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.

  • Article
  • Published:

Notch signaling respecifies the hemangioblast to a cardiac fate

Abstract

To efficiently generate cardiomyocytes from embryonic stem (ES) cells in culture it is essential to identify key regulators of the cardiac lineage and to develop methods to control them. Using a tet-inducible mouse ES cell line to enforce expression of a constitutively activated form of the Notch 4 receptor, we show that signaling through the Notch pathway can efficiently respecify hemangioblasts to a cardiac fate, resulting in the generation of populations consisting of >60% cardiomyocytes. Microarray analyses reveal that this respecification is mediated in part through the coordinated regulation of the BMP and Wnt pathways by Notch signaling. Together, these findings have uncovered a potential role for the Notch pathway in cardiac development and provide an approach for generating large numbers of cardiac progenitors from ES cells.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: The role of Notch signaling in cardiac differentiation from embryoid body–derived Bry-GFP+/Flk-1 cardiac mesodermal cells.
Figure 2: Effects of Notch4 overexpression on the embryoid body–derived Bry-GFP+/Flk-1+ population.
Figure 3: Respecification by Notch signaling is restricted to the hemangioblast stage of embryoid body development.
Figure 4: Effect of Notch4 expression on BL-CFC–derived blast colony development.
Figure 5: Microarray-based expression analysis of Bry-GFP+/Flk-1+ cells after Dox induction.
Figure 6: Effects of cytokines on the cardiac differentiation from Bry-GFP+/Flk-1+ and Bry-GFP+/Flk-1 cells.
Figure 7

Similar content being viewed by others

Accession codes

Accessions

Gene Expression Omnibus

References

  1. Murry, C.E. & Keller, G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132, 661–680 (2008).

    Article  CAS  PubMed  Google Scholar 

  2. Parameswaran, M. & Tam, P.P. Regionalisation of cell fate and morphogenetic movement of the mesoderm during mouse gastrulation. Dev. Genet. 17, 16–28 (1995).

    Article  CAS  PubMed  Google Scholar 

  3. Kinder, S.J. et al. The orderly allocation of mesodermal cells to the extraembryonic structures and the anteroposterior axis during gastrulation of the mouse embryo. Development 126, 4691–4701 (1999).

    CAS  PubMed  Google Scholar 

  4. Choi, K., Kennedy, M., Kazarov, A., Papadimitriou, J.C. & Keller, G. A common precursor for hematopoietic and endothelial cells. Development 125, 725–732 (1998).

    CAS  PubMed  Google Scholar 

  5. Huber, T.L., Kouskoff, V., Fehling, H.J., Palis, J. & Keller, G. Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature 432, 625–630 (2004).

    Article  CAS  PubMed  Google Scholar 

  6. Kennedy, M., D'Souza, S.L., Lynch-Kattman, M., Schwantz, S. & Keller, G. Development of the hemangioblast defines the onset of hematopoiesis in human ES cell differentiation cultures. Blood 109, 2679–2687 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Kattman, S.J., Huber, T.L. & Keller, G.M. Multipotent flk-1+ cardiovascular progenitor cells give rise to the cardiomyocyte, endothelial, and vascular smooth muscle lineages. Dev. Cell 11, 723–732 (2006).

    Article  CAS  PubMed  Google Scholar 

  8. Moretti, A. et al. Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification. Cell 127, 1151–1165 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Wu, S.M. et al. Developmental origin of a bipotential myocardial and smooth muscle cell precursor in the mammalian heart. Cell 127, 1137–1150 (2006).

    Article  CAS  PubMed  Google Scholar 

  10. Yang, L. et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453, 524–528 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Fehling, H.J. et al. Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation. Development 130, 4217–4227 (2003).

    Article  CAS  PubMed  Google Scholar 

  12. Kouskoff, V. Lacaud, Gl, Schwantz, S., Fehling, H.J., and Keller, G. Sequential development of hematopoietic and cardiac mesoderm during embryonic stem cell differentiation. Proc. Natl. Acad. Sci. USA 102, 13170–13175 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Ema, M., Takahashi, S. & Rossant, J. Deletion of the selection cassette, but not cis-acting elements, in targeted Flk1-lacZ allele reveals Flk1 expression in multipotent mesodermal progenitors. Blood 107, 111–117 (2006).

    Article  CAS  PubMed  Google Scholar 

  14. Schultheiss, T., Burch, J. & Lassar, A. A role for bone morphogenetic proteins in the induction of cardiac myogenesis. Genes Dev. 11, 451–462 (1997).

    Article  CAS  PubMed  Google Scholar 

  15. Andreé, B., Duprez, D., Vorbusch, B., Arnold, H. & Brand, T. BMP-2 induces ectopic expression of cardiac lineage markers and interferes with somite formation in chicken embryos. Mech. Dev. 70, 119–131 (1998).

    Article  PubMed  Google Scholar 

  16. Zhang, H. & Bradley, A. Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development. Development 122, 2977–2986 (1996).

