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Fgf8 and Fgf10 are expressed in the anterior part of the second heart field (SHF) that constitutes a population of cardiac progenitor cells contributing to the arterial pole of the heart. Previous studies of hypomorphic and conditional Fgf8 mutants show disrupted outflow tract (OFT) and right ventricle (RV) development, whereas Fgf10 mutants do not have detectable OFT defects.
Our aim was to investigate functional overlap between Fgf8 and Fgf10 during formation of the arterial pole‥
We generated mesodermal Fgf8/Fgf10 compound mutants with MesP1Cre. The OFT/RV morphology in these mutants was affected with variable penetrance, however, the incidence of embryos with severely affected OFT/RV morphology was significantly increased in response to decreasing Fgf8 and Fgf10 gene dosage. Fgf8 expression in the pharyngeal arch ectoderm is important for development of the pharyngeal arch arteries (PAAs) and their derivatives. We now show that Fgf8 deletion in the mesoderm alone leads to PAA phenotypes, and that these vascular phenotypes are exacerbated by loss of Fgf10 function in the mesodermal core of the arches.
These results show functional overlap of FGF8 and FGF10 signaling from SHF mesoderm during development of the OFT/RV, and from pharyngeal arch mesoderm during PAA formation, highlighting the sensitivity of these key aspects of cardiovascular development to FGF dosage.
Malformations of the arterial pole of the heart account for more than 30% of human congenital heart defects. 1 Remodeling of the outflow tract (OFT) plays a critical role in the maturation of the arterial pole. As the heart matures, cushion tissue is formed in the OFT, as a result of an epithelial-mesenchymal transformation (EMT).2 Rotation and shortening of the OFT are accompanied by fusion of the cushions to form a septum that divides the OFT into the aorta and pulmonary trunk. Subsequent morphogenetic events result in alignment of the aorta with the left ventricle and the pulmonary trunk with the right ventricle (RV).3 The OFT is derived from the anterior part of the second heart field (SHF), which in the mouse embryo, also contributes to the RV and ventricular septum.4–6 This field of splanchnic mesoderm initially lies medial to the cardiac crescent and then dorsally and anteriorly to the heart tube. In addition to myocardium, the SHF contributes smooth muscle to the base of the great arteries. 6,7 A second source of cells, namely cardiac neural crest (CNC), migrates from the neuroectoderm of the dorsal neural tube into the OFT and contributes to cushion formation and correct septation and alignment of the aortic and pulmonary outflows.8–10 CNC also interacts with the mesodermal cells of the SHF.11 When CNC is ablated in the chick embryo, OFT elongation and looping of the cardiac tube is perturbed leading to persistent truncus arteriosus (PTA) and misalignment of the great arteries relative to the ventricles.10 Many signaling pathways in the SHF, CNC and pharyngeal region control development of the arterial pole of the heart by affecting specification, proliferation, survival and differentiation of progenitor cells.12
SHF mesoderm also plays a role in the formation of the blood vessels that channel blood from the aorta and pulmonary trunk to the body and lungs. These arteries form within the 3rd, 4th and 6th pharyngeal arches, which are bilateral embryonic structures consisting of surface ectoderm, inner endoderm and a mesodermal core expressing SHF markers, that becomes surrounded by CNC. The endothelium of the pharyngeal arch arteries (PAAs) is formed from the mesodermal core of the arches, whereas the vascular smooth muscle is predominantly CNC-derived.8,13,14 The PAAs form progressively following the anterior/posterior gradient of pharyngeal arch development, and from E10.5 in the mouse embryo, remodeling leads to the mature PAA system. Many different cellular and genetic perturbations disrupt formation and /or remodeling of the PAAs.10
