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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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Related in: MedlinePlus

Overexpression of Wnt signaling rescues Cdh2 mutant phenotype. (A) Representative traverse section showing Wnt signaling activity in AHF progenitors as revealed by TCL/LEF-lacZ mice. Black arrows denote cells that have active Wnt signaling within pharyngeal mesoderm, whereas red arrows denote cells with inactive Wnt signaling within heart tube. Scale bar, 500 μm. (B) Representative images of AHF-CPCs isolated from wild type (AHF-Cre; AHF-GFP; Cdh2fl/+) and mutant (AHF-Cre; AHF-GFP; Cdh2fl/fl) stained red for either nuclear β-catenin or membrane-bound β-catenin. The average mean intensities were tabulated from 60 fields. Nuclei are marked by DAPI. A cut-off value >10 was imposed to remove background. Scale bar, 50 μm. Error bars indicate SD, n = 3 replicates, **P < 0.001, evaluated by Student's t-test. (C) Quantitative PCR analysis of mRNA isolated from sorted progenitor cells (AHF-GFP+ CPCs) carrying AHF-GFP reporter transgene. The expression levels show that Wnt signaling ligands, receptors and effector genes (Axin2 and Lef1) were downregulated in Cdh2-deficient progenitor cells, while upregulation of differentiation genes such as Mlc2a and Tnnt2 were observed. Samples were normalized against the housekeeping gene, β-actin. Error bars indicate SD, n = 3 replicates. (D) Representative sagittal sections of single (AHF-Cre; Cdh2fl/+; bCat(e3)fl/+) and double (AHF-Cre; Cdh2fl/fl; bCat(e3)fl/+) mutants at E9.5. Sections stained for cTnT (red) and Isl1 (green) revealed similar expression patterns. Premature differentiation of Isl1+ CPCs into cTnT+ cells at the pharyngeal mesoderm was not observed in the double mutant as compared to single N-cadherin mutant (AHF-Cre; Cdh2fl/fl) (Figure 3D). Nuclei are marked by DAPI. Scale bar, 100 μm. (E) Graphical representation of the number of cardiomyocytes (cTnT+ cells) in the AHF in various mutants (AHF-Cre; Cdh2fl/fl, AHF-Cre; Cdh2fl/+; bCat(e3)fl/+ and AHF-Cre; Cdh2fl/fl; bCat(e3)fl/+) compared to control at E9.5 (somite number = 23-29). GOF, gain of function; LOF, loss of function. cTnT+ cells within the AHF, the region between OFT and the 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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fig4: Overexpression of Wnt signaling rescues Cdh2 mutant phenotype. (A) Representative traverse section showing Wnt signaling activity in AHF progenitors as revealed by TCL/LEF-lacZ mice. Black arrows denote cells that have active Wnt signaling within pharyngeal mesoderm, whereas red arrows denote cells with inactive Wnt signaling within heart tube. Scale bar, 500 μm. (B) Representative images of AHF-CPCs isolated from wild type (AHF-Cre; AHF-GFP; Cdh2fl/+) and mutant (AHF-Cre; AHF-GFP; Cdh2fl/fl) stained red for either nuclear β-catenin or membrane-bound β-catenin. The average mean intensities were tabulated from 60 fields. Nuclei are marked by DAPI. A cut-off value >10 was imposed to remove background. Scale bar, 50 μm. Error bars indicate SD, n = 3 replicates, **P < 0.001, evaluated by Student's t-test. (C) Quantitative PCR analysis of mRNA isolated from sorted progenitor cells (AHF-GFP+ CPCs) carrying AHF-GFP reporter transgene. The expression levels show that Wnt signaling ligands, receptors and effector genes (Axin2 and Lef1) were downregulated in Cdh2-deficient progenitor cells, while upregulation of differentiation genes such as Mlc2a and Tnnt2 were observed. Samples were normalized against the housekeeping gene, β-actin. Error bars indicate SD, n = 3 replicates. (D) Representative sagittal sections of single (AHF-Cre; Cdh2fl/+; bCat(e3)fl/+) and double (AHF-Cre; Cdh2fl/fl; bCat(e3)fl/+) mutants at E9.5. Sections stained for cTnT (red) and Isl1 (green) revealed similar expression patterns. Premature differentiation of Isl1+ CPCs into cTnT+ cells at the pharyngeal mesoderm was not observed in the double mutant as compared to single N-cadherin mutant (AHF-Cre; Cdh2fl/fl) (Figure 3D). Nuclei are marked by DAPI. Scale bar, 100 μm. (E) Graphical representation of the number of cardiomyocytes (cTnT+ cells) in the AHF in various mutants (AHF-Cre; Cdh2fl/fl, AHF-Cre; Cdh2fl/+; bCat(e3)fl/+ and AHF-Cre; Cdh2fl/fl; bCat(e3)fl/+) compared to control at E9.5 (somite number = 23-29). GOF, gain of function; LOF, loss of function. cTnT+ cells within the AHF, the region between OFT and the 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: First and foremost, we confirmed the presence of canonical Wnt/β-catenin signaling activity in AHF-CPCs with a BAT-GAL reporter mouse line, where lacZ gene is expressed under the control of β-catenin/TCF responsive elements12. Analysis of the X-gal-stained embryos at E9.5 indicated the presence of an active canonical Wnt/β-catenin signaling pathway in the AHF progenitor cells (Figure 4A).