    CAS  PubMed  Google Scholar 

  17. Marvin, M.J., Di Rocco, G., Gardiner, A., Bush, S.M. & Lassar, A.B. Inhibition of Wnt activity induces heart formation from posterior mesoderm. Genes Dev. 15, 316–327 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Nostro, M.C., Cheng, X., Keller, G.M. & Gadue, P. Wnt, activin, and BMP signaling regulate distinct stages in the developmental pathway from embryonic stem cells to blood. Cell Stem Cell 2, 60–71 (2007).

    Article  Google Scholar 

  19. Ueno, S. et al. Biphasic role for Wnt/beta-catenin signaling in cardiac specification in zebrafish and embryonic stem cells. Proc. Natl. Acad. Sci. USA 104, 9685–9690 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Naito, A.T. et al. Developmental stage-specific biphasic roles of Wnt/beta-catenin signaling in cardiomyogenesis and hematopoiesis. Proc. Natl. Acad. Sci. USA 103, 19812–19817 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Qyang, Y. et al. The renewal and differentiation of Isl1+ cardiovascular progenitors are controlled by wnt/β-catenin pathway. Cell Stem Cell 1, 165–179 (2007).

    Article  CAS  PubMed  Google Scholar 

  22. Schneider, V.A. & Mercola, M. Wnt antagonism initiates cardiogenesis in Xenopus laevis . Genes Dev. 15, 304–315 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Rones, M.S., McLaughlin, K.A., Raffin, M. & Mercola, M. Serrate and Notch specify cell fates in the heart field by suppressing cardiomyogenesis. Development 127, 3865–3876 (2000).

    CAS  PubMed  Google Scholar 

  24. Schroeder, T. et al. Recombination signal sequence-binding protein Jkappa alters mesodermal cell fate decisions by suppressing cardiomyogenesis. Proc. Natl. Acad. Sci. USA 100, 4018–4023 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Nemir, M., Croquelois, A., Pedrazzini, T. & Radtke, F. Induction of Cardiogenesis in embryonic stem cells via downregulation of Notch1 signaling. Circ. Res. 98, 1471–1478 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Williams, R., Lendahl, U. & Lardelli, M. Complementary and combinatorial patterns of Notch gene family expression during early mouse development. Mech. Dev. 53, 357–368 (1995).

    Article  CAS  PubMed  Google Scholar 

  27. Uyttendaele, H. et al. Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene. Development 122, 2251–2259 (1996).

    CAS  PubMed  Google Scholar 

  28. Shirayoshi, Y. et al. Proto-oncogene of int-3, a mouse Notch homologue, is expressed in endothelial cells during early embryogenesis. Genes Cells 2, 213–224 (1997).

    Article  CAS  PubMed  Google Scholar 

  29. Joutel, A. et al. The ectodomain of the Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients. J. Clin. Invest. 105, 597–605 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Loomes, K.M. et al. Characterization of Notch receptor expression in the developing mammalian heart and liver. Am. J. Med. Genet. 112, 181–189 (2002).

    Article  PubMed  Google Scholar 

  31. Kyba, M., Perlingeiro, R.C. & Daley, G.Q. HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic stem cell and yolk sac hematopoietic progenitors. Cell 109, 29–37 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Das, I. et al. Notch oncoproteins depend on gamma-secretase/presenilin activity for processing and function. J. Biol. Chem. 279, 30771–30780 (2004).

    Article  CAS  PubMed  Google Scholar 

  33. Lints, T.J., Parsons, L.M., Hartley, L., Lyons, I. & Harvey, R.P. Nkx-2.5: a novel murine homeobox gene expressed in early heart progenitor cells and their myogenic descendants. Development 119, 419–431 (1993).

    CAS  PubMed  Google Scholar 

  34. Schmitt, T.M. et al. Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat. Immunol. 5, 410–417 (2004).

    Article  CAS  PubMed  Google Scholar 

  35. Dudley, A.T. & Robertson, E.J. Overlapping expression domains of bone morphogenetic protein family members potentially account for limited tissue defects in BMP7 deficient embryos. Dev. Dyn. 208, 349–362 (1997).

    Article  CAS  PubMed  Google Scholar 

  36. Kim, R.Y., Robertson, E.J. & Solloway, M.J. Bmp6 and Bmp7 are required for cushion formation and septation in the developing mouse heart. Dev. Biol. 235, 449–466 (2001).

    Article  CAS  PubMed  Google Scholar 

  37. Wang, H. et al. Wnt2 coordinates the commitment of mesoderm to hematopoietic, endothelial, and cardiac lineages in embryoid bodies. J. Biol. Chem. 282, 782–791 (2007).

    Article  CAS  PubMed  Google Scholar 

  38. Alexandrovich, A. et al. Wnt2 is a direct downstream target of GATA6 during early cardiogenesis. Mech. Dev. 123, 297–311 (2006).

    Article  CAS  PubMed  Google Scholar 

  39. Pandur, P., Lasche, M., Eisenberg, L.M. & Kuhl, M. Wnt-11 activation of a non-canonical Wnt signalling pathway is required for cardiogenesis. Nature 418, 636–641 (2002).

    Article  CAS  PubMed  Google Scholar 

  40. Shawber, C. et al. Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway. Development 122, 3765–3773 (1996).

    CAS  PubMed  Google Scholar 

  41. Nofziger, D., Miyamoto, A., Lyons, K.M. & Weinmaster, G. Notch signaling imposes two distinct blocks in the differentiation of C2C12 myoblasts. Development 126, 1689–1702 (1999).

    CAS  PubMed  Google Scholar 

  42. Wilson-Rawls, J., Molkentin, J.D., Black, B.L. & Olson, E.N. Activated notch inhibits myogenic activity of the MADS-Box transcription factor myocyte enhancer factor 2C. Mol. Cell. Biol. 19, 2853–2862 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Guan, E. et al. T cell leukemia-associated human Notch/TAN-1 has IB-like activity and physically interacts with NF-κB proteins in T cells. J. Exp. Med. 183, 2025–2032 (1996).

    Article  CAS  PubMed  Google Scholar 

  44. Axelrod, J.D., Matsuno, K., Artavanis-Tsakonas, S. & Perrimon, N. Interaction between Wingless and Notch signaling pathways mediated by Dishevelled. Science 271, 1826–1832 (1996).

    Article  CAS  PubMed  Google Scholar 

  45. Blokzijl, A. et al. Cross-talk between the Notch and TGF-beta signaling pathways mediated by interaction of the Notch intracellular domain with Smad3. J. Cell Biol. 163, 723–728 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Dahlqvist, C. et al. Functional Notch signaling is required for BMP4-induced inhibition of myogenic differentiation. Development 130, 6089–6099 (2003).

    Article  CAS  PubMed  Google Scholar 

  47. Sun, Y. et al. Notch4 intracellular domain binding to Smad3 and inhibition of the TGF-beta signaling. Oncogene 24, 5365–5374 (2005).

    Article  CAS  PubMed  Google Scholar 

  48. Brady, G., Barbara, M. & Iscove, N.N. Representative in vitro cDNA amplification from individual hematopoietic cells and colonies. Methods Mol. Cell. Biol. 2, 17–25 (1990).

    CAS  Google Scholar 

  49. Robertson, S.M., Kennedy, M., Shannon, J.M. & Keller, G. A transitional stage in the commitment of mesoderm to hematopoiesis requiring the transcription factor SCL/tal-1. Development 127, 2447–2459 (2000).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We would like to thank members of the Keller laboratory for discussions and critical reading of the manuscript, Stefan Irion (McEwen Center for Regenerative Medicine) for providing the Bry-GFP/Ainv ES cell line, Kitajewski for providing the activated form of Notch4 cDNA (int-3)27 tagged with hemagglutinin (HA) sequence, M. Kyba (Lillehei Heart Institute, University of Minnesota) for the tet-on inducible ES cell line, Ainv18 and Zuniga-Pflucker (University of Toronto, Sunnybrook Research Institute) for the OP9-DL1 cell line. This work was supported by National Institutes of Health grants R01 HL71800, R01 HL 48834.

Author information

Authors and Affiliations

Authors

Contributions

V.C.C. and G.K. conceived the experiments and V.C.C. designed experimental details. R.S. and D.J. performed microarray analyses. X.C. generated the Notch1-inducible ES cell line and participated in experimental design. V.C.C. performed all remaining experiments. The manuscript was written by V.C.C. and G.K.

Corresponding author

Correspondence to Gordon Keller.

Supplementary information

Supplementary Text and Figures

Figures 1–4 (PDF 173 kb)

Supplementary Video 1

Day 3.25 Bry-GFP+/Flk-1+ cell reaggregated 24 hours in the presence of Dox and plated in the cardiac cultures (Original magnification 40x; QuickTime movie; 2.0 MB). (MOV 1991 kb)

Supplementary Video 2

Day 3.25 Bry-GFP+/Flk-1+ cells reaggregated in the absence of Dox and plated in the cardiac cultures (Original magnification 40x; QuickTime movie; 2.0 MB) (MOV 2049 kb)

Supplementary Video 3

Compact colony with contracting cells generated in the blast colony assay in the presence of Dox (Original magnification 200x; QuickTime movie; 0.13 MB) (MOV 133 kb)

Supplementary Video 4

Mixed lineage colony with contracting core surrounded by red outer cells (Original magnification 200x; QuickTime movie; 0.19 MB) (MOV 189 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chen, V., Stull, R., Joo, D. et al. Notch signaling respecifies the hemangioblast to a cardiac fate. Nat Biotechnol 26, 1169–1178 (2008). https://doi.org/10.1038/nbt.1497

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.1497

This article is cited by

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