Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

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

Nature volume 474, pages 645–648 (2011)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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.

Similar content being viewed by others

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Data deposits

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

References

  1. Dyer, L. A. & Kirby, M. L. The role of secondary heart field in cardiac development. Dev. Biol. 336, 137–144 (2009)

    Article  CAS  Google Scholar 

  2. Rochais, F., Mesbah, K. & Kelly, R. G. Signaling pathways controlling second heart field development. Circ. Res. 104, 933–942 (2009)

    Article  CAS  Google Scholar 

  3. Vincent, S. D. & Buckingham, M. E. How to make a heart: the origin and regulation of cardiac progenitor cells. Curr. Top. Dev. Biol. 90, 1–41 (2010)

    Article  Google Scholar 

  4. Olson, E. N. Gene regulatory networks in the evolution and development of the heart. Science 313, 1922–1927 (2006)

    Article  ADS  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  6. Meilhac, S. M. et al. The clonal origin of myocardial cells in different regions of the embryonic mouse heart. Dev. Cell 6, 685–698 (2004)

    Article  CAS  Google Scholar 

  7. Grimes, A. C. et al. Solving an enigma: arterial pole development in the zebrafish heart. Dev. Biol. 290, 265–276 (2006)

    Article  CAS  Google Scholar 

  8. Brade, T. et al. The amphibian second heart field: Xenopus islet-1 is required for cardiovascular development. Dev. Biol. 311, 297–310 (2007)

    Article  CAS  Google Scholar 

  9. de Pater, E. et al. Distinct phases of cardiomyocyte differentiation regulate growth of the zebrafish heart. Development 136, 1633–1641 (2009)

    Article  CAS  Google Scholar 

  10. Grimes, A. C. et al. Phylogeny informs ontogeny: a proposed common theme in the arterial pole of the vertebrate heart. Evol. Dev. 12, 552–567 (2010)

    Article  Google Scholar 

  11. Rifkin, D. B. Latent transforming growth factor-β (TGF-β) binding proteins: orchestrators of TGF-β availability. J. Biol. Chem. 280, 7409–7412 (2005)

    Article  CAS  Google Scholar 

  12. Kelly, R. G., Brown, N. A. & Buckingham, M. E. The arterial pole of the mouse heart forms from Fgf10-expressing cells in pharyngeal mesoderm. Dev. Cell 1, 435–440 (2001)

    Article  CAS  Google Scholar 

  13. Chen, J. N. et al. Left-right pattern of cardiac BMP4 may drive asymmetry of the heart in zebrafish. Development 124, 4373–4382 (1997)

    CAS  PubMed  Google Scholar 

  14. Miao, M. et al. Differential expression of two tropoelastin genes in zebrafish. Matrix Biol. 26, 115–124 (2007)

    Article  CAS  Google Scholar 

  15. Ilagan, R. et al. Fgf8 is required for anterior heart field development. Development 133, 2435–2445 (2006)

    Article  CAS  Google Scholar 

  16. Prall, O. W. et al. An Nkx2-5/Bmp2/Smad1 negative feedback loop controls heart progenitor specification and proliferation. Cell 128, 947–959 (2007)

    Article  CAS  Google Scholar 

  17. von Both, I. et al. Foxh1 is essential for development of the anterior heart field. Dev. Cell 7, 331–345 (2004)

    Article  CAS  Google Scholar 

  18. Mosimann, C. et al. Ubiquitous transgene expression and Cre-based recombination driven by the ubiquitin promoter in zebrafish. Development 138, 169–177 (2010)

    Article  Google Scholar 

  19. Waldo, K. L. et al. Conotruncal myocardium arises from a secondary heart field. Development 128, 3179–3188 (2001)

    CAS  PubMed  Google Scholar 

  20. Zaffran, S. et al. Right ventricular myocardium derives from the anterior heart field. Circ. Res. 95, 261–268 (2004)

    Article  CAS  Google Scholar 

  21. Todorovic, V. et al. Long form of latent TGF-β binding protein 1 (Ltbp1L) is essential for cardiac outflow tract septation and remodeling. Development 134, 3723–3732 (2007)

    Article  CAS  Google Scholar 

  22. Li, H. Y. et al. Dihydropyrrolopyrazole transforming growth factor-β type I receptor kinase domain inhibitors: a novel benzimidazole series with selectivity versus transforming growth factor-β type II receptor kinase and mixed lineage kinase-7. J. Med. Chem. 49, 2138–2142 (2006)

    Article  CAS  Google Scholar 

  23. Mjaatvedt, C. H. et al. The outflow tract of the heart is recruited from a novel heart-forming field. Dev. Biol. 238, 97–109 (2001)

    Article  CAS  Google Scholar 

  24. Kang, J. et al. Isl1 is a direct transcriptional target of Forkhead transcription factors in second-heart-field-derived mesoderm. Dev. Biol. 334, 513–522 (2009)

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Cardiovascular Research Center, Massachusetts General Hospital, Charlestown, 02129, Massachusetts, USA

    Yong Zhou, Timothy J. Cashman, Kathleen R. Nevis, Pablo Obregon, Aihua Gu, Samuel Sondalle, Caroline E. Burns & C. Geoffrey Burns

  2. Harvard Medical School, Boston, Massachusetts 02115, USA ,

    Yong Zhou, Timothy J. Cashman, Kathleen R. Nevis, Pablo Obregon, Yan Liu, Aihua Gu, Christian Mosimann, Samuel Sondalle, Caroline E. Burns & C. Geoffrey Burns

  3. Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin, Madison, 53705, Wisconsin, USA

    Sara A. Carney, Richard E. Peterson & Warren Heideman

  4. Nephrology Division, Massachusetts General Hospital, Charlestown, 02129, Massachusetts, USA

    Yan Liu

  5. School of Public Health, Nanjing Medical University, Nanjing, 210029, China

    Aihua Gu

  6. Stem Cell Program and Division of Hematology/Oncology, Children’s Hospital Boston, 02115, Massachusetts, USA

    Christian Mosimann

  7. Harvard Stem Cell Institute, Cambridge, 02138, Massachusetts, USA

    Samuel Sondalle & Caroline E. Burns

Authors
  1. Yong Zhou
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Timothy J. Cashman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kathleen R. Nevis
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pablo Obregon
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sara A. Carney
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yan Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Aihua Gu
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Christian Mosimann
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Samuel Sondalle
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Richard E. Peterson
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Warren Heideman
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Caroline E. Burns
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. C. Geoffrey Burns
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-10 with legends, Supplementary Methods and additional references. (PDF 25227 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature10094

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing