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Latent TGF-β binding protein 3 identifies a second heart field in zebrafish

Abstract

The four-chambered mammalian heart develops from two fields of cardiac progenitor cells distinguished by their spatiotemporal patterns of differentiation and contributions to the definitive heart1,2,3. The first heart field differentiates earlier in lateral plate mesoderm, generates the linear heart tube and ultimately gives rise to the left ventricle. The second heart field (SHF) differentiates later in pharyngeal mesoderm, elongates the heart tube, and gives rise to the outflow tract and much of the right ventricle. Because hearts in lower vertebrates contain a rudimentary outflow tract but not a right ventricle4, the existence and function of SHF-like cells in these species has remained a topic of speculation4,5,6,7,8,9,10. Here we provide direct evidence from Cre/Lox-mediated lineage tracing and loss-of-function studies in zebrafish, a lower vertebrate with a single ventricle, that latent TGF-β binding protein 3 (ltbp3) transcripts mark a field of cardiac progenitor cells with defining characteristics of the anterior SHF in mammals. Specifically, ltbp3+ cells differentiate in pharyngeal mesoderm after formation of the heart tube, elongate the heart tube at the outflow pole, and give rise to three cardiovascular lineages in the outflow tract and myocardium in the distal ventricle. In addition to expressing Ltbp3, a protein that regulates the bioavailability of TGF-β ligands11, zebrafish SHF cells co-express nkx2.5, an evolutionarily conserved marker of cardiac progenitor cells in both fields4. Embryos devoid of ltbp3 lack the same cardiac structures derived from ltbp3+ cells due to compromised progenitor proliferation. Furthermore, small-molecule inhibition of TGF-β signalling phenocopies the ltbp3-morphant phenotype whereas expression of a constitutively active TGF-β type I receptor rescues it. Taken together, our findings uncover a requirement for ltbp3–TGF-β signalling during zebrafish SHF development, a process that serves to enlarge the single ventricular chamber in this species.

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Figure 1: ltbp3 and nkx2.5 transcripts mark extra-cardiac cells contiguous to the outflow pole of the zebrafish heart tube.
Figure 2: Knocking down Ltbp3 causes multi-lineage cardiovascular defects in the ventricle and OFT.
Figure 3: ltbp3 + cells give rise to three cardiovascular lineages in the zebrafish ventricle and OFT.
Figure 4: Diminished TGF-β signalling underlies the ltbp3 morphant cardiovascular phenotypes.

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GenBank/EMBL/DDBJ

Data deposits

The zebrafish ltbp3 cDNA sequence has been deposited into GenBank under accession number JF731042.

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Acknowledgements

We thank M. Whitman for advice on pSmad2 staining, D. Hami and M. Kirby for providing Eln2 antisera and their immunohistochemistry protocol, R. Cornell for providing tfAP2a and tfAP2c morpholinos, M. Whitman, A. Srinivasan, D. Langenau, E. Provost, S. Leach, R. Anderson, D. Stainier, I. Woods, and A. Schier for providing plasmids. J. W. Xiong for providing clom39 fish, B. Barut and L. Zon for providing bacterial artificial chromosomes (BACs), and the MGH Nephrology Division for access to their confocal microscopy facilities. S.C., R.E.P. and W.H. were supported by NIH grant R01 ES012716 from the National Institute of Environmental Health Sciences. C.M. received support through an EMBO long-term fellowship and an HFSP long-term fellowship. This work was funded by the Cardiovascular Research Center at Massachusetts General Hospital, a Claflin Distinguished Scholar Award and Harvard Stem Cell Institute Seed Grant to C.E.B., and by awards from the National Heart Lung and Blood Institute (5R01HL096816), American Heart Association (Grant in Aid no. 10GRNT4270021), and Harvard Stem Cell Institute (Seed Grant) to C.G.B.

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Contributions

Y.Z. performed the majority of the experiments and analysed data; T.J.C., K.R.N., P.O., Y.L., A.G. and S.S. performed experiments and analysed data; C.M. provided the ubiquitin promoter prior to publication; S.A.C., R.E.P., W.H. and C.G.B. discovered that ltbp3 transcripts are enriched in a cardiac fraction on day 3 post-fertilization; C.G.B. performed experiments including BAC recombineering; C.E.B. and C.G.B. co-directed the study, analysed data, and wrote the paper with input from all authors.

Corresponding authors

Correspondence to Caroline E. Burns or C. Geoffrey Burns.

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The authors declare no competing financial interests.

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Zhou, Y., Cashman, T., Nevis, K. et al. Latent TGF-β binding protein 3 identifies a second heart field in zebrafish. Nature 474, 645–648 (2011). https://doi.org/10.1038/nature10094

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