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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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Loss of N-cadherin impairs proliferation and induces premature differentiation of AHF progenitor cells. (A) Representative sagittal section of control and mutant embryos at E9.5 immunostained for Isl1 (green) and phospho-Histone 3 (red); arrows point to Isl1/PiH3 double-positive cells in the OFT and AHF (enlarged image). Nuclei are marked by DAPI. Scale bar, 200 μm. (B) Quantification of cell proliferation indicated by mitotic index in the anterior heart field and neural tube regions of wild-type and mutants at E9.5. Error bars indicate SD, n = 4 experiments, **P = 0.01, evaluated by Student's t-test. (C) Representative transverse sections of control and mutant embryos at E9.0 immunostained for Isl1 (green) and cleaved Caspase 3 (red) in outflow tract (OFT) and neural tube (NT). Nuclei are marked by DAPI. Scale bar, 200 μm. (D) Sagittal sections of control and mutant embryos at E9.0 immunostained for cardiac troponin T (cTnT) (red). Note the presence of ectopic cTnT+ cells (arrows) in the AHF. Nuclei are marked by DAPI. Scale bar, 200 μm. (E) Quantification of cTnT+ cells in the AHF of the control and mutant embryos at E9.0. cTnT+ cells within the AHF, the region between OFT and anterior portion of pharyngeal mesoderm were counted. A total of 7-9 serial sections for each embryo were used. Error bars indicate SD, n = 6 embryos. ***P = 0.001, evaluated by Student's t-test.
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fig3: Loss of N-cadherin impairs proliferation and induces premature differentiation of AHF progenitor cells. (A) Representative sagittal section of control and mutant embryos at E9.5 immunostained for Isl1 (green) and phospho-Histone 3 (red); arrows point to Isl1/PiH3 double-positive cells in the OFT and AHF (enlarged image). Nuclei are marked by DAPI. Scale bar, 200 μm. (B) Quantification of cell proliferation indicated by mitotic index in the anterior heart field and neural tube regions of wild-type and mutants at E9.5. Error bars indicate SD, n = 4 experiments, **P = 0.01, evaluated by Student's t-test. (C) Representative transverse sections of control and mutant embryos at E9.0 immunostained for Isl1 (green) and cleaved Caspase 3 (red) in outflow tract (OFT) and neural tube (NT). Nuclei are marked by DAPI. Scale bar, 200 μm. (D) Sagittal sections of control and mutant embryos at E9.0 immunostained for cardiac troponin T (cTnT) (red). Note the presence of ectopic cTnT+ cells (arrows) in the AHF. Nuclei are marked by DAPI. Scale bar, 200 μm. (E) Quantification of cTnT+ cells in the AHF of the control and mutant embryos at E9.0. cTnT+ cells within the AHF, the region between OFT and anterior portion of pharyngeal mesoderm were counted. A total of 7-9 serial sections for each embryo were used. Error bars indicate SD, n = 6 embryos. ***P = 0.001, evaluated by Student's t-test.

Mentions: We reasoned that the decreased number of CPCs could be due to disrupted proliferation or augmented apoptosis as a result of N-cadherin deletion. To test this, immunostaining for phosphorylated histone 3 (PiH3), a mitotic marker, was performed to assess the proliferation of the Isl1+ CPCs in E9.5 embryos. We found that the mitotic index of the CPCs (Isl1 and PiH3 dual-positive cells) in the mutant embryos (AHF-Cre; Cdh2fl/fl) was significantly lower than that in the control embryos, although the mitotic index in the neuroepithelial cells of brain remained the same for both the wild-type and mutant embryos (Figure 3A and 3B), indicating that the decrease in proliferation was specific to the Isl+ cells within AHF.


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)

Loss of N-cadherin impairs proliferation and induces premature differentiation of AHF progenitor cells. (A) Representative sagittal section of control and mutant embryos at E9.5 immunostained for Isl1 (green) and phospho-Histone 3 (red); arrows point to Isl1/PiH3 double-positive cells in the OFT and AHF (enlarged image). Nuclei are marked by DAPI. Scale bar, 200 μm. (B) Quantification of cell proliferation indicated by mitotic index in the anterior heart field and neural tube regions of wild-type and mutants at E9.5. Error bars indicate SD, n = 4 experiments, **P = 0.01, evaluated by Student's t-test. (C) Representative transverse sections of control and mutant embryos at E9.0 immunostained for Isl1 (green) and cleaved Caspase 3 (red) in outflow tract (OFT) and neural tube (NT). Nuclei are marked by DAPI. Scale bar, 200 μm. (D) Sagittal sections of control and mutant embryos at E9.0 immunostained for cardiac troponin T (cTnT) (red). Note the presence of ectopic cTnT+ cells (arrows) in the AHF. Nuclei are marked by DAPI. Scale bar, 200 μm. (E) Quantification of cTnT+ cells in the AHF of the control and mutant embryos at E9.0. cTnT+ cells within the AHF, the region between OFT and anterior portion of pharyngeal mesoderm were counted. A total of 7-9 serial sections for each embryo were used. Error bars indicate SD, n = 6 embryos. ***P = 0.001, evaluated by Student's t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: Loss of N-cadherin impairs proliferation and induces premature differentiation of AHF progenitor cells. (A) Representative sagittal section of control and mutant embryos at E9.5 immunostained for Isl1 (green) and phospho-Histone 3 (red); arrows point to Isl1/PiH3 double-positive cells in the OFT and AHF (enlarged image). Nuclei are marked by DAPI. Scale bar, 200 μm. (B) Quantification of cell proliferation indicated by mitotic index in the anterior heart field and neural tube regions of wild-type and mutants at E9.5. Error bars indicate SD, n = 4 experiments, **P = 0.01, evaluated by Student's t-test. (C) Representative transverse sections of control and mutant embryos at E9.0 immunostained for Isl1 (green) and cleaved Caspase 3 (red) in outflow tract (OFT) and neural tube (NT). Nuclei are marked by DAPI. Scale bar, 200 μm. (D) Sagittal sections of control and mutant embryos at E9.0 immunostained for cardiac troponin T (cTnT) (red). Note the presence of ectopic cTnT+ cells (arrows) in the AHF. Nuclei are marked by DAPI. Scale bar, 200 μm. (E) Quantification of cTnT+ cells in the AHF of the control and mutant embryos at E9.0. cTnT+ cells within the AHF, the region between OFT and anterior portion of pharyngeal mesoderm were counted. A total of 7-9 serial sections for each embryo were used. Error bars indicate SD, n = 6 embryos. ***P = 0.001, evaluated by Student's t-test.
Mentions: We reasoned that the decreased number of CPCs could be due to disrupted proliferation or augmented apoptosis as a result of N-cadherin deletion. To test this, immunostaining for phosphorylated histone 3 (PiH3), a mitotic marker, was performed to assess the proliferation of the Isl1+ CPCs in E9.5 embryos. We found that the mitotic index of the CPCs (Isl1 and PiH3 dual-positive cells) in the mutant embryos (AHF-Cre; Cdh2fl/fl) was significantly lower than that in the control embryos, although the mitotic index in the neuroepithelial cells of brain remained the same for both the wild-type and mutant embryos (Figure 3A and 3B), indicating that the decrease in proliferation was specific to the Isl+ cells within AHF.

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