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N-cadherin prevents the premature differentiation of anterior heart field progenitors in the pharyngeal mesodermal microenvironment.

Soh BS, Buac K, Xu H, Li E, Ng SY, Wu H, Chmielowiec J, Jiang X, Bu L, Li RA, Cowan C, Chien KR - Cell Res. (2014)

Bottom Line: The cardiac progenitor cells (CPCs) in the anterior heart field (AHF) are located in the pharyngeal mesoderm (PM), where they expand, migrate and eventually differentiate into major cell types found in the heart, including cardiomyocytes.Through in silico data mining, genetic loss-of-function studies, and in vivo genetic rescue studies, we identified N-cadherin and interaction with canonical Wnt signals as a critical component of the microenvironment that facilitates the expansion of AHF-CPCs in the PM.CPCs in N-cadherin mutant embryos were observed to be less proliferative and undergo premature differentiation in the PM.

View Article: PubMed Central - PubMed

Affiliation: 1] Cardiovascular Research Center, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA [2] Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Medical School, 7 Divinity Avenue, Cambridge, MA 02138, USA [3] Stem Cell & Regenerative Medicine Consortium, and the Department of Physiology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China [4] Department of Cell and Molecular Biology and Medicine, Karolinska Institute, 171 77 Stockholm, Sweden.

ABSTRACT
The cardiac progenitor cells (CPCs) in the anterior heart field (AHF) are located in the pharyngeal mesoderm (PM), where they expand, migrate and eventually differentiate into major cell types found in the heart, including cardiomyocytes. The mechanisms by which these progenitors are able to expand within the PM microenvironment without premature differentiation remain largely unknown. Through in silico data mining, genetic loss-of-function studies, and in vivo genetic rescue studies, we identified N-cadherin and interaction with canonical Wnt signals as a critical component of the microenvironment that facilitates the expansion of AHF-CPCs in the PM. CPCs in N-cadherin mutant embryos were observed to be less proliferative and undergo premature differentiation in the PM. Notably, the phenotype of N-cadherin deficiency could be partially rescued by activating Wnt signaling, suggesting a delicate functional interaction between the adhesion role of N-cadherin and Wnt signaling in the early PM microenvironment. This study suggests a new mechanism for the early renewal of AHF progenitors where N-cadherin provides additional adhesion for progenitor cells in the PM, thereby allowing Wnt paracrine signals to expand the cells without premature differentiation.

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N-cadherin expression in embryonic stem cell-derived AHF cardiac progenitors. (A) “Cell-cell adhesion” genes enriched in embryonic stem cell-derived cardiac progenitor cells identified through the analysis of the microarray dataset reported by Domian et al.20. (B) Quantitative PCR analysis of N-cadherin expression (Cdh2) in cardiac progenitor cells (CPCs) derived from day 6 embryoid bodies (EBs) compared to non-cardiac cells (control) isolated from the same EBs. Error bars indicate SD, n = 3 experiments, *P < 0.05, evaluated by Student's t-test. (C) Representative sagittal section of wild-type mouse embryo (E9.5) immunostained for E-cadherin (red). Enlarged image shows a lack of E-cadherin expression in the PM (indicated by white arrows). Nuclei were marked by DAPI. Scale bar, 200 μm. (D) Sagittal section of wild-type mouse embryo (E9.5) immunostained for N-cadherin (red) and Mef2c (green). N-cadherin expression increases anteriorly towards the outflow tract (OFT) (yellow arrows). Coincident with the increase in N-cadherin expression was an increase in Mef2c expression, an important myocardial cell differentiation marker. Nuclei were marked by DAPI. Scale bar, 200 μm. (E) Immunofluorescence on sagittal section of wild-type mouse embryo at E9.0. N-cadherin (red) is weakly expressed in the Isl1+ (green) CDCs in the AHF (enlarged image). Arrows indicate strong N-cadherin expression in differentiated cardiomyocytes within the heart tube. Section orientation is as shown: A, anterior; P, posterior; D, Dorsal; V, ventral. Scale bar, 20 μm. (F) N-cadherin expression in CPCs compared to cardiomyocytes (CMs). The analysis was based on microarray datasets reported by Domian et al.20.
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fig1: N-cadherin expression in embryonic stem cell-derived AHF cardiac progenitors. (A) “Cell-cell adhesion” genes enriched in embryonic stem cell-derived cardiac progenitor cells identified through the analysis of the microarray dataset reported by Domian et al.20. (B) Quantitative PCR analysis of N-cadherin expression (Cdh2) in cardiac progenitor cells (CPCs) derived from day 6 embryoid bodies (EBs) compared to non-cardiac cells (control) isolated from the same EBs. Error bars indicate SD, n = 3 experiments, *P < 0.05, evaluated by Student's t-test. (C) Representative sagittal section of wild-type mouse embryo (E9.5) immunostained for E-cadherin (red). Enlarged image shows a lack of E-cadherin expression in the PM (indicated by white arrows). Nuclei were marked by DAPI. Scale bar, 200 μm. (D) Sagittal section of wild-type mouse embryo (E9.5) immunostained for N-cadherin (red) and Mef2c (green). N-cadherin expression increases anteriorly towards the outflow tract (OFT) (yellow arrows). Coincident with the increase in N-cadherin expression was an increase in Mef2c expression, an important myocardial cell differentiation marker. Nuclei were marked by DAPI. Scale bar, 200 μm. (E) Immunofluorescence on sagittal section of wild-type mouse embryo at E9.0. N-cadherin (red) is weakly expressed in the Isl1+ (green) CDCs in the AHF (enlarged image). Arrows indicate strong N-cadherin expression in differentiated cardiomyocytes within the heart tube. Section orientation is as shown: A, anterior; P, posterior; D, Dorsal; V, ventral. Scale bar, 20 μm. (F) N-cadherin expression in CPCs compared to cardiomyocytes (CMs). The analysis was based on microarray datasets reported by Domian et al.20.

