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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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Increased levels of Isl1 promote myocardial differentiation. a, Schematic diagram of differentiation of Myh7-GFP ES cells with Isl1 overexpression. b, c, Relative expression levels of Isl1 on ED6 EBs (b), and endothelial (Flk1), cardiac sarcomeric (Actc1, Mlc2v, Myh7) and smooth muscle (Sma) genes on day 8 EBs (c), determined by qPCR. d, FACS analyses on ED 9 EBs to identify % of cells entering myocardial-lineage. Data are mean ± s. e. m.; n=3; *P < 0.005.
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Figure 4: Increased levels of Isl1 promote myocardial differentiation. a, Schematic diagram of differentiation of Myh7-GFP ES cells with Isl1 overexpression. b, c, Relative expression levels of Isl1 on ED6 EBs (b), and endothelial (Flk1), cardiac sarcomeric (Actc1, Mlc2v, Myh7) and smooth muscle (Sma) genes on day 8 EBs (c), determined by qPCR. d, FACS analyses on ED 9 EBs to identify % of cells entering myocardial-lineage. Data are mean ± s. e. m.; n=3; *P < 0.005.

Mentions: Given that Isl1 loss-of-function suppressed cardiomyocyte differentiation, we sought to determine if Isl1 conversely plays an instructive role in myocardial lineage formation. Isl1 expression levels were upregulated from ED4–5 EBs (Supp. fig. 3a). To prematurely increase Isl1 expression levels in a temporally and physiologically relevant way, we transiently transfected an Isl1 expression construct (30 ng/105 cells) into dissociated ED2 EB cells and re-aggregated them for further differentiation (Fig. 4a). This resulted in about a twofold increase in Isl1 levels at ED6 (Fig. 4b). Myocardial differentiation was monitored by sarcomeric gene (e.g., Myh7, Mlc2v, Actc1) expression over the course of EB differentiation. Sarcomeric gene expression levels did not change during the early phase of CPC differentiation (data not shown). However, by ED8, Isl1-transfected EBs expressed higher levels of cardiac muscle genes than control EBs (Fig. 4c). To determine the effect of excess Isl1 on the number of cardiomyocytes, we utilized the Myh7-GFP ES cell line to quantify cardiomyocytes. We observed a 25% increase in Myh7+ cells in Isl1-overexpressed EBs (Fig. 4d, Supp. fig. 3b). This suggests that Isl1 can promote myocardial differentiation of CPCs in an instructive manner.


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)

Increased levels of Isl1 promote myocardial differentiation. a, Schematic diagram of differentiation of Myh7-GFP ES cells with Isl1 overexpression. b, c, Relative expression levels of Isl1 on ED6 EBs (b), and endothelial (Flk1), cardiac sarcomeric (Actc1, Mlc2v, Myh7) and smooth muscle (Sma) genes on day 8 EBs (c), determined by qPCR. d, FACS analyses on ED 9 EBs to identify % of cells entering myocardial-lineage. Data are mean ± s. e. m.; n=3; *P < 0.005.
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Figure 4: Increased levels of Isl1 promote myocardial differentiation. a, Schematic diagram of differentiation of Myh7-GFP ES cells with Isl1 overexpression. b, c, Relative expression levels of Isl1 on ED6 EBs (b), and endothelial (Flk1), cardiac sarcomeric (Actc1, Mlc2v, Myh7) and smooth muscle (Sma) genes on day 8 EBs (c), determined by qPCR. d, FACS analyses on ED 9 EBs to identify % of cells entering myocardial-lineage. Data are mean ± s. e. m.; n=3; *P < 0.005.
Mentions: Given that Isl1 loss-of-function suppressed cardiomyocyte differentiation, we sought to determine if Isl1 conversely plays an instructive role in myocardial lineage formation. Isl1 expression levels were upregulated from ED4–5 EBs (Supp. fig. 3a). To prematurely increase Isl1 expression levels in a temporally and physiologically relevant way, we transiently transfected an Isl1 expression construct (30 ng/105 cells) into dissociated ED2 EB cells and re-aggregated them for further differentiation (Fig. 4a). This resulted in about a twofold increase in Isl1 levels at ED6 (Fig. 4b). Myocardial differentiation was monitored by sarcomeric gene (e.g., Myh7, Mlc2v, Actc1) expression over the course of EB differentiation. Sarcomeric gene expression levels did not change during the early phase of CPC differentiation (data not shown). However, by ED8, Isl1-transfected EBs expressed higher levels of cardiac muscle genes than control EBs (Fig. 4c). To determine the effect of excess Isl1 on the number of cardiomyocytes, we utilized the Myh7-GFP ES cell line to quantify cardiomyocytes. We observed a 25% increase in Myh7+ cells in Isl1-overexpressed EBs (Fig. 4d, Supp. fig. 3b). This suggests that Isl1 can promote myocardial differentiation of CPCs in an instructive manner.

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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