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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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Isl1 targets Myocd and β-Catenin regulates Bhlhb2 to repress Smyd1. a, Relative expression levels of Myocd and Smyd1 in FACS-purified control and Isl1 knockdown (KD) CPCs, determined by qPCR (mean ± s. d.; n=4; *P < 0.005). b–i, Control (b–e) and Isl1- (f–i) embryos at E 9.5 after in situ hybridization with Myocd (b–d, f–h), or Smyd1 (e, i) riboprobes. c, g, Lateral views focused on heart (h) and pharyngeal arch (pa) regions. d, h, Transverse section through the outfow tract. Asterisks indicate pre-cardiac mesoderm. Scale bars, 100 µm. j, Location of the conserved island containing Isl1 binding site (red) in the Myocd locus. k, Relative luciferase activity determined with luciferase reporters linked to the conserved island with the intact Isl1 site (Myocd-luc) or with a mutant Isl1 site (Myocd-lucmt) in the presence or absence of Isl1 (mean ± s. d.; n=3; *P < 0.005). l, Chromatin immunoprecipitation (ChIP) assay shows specific PCR amplification of the Isl1 consensus site shown in j, representing association with Isl1 protein. m, Electophoretic mobility shift assay with in-vitro synthesized Isl1 protein and radiolabeled probes (Probe) spanning the Isl1 site shown in j. Unlabeled probes were used as competitors. WT, wildtype; MT, mutant. n, Relative expression levels of Bhlhb2 in CPCs with stabilized β-Catenin, determined by qPCR (mean ±s. d.; n=3; *P < 0.005).. o, Relative expression levels of Smyd1 and Isl1 after transfecting FACS-purified CPCs with Bhlhb2 and differentiating them for 3 days (mean ± s. d.; n=3; *P < 0.005). p, The Bhlhb2 locus showing four conserved Lef/Tcf binding sites. q, ChIP assays performed with Lef/Tcf consensus sites shown in p. β-Catenin forms complexes with sites A and D as revealed by amplification of those sites. r, Relative luciferase activity determined with luciferase reporters containing the intact Lef/Tcf site D (Bhlhb2D-luc) or with a mutant Lef/Tcf site D (Bhlhb2D-lucmt) in the presence or absence of β-Catenin or BIO (2µM). Data are mean ± s. d.; n=3; *P < 0.005. s, A molecular cascade involving Notch1/ β-Catenin/ Isl1 during CPC fate determination. Notch1 functions to negatively regulate accumulation of free β-Catenin, which regulates Myocd and Smyd1 through Isl1 and Bhlhb2, respectively, to determine CPC fates. Relationships indicated may be direct or indirect.
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Figure 5: Isl1 targets Myocd and β-Catenin regulates Bhlhb2 to repress Smyd1. a, Relative expression levels of Myocd and Smyd1 in FACS-purified control and Isl1 knockdown (KD) CPCs, determined by qPCR (mean ± s. d.; n=4; *P < 0.005). b–i, Control (b–e) and Isl1- (f–i) embryos at E 9.5 after in situ hybridization with Myocd (b–d, f–h), or Smyd1 (e, i) riboprobes. c, g, Lateral views focused on heart (h) and pharyngeal arch (pa) regions. d, h, Transverse section through the outfow tract. Asterisks indicate pre-cardiac mesoderm. Scale bars, 100 µm. j, Location of the conserved island containing Isl1 binding site (red) in the Myocd locus. k, Relative luciferase activity determined with luciferase reporters linked to the conserved island with the intact Isl1 site (Myocd-luc) or with a mutant Isl1 site (Myocd-lucmt) in the presence or absence of Isl1 (mean ± s. d.; n=3; *P < 0.005). l, Chromatin immunoprecipitation (ChIP) assay shows specific PCR amplification of the Isl1 consensus site shown in j, representing association with Isl1 protein. m, Electophoretic mobility shift assay with in-vitro synthesized Isl1 protein and radiolabeled probes (Probe) spanning the Isl1 site shown in j. Unlabeled probes were used as competitors. WT, wildtype; MT, mutant. n, Relative expression levels of Bhlhb2 in CPCs with stabilized β-Catenin, determined by qPCR (mean ±s. d.; n=3; *P < 0.005).. o, Relative expression levels of Smyd1 and Isl1 after transfecting FACS-purified CPCs with Bhlhb2 and differentiating them for 3 days (mean ± s. d.; n=3; *P < 0.005). p, The Bhlhb2 locus showing four conserved Lef/Tcf binding sites. q, ChIP assays performed with Lef/Tcf consensus sites shown in p. β-Catenin forms complexes with sites A and D as revealed by amplification of those sites. r, Relative luciferase activity determined with luciferase reporters containing the intact Lef/Tcf site D (Bhlhb2D-luc) or with a mutant Lef/Tcf site D (Bhlhb2D-lucmt) in the presence or absence of β-Catenin or BIO (2µM). Data are mean ± s. d.; n=3; *P < 0.005. s, A molecular cascade involving Notch1/ β-Catenin/ Isl1 during CPC fate determination. Notch1 functions to negatively regulate accumulation of free β-Catenin, which regulates Myocd and Smyd1 through Isl1 and Bhlhb2, respectively, to determine CPC fates. Relationships indicated may be direct or indirect.

Mentions: In addition to Isl1, Myocd and Smyd1 are important genes for cardiogenesis23–27 that were downregulated in CPCs with increased β-Catenin (Fig. 2c–g, i–k). Myocd is a potent coactivator for serum response factor regulation of smooth muscle24 and cardiac gene expression27. Smyd1 is a muscle-restricted histone methyltransferase essential for cardiomyocyte differentiation in vivo23, 25. To determine whether Isl1 regulates these genes in CPCs, we used Nkx2.5-GFP+ CPCs purified from the stable Isl1-KD ES cell line. Smyd1 levels did not change, but Myocd levels were significantly reduced in the Isl1-KD CPCs (Fig. 5a). To determine if this is also the case in vivo, we performed in situ hybridization for Myocd transcripts in Isl1- embryos. In agreement with in vitro data, Myocd levels were severely compromised in Isl1- embryos, while Smyd1 levels did not change (Fig. 5b–i). This suggests that Isl1 is required for normal Myocd expression.


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)

Isl1 targets Myocd and β-Catenin regulates Bhlhb2 to repress Smyd1. a, Relative expression levels of Myocd and Smyd1 in FACS-purified control and Isl1 knockdown (KD) CPCs, determined by qPCR (mean ± s. d.; n=4; *P < 0.005). b–i, Control (b–e) and Isl1- (f–i) embryos at E 9.5 after in situ hybridization with Myocd (b–d, f–h), or Smyd1 (e, i) riboprobes. c, g, Lateral views focused on heart (h) and pharyngeal arch (pa) regions. d, h, Transverse section through the outfow tract. Asterisks indicate pre-cardiac mesoderm. Scale bars, 100 µm. j, Location of the conserved island containing Isl1 binding site (red) in the Myocd locus. k, Relative luciferase activity determined with luciferase reporters linked to the conserved island with the intact Isl1 site (Myocd-luc) or with a mutant Isl1 site (Myocd-lucmt) in the presence or absence of Isl1 (mean ± s. d.; n=3; *P < 0.005). l, Chromatin immunoprecipitation (ChIP) assay shows specific PCR amplification of the Isl1 consensus site shown in j, representing association with Isl1 protein. m, Electophoretic mobility shift assay with in-vitro synthesized Isl1 protein and radiolabeled probes (Probe) spanning the Isl1 site shown in j. Unlabeled probes were used as competitors. WT, wildtype; MT, mutant. n, Relative expression levels of Bhlhb2 in CPCs with stabilized β-Catenin, determined by qPCR (mean ±s. d.; n=3; *P < 0.005).. o, Relative expression levels of Smyd1 and Isl1 after transfecting FACS-purified CPCs with Bhlhb2 and differentiating them for 3 days (mean ± s. d.; n=3; *P < 0.005). p, The Bhlhb2 locus showing four conserved Lef/Tcf binding sites. q, ChIP assays performed with Lef/Tcf consensus sites shown in p. β-Catenin forms complexes with sites A and D as revealed by amplification of those sites. r, Relative luciferase activity determined with luciferase reporters containing the intact Lef/Tcf site D (Bhlhb2D-luc) or with a mutant Lef/Tcf site D (Bhlhb2D-lucmt) in the presence or absence of β-Catenin or BIO (2µM). Data are mean ± s. d.; n=3; *P < 0.005. s, A molecular cascade involving Notch1/ β-Catenin/ Isl1 during CPC fate determination. Notch1 functions to negatively regulate accumulation of free β-Catenin, which regulates Myocd and Smyd1 through Isl1 and Bhlhb2, respectively, to determine CPC fates. Relationships indicated may be direct or indirect.
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Related In: Results  -  Collection