Mentions: The AHF progenitors reside within the PM before they migrate to the elongating heart tube. In an attempt to identify the components of the microenvironment of the Isl1+ CPCs in the AHF, we analyzed microarray data containing the transcriptional profiles of CPCs derived from mouse ES cells. The generation of the microarray dataset has been previously described20. Focusing on genes categorized under the gene ontology term “cell-cell adhesion” (GO: 160037), and comparing their expressions in the Isl1+ CPCs to those in non-cardiac cells, Cdh2 was found to be among the top genes that were significantly enriched in the CPCs (Figure 1A and 1B). The gene Cdh2 encodes for N-cadherin, a Ca2+-dependent adhesion molecule. N-cadherin has been implicated in epithelial-mesenchymal transition (EMT), a process involved in many developmental and pathological events, including the formation of splanchnic mesoderm from primitive streaks21.


N-cadherin prevents the premature differentiation of anterior heart field progenitors in the pharyngeal mesodermal microenvironment.

Soh BS, Buac K, Xu H, Li E, Ng SY, Wu H, Chmielowiec J, Jiang X, Bu L, Li RA, Cowan C, Chien KR - Cell Res. (2014)

N-cadherin expression in embryonic stem cell-derived AHF cardiac progenitors. (A) “Cell-cell adhesion” genes enriched in embryonic stem cell-derived cardiac progenitor cells identified through the analysis of the microarray dataset reported by Domian et al.20. (B) Quantitative PCR analysis of N-cadherin expression (Cdh2) in cardiac progenitor cells (CPCs) derived from day 6 embryoid bodies (EBs) compared to non-cardiac cells (control) isolated from the same EBs. Error bars indicate SD, n = 3 experiments, *P < 0.05, evaluated by Student's t-test. (C) Representative sagittal section of wild-type mouse embryo (E9.5) immunostained for E-cadherin (red). Enlarged image shows a lack of E-cadherin expression in the PM (indicated by white arrows). Nuclei were marked by DAPI. Scale bar, 200 μm. (D) Sagittal section of wild-type mouse embryo (E9.5) immunostained for N-cadherin (red) and Mef2c (green). N-cadherin expression increases anteriorly towards the outflow tract (OFT) (yellow arrows). Coincident with the increase in N-cadherin expression was an increase in Mef2c expression, an important myocardial cell differentiation marker. Nuclei were marked by DAPI. Scale bar, 200 μm. (E) Immunofluorescence on sagittal section of wild-type mouse embryo at E9.0. N-cadherin (red) is weakly expressed in the Isl1+ (green) CDCs in the AHF (enlarged image). Arrows indicate strong N-cadherin expression in differentiated cardiomyocytes within the heart tube. Section orientation is as shown: A, anterior; P, posterior; D, Dorsal; V, ventral. Scale bar, 20 μm. (F) N-cadherin expression in CPCs compared to cardiomyocytes (CMs). The analysis was based on microarray datasets reported by Domian et al.20.
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fig1: N-cadherin expression in embryonic stem cell-derived AHF cardiac progenitors. (A) “Cell-cell adhesion” genes enriched in embryonic stem cell-derived cardiac progenitor cells identified through the analysis of the microarray dataset reported by Domian et al.20. (B) Quantitative PCR analysis of N-cadherin expression (Cdh2) in cardiac progenitor cells (CPCs) derived from day 6 embryoid bodies (EBs) compared to non-cardiac cells (control) isolated from the same EBs. Error bars indicate SD, n = 3 experiments, *P < 0.05, evaluated by Student's t-test. (C) Representative sagittal section of wild-type mouse embryo (E9.5) immunostained for E-cadherin (red). Enlarged image shows a lack of E-cadherin expression in the PM (indicated by white arrows). Nuclei were marked by DAPI. Scale bar, 200 μm. (D) Sagittal section of wild-type mouse embryo (E9.5) immunostained for N-cadherin (red) and Mef2c (green). N-cadherin expression increases anteriorly towards the outflow tract (OFT) (yellow arrows). Coincident with the increase in N-cadherin expression was an increase in Mef2c expression, an important myocardial cell differentiation marker. Nuclei were marked by DAPI. Scale bar, 200 μm. (E) Immunofluorescence on sagittal section of wild-type mouse embryo at E9.0. N-cadherin (red) is weakly expressed in the Isl1+ (green) CDCs in the AHF (enlarged image). Arrows indicate strong N-cadherin expression in differentiated cardiomyocytes within the heart tube. Section orientation is as shown: A, anterior; P, posterior; D, Dorsal; V, ventral. Scale bar, 20 μm. (F) N-cadherin expression in CPCs compared to cardiomyocytes (CMs). The analysis was based on microarray datasets reported by Domian et al.20.
Mentions: The AHF progenitors reside within the PM before they migrate to the elongating heart tube. In an attempt to identify the components of the microenvironment of the Isl1+ CPCs in the AHF, we analyzed microarray data containing the transcriptional profiles of CPCs derived from mouse ES cells. The generation of the microarray dataset has been previously described20. Focusing on genes categorized under the gene ontology term “cell-cell adhesion” (GO: 160037), and comparing their expressions in the Isl1+ CPCs to those in non-cardiac cells, Cdh2 was found to be among the top genes that were significantly enriched in the CPCs (Figure 1A and 1B). The gene Cdh2 encodes for N-cadherin, a Ca2+-dependent adhesion molecule. N-cadherin has been implicated in epithelial-mesenchymal transition (EMT), a process involved in many developmental and pathological events, including the formation of splanchnic mesoderm from primitive streaks21.

Bottom Line: The cardiac progenitor cells (CPCs) in the anterior heart field (AHF) are located in the pharyngeal mesoderm (PM), where they expand, migrate and eventually differentiate into major cell types found in the heart, including cardiomyocytes.Through in silico data mining, genetic loss-of-function studies, and in vivo genetic rescue studies, we identified N-cadherin and interaction with canonical Wnt signals as a critical component of the microenvironment that facilitates the expansion of AHF-CPCs in the PM.CPCs in N-cadherin mutant embryos were observed to be less proliferative and undergo premature differentiation in the PM.

View Article: PubMed Central - PubMed

Affiliation: 1] Cardiovascular Research Center, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA [2] Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Medical School, 7 Divinity Avenue, Cambridge, MA 02138, USA [3] Stem Cell & Regenerative Medicine Consortium, and the Department of Physiology, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China [4] Department of Cell and Molecular Biology and Medicine, Karolinska Institute, 171 77 Stockholm, Sweden.

ABSTRACT
The cardiac progenitor cells (CPCs) in the anterior heart field (AHF) are located in the pharyngeal mesoderm (PM), where they expand, migrate and eventually differentiate into major cell types found in the heart, including cardiomyocytes. The mechanisms by which these progenitors are able to expand within the PM microenvironment without premature differentiation remain largely unknown. Through in silico data mining, genetic loss-of-function studies, and in vivo genetic rescue studies, we identified N-cadherin and interaction with canonical Wnt signals as a critical component of the microenvironment that facilitates the expansion of AHF-CPCs in the PM. CPCs in N-cadherin mutant embryos were observed to be less proliferative and undergo premature differentiation in the PM. Notably, the phenotype of N-cadherin deficiency could be partially rescued by activating Wnt signaling, suggesting a delicate functional interaction between the adhesion role of N-cadherin and Wnt signaling in the early PM microenvironment. This study suggests a new mechanism for the early renewal of AHF progenitors where N-cadherin provides additional adhesion for progenitor cells in the PM, thereby allowing Wnt paracrine signals to expand the cells without premature differentiation.

Show MeSH
Related in: MedlinePlus