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)

Overexpression of Wnt signaling rescues Cdh2 mutant phenotype. (A) Representative traverse section showing Wnt signaling activity in AHF progenitors as revealed by TCL/LEF-lacZ mice. Black arrows denote cells that have active Wnt signaling within pharyngeal mesoderm, whereas red arrows denote cells with inactive Wnt signaling within heart tube. Scale bar, 500 μm. (B) Representative images of AHF-CPCs isolated from wild type (AHF-Cre; AHF-GFP; Cdh2fl/+) and mutant (AHF-Cre; AHF-GFP; Cdh2fl/fl) stained red for either nuclear β-catenin or membrane-bound β-catenin. The average mean intensities were tabulated from 60 fields. Nuclei are marked by DAPI. A cut-off value >10 was imposed to remove background. Scale bar, 50 μm. Error bars indicate SD, n = 3 replicates, **P < 0.001, evaluated by Student's t-test. (C) Quantitative PCR analysis of mRNA isolated from sorted progenitor cells (AHF-GFP+ CPCs) carrying AHF-GFP reporter transgene. The expression levels show that Wnt signaling ligands, receptors and effector genes (Axin2 and Lef1) were downregulated in Cdh2-deficient progenitor cells, while upregulation of differentiation genes such as Mlc2a and Tnnt2 were observed. Samples were normalized against the housekeeping gene, β-actin. Error bars indicate SD, n = 3 replicates. (D) Representative sagittal sections of single (AHF-Cre; Cdh2fl/+; bCat(e3)fl/+) and double (AHF-Cre; Cdh2fl/fl; bCat(e3)fl/+) mutants at E9.5. Sections stained for cTnT (red) and Isl1 (green) revealed similar expression patterns. Premature differentiation of Isl1+ CPCs into cTnT+ cells at the pharyngeal mesoderm was not observed in the double mutant as compared to single N-cadherin mutant (AHF-Cre; Cdh2fl/fl) (Figure 3D). Nuclei are marked by DAPI. Scale bar, 100 μm. (E) Graphical representation of the number of cardiomyocytes (cTnT+ cells) in the AHF in various mutants (AHF-Cre; Cdh2fl/fl, AHF-Cre; Cdh2fl/+; bCat(e3)fl/+ and AHF-Cre; Cdh2fl/fl; bCat(e3)fl/+) compared to control at E9.5 (somite number = 23-29). GOF, gain of function; LOF, loss of function. cTnT+ cells within the AHF, the region between OFT and the 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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fig4: Overexpression of Wnt signaling rescues Cdh2 mutant phenotype. (A) Representative traverse section showing Wnt signaling activity in AHF progenitors as revealed by TCL/LEF-lacZ mice. Black arrows denote cells that have active Wnt signaling within pharyngeal mesoderm, whereas red arrows denote cells with inactive Wnt signaling within heart tube. Scale bar, 500 μm. (B) Representative images of AHF-CPCs isolated from wild type (AHF-Cre; AHF-GFP; Cdh2fl/+) and mutant (AHF-Cre; AHF-GFP; Cdh2fl/fl) stained red for either nuclear β-catenin or membrane-bound β-catenin. The average mean intensities were tabulated from 60 fields. Nuclei are marked by DAPI. A cut-off value >10 was imposed to remove background. Scale bar, 50 μm. Error bars indicate SD, n = 3 replicates, **P < 0.001, evaluated by Student's t-test. (C) Quantitative PCR analysis of mRNA isolated from sorted progenitor cells (AHF-GFP+ CPCs) carrying AHF-GFP reporter transgene. The expression levels show that Wnt signaling ligands, receptors and effector genes (Axin2 and Lef1) were downregulated in Cdh2-deficient progenitor cells, while upregulation of differentiation genes such as Mlc2a and Tnnt2 were observed. Samples were normalized against the housekeeping gene, β-actin. Error bars indicate SD, n = 3 replicates. (D) Representative sagittal sections of single (AHF-Cre; Cdh2fl/+; bCat(e3)fl/+) and double (AHF-Cre; Cdh2fl/fl; bCat(e3)fl/+) mutants at E9.5. Sections stained for cTnT (red) and Isl1 (green) revealed similar expression patterns. Premature differentiation of Isl1+ CPCs into cTnT+ cells at the pharyngeal mesoderm was not observed in the double mutant as compared to single N-cadherin mutant (AHF-Cre; Cdh2fl/fl) (Figure 3D). Nuclei are marked by DAPI. Scale bar, 100 μm. (E) Graphical representation of the number of cardiomyocytes (cTnT+ cells) in the AHF in various mutants (AHF-Cre; Cdh2fl/fl, AHF-Cre; Cdh2fl/+; bCat(e3)fl/+ and AHF-Cre; Cdh2fl/fl; bCat(e3)fl/+) compared to control at E9.5 (somite number = 23-29). GOF, gain of function; LOF, loss of function. cTnT+ cells within the AHF, the region between OFT and the 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: First and foremost, we confirmed the presence of canonical Wnt/β-catenin signaling activity in AHF-CPCs with a BAT-GAL reporter mouse line, where lacZ gene is expressed under the control of β-catenin/TCF responsive elements12. Analysis of the X-gal-stained embryos at E9.5 indicated the presence of an active canonical Wnt/β-catenin signaling pathway in the AHF progenitor cells (Figure 4A).

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