Fgf10 was identified as a gene expressed in cells that contribute to the OFT and RV in the mouse embryo, leading to the concept of the SHF.15 However Fgf10 null mutants have no evident SHF or PAA defects.16 Fgf8 expression overlaps with that of Fgf10 in the SHF from the cardiac crescent stage.15 Their expression is restricted to the anterior part of the SHF and is regulated by retinoic acid signaling on the anterior-posterior axis.17 Fgf10 transcription extends from the anterior SHF into the mesodermal core of the pharyngeal arches where Fgf8 transcripts are difficult to detect. However, Fgf8LacZ/+, Fgf8GFP/+ and Fgf8 enhancer-LacZ transgenic mice show LacZ or GFP expression in the anterior SHF and the mesodermal core of the arches, implying that Fgf8 is transcribed there.18–20 Unlike Fgf10, Fgf8 is also expressed in pharyngeal endoderm and ectoderm, where its expression is maintained, at least until E9.5.21 Fgf8 mutants die at gastrulation,22 however Fgf8 hypomorphs survive to birth with variable OFT defects including PTA, transposition of the great arteries (TGA) and pharyngeal arch-related phenotypes. The 4th PAA is most severely affected resulting in interrupted or right aortic arch.23,24 Apoptosis of CNC was noted in these hypomorphs, suggesting FGF8 signaling defects in surrounding tissues that directly or indirectly affect CNC, since Fgf8 is not expressed in these cells. Subsequently, conditional mutagenesis of Fgf8 with a battery of Cre recombinases, directed to pharyngeal ectoderm, pharyngeal endoderm and SHF/pharyngeal arch mesoderm has allowed tissue-specific phenotypes to be determined.13,19,20,25 Deletion of Fgf8 in the pharyngeal ectoderm leads to CNC apoptosis, affecting PAA development.25 The role of mesodermal FGF8 in PAA development has not been reported. Mesodermal and endodermal FGF8 are critical for OFT development, affecting the proliferation, survival and transcriptional activity of SHF cells and their derivatives, in addition to the function of the CNC. Deletion of Fgf8 in early cardiogenic mesoderm with MesP1Cre causes initial OFT/RV hypoplasia and OFT alignment defects in survivors at incomplete penetrance.19 This implies that FGF10 may compensate for loss of FGF8 in the precardiac mesoderm.
In this study, we generated compound Fgf8 and Fgf10 mutants in the cardiac and pharyngeal mesoderm, using MesP1Cre. We show that PAA development is perturbed by mesodermal Fgf8 deletion. The incidence and severity of PAA and of OFT defects increased with decreasing Fgf8 and Fgf10 gene dosage, resulting in severe defects in double Fgf8;Fgf10 homozygous conditional mutants. These results reveal functional overlap of mesodermal FGF8 and FGF10 during SHF/OFT and PAA development, uncovering for the first time a role for FGF10 in the formation of the arterial pole of the heart. They also illustrate the sensitivity of these processes to incremental reductions in the level of FGF.
The Fgf8-conditional mutant, Fgf10 mutant and MesP1Cre alleles are as previously described.19,26,27 The MesP1Cre/+ line was bred onto an Fgf10+/− background and then onto Fgf8flox/+, which was crossed with an Fgf8flox/flox;Fgf10+/− line to obtain compound mutants. Fgf8 and Fgf10 genotyping by PCR was performed using the following primers; P1, P2 and 5’-GAGCTTGCTGGGGGAAACTTCCTGACTAGG-3’ for Fgf10,26 and 5’-TGCCTAAGGGGAGAAGGCTGG-3’, 5’-AAATTTAAGCTGTGTAGATTCCATAG-3’ and 5’-GATTTCAGGAGAACAGACCAGAG-3’ primers for Fgf8. Numbers of embryos obtained at different stages are summarized in Online Table I. Total numbers of embryos examined for each genotype at E10.5 and E15.5 to18.5 are given in Table 1 and Table 2, respectively.
Right sided views of the OFT and RV in embryos at E9.5 were scored according to four parameters, OFT length, the angle between the proximal and distal regions of the OFT, the size of the RV, and the extent of looping estimated by the angle of the OFT across the ventral body of the embryo. Scoring was performed by two independent observers blinded to genotype. There was remarkably little inter-observer variability in the scores of individual embryos of all genotypes (not shown). Embryos were classified as normal, mild, moderate and severe depending on the score (2 points for each parameter, with a maximum of 8 points. < 2 points, severe; < 4 points, moderate; < 6 points, mild; ≥ 6, normal).
10µm longitudinal sections were made in the OFT and pharyngeal arch region after paraffin embedding of E10.5 embryos. To visualize PAA formation, India ink was injected into the left ventricle or umbilical vein of E10.5 embryos using drawn glass pipettes.
The 3rd to 6th pharyngeal arch region was dissected from embryos at 24 to 26 somite stages, and stored in RNAlater (Qiagen) at −20°C until use. Total RNA was isolated from a pool of six samples for each genotype using RNeasy Mini Kit (Qiagen). The first strand cDNA was synthesized using SuperScript II Reverse Transcriptase (Invitrogen). Quantitative PCR was performed with Power SYBR Green PCR Master Mix (Applied Biosystems) with the StepOnePlus Real-Time PCR system (Applied Biosystems). Reverse transcription and quantitative PCR were repeated three times, and GAPDH was used to normalize gene expression. Primer sequences were 5'-TGAAAGGCGGATACTTGGAC-3' and 5'-TGTCCGGTACCTGAGCTTCT-3' for Pea3, 5’-GGACACAGATCTGGCTCACGA-3’ and 5’-CGTGGCTACAGGACGACAAC-3’ for Erm, Isl1 primers were as designed by Liao et al.28
Fgf10 expression is observed in the anterior SHF and pharyngeal mesoderm, whereas Fgf8 is expressed in pharyngeal endoderm and ectoderm in addition to these mesodermal tissues. To generate compound mutants for Fgf8 and Fgf10 in this mesoderm, we deleted the Fgf8 conditional allele (Fgf8flox) with MesP1Cre/+. Fgf10 null homozygotes are not viable after birth, and we therefore used the Fgf10 heterozygotes (Fgf10+/−) for crosses (see Online Table I, Fgf8flox/+;Fgf10+/−;MesP1Cre/+ ×Fgf8flox/flox;Fgf10+/−). In the following text, MesP1Cre/+ is included in all genotypes.
In Fgf8;Fgf10 double heterozygous (Fgf8flox/+;Fgf10+/−) (Figure 1B) and Fgf8flox/+;Fgf10−/− embryos (Figure 1C), OFT and RV morphology is normal at E9.5, similar to that of Fgf8flox/+ embryos (Figure 1A). Fgf8flox/flox embryos have hypomorphic OFTs and RVs (Figure 1D–F), as previously reported with a MesP1Cre at E9.5.19 When Fgf10 dosage was decreased in addition to Fgf8 deletion (Figure 1G–L), more severe phenotypes were observed including hypoplasia of the 2nd and 3rd pharyngeal arches (Figure 1I,L). This was also illustrated by sections of the OFT at E10.5 showing normal presence of CNC and initiation of EMT prior to cushion formation in control Fgf8;Fgf10 double heterozygotes (Figure 1M). In contrast, CNC invasion and EMT were notably compromised in Fgf8;Fgf10 double homozygous mutants (Figure 1O), and already affected in Fgf8flox/flox mutants (Figure 1N).
To confirm and quantify this, we scored OFT and RV morphology on the basis of OFT length, the angle between the proximal and distal regions of the OFT, RV size and extent of looping, by a blind test. Phenotypes were classified as normal, mild, moderate and severe, depending on the score (Figure 2A). The number of abnormal Fgf8flox/flox embryos was less than in our previous report, probably because in that case the Fgf8flox/− cross was analyzed.19 The important point, shown in Figure 2A, is that increasingly affected embryos were observed as Fgf dosage is reduced. No severe phenotypes were observed with Fgf8 deletion alone, whereas no normal hearts were observed in Fgf8;Fgf10 double homozygous mutants. This result shows that FGF10 functions with FGF8 in OFT and RV development. To verify that FGF signaling was attenuated in Fgf8;Fgf10 double homozygous mutants, we performed quantitative PCR analysis for FGF target genes, Pea3 and Erm, using RNA isolated from the pharyngeal region (including SHF mesoderm, endoderm and ectoderm) of compound mutant embryos (Figure 2B). We could not detect differences between Fgf8flox/+;Fgf10+/− and Fgf8flox/flox;Fgf10+/− mutants, suggesting that FGF8 in pharyngeal endoderm and ectoderm, together with FGF10 from one allele of Fgf10 expressed in mesoderm, lead to levels of Pea3 and Erm transcription in the pharyngeal region that obscure the reduction in the mesoderm. However, significant downregulation of the signaling read out from all FGF sources was detected in Fgf8;Fgf10 double homozygous mutants (Figure 2B). These results demonstrate the effect on FGF signaling readout of removing both alleles of Fgf10 on an Fgf8 mesodermal null background. Transcripts for Isl1, a key transcription factor required in the SHF,29 were also downregulated in the double homozygous mutants (Figure 2B). Apoptosis and proliferation were altered in Isl1-positive SHF cells in the double homozygous mutants (Online Figure I, A–D), as reported for Fgf8flox/− mutants,19 thus explaining the reduction in Isl1 transcripts.
