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A regulatory pathway involving Notch1/beta-catenin/Isl1 determines cardiac progenitor cell fate.

Kwon C, Qian L, Cheng P, Nigam V, Arnold J, Srivastava D - Nat. Cell Biol. (2009)

Bottom Line: Surprisingly, disruption of Isl1, normally expressed transiently in CPCs before their differentiation, resulted in expansion of CPCs in vivo and in an embryonic stem (ES) cell system.Furthermore, Isl1 was required for CPC differentiation into cardiomyocyte and smooth muscle cells, but not endothelial cells.These findings reveal a regulatory network controlling CPC expansion and cell fate that involves unanticipated functions of beta-catenin, Notch1 and Isl1 that may be leveraged for regenerative approaches involving CPCs.

View Article: PubMed Central - PubMed

Affiliation: Gladstone Institute of Cardiovascular Disease and Departments of Pediatrics and Biochemistry & Biophysics, University of California, San Francisco, 1650 Owens Street, San Francisco, CA 94158, USA. ckwon@gladstone.ucsf.edu

ABSTRACT
Regulation of multipotent cardiac progenitor cell (CPC) expansion and subsequent differentiation into cardiomyocytes, smooth muscle or endothelial cells is a fundamental aspect of basic cardiovascular biology and cardiac regenerative medicine. However, the mechanisms governing these decisions remain unclear. Here, we show that Wnt/beta-catenin signalling, which promotes expansion of CPCs, is negatively regulated by Notch1-mediated control of phosphorylated beta-catenin accumulation within CPCs, and that Notch1 activity in CPCs is required for their differentiation. Notch1 positively, and beta-catenin negatively, regulated expression of the cardiac transcription factors, Isl1, Myocd and Smyd1. Surprisingly, disruption of Isl1, normally expressed transiently in CPCs before their differentiation, resulted in expansion of CPCs in vivo and in an embryonic stem (ES) cell system. Furthermore, Isl1 was required for CPC differentiation into cardiomyocyte and smooth muscle cells, but not endothelial cells. These findings reveal a regulatory network controlling CPC expansion and cell fate that involves unanticipated functions of beta-catenin, Notch1 and Isl1 that may be leveraged for regenerative approaches involving CPCs.

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Notch1 loss-of-function causes CPC expansion and increases free β-Catenin levels. a–f, Control embryos. g–l,Isl1Cre, Notch1flox/flox embryos (N1-KO). a,g, Lateral views of ED10.5 embryos. b, c, h, i, Lateral (b,h) or frontal (c,i) view of embryos focused on cardiac regions showing absence of right ventricle (rv) in mutants. d,e, j, k, Transverse sections (H&E) of embryos (d, j) with enlargement of boxed areas (e, k) showing hyperplasia of precardiac progenitors (asterisk). f, l, Phosphohistone3 (Ph3, red) and Isl1 (green) immunostaining of transverse sections through the precardiac region. To compensate for the severe downregulation of Isl1 in Notch1 mutant embryos, Isl1 signals were amplified with the TSA system. DAPI (blue) was used to counterstain the nuclei. m, Percentage of ph3-positive cells in precardiac mesoderm region shown in e and k (mean ± s. d.; n=4; *P < 0.01). n, Western analyses of FACS-purified CPCs transfected with control siRNA (C) or Notch1 siRNA (N1-KD) using Notch1, free or total β-Catenin antibodies. Free β-Catenin antibodies detect dephosphorylated β-Catenin, the effector molecule of the Wnt/β-Catenin signaling pathway. GAPDH antibody was used as a control. o, Relative number of cells on the 2nd day after transfecting CPCs with control or Notch1 siRNA (mean ± s. d.; n=6; *P < 0.01). p, Top/Fop flash activity in CPCs transfected with indicated siRNA. Top flash is a luciferase reporter with Tcf binding sites to read Wnt/β-Catenin signaling activity. Fop flash contains mutated Tcf binding sites. Luciferase values were normalized to Renilla activity (mean ± s. d.; n=3; *P < 0.01). h, heart; pa, pharyngeal arch; ot, outflow tract; lv, left ventricle. Scale bars, 250 µm (a, g) or 100 µm (b–e, h–k).
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Figure 1: Notch1 loss-of-function causes CPC expansion and increases free β-Catenin levels. a–f, Control embryos. g–l,Isl1Cre, Notch1flox/flox embryos (N1-KO). a,g, Lateral views of ED10.5 embryos. b, c, h, i, Lateral (b,h) or frontal (c,i) view of embryos focused on cardiac regions showing absence of right ventricle (rv) in mutants. d,e, j, k, Transverse sections (H&E) of embryos (d, j) with enlargement of boxed areas (e, k) showing hyperplasia of precardiac progenitors (asterisk). f, l, Phosphohistone3 (Ph3, red) and Isl1 (green) immunostaining of transverse sections through the precardiac region. To compensate for the severe downregulation of Isl1 in Notch1 mutant embryos, Isl1 signals were amplified with the TSA system. DAPI (blue) was used to counterstain the nuclei. m, Percentage of ph3-positive cells in precardiac mesoderm region shown in e and k (mean ± s. d.; n=4; *P < 0.01). n, Western analyses of FACS-purified CPCs transfected with control siRNA (C) or Notch1 siRNA (N1-KD) using Notch1, free or total β-Catenin antibodies. Free β-Catenin antibodies detect dephosphorylated β-Catenin, the effector molecule of the Wnt/β-Catenin signaling pathway. GAPDH antibody was used as a control. o, Relative number of cells on the 2nd day after transfecting CPCs with control or Notch1 siRNA (mean ± s. d.; n=6; *P < 0.01). p, Top/Fop flash activity in CPCs transfected with indicated siRNA. Top flash is a luciferase reporter with Tcf binding sites to read Wnt/β-Catenin signaling activity. Fop flash contains mutated Tcf binding sites. Luciferase values were normalized to Renilla activity (mean ± s. d.; n=3; *P < 0.01). h, heart; pa, pharyngeal arch; ot, outflow tract; lv, left ventricle. Scale bars, 250 µm (a, g) or 100 µm (b–e, h–k).

