Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development

Abstract

Cardiac development arises from two sources of mesoderm progenitors, the first heart field (FHF) and the second (SHF). Mesp1 has been proposed to mark the most primitive multipotent cardiac progenitors common for both heart fields. Here, using clonal analysis of the earliest prospective cardiovascular progenitors in a temporally controlled manner during early gastrulation, we found that Mesp1 progenitors consist of two temporally distinct pools of progenitors restricted to either the FHF or the SHF. FHF progenitors were unipotent, whereas SHF progenitors were either unipotent or bipotent. Microarray and single-cell PCR with reverse transcription analysis of Mesp1 progenitors revealed the existence of molecularly distinct populations of Mesp1 progenitors, consistent with their lineage and regional contribution. Together, these results provide evidence that heart development arises from distinct populations of unipotent and bipotent cardiac progenitors that independently express Mesp1 at different time points during their specification, revealing that the regional segregation and lineage restriction of cardiac progenitors occur very early during gastrulation.

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Figure 1: Mesp1–rtTA transgenic mice faithfully recapitulate Mesp1 endogenous expression.
Figure 2: Two temporally distinct populations of Mesp1 progenitors contribute to the development of the FHF and SHF.
Figure 3: Bio-statistical modelling of the the multicolour-labelled hearts.
Figure 4: Clonal analysis of lineage differentiation of Mesp1-derived progenitors in vivo.
Figure 5: Molecular signature of early and late Mesp1-expressing cells in vivo.
Figure 6: Different temporal expression of Mesp1 direct target genes.
Figure 7: Revised model of the early step of cardiovascular progenitor specification and lineage commitment during mouse development.

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Acknowledgements

We thank F. Bollet-Quivogne and J-M. Vanderwinden for their help with confocal imaging and M. Tarabichi for his help with gene set enrichment analysis. We thank M. Buckingham and A. Joyner for kindly providing the probes for in situ hybridization. We also thank the Genomic core facility of the EMBL, Heidelberg for their help with the ChIP-Seq. F.L. has been sequentially supported by the FNRS and the EMBO long-term fellowship. S.C. is supported by a fellowship of the FRS/FRIA. X.L. is supported by the FNRS. A.B. is supported by the FRS/FNRS. S.R. and B.D.S. are supported by the Wellcome Trust (grant number 098357/Z/12/Z). C.B. is an investigator of WELBIO. This work was supported by the FNRS, the ULB foundation, the Fondation contre le Cancer, the European Research Council (ERC), and the foundation Bettencourt Schueller (C.B. and F.L.).

Author information

C.B., F.L., S.C. and X.L. designed the experiments and performed data analysis. F.L. and S.C. performed most of the experiments. X.L. performed the single-cell PCR analysis. Y.A. generated the Mesp1–rtTA transgenic mice. A.R. and H. A. performed microarrays. C.P. provided technical assistance. C.D. helped with FACS isolation of Mesp1-expressing cells. A.B. helped in the design and initial characterization of the Mesp1–rtTA transgene. B.D.S. and S.R. performed the bio-statistical analysis of the clonal fate data. C.B. and F.L. wrote the manuscript.

Correspondence to Cédric Blanpain.

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Integrated supplementary information

Supplementary Figure 1 Mesp1-rtTA/tetO-Cre transgenic mice induced Cre expression similarly to Mesp1-Cre Knock-in mice.

(a,b) Sections of E12.5 Mesp1-Cre/Rosa-tdTomato (a) and Mesp1-rtTA/TetO-Cre/Rosa-tdTomato hearts (induced by Dox administration between E6.25 and E7.5) (b) and co-stained with DAPI. Both transgenic hearts have a similar expression of the tdTomato with a negative region in the OFT that derive from Mesp1 negative neural crest cells (asterisks). ce Doxycycline injection has no effect on Mesp1 expression during early mouse embryonic development. (cd) In situ hybridization for Mesp1 expression in early embryo at E6.5. The detection of Mesp1 mRNA in the primitive streak (PS) and the nascent lateral mesoderm is similar in embryo that did not receive DOX (c) and in embryos injected with DOX (+ DOX) (d). A, anterior; P, posterior. e Expression of Mesp1 analysed by RT-qPCR in early embryos (E6.75) without (n = 9) or after doxycycline injections (n = 6). These data show no difference in Mesp1 expression after DOX injection. (fg) In situ hybridization for Cre expression in early embryos at E6.75. The detection of Cre mRNA in the primitive streak (PS) and the nascent lateral mesoderm is similar in Mesp1-Cre knock-in (f) and in Mesp1-rtTA/TetO-Cre transgenic embryos injected with DOX at E6.25 (+ DOX) (g). Cre expression is similar to the endogenous Mesp1 expression in wild type embryos (h). A, anterior; P, posterior. Scale bar, 500 μm.

Supplementary Figure 2 Temporal Dox administration in Mes1-rtTA/TetO-Cre/Rosa-Confetti embryos.