PAAs, visualized by ink injection, were examined in Fgf8;Fgf10 compound mutants at E10.5 (Figure 3). At this stage, normal mouse embryos have 3rd, 4th and 6th bilateral PAAs (Figure 3A–C). PAA development was affected in some Fgf8flox/flox embryos (Figure 3D–F,M). Ectodermal Fgf8 ablation causes bilateral 4th PAA hypoplasia or aplasia.25 With mesodermal deletion of Fgf8, we observed not only 4th PAA defects, but also effects on the development of the 3rd and 6th PAAs, and abnormal maintenance of the 2nd PAA (Figure 3M). Fgf10−/− mutants did not show any PAA defects, as reported previously (data not shown).30 However, deletion of one allele of Fgf10 increased the proportion of PAA defects in Fgf8flox/flox embryos (Figure 3G–I,M), and this incidence was further increased in Fgf8;Fgf10 double homozygous mutants (Figure 3J–M). Apoptosis in CNC was observed at the level of the developing 4th PAA, in Fgf8flox/flox;Fgf10+/− mutants (Online Figure I, E,F), as reported for Fgf8 hypomorphic, Fgf8flox/−;AP2aIresCre, and Fgf8flox/−;Hoxa3Cre mutants.23,25 Sections also illustrated the severe PAA defects seen in Fgf8;Fgf10 double homozygous mutants (Figure 3O), compared to control Fgf8;Fgf10 double heterozygous embryos (Figure 3N). These results, summarized in Table 1, show that mesodermal FGF8 and FGF10 are critical for PAA development.
The adult configuration of the heart and great vessels is largely established by E15.5: the ventricles are separated by the ventricular septum; the OFT has given rise to myocardium at the base of the aorta and pulmonary trunk, and the 3rd, 4th and 6th PAAs have been remodeled into the common carotid and subclavian arteries, and contributed to part of the aortic arch and ductus arteriosus (Figure 4A,B). In the heart of Fgf8flox/flox embryos, as previously reported,19 TGA, double outlet right ventricle (DORV) and ventricular septal defects (VSD) were observed, although in a minority of cases (Figure 4F,G, Table 2). In Fgf8flox/flox;Fgf10 compound mutants, these heart defects became more frequent as Fgf10 dosage is reduced (Figure 4J,L,M, Table 2). Defects in PAA derivatives reflect the PAA abnormalities seen in mutants at E10.5. We observed absence of the left common carotid artery (LCC) (Figure 4E,H,K) which is due to loss of the left 3rd PAA, aberrant origin of the right subclavian artery (ARSA) (Figure 4F,I,K) which is due to loss of the right 4th PAA, right aortic arch (RtAA) (Figure 4I) which is caused by loss of the left 4th PAA (the aortic arch is probably replaced by the right 4th PAA) and narrow aortic arch (Figure 4L), due to a hypoplastic left 4th PAA. Consistent with earlier defects of the heart and PAA, the incidence of these later defects in surviving embryos was significantly increased when Fgf10 gene dosage was reduced, in conjunction with mesodermal deletion of Fgf8 (Table 2). These results confirm the functional overlap of mesodermal FGF8 and FGF10 in the development of the arterial pole of the heart and PAA, seen at earlier stages.
The phenotypic analysis that we present establishes a role for FGF10 as well as FGF8 in the formation of the arterial pole of the heart and demonstrates that this process is highly sensitive to FGF dosage. Furthermore we show that the level of FGF8 and FGF10 signaling is not only critical for OFT development, but also for the PAAs and their derivatives. Unexpectedly, mesodermal, as well as ectodermal25, expression of Fgf8 is important for the correct formation of these arteries and thus for cardiac function.
Most of the mutant embryos survive to late fetal stages, permitting analysis of the definitive morphology of the arterial pole. This is in contrast to previously reported observations on Fgf8flox/−;MesP1Cre/+ embryos of which 65% died by E10.5;19 this difference probably reflects the mesodermal specificity of deletion of the floxed alleles, which in our case (Fgf8flox/flox;MesP1Cre/+) are entirely responsible for generating the mutant phenotype. At early stages, in Fgf8 and in Fgf8;Fgf10 mutants, we observe hypoplasia of both the OFT and RV which also derives from SHF cells expressing Fgf815,20 as well as Fgf10.15 This reflects loss of proliferation and apoptosis of progenitor cells, observed when FGF signaling is abrogated, either due to mutation of mesodermal Fgf8 and Fgf10 (in this study)19 or to interference with FGF signal reception in the SHF.31,32 A reduction in SHF cells is also indicated by a decrease in Isl1 expression, reported for the more severe Fgf8flox/−;MesP1Cre phenotype19 and notable in Fgf8flox/flox;Fgf10−/− double mutants. As the OFT matures leading to EMT, cushion formation is initiated and CNC migrates into the OFT. These processes are affected when FGF signaling in mesoderm is abrogated,31,32 and the phenotypes again are striking in the Fgf8flox/flox;Fgf10−/− double mutants. The effects of FGF signaling from the SHF on CNC are indirect, since the arterial pole of the heart develops normally when FGF signaling in CNC is diminished.31,32 We propose that the BMP/TGFβ pathway mediates the action of mesodermal FGF signaling on CNC, since components of this pathway are downregulated in Fgf8 mutants.31 This would also have effects on the Nkx2.5 transcriptional network in the SHF.33 Further identification of targets of Pea3/Erm, transcriptional effectors of FGF signaling, should provide more insight into the FGF regulatory network. Failure of CNC migration into the OFT probably results from abnormal cell death in the pharyngeal arches,20,23–25 Upregulation of FGF signaling observed with CNC ablation34 or in Tbx3 mutants,35 also disrupts OFT development, demonstrating a critical requirement for precise levels of FGF signaling during arterial pole formation. Since CNC contributes the smooth muscle of the nascent PAAs and their mature derivatives, CNC death in Fgf8;Fgf10;MesP1Cre compound mutants contributes to defects in the stabilization and remodeling of these arteries. In Fgf8flox/−;cAP2αIresCre mutants where Fgf8 was deleted in pharyngeal ectoderm, major defects in the 4th PAA were documented, with minor effects on 3rd and 6th PAAs.25 Fgf8 deletion in the mesodermal core of the arches causes similar effects on 3rd, 4th and 6th PAAs. The increase in PAA defects in Fgf8flox/flox;Fgf10−/− double mutants reveals an unsuspected role for FGF10 in PAA development. We also observe some cases of 2nd PAA persistence at E10.5, probably reflecting a developmental delay. Mesodermal FGF signaling is clearly also necessary and the timing of this requirement may precede the appearance of CNC. MesP1Cre is activated at about E6.5, well before somites begin to form, whereas the Mef2c regulatory element that drives Mef2cCre expression in the SHF, including pharyngeal arch mesoderm, is activated later, from 2–3 somite stages.19 Since PAA formation and remodeling are normal in Fgf8flox/−;Mef2cCre embryos (16/16: AM unpublished data) the MesP1Cre phenotypes indicate that mesodermal FGF signaling is required at early stages to support PAA development, before CNC arrive in the arches.
Cardiac defects in surviving Fgf8;Fgf10 mutants, present as alignment defects of the aorta and pulmonary trunk (TGA) together with DORV and VSD, reflecting earlier malpositioning of the OFT and affected CNC. These defects also predominate when mesodermal Fgf8 is mutated (Table 2), whereas PTA is seen when Fgf8 is deleted in both mesoderm and pharyngeal endoderm13,19. The defects that we observe later in carotid and subclavian arteries and in the aortic arch reflect earlier defects in PAA development. When Fgf8 is deleted in pharyngeal ectoderm,25 the range and frequency of defects is different from those observed with mesodermal deletion of Fgf8, or when Fgf10 is also mutated. In addition to dose dependence, this result reveals the importance of paracrine effects of FGF signaling during PAA development.
Deletion of Fgf8 and Fgf10 does not result in total loss of the structures that depend on FGF signaling. This may be because other signaling pathways, such as TGFβ/BMP, which lies downstream of FGF8,19 also function independently to promote formation of the arterial pole of the heart and the PAAs. It may also reflect the ability of other FGFs to compensate in the SHF and pharyngeal arches. Fgf15, for example, is expressed in pharyngeal endoderm and Fgf15 mutants have cardiac defects (DORV, overriding aorta and VSD).36 Furthermore double Fgf15;Tbx1 heterozygotes show aberrant origin of the right subclavian artery, suggesting that FGF15 can play a role in the development of the arterial pole of the heart and PAAs.36 Loss of Fgf3, which is also expressed in pharyngeal endoderm, does not cause heart or PAA phenotypes, but Fgf3−/−;Tbx1+/− mutants have an aberrant origin of the right subclavian artery and interrupted aortic arch.37
In considering the relative roles of FGF8 and FGF10, the former is more widely produced, by pharyngeal endoderm and pharyngeal ectoderm, as well as by mesoderm in the anterior SHF and in its extension into the core of the pharyngeal arches, whereas FGF10 production is predominantly limited to the mesoderm. Both FGFs bind to FGFR2, although FGF10 binds preferentially to the IIIb isoform whereas FGF8 binds to IIIc and also has affinity for FGFR1, FGFR3IIIc and FGFR4.38–40 Mutational analysis of Fgfr1 and Fgfr2 suggests that FGFR1 is the dominant receptor in the SHF,31 although FGFR2 is also active and indeed in a different genetic background may be more important.32 Mutation of Fgfr1 and Fgfr2 in mesoderm compromises OFT development, as does over-expression of Sprouty2, an inhibitor of intracellular FGF signaling.31 Examination of Fgfr2-IIIb mutants shows later cardiac defects,16 but no striking effects on the early OFT, although RV hypoplasia, overriding aorta, DORV and VSD were noted. The question of in vivo FGF/receptor specificity is complex and the only clear conclusion to date is that FGFR1 and FGFR2 are required for FGF8 and FGF10 signaling, playing an essential role in the control of SHF progenitor cell proliferation and downstream signaling cascades31 during OFT morphogenesis.
The Tbx1 mutant phenotype, which recapitulates cardiovascular aspects of human DiGeorge syndrome,41–43 overlaps with that of Fgf8 hypomorphs.23,24 Tbx1 is expressed in the mesoderm of the anterior SHF, including the mesodermal core, endoderm and ectoderm of the pharyngeal arches,44,45 like Fgf8. Genetic analysis suggests that Tbx1 lies upstream of Fgf8 in pharyngeal endoderm46 and also affects Fgf8 and Fgf10 expression in SHF mesoderm.18,46,47 Phenotypic analysis of embryos after tissue specific deletion of Tbx1 with mesodermal Cre lines has shown that mesodermal Tbx1 is critical for SHF and OFT development.48 This is similar to FGF19 and to FGF10 as we now show.
Our findings demonstrate for the first time that mesodermal FGF10, as well as FGF8, is important for formation of the arterial pole of the heart and the PAAs, providing new insight into the cardiovascular abnormalities seen in DiGeorge syndrome. Furthermore, mutations in Fgf10 and Fgf8 that affect their expression in pharyngeal mesoderm may underlie human congenital heart and vascular defects.
Over 30% of congenital heart defects affect development of the arterial pole of the heart. In the mammalian embryo, cells that will contribute to this part of the heart derive from mesoderm of the second heart field. Loss of Fgf8 function in these cells leads to arterial pole defects. Fgf10 is also expressed in the second heart field, yet Fgf10 mutants do not have detectable outflow tract or great vessel defects. This may be due to compensation by FGF8. In order to address this question, we examined compound mutants, and show that arterial pole defects are more severe in the absence of both Fgf8 and Fgf10 function in the second heart field. This is also the case for arch arteries that contribute connecting vessels such as the subclavian and common carotid arteries, revealing for the first time a role for mesodermal FGF8 and FGF10 in vascular development. Our compound mutant analysis also demonstrates that this requirement is highly dosage sensitive, such that progressive reduction in the number of functional alleles of Fgf8 and Fgf10 leads to increasingly severe defects. These findings identify Fgf8 and Fgf10 as candidate genes for congenital cardiovascular malformations in the human population.
We thank Y. Saga for the MesP1Cre/+ line and N. Itoh and S. Kato for Fgf10+/− mice. We are grateful to C. Bodin and C. Cimper for help with histology, E. Pecnard and S. Coqueran for genotyping, E.J. Park for scoring of OFT/RV morphology of embryos, and to members of our lab and S. Zaffran for helpful discussions.
Sources of Funding
The work in M.B.’s lab was supported by the Pasteur Institute and the CNRS, with grants from the E.U. Integrated Project “Heart Repair” LHSM-CT2005-018630 (also to R.K.) and the CardioCell LT2009-223372 project. The work in A.M.’s lab was supported by the NICHD. Y.W. had a fellowship from the E.U. Integrated Project “Heart Repair”. S.M.-T. had a fellowship from the Naito Foundation to work in M.B.’s lab for six months.
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