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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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Impaired development of AHF derivatives in AHF-specific Cdh2 mutant. (A) The percentage of surviving embryos dissected at various embryonic stages compared to expected Mendelian ratio. Note that mutant embryos are dying between E10.5 and E11.5. Viability was measured by the presence of a heartbeat. (B) AHF-specific Cdh2 mutant embryos exhibit hypoplastic cardiac outflow tract (OFT) compared to controls at E9.5. H&E staining of the sagittal sections revealed smaller OFT in the mutant at E9.5 (enlarged image). Cardiomyocytes are stained dark red. Scale bar, 200 μm. (C) Whole embryo images of AHF-GFP expression in control and mutant embryos. Note reduