 A and B). Ab- ferentiate by expressing Gata4, Baf60c, and Nkx2-5 in the cardiac lation of RBPJ (Fig. S1 C–D′) strongly attenuated canonical Wnt crescent and cardiac tube. Cells of the SHF express Isl1, which fl signaling, as seen by the reduced expression of the Wnt target marks undifferentiated cells (5). The development of the out ow genes Axin2 and Lef1, particularly in the rudimentary anterior tract involves Isl1-expressing cardiac progenitors from the ante- SHF and SHF derivatives at E8.5–9.25 (Fig. S1 E–L and Table rior SHF, endocardial cells, and cardiac neural crest cells (6). S2). Moreover, a major reduction in Lef1 and Isl1 coexpression Mesodermal progenitor cells pass through several developmen- occurred in the splanchnic mesoderm (Fig. S1 M–P). Axin2 ex- tal stages before they become fully differentiated cardiomyocytes. fi pression also was visualized in mice that carried a Wnt reporter, During a rst phase, mesodermal progenitors express a core the heterozygous Axin2LacZ allele (19, 21). LacZ was expressed in complex of essential transcription factors and chromatin remod- splanchnic mesoderm and SHF derivatives in control mice (13) elers—Gata4, Brg1, and Baf60c—which induce transcription factors such as Nkx2-5, Tbx5/20, and Isl1 that define cells as cardiac progenitors (5, 7). During predifferentiation of cardiac progenitors into cardioblasts, these transcription factors induce the expression Author contributions: A.K. and W.B. designed research; A.K. and M.M. performed of Mef2c and Hand1/2 and also early cardiac muscle-specificgenes, research; 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. e.g., the genes that encode cardiac actin and myosin light chain 2a fl (8–10). In the differentiation phase, down-regulation of Isl1 and The authors declare no con ict of interest. interaction of Mef2c/Hand1/2 with the core complex induce the This article is a PNAS Direct Submission. expression of cardiac muscle-specific genes for structural proteins Data deposition: The data reported in this paper have been deposited in the Gene Ex- such as troponin T2 and myosin heavy chains (11, 12). pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE36804). How the Notch, Wnt, and Bmp developmental signaling path- 1To whom correspondence should be addressed. E-mail: [email protected]. ways control heart development and the heart-specific transcrip- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tion factors has been studied. Notch signals repress differentiation 1073/pnas.1121236109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1121236109 PNAS Early Edition | 1of6 Downloaded by guest on September 28, 2021 MesP1-cre; MesP1-cre; Control RBPJ lox/lox Control RBPJ lox/lox A B E F OFT RV OFT H/E Isl1 RV LV RV LV RV

C D G H

OFT OFT

RV Hand2 RV Axin2-LacZ 52.9E 52.9E MesP1-cre; MesP1-cre; MesP1-cre; lox/lox lox/+ Control lox/lox RBPJ ; RBPJ ; RBPJ Axin2-/- Axin2-/- I J K L OFT OFT OFT OFT RV RV LV RV LV RV LV LV Nkx2-5 MN O P

Fig. 1. The Axin2 mutation restores Wnt and Bmp signaling and substantially rescues the RBPJ phenotype in the heart.

/ cre (A and B) H&E staining of transverse sections of hearts of M’ N’ O’ P’ control and RPPJ mutants at E9.75. Right ventricles (RV) are E9.25 Isl1 marked by arrows. LV, left ventricle. (C and D) Whole-mount in FG FG FG situ hybridization for Hand2 in controls and RBPJ mutants at FG E9.75. (E and F) In situ hybridization on sections for Isl1 at E9.25. (G and H) Canonical Wnt activation as determined on transverse sections of whole-mount in situ hybridizations for Axin2-LacZ in controls (Axin2LacZ/+) and RBPJ mutants (RBPJ/ Axin2LacZ/+) at E9.25. Right ventricles (RV; see Inset magnifica- LV S LV fl Q R LV T tions) and out ow tract myocardium (OFT) are marked by RV RV LV RV arrows and arrowheads, respectively, and splanchnic meso- / Nkx2-5 RV derm is marked by asterisks. (I–L) Whole-mount in situ hybridization for Nkx2-5 in controls, RBPJ single-mutant, RBPJ/ Axin2 double-mutant, and Axin2 single-mutant embryos at TropT2 E9.25. (M–T) Immunofluorescence analysis for Isl1 (red) and cre Nkd2 (green) (M–P), and TropT2 (red) and Nkx2-5 (green) (Q–T)in U PCDH7 V HEART WNT BMP fi Gpr83 indicated genotypes. See also magni cations of splanchnic H SRF mesoderm (M′–P′). FG, foregut. (U) Heatmap of gene-expression E Ac tc 1 A Me f2 c arrays of RBPJ/Axin2 double mutants vs. controls and RBPJ and R TNC T Wnt2 Axin2 single mutants. The restored expression of particular Wnt5a W Wnt9b genes is shown in orange for cardiac-specific genes, green for N Dkk1 T Le f1 Wnt genes, and magenta for Bmp-controlled genes. (V)qRT-PCR LRP6 B Smad6 analyses of mRNA expression at E9.25 in the heart region of M Bmp4 P controls (gray bars), Axin2 single (blue bars), RBPJ single mutants Msx2

BmpRIa Relative expression (AU) (magenta bars), and RBPJ/Axin2 double mutants (blue/magenta- Noggin SMA SRF TropT2 Axin2 Isl1 Wnt5a Bmp4 Bmp7 Noggin RBPJ Axin2 < < mutant mutantmutant striped bars). Error bars represent SEM (n =4).*P 0.05, **P Control control Axin2 mutant RBPJ mutant RBPJ/Axin2 mutant RBPJ/Axin2 0.01, ***P < 0.005. AU, arbitrary units.

but was reduced at these sites in the RBPJ mutants (Fig. 1 G and by immunohistology of Cre+ cells (Fig. S2 A–B′′). Canonical Wnt H). These data indicate cross-talk between Notch/RBPJ and ca- signals previously had been shown to be essential for pro- nonical Wnt signaling, operating particularly in the SHF, in early liferation of Isl1-expressing SHF progenitors (5, 13). However, heart morphogenesis. proliferation in Isl1-expressing cells was little affected in RBPJ − We confirmed by genetic means that Notch/RBPJ and Wnt/ single mutants and RBPJ/Axin2+/ mutants (Fig. S2 C and D). − β-catenin interact during SHF morphogenesis, and we generated However, in RBPJ/Axin2+/ mutants, a pronounced rescue of the − − compound MesP1-cre;RBPJlox/lox;Axin2 / mutants (hereafter number of Isl1-expressing cells in the splanchnic mesoderm and − − “RBPJ/Axin2 double mutants”). Remarkably, the Axin2 / mu- of ventricular cardiomyocyte differentiation was observed, evi- tation substantially but not completely rescued the heart phe- denced by the restored expression of troponin T2 (Fig. 1 M–T notypes at E9.25 in RBPJ mutants (Fig. 1 I–L and Table S1). In and Fig. S2E). Thus, coexpression of Nkx2-5 and troponin T2 particular, the size of the right ventricles and outflow tracts and were not observed in RBPJ mutant hearts, whereas robust coex- also the expression of Nkx2-5 were normalized (Fig. 1 J and K). pression was seen in RBPJ/Axin2 double mutants (Fig. 1 Q–T). Elimination of Axin2 alone did not produce heart hyperplasia We used gene-expression profiling of dissected cardiac tissue (Fig. 1L). Pronounced activation of canonical Wnt signaling by (heart, including SHF) to study the mechanism of the rescue of β-galactosidase expression occurred in splanchnic mesoderm and the cardiac differentiation program in RBPJ/Axin2 mutants (Fig. ventricular walls of RBPJ/Axin2LacZ/LacZ mutants, as analyzed 1U). Compared with RBPJ single-mutant tissues, the expression of

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1121236109 Klaus et al. Downloaded by guest on September 28, 2021 many down-regulated genes was largely restored in RBPJ/Axin2 during progenitor specification, Nkx2-5, Isl1, SRF,andTbx20 were double mutants (compare the third and second columns in Fig. 1U similarly down- and up-regulated (Figs. 1 I–L and V and Figs. 2 E– and see Tables S1 and S3). Among these genes were those re- H and Table S2). The expression of factors that mark prediffer- quired for cardiac differentiation, e.g., cardiac actin (Actc1), SRF, entiation, Hand2 and Mef2c, also was down-regulated in RBPJ and Mef2c (identified in orange in Fig. 1U). Moreover, the ex- mutants and was rescued by Axin2 ablation (Fig. 2 I–P). Elimi- pression of >20 genes that encode components and targets of nation of one allele of Axin2 sufficed to rescue, in part, the ex- canonical Wnt signaling, e.g., Wnt2, Dkk1,andLef1 (identified in pression of Mef2c (quantifications are given in Fig. 2P). In situ green in Fig. 1U; see also Table S3, and for Wnt target genes see hybridization also confirmed the reduced expression of Lef1, www.stanford.edu/group/nusselab/cgi-bin/wnt/) were up-regulated Wnt2, and the Bmp target gene Smad6 in SHF derivatives of RBPJ also. Surprisingly, the expression of many genes encoding mem- mutants and their up-regulation in RBPJ/Axin2 double mutants bers of the Bmp pathway (Bmp4, BmpR1a) and Bmp target genes (Fig. 2 Q–X and Fig. S2 F–I). The expression of the Notch/RBPJ (Smad6, Msx2) (indentified in magenta in Fig. 1U; see also Table target gene Hey3 was not restored, but Hey1 and Hey2 were nor- S3) was rescued also. We confirmed restoration of expression of malized by Axin2 ablation (Fig. S2 J–P and Table S3)(6).The selected cardiac differentiation genes (SMA, SRF,andtroponin expression of Tenascin C (Fig. S2 Q–T) and of other genes not yet T2) and of Wnt and Bmp target genes by quantitative RT-PCR described as being involved in SHF morphogenesis [e.g., proto- (qRT-PCR) (Fig. 1V). Furthermore, Bmp4 but not Bmp7 expres- cadherin (PCDH7)(Fig. S2 U–X) and G protein-coupled receptor sion was restored (Fig. 1V; see also below). The majority of these Gpr83, Nkd2, and others (identified in Table S3)] was normalized. genes were not or were only slightly affected in Axin2 single Thus, Wnt/β-catenin and further signals regulated by Wnt are mutants [Fig. 1U (compare first and fourth columns) and V]. Thus, required for the expression of transcription factors that are es- our genetic data indicate that Wnt/β-catenin acts downstream of sential for specification of cardiac progenitors as well as for dif- RBPJ/Notch and controls Bmp signals in morphogenesis and ferentiation, i.e., the induction of heart-specific structural proteins. differentiation of cardiac cells. Axin2 Mutation Restores Expression of Crucial Transcription Factors in Wnt/β-Catenin and Bmp Signals Downstream of Notch/RBPJ Are FACS-Sorted Cardiac Progenitors of RBPJ Mutants. Our analyses Crucial for Different Stages of Cardiac Progenitor Differentiation. show that Axin2 ablation rescues cardiac development of RBPJ The minimal core complex, Gata4 and Baf60c, that induces mutants during the formation of cardiac progenitor cells. To cardiac progenitor specification was down-regulated in the RBPJ confirm the stage and cell type precisely, we used FACS to iso- mutants and was rescued by Axin2 ablation, as confirmed by in situ late cardiac progenitors. A reporter line that expresses EYFP in hybridization (Fig. 2 A–D; see also below). The factors produced a cre-inducible manner was crossed into controls and mutants BIOLOGY DEVELOPMENTAL

MesP1-cre; MesP1-cre; MesP1-cre; RBPJ lox/lox ; RBPJ lox/+ ; Control lox/lox RBPJ Axin2-/- Axin2-/- A BDC

E FHG Tbx20 Baf60c

I JLK Hand2

M N O P Mef2c

Relative expression (AU) Mef2c Q RS T Fig. 2. Canonical Wnt and Bmp signaling downstream of Notch/RPBJ are crucial for the different stages of cardiac differentiation at E9.25. (A–L) In situ hybridization on transverse Lef1 sections of hearts for Baf60c, Tbx20,andHand2 in indicated Relative expression (AU) Lef1 genotypes at E9.25. (M–X) Whole-mount in situ hybridizations of embryos or on transverse sections of hearts for Mef2c,(M, N, U V W X O), Lef1 (Q, R, S), and Smad6 (U, V, W) (outﬂow tract/right ventricles are marked by arrows) and corresponding qRT-PCR − analyses (P, T, X)incontrols,RBPJ single mutants, RBPJ/Axin2+/ ,

Smad6 RBPJ/Axin2 double mutants, and Axin2 single mutants. Error Relative expression (AU) Smad6 bars represent SEM (n = 4). *P < 0.05, **P < 0.01, ***P < 0.005. lortnoc Axin2 -/- mutant RBPJ mutant +/- tnatum2nixA/JPBR RBPJ/Axin2 -/- mutant AU, arbitrary units.

Klaus et al. PNAS Early Edition | 3of6 Downloaded by guest on September 28, 2021 that carry MesP1-cre (26). Reporter expression was observed in differentiated cardiac cells (blue bars in Fig. 3C), suggesting that MesP1-derived mesodermal cardiac progenitor cells that express P2 cells are cardioblasts and/or cardiomyocytes. Immunofluo- Isl1, in particular in the splanchnic mesoderm and distal outflow rescence showed that isolated P1 progenitors expressed Nkx2-5, tracts, and in cells of the left and right ventricles (arrowheads in whereas P2 cells had high levels of cardiac troponin (Fig. 3 D and Fig. 3A; merged yellow fluorescence marks coexpression of Isl1 E). When isolated P1 and P2 cells were cultured for 7 d, P1 cells and GFP). Two distinct EYFP+ cell populations from the car- underwent cardiac differentiation in the presence of Activin A fl diac tissue (heart, including SHF) were isolated: small-diameter and Bmp4 (27), as analyzed by immuno uorescence for Nkx2-5 – ′ P1 cells (outlined in orange in Fig. 3B) and large-diameter P2 and troponinT2 (Fig. S3 A B ). At the end of the culture period, cells (outlined in blue in Fig. 3B). The identity of the sorted cells cardiac troponin in P1 and P2 cells was equal. Thus, P1 cells are obtained from control hearts was verified by analyses of Isl1 and indeed cardiac progenitors that retain the capacity to differen- αMHC expression. RT-PCR showed that P1 cells strongly ex- tiate. P1 progenitor cells strongly expressed Notch and Wnt pressed the progenitor-specific factors Isl1, Tbx20, and SRF, pathway components and target genes, in particular Jag2 and Notch4 and Wnt2, Axin2, and Lef1, but these genes were seen to suggesting that they represent cardiac progenitors (orange bars a lesser extent in P2 cells (Fig. S3C). These genes were expressed in Fig. 3C). In contrast, P2 cells expressed reduced levels of Isl1, α in cardiac tissue of RBPJ/Axin2 double mutants but not in RBPJ Tbx20, and SRF but high levels of MLC2a and MHC typical for mutants (Table S3). Other Notch pathway components, i.e., Dll4, Jag1, and Notch1/2, were highly expressed in the differentiated P2 cardiac cells (Fig. S3C). We used P1 and P2 cell populations +/− lox/+ lox/+ of RBPJ single-mutant and RBPJ/Axin2 mutant hearts to de- A MesP1-cre; EYFP B MesP1-cre; EYFP D fine potential differences in their response to the mutations. P2 cells Remarkably, loss of RBPJ in P1 and P2 cells abolished Nkx2-5,

TropT2 Isl1, Tbx20, and Mef2c expression (yellow bars in Fig. 3F and LV P1 light blue bars in Fig. 3G). However, loss of Axin2 (one allele E sufﬁced) substantially normalized the expression of Nkx2-5, Isl1, cell size P1 progenitors Nkx2-5 Tbx20, and Mef2c in P1 and P2 cells of RBPJ mutants (striped RV bars in Fig. 3 F and G). Furthermore, Bmp4 expression was re- +/−

GFP P2 YFP+ stored in RBPJ/Axin2 mutant P1 cells (Fig. 3 F and G Right; see also below and Fig. S4 B–D). Thus, Axin2 ablation restored cardioblasts/ C cardiomyocytes the expression of the crucial transcription factors Nkx2-5, Isl1, control P1 cells Tbx20, and Mef2c and the expression of Bmp4 in RBPJ mutant control P2 cells progenitors cardiac cells, indicating that the sequential regulation of cardiac- speciﬁc transcription factors mediated by RBPJ, Wnt, and Bmp signals occurs in mesoderm-derived cardiac progenitors as well as in the early differentiated cardiac cells.