(a) While clonal dose of DOX (0.575 μg g−1) induces labelling in Mesp1-rtTA/TetO-Cre/Rosa-Confetti embryos at E6.25 (n = 53), at E6.75 (n = 118) or at E7.25 (n = 65), this dose was not sufficient to induce labelling at E5.75 (n = 13). A much higher dose of Dox (25 μg g−1) was required to produce labelling at a clonal density at E5.75 (n = 90). This 40 fold increase of DOX is likely to persist at a concentration sufficient to activate the Cre at the time of endogenous Mesp1 expression. This high dose of DOX never labelled any heart after administration at E8.5 or E9.5 (n = 24) supporting the absence of transgene expression after the end of endogenous Mesp1 expression. (b,c) Examples of Mesp1-rtTA/TetO-Cre/Rosa-Confetti unicolour labelled hearts at E12.5 induced at E5.75 after administration of high dose of Doxycycline (25 μg g−1). Note that each cluster is localized within the LV, FHF derivative and no labelling was detected other compartments. OFT, outflow tract; RV, right ventricle; LV, left ventricle; RA, right atrium; LA, left atrium; IFT, inflow tract. Scale bars, 200 μm. The number on the upper right in each panel refers to the ID of the labelled heart. (d) Quantification of the regional (FHF and SHF) contribution of patches of Mesp1 labelled cells in unicolour hearts induced at E5.75 with the high dose of Doxycycline (25 μg g−1), shows the exclusive labelling of the FHF (red) similarly to was found at E6.25 (Fig. 2m).

Supplementary Figure 3 Biostatistical modeling of the clonal fate data.

(a) The likelihood function F gives the probability of the experimental data for different values of the induction frequency pN and the fragmentation rate f. The numeric values have been rescaled such that the maximum of the likelihood function corresponds to 1. Colour denotes the value of F, such that red signifies a large value and blue signifies small value. Lines of equal values are indicated on the bottom of the figure. One sees that the maximum value of F is relatively featureless along a curve in the pN-f-plane. To infer the values of pN and f we must therefore refer to an independent measurement of one of the two parameters. (b) The multicolour labelling strategy allows us to independently infer the induction frequency pN = 1.3 by evaluating the abundances of hearts with a given number of colours. With this, we are left with a slice through the pN-f-plane and the fragmentation rate can be determined with a higher accuracy. (c) Monoclonal datasets (n = 89) identify two subpopulations in Mesp1 expressing cells: FHF progenitors, which contribute to the LV and SHF progenitors, which contribute to OFT and IFT. The plot shows the probabilities of monoclonal fragments in the different heart compartments. (d) Values for the induction frequency, pN, and the fragmentation rate, f, for the two FHF (n = 188) and SHF (n = 102) precursors. While the overall induction frequency is higher for FHF precursors, which we attribute to highest expression of Mesp1 at the early time points, the fragmentation rate is higher for SHF precursors. (e) We may use the distribution of monoclonal fragments (c) to predict the distribution of fragments in all hearts (n = 263). We find an excellent agreement with the notable exception of the RV, which might suggest the existence of an independent pool of progenitors contributing to RV morphogenesis. Error bars indicate one sigma (c and e) or 95% (d) confidence intervals.

Supplementary Figure 4 Late Mesp1 progenitors also contribute to the head.

(aa’) Example of a Mesp1-rtTA/TetO-Cre/Rosa-Confetti embryo with co-labelled head (a) and heart (a’) at E12.5. Scale bars, 200 μm. (b–b’) Sections of a Mesp1-rtTA/TetO-Cre/Rosa-Confetti labelled embryo showing labelling in the head in an extraocular muscle (EOM) (b) as well as in the right ventricle RV (b’). Scale bars, 200 μm. (c) Temporal appearance of head muscle labelling inferred from all datasets (n = 105 independent embryos translating to n = 181 embryos by colour). Plotted is the fraction of head muscle labelling induced at each induction time for a given colour. Head muscles are preferentially labelled at the late time points. (d) Regional contribution of head progenitors in monoclonal datasets (n = 5), showing the co-labelling of the head with the heart and preferentially SHF derivatives or the right ventricle (RV). OFT, outflow tract; RV, right ventricle; LV, left ventricle; RA, right atrium; LA, left atrium. Errors bars indicate one sigma confidence intervals.

Supplementary Figure 5 Cohesive versus dispersive mode of growth of the myocardium and the endocardium.

(ab) Sections of E12.5 Mesp1-rtTA/tetO-Cre/Rosa-Confetti unicolour hearts. (a) Example of a compact myocardial YFP-labelled clone showing cohesive growth of the myocardium. (b) Example of a dispersed endocardial RFP-unicolour clone showing dispersive mode of growth of the endocardium. Scale bars, 200 μm. The number on the upper right in each panel refers to the ID of the labelled heart.

Supplementary Figure 6 Semi-quantification of single cell RT-PCR analysis.

Examples showing strong, weak and no gene expression in single cells.

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Lescroart, F., Chabab, S., Lin, X. et al. Early lineage restriction in temporally distinct populations of Mesp1 progenitors during mammalian heart development. Nat Cell Biol 16, 829–840 (2014). https://doi.org/10.1038/ncb3024

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