Mentions: To examine the CPC-autonomous role of Notch1 signaling in vivo, we deleted Notch1 in precardiac mesodermal progenitors by crossing Notch1flox mice19 with mice containing Cre recombinase in the Isl1 locus (Isl1Cre)20, resulting in Cre-mediated recombination in early CPCs by E7.75. The resulting Notch1- embryos failed to populate the developing right ventricle segment, which is derived from Isl1+ CPCs (Fig. 1a–c, g–i). Strikingly, the affected Isl1+ CPC pool dorsal to the developing heart was expanded with an increased percentage of proliferating cells marked by a phosphohistone H3 (PH3) antibody (Fig. 1d–f, j–m). The accumulation and proliferation of CPCs behind the developing heart was similar to the effect of stabilized β-Catenin on CPCs2, although in the latter CPCs also migrated into the heart.


A regulatory pathway involving Notch1/beta-catenin/Isl1 determines cardiac progenitor cell fate.

Kwon C, Qian L, Cheng P, Nigam V, Arnold J, Srivastava D - Nat. Cell Biol. (2009)

Notch1 loss-of-function causes CPC expansion and increases free β-Catenin levels. a–f, Control embryos. g–l,Isl1Cre, Notch1flox/flox embryos (N1-KO). a,g, Lateral views of ED10.5 embryos. b, c, h, i, Lateral (b,h) or frontal (c,i) view of embryos focused on cardiac regions showing absence of right ventricle (rv) in mutants. d,e, j, k, Transverse sections (H&E) of embryos (d, j) with enlargement of boxed areas (e, k) showing hyperplasia of precardiac progenitors (asterisk). f, l, Phosphohistone3 (Ph3, red) and Isl1 (green) immunostaining of transverse sections through the precardiac region. To compensate for the severe downregulation of Isl1 in Notch1 mutant embryos, Isl1 signals were amplified with the TSA system. DAPI (blue) was used to counterstain the nuclei. m, Percentage of ph3-positive cells in precardiac mesoderm region shown in e and k (mean ± s. d.; n=4; *P < 0.01). n, Western analyses of FACS-purified CPCs transfected with control siRNA (C) or Notch1 siRNA (N1-KD) using Notch1, free or total β-Catenin antibodies. Free β-Catenin antibodies detect dephosphorylated β-Catenin, the effector molecule of the Wnt/β-Catenin signaling pathway. GAPDH antibody was used as a control. o, Relative number of cells on the 2nd day after transfecting CPCs with control or Notch1 siRNA (mean ± s. d.; n=6; *P < 0.01). p, Top/Fop flash activity in CPCs transfected with indicated siRNA. Top flash is a luciferase reporter with Tcf binding sites to read Wnt/β-Catenin signaling activity. Fop flash contains mutated Tcf binding sites. Luciferase values were normalized to Renilla activity (mean ± s. d.; n=3; *P < 0.01). h, heart; pa, pharyngeal arch; ot, outflow tract; lv, left ventricle. Scale bars, 250 µm (a, g) or 100 µm (b–e, h–k).
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Figure 1: Notch1 loss-of-function causes CPC expansion and increases free β-Catenin levels. a–f, Control embryos. g–l,Isl1Cre, Notch1flox/flox embryos (N1-KO). a,g, Lateral views of ED10.5 embryos. b, c, h, i, Lateral (b,h) or frontal (c,i) view of embryos focused on cardiac regions showing absence of right ventricle (rv) in mutants. d,e, j, k, Transverse sections (H&E) of embryos (d, j) with enlargement of boxed areas (e, k) showing hyperplasia of precardiac progenitors (asterisk). f, l, Phosphohistone3 (Ph3, red) and Isl1 (green) immunostaining of transverse sections through the precardiac region. To compensate for the severe downregulation of Isl1 in Notch1 mutant embryos, Isl1 signals were amplified with the TSA system. DAPI (blue) was used to counterstain the nuclei. m, Percentage of ph3-positive cells in precardiac mesoderm region shown in e and k (mean ± s. d.; n=4; *P < 0.01). n, Western analyses of FACS-purified CPCs transfected with control siRNA (C) or Notch1 siRNA (N1-KD) using Notch1, free or total β-Catenin antibodies. Free β-Catenin antibodies detect dephosphorylated β-Catenin, the effector molecule of the Wnt/β-Catenin signaling pathway. GAPDH antibody was used as a control. o, Relative number of cells on the 2nd day after transfecting CPCs with control or Notch1 siRNA (mean ± s. d.; n=6; *P < 0.01). p, Top/Fop flash activity in CPCs transfected with indicated siRNA. Top flash is a luciferase reporter with Tcf binding sites to read Wnt/β-Catenin signaling activity. Fop flash contains mutated Tcf binding sites. Luciferase values were normalized to Renilla activity (mean ± s. d.; n=3; *P < 0.01). h, heart; pa, pharyngeal arch; ot, outflow tract; lv, left ventricle. Scale bars, 250 µm (a, g) or 100 µm (b–e, h–k).
Mentions: To examine the CPC-autonomous role of Notch1 signaling in vivo, we deleted Notch1 in precardiac mesodermal progenitors by crossing Notch1flox mice19 with mice containing Cre recombinase in the Isl1 locus (Isl1Cre)20, resulting in Cre-mediated recombination in early CPCs by E7.75. The resulting Notch1- embryos failed to populate the developing right ventricle segment, which is derived from Isl1+ CPCs (Fig. 1a–c, g–i). Strikingly, the affected Isl1+ CPC pool dorsal to the developing heart was expanded with an increased percentage of proliferating cells marked by a phosphohistone H3 (PH3) antibody (Fig. 1d–f, j–m). The accumulation and proliferation of CPCs behind the developing heart was similar to the effect of stabilized β-Catenin on CPCs2, although in the latter CPCs also migrated into the heart.

Bottom Line: Surprisingly, disruption of Isl1, normally expressed transiently in CPCs before their differentiation, resulted in expansion of CPCs in vivo and in an embryonic stem (ES) cell system.Furthermore, Isl1 was required for CPC differentiation into cardiomyocyte and smooth muscle cells, but not endothelial cells.These findings reveal a regulatory network controlling CPC expansion and cell fate that involves unanticipated functions of beta-catenin, Notch1 and Isl1 that may be leveraged for regenerative approaches involving CPCs.

View Article: PubMed Central - PubMed

Affiliation: Gladstone Institute of Cardiovascular Disease and Departments of Pediatrics and Biochemistry & Biophysics, University of California, San Francisco, 1650 Owens Street, San Francisco, CA 94158, USA. ckwon@gladstone.ucsf.edu

ABSTRACT
Regulation of multipotent cardiac progenitor cell (CPC) expansion and subsequent differentiation into cardiomyocytes, smooth muscle or endothelial cells is a fundamental aspect of basic cardiovascular biology and cardiac regenerative medicine. However, the mechanisms governing these decisions remain unclear. Here, we show that Wnt/beta-catenin signalling, which promotes expansion of CPCs, is negatively regulated by Notch1-mediated control of phosphorylated beta-catenin accumulation within CPCs, and that Notch1 activity in CPCs is required for their differentiation. Notch1 positively, and beta-catenin negatively, regulated expression of the cardiac transcription factors, Isl1, Myocd and Smyd1. Surprisingly, disruption of Isl1, normally expressed transiently in CPCs before their differentiation, resulted in expansion of CPCs in vivo and in an embryonic stem (ES) cell system. Furthermore, Isl1 was required for CPC differentiation into cardiomyocyte and smooth muscle cells, but not endothelial cells. These findings reveal a regulatory network controlling CPC expansion and cell fate that involves unanticipated functions of beta-catenin, Notch1 and Isl1 that may be leveraged for regenerative approaches involving CPCs.

Show MeSH
Related in: MedlinePlus