Nkx2-5, Isl1, and Baf60c are Wnt/β-Catenin Target Genes, and Gata4 Relative expression (AU) and SRF Are Bmp4 Targets in Cardiac Progenitors. We next defined the heart-specific transcription factors controlled by canonical Wnt and/or Bmp signaling at this developmental stage. Whole control (P1) E8.75 embryos were cultured in the presence or absence F RBPJ mutant (P1) RBPJ/Axin2 +/- mutant (P1) of Noggin, a soluble Bmp inhibitor (Fig. 4A) (14). Analysis of mRNA from dissected cardiac tissue (heart, including SHF) of control embryos treated with Noggin revealed decreased expression of all cardiac-specific transcription factors and also reduced Lef1, indicating that Noggin also affected Wnt signaling − (Fig. S4A). However, analysis of RBPJ/Axin2+/ mutants, in which Wnt signaling is up-regulated, showed that Noggin treatment strongly reduced the expression of Gata4, SRF, Mef2c, and Msx2 (Fig. 4A′), indicating that Gata4, SRF, and Mef2c are controlled by Bmp signaling in the presence of Wnt signaling. In G control (P2) RBPJ mutant (P2) contrast, Nkx2-5, Baf60c, and Isl1 expression was not affected RBPJ/Axin2 +/- mutant (P2) significantly 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 in

Relative expression (AU) Relative expression (AU) RBPJ single mutants. Bmp4 but not Bmp2 and Bmp7 expression was restored in the heart of RBPJ/Axin2 double mutants (Fig. S4 – β B O; see also Fig. 1V). It should be noted that in Bmp4 mutants Fig. 3. Wnt/ -catenin activation by Axin2 ablation restores cardiac commit- Bmp7 Bmp7 ment and Bmp signaling in SHF progenitor cells at E9.25. (A) Immunofluo- was expressed in the entire heart, suggesting that rescence analysis of GFP (green) and Isl1 (red) on a transverse section of control might substitute for Bmp4 loss. To assess the role of the restored embryos (arrowheads mark merged yellow fluorescence in MesP1-derived SHF Bmp4 expression in the RBPJ/Axin2 double mutants and to cor- cells). (B)FACSofYFP+ cells of MesP1-cre; EYFPlox/+ embryos at E9.25 showing roborate the above assignment of signal-dependent expression of two populations of YFP+ cells: P1 progenitors (outlined in orange) and P2 cells cardiac transcription factors, we introduced a conditional Bmp4 + mutation (28) into the RBPJ/Axin2 mutant background (MesP1- (outlined in blue). (C) Relative mRNA expression levels in the YFP populations lox/lox lox/lox +/− “ ” (P1, orange; P2, blue) of controls show that P1 cells express higher levels of cre;RBPJ ;Bmp4 ;Axin2 ; hereafter triple mutants ). cardiac progenitor genes, and P2 cells show a marked increase in muscle- Bmp signaling indeed was lost in the mutants, and Bmp4 expression specific genes. (D and E) Immunofluorescence analysis of GFP (blue), Nkx2-5 as well as pSmad1/5/8-Isl1 costaining were abrogated in the SHF of (green), and troponin T2 (red) in P1 cells (D) and P2 cells (E). (F and G) qRT-PCR the triple mutants at E9.25 (Fig. S4 P–T′ and Table S1). However, analysis of mRNA in the cardiac progenitor population (P1 cells) (F)andinP2 the quantity of Isl1-expressing cells was unchanged, demonstrating − cells (G)ofcontrols(MesP1-cre;EYFPlox/+;RBPJlox/+;Axin2+/ ), RBPJ single that the low pSmad1/5/8 expression was not caused by a loss of SHF mutants (MesP1-cre;EYFPlox/+;RBPJlox/lox), and double mutants (MesP1-cre; cells (Fig. S4U). It should be noted that Smad1/5/8 phosphorylation − EYFPlox/+;RBPJlox/lox;Axin2+/ ). Error bars represent SEM (n =3–6). *P < 0.05, was detectable in the ventricular walls of the triple mutants, in- **P < 0.01, ***P < 0.005. AU, arbitrary units. dicating that these differentiated cells receive signals provided by

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1121236109 Klaus et al. Downloaded by guest on September 28, 2021 Fig. 4. Wnt/β-catenin and Bmp A A’ RBPJ/Axin2 +/- B signaling target different sets RBPJ/Axin2 +/- (+nog) of cardiac-speciﬁc transcription Gata4 factors to maintain differen- SRF tiation of cardiac progenitors E8.75 Mef2c within the distal outﬂow tract. embryo (A) Scheme of mouse embryo 12h culture Msx2 culture. Culture for 12 h in the ±Noggin Nkx2-5 absence or presence of Noggin was from E8.75–9.25. (A′) qRT- Baf60c Relative expression (AU) PCR analysis of mRNA levels of Isl1 the indicated genes of RBPJ/ E9.25 4ataG SRF Mef2c Msx2 Lef1 Nkx2-5 Baf60c 1lsI − GAPDH Axin2+/ double mutants in the +/- +/- MesP1-cre; +/- +/- absence (plain green bars) and MesP1-cre; lox/lox MesP1-cre; trol trol lox/lox RBPJ ; lox/lox n n Control MesP1-cre; RBPJ ; Bmp4 ; presence of Noggin (+Noggin; lox/lox lox/lox Co mutant Co mutant RBPJ Axin2 +/- Bmp4 ; Axin2 +/- +/- striped green bars). A 2.5- to Axin2 RBPJ/Axin2 RBPJ/Axin2 Bmp4 mutant / RBPJ/Axin2 RBPJ/Axin2 Bmp4 mutant / threefold decrease of Gata4, C D FE G MesP1-cre; SRF, and Mef2c expression was MesP1-cre; lox/lox MesP1-cre; lox/lox RBPJ ; lox/lox RBPJ ; lox/lox Bmp4 ; Gata4 Control observed by treatment with +/- Bmp4 ; +/- Axin2 +/- Axin2 Noggin. Error bars represent Axin2 < < H I KJ L M N O P SEM (n = 4). *P 0.05, **P LV

OFT TropT2 0.01, ***P < 0.005. AU, arbitrary / Isl1 units. (B) Relative expression of RV