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Figure 5: Isl1 targets Myocd and β-Catenin regulates Bhlhb2 to repress Smyd1. a, Relative expression levels of Myocd and Smyd1 in FACS-purified control and Isl1 knockdown (KD) CPCs, determined by qPCR (mean ± s. d.; n=4; *P < 0.005). b–i, Control (b–e) and Isl1- (f–i) embryos at E 9.5 after in situ hybridization with Myocd (b–d, f–h), or Smyd1 (e, i) riboprobes. c, g, Lateral views focused on heart (h) and pharyngeal arch (pa) regions. d, h, Transverse section through the outfow tract. Asterisks indicate pre-cardiac mesoderm. Scale bars, 100 µm. j, Location of the conserved island containing Isl1 binding site (red) in the Myocd locus. k, Relative luciferase activity determined with luciferase reporters linked to the conserved island with the intact Isl1 site (Myocd-luc) or with a mutant Isl1 site (Myocd-lucmt) in the presence or absence of Isl1 (mean ± s. d.; n=3; *P < 0.005). l, Chromatin immunoprecipitation (ChIP) assay shows specific PCR amplification of the Isl1 consensus site shown in j, representing association with Isl1 protein. m, Electophoretic mobility shift assay with in-vitro synthesized Isl1 protein and radiolabeled probes (Probe) spanning the Isl1 site shown in j. Unlabeled probes were used as competitors. WT, wildtype; MT, mutant. n, Relative expression levels of Bhlhb2 in CPCs with stabilized β-Catenin, determined by qPCR (mean ±s. d.; n=3; *P < 0.005).. o, Relative expression levels of Smyd1 and Isl1 after transfecting FACS-purified CPCs with Bhlhb2 and differentiating them for 3 days (mean ± s. d.; n=3; *P < 0.005). p, The Bhlhb2 locus showing four conserved Lef/Tcf binding sites. q, ChIP assays performed with Lef/Tcf consensus sites shown in p. β-Catenin forms complexes with sites A and D as revealed by amplification of those sites. r, Relative luciferase activity determined with luciferase reporters containing the intact Lef/Tcf site D (Bhlhb2D-luc) or with a mutant Lef/Tcf site D (Bhlhb2D-lucmt) in the presence or absence of β-Catenin or BIO (2µM). Data are mean ± s. d.; n=3; *P < 0.005. s, A molecular cascade involving Notch1/ β-Catenin/ Isl1 during CPC fate determination. Notch1 functions to negatively regulate accumulation of free β-Catenin, which regulates Myocd and Smyd1 through Isl1 and Bhlhb2, respectively, to determine CPC fates. Relationships indicated may be direct or indirect.
Mentions: In addition to Isl1, Myocd and Smyd1 are important genes for cardiogenesis23–27 that were downregulated in CPCs with increased β-Catenin (Fig. 2c–g, i–k). Myocd is a potent coactivator for serum response factor regulation of smooth muscle24 and cardiac gene expression27. Smyd1 is a muscle-restricted histone methyltransferase essential for cardiomyocyte differentiation in vivo23, 25. To determine whether Isl1 regulates these genes in CPCs, we used Nkx2.5-GFP+ CPCs purified from the stable Isl1-KD ES cell line. Smyd1 levels did not change, but Myocd levels were significantly reduced in the Isl1-KD CPCs (Fig. 5a). To determine if this is also the case in vivo, we performed in situ hybridization for Myocd transcripts in Isl1- embryos. In agreement with in vitro data, Myocd levels were severely compromised in Isl1- embryos, while Smyd1 levels did not change (Fig. 5b–i). This suggests that Isl1 is required for normal Myocd expression.

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