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.
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Gene Expression Omnibus
Murry, C.E. & Keller, G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell 132, 661–680 (2008).
Parameswaran, M. & Tam, P.P. Regionalisation of cell fate and morphogenetic movement of the mesoderm during mouse gastrulation. Dev. Genet. 17, 16–28 (1995).
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).
Choi, K., Kennedy, M., Kazarov, A., Papadimitriou, J.C. & Keller, G. A common precursor for hematopoietic and endothelial cells. Development 125, 725–732 (1998).
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).
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).
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).
Moretti, A. et al. Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification. Cell 127, 1151–1165 (2006).
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).
Yang, L. et al. Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population. Nature 453, 524–528 (2008).
Fehling, H.J. et al. Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation. Development 130, 4217–4227 (2003).
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).
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).
Schultheiss, T., Burch, J. & Lassar, A. A role for bone morphogenetic proteins in the induction of cardiac myogenesis. Genes Dev. 11, 451–462 (1997).
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).
Zhang, H. & Bradley, A. Mice deficient for BMP2 are nonviable and have defects in amnion/chorion and cardiac development. Development 122, 2977–2986 (1996).
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).
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).
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).
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).
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).
Schneider, V.A. & Mercola, M. Wnt antagonism initiates cardiogenesis in Xenopus laevis . Genes Dev. 15, 304–315 (2001).
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).
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).
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).
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).
Uyttendaele, H. et al. Notch4/int-3, a mammary proto-oncogene, is an endothelial cell-specific mammalian Notch gene. Development 122, 2251–2259 (1996).
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).
Joutel, A. et al. The ectodomain of the Notch3 receptor accumulates within the cerebrovasculature of CADASIL patients. J. Clin. Invest. 105, 597–605 (2000).
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).
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).
Das, I. et al. Notch oncoproteins depend on gamma-secretase/presenilin activity for processing and function. J. Biol. Chem. 279, 30771–30780 (2004).
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).
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).
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).
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).
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).
Alexandrovich, A. et al. Wnt2 is a direct downstream target of GATA6 during early cardiogenesis. Mech. Dev. 123, 297–311 (2006).
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).
Shawber, C. et al. Notch signaling inhibits muscle cell differentiation through a CBF1-independent pathway. Development 122, 3765–3773 (1996).
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).
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).
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).
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).
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).
Dahlqvist, C. et al. Functional Notch signaling is required for BMP4-induced inhibition of myogenic differentiation. Development 130, 6089–6099 (2003).
Sun, Y. et al. Notch4 intracellular domain binding to Smad3 and inhibition of the TGF-beta signaling. Oncogene 24, 5365–5374 (2005).
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).
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).
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.
Figures 1–4 (PDF 173 kb)
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)
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)
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)
Mixed lineage colony with contracting core surrounded by red outer cells (Original magnification 200x; QuickTime movie; 0.19 MB) (MOV 189 kb)
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
Re-enforcing hypoxia-induced polyploid cardiomyocytes enter cytokinesis through activation of β-catenin
Scientific Reports (2019)
Nature Reviews Cardiology (2018)
Stem Cell-Derived Exosomes, Autophagy, Extracellular Matrix Turnover, and miRNAs in Cardiac Regeneration during Stem Cell Therapy
Stem Cell Reviews and Reports (2017)
Basic Research in Cardiology (2015)
Nature Communications (2013)