indicated mRNAs in FACS P1 E9.25 Nkx2-5 E9.5 progenitor cells and P2 cells of controls, 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 magnificationsoftheSHFasindicatedintheschemeatleft).(M–P) Immunofluorescence analysis − − − in control (M), RBPJ/Axin2+/ (Ν), RBPJ/Bmp4/Axin2+/ triple-mutant (O), and Bmp4/Axin2+/ mutant (P) embryos for TropT2 (red) and Nkx2-5 (green) at E9.5. Bars mark the area of undifferentiated cells in the distal outflow tracts. BIOLOGY DEVELOPMENTAL a Bmp ligand other than Bmp4. In accordance with the embryo of Bmp signaling in this developmental context. The rescue by culture in the presence of Noggin, FACS-sorted P1 progenitors of Axin2 was virtually complete with respect to the restoration of the triple-mutant embryos displayed strongly reduced Gata4, SRF, canonical Wnt signaling in the heart, as assessed by the expression and Mef2c expression (Fig. 4BUpperLeft; the Bmp target gene of the endogenous Axin2 promoter (Axin2LacZ) and of Wnt target Msx2 served as a control). In contrast, the additional ablation of genes such as Lef1 or by the expression of cardiac-specificgenesin Bmp4 had no significant effect on the expression of Nkx2-5, Isl1, isolated cardiac progenitors. However, cardiac morphogenesis was and Baf60c, as shown by comparison of triple-mutant and double- attenuated, indicating that, in addition to Wnt signaling, Notch mutant P1 cells (Fig. 4B Left and Fig. S5A). In differentiated P2 signaling may control other essential target genes, e.g., Bmp2 and cells, the expression of Gata4, SRF,andMef2c was unchanged (Fig. Bmp7 (shown in this study) or Fgf (5, 23). Outflow tract morpho- 4BRightand Fig. S5B). The ventricular wall contained differenti- genesis is known to depend on signals provided by cardiac pro- ated pSmad1/5/8+ cardiac cells, which are a probable source of P2 genitors from the anterior SHF as well as endocardial, neural crest, cells (Fig. S4 P–T). In situ hybridization confirmed that the ex- andendodermalcells(5,6,29).InRBPJ/Axin2 double mutants, pression of Gata4, Mef2c,andHand2 was down-regulated in the diffusible morphogens (Wnt ligands and Bmp4, here shown by SHF at E9.25, but the expression of Isl1, Nkx2-5,andTbx20 was not gene profiling and by in situ hybridization) may affect gene ex- − affected (Fig. 4 C–L, Fig. S5 C–R,andTable S2). The change in the pression and cell death in MesP1 cells, such as neural crest or expression of the cardiac transcription factors was accompanied at endodermal cells. Thus the up-regulated expression of such E9.5 by a marked arrest of differentiation in the distal outflow tract morphogens in double mutants also might contribute indirectly to of the triple mutants, a possible consequence of the loss of Hand2 the rescued development of nonmesoderm-derived cells (29). In and Mef2c in the anterior SHF (Fig. 4 M–P; bars mark the area of previous analyses of mice that carried a conditional Jagged1 mu- cells lacking troponin T2 in the distal outflow tract). tation or that expressed a dominant-negative Maml using Mef2c-cre In summary, we used genetics and embryo culture to show that also exhibit SHF morphogenesis affects and reduced Bmp and Fgf Wnt signaling controls the expression of Nkx2-5, Baf60c, and signaling (23). In contrast, in studies using Isl1-cre, elevated Wnt/ Isl1, whereas Bmp signaling regulates Gata4, SRF, and Mef2c. β-catenin signaling has been observed after conditional mutation of Wnt and Bmp signaling act downstream of Notch in cardiac Notch1 but not RBPJ (24, 25). The difference in phenotypes might progenitors. We also unraveled a regulatory hierarchy in outflow be caused by the use of distinct cre lines to target cardiac cells or by tract development and showed that the Bmp- and Wnt-de- effects that are not mediated by canonical Notch signaling. pendent expression of Hand2 and Mef2c in the anterior SHF The question arises as to how the two developmental signaling resulted in arrested differentiation. systems, Wnt/β-catenin and Bmp, control the expression of the transcription factors required for cardiac differentiation. Our Discussion results suggest a model in which Wnt/β-catenin regulates the Here we used mouse genetics, FACS enrichment of cardiac crucial transcription factors Baf60c, Nkx2-5,andIsl1 at an early progenitors, and embryo culture with pharmacological inhibitors stage of progenitor formation (Fig. S6). At this stage Wnt/β-cat- to unravel the regulatory hierarchies of Wnt/β-catenin, Bmp, and enin also regulates Bmp4 signaling, which in turn activates Gata4 Notch signaling during cardiac progenitor differentiation and and SRF. Together, these two sets of effectors activate the tran- outflow tract development. The study may have implications for scription factors Mef2c and Hand2, thus allowing cardioblast for- understanding and modeling heart development. mation. To corroborate the nature of genes that are regulated by We show that cardiac defects of a conditional RBPJ mutation either Wnt/β-catenin or Bmp signaling, we used embryo culture at E9 could be rescued substantially by ablating the negative in the presence of the Bmp inhibitor Noggin and introduced a Wnt regulator Axin2, indicating that Wnt/β-catenin is a central conditional Bmp4 mutation into RBPJ/Axin2 mice. Both experi- downstream effector of Notch signaling in cardiac progenitor dif- ments confirmed the distinct regulation of the two sets of non- ferentiation and outflow tract morphogenesis (Fig. S6). Moreover, overlapping transcription factors by Wnt/β-catenin and Bmp the expression of Bmp4 and Bmp-controlled genes was restored in signaling (Fig. S6). Analyses of Bmp4 mutants indicate that Bmp4 such double mutants, suggesting that Wnt/β-catenin acts upstream signals control the expression of Gata4 and SRF; however, residual

Klaus et al. PNAS Early Edition | 5of6 Downloaded by guest on September 28, 2021 Bmp7 expression observed in such mutants cannot take over the culture of isolated cardiac progenitors will be useful for bio- role of Bmp4. Apparently, Notch signaling regulates Bmp2 and chemical investigations to define the mechanisms by which Bmp7 independently of Wnt, because the expression of Bmp2 and Notch, Bmp, and Wnt cooperate to control the production of the Bmp7 is not rescued in RBPJ/Axin2 mice, whereas Bmp4 expres- essential core transcription complexes of the embryonic heart. sion is rescued in a Wnt-dependent manner. In RBPJ/Axin2 double mutants, the initiation of Hand2 and Mef2c expression is rescued, Materials and Methods and this rescue depends on Bmp4, as assessed by the analysis of A detailed description of materials and methods used in this study is given in RBPJ/Axin2/Bmp4 triple mutants. In Bmp4 single mutants, how- SI Materials and Methods. Methods are briefly explained below. ever, Mef2c and Hand2 are not misexpressed. These observations fl The different mouse strains, RBPJlox,Axin2LacZ,Bmp4lox, MesP1-cre, and cre- might re ect the fact that Bmp2 and Bmp7 are expressed in Bmp4 lox loxP-STOP-loxP-EYFP mutants, whereas all these Bmp ligands are silenced in RBPJ/ inducible EYFP [Gt(ROSA) ] and the conditions for breeding fi mice and genotyping primers have been described previously (4, 19, 26, 28, 30). Axin2/Bmp4 mutants. We nd that Wnt signaling promotes Nkx2- fi 5, Isl1,andBaf60c. That Wnt controls Nkx2-5 has been noted Mutant embryos were identi ed by PCR using amnion or yolk sac tissue. previously, and it has been suggested that this control is mediated Whole E8.75–9.25 embryos were cultured in RPMI1640 (Lonza), 6.7% (vol/ by a down-regulation of HDAC1 (15). Several effects of Bmp vol) FBS (Invitrogen), 10% (vol/vol) rat serum (Sigma), 0.3% D-glucose-mono- signaling in cardiomyocyte differentiation have been described hydrate (Merck), 1.5% (vol/vol) penicillin-streptomycin (Invitrogen), and 16mM previously High Bmp activity is required for Gata4 expression and HEPES (Sigma) in the absence or presence of recombinant Noggin (350 ng/μL) for the initiation of Nkx2-5 expression at E8, but our data indicate (R&D Systems), as adapted from methods described previously (31). that at E9 the maintenance of Nkx2-5 no longer is Bmp dependent Embryos were fixed and used for whole-mount in situ hybridizations or (9, 13, 14). Furthermore, miRNA-dependent fine-tuning of Isl1 fixed, embedded, and sectioned for immunofluorescence, in situ hybridiza- also is regulated by Bmp signals (12). tion, or H&E staining. Several laboratories have identified cardiac progenitor and For microarray analyses, total RNA of E9.25 hearts, including SHF from stem cells by using various protocols for sorting and culture (27). three or four independent controls, RBPJ single-mutant embryos, and RBPJ/ + We isolated MesP1-derived EYFP cardiac progenitor cells of Axin2 double-mutant embryos were isolated using TRIzol (Invitrogen) and the dissected tissue (heart, including SHF) by FACS to in- the RNeasy Mini cleanup kit (Qiagen). Total RNA (500 ng) was labeled and vestigate gene expression in cells that carried the cre-mediated hybridized on the MouseRef-8 v2 array (Illumina) as specified by the man- RBPJ mutation. Cardiac progenitors (P1 cells) from control mice ufacturer and data were analyzed (accession number GSE36804). expressed Nkx2-5 and produced troponin T2 after 1 wk of cul- For FACS, cardiac tissue (including SHF) was collected at E9.25, trypsinized ture in differentiation medium (27). This result demonstrates for 5 min at 37 °C, and centrifuged at 150 × g for 3 min at 4 °C. Pellets were that P1 cells indeed represent cardiac progenitors that retain the suspended in 500 μL DMEM (Invitrogen), washed once with PBS, and passed capacity to differentiate into cardiomyocytes. P1 cells exhibited through a cell-strainer cap (35μm) (BD Biosciences). Yellow fluorescent cells low Wnt and Bmp4 signaling in the absence of RBPJ, as assessed were sorted using FACS Aria (BD Biosciences), and apoptotic cells were ex- by the analysis of the respective target genes, whereas Wnt and cluded by eliminating 7-AAD+ cells (BD Biosciences). Bmp4 signaling levels were high in controls and in RBPJ/Axin2 Cells isolated by FACS were used for RNA isolation and subsequent qRT- double mutants. The expression of Wnt- and Bmp4-dependent PCR analysis or were cultured for immunofluorescence analysis. heart-specific transcription factors also was restored in P1 cells. The restoration did depend on Bmp4, because the expression ACKNOWLEDGMENTS. We thank Dr. C. Birchmeier (Max Delbrueck Center of Mef2c and Hand2 was down-regulated in RBPJ/Axin2/Bmp4 for Molecular Medicine) for helpful discussions and critical reading of the triple-mutant P1 cells. Thus, the sequential activation of Notch, manuscript, Dr. T. Honjo (Kyoto University) for providing RBPJ floxed mice, Wnt, and Bmp signaling is required during specification and and Dr. H. P. Rahn’s FACS facility at the Max Delbrueck Center for Molecular differentiation of cardiac progenitor cells. In the future, the Medicine for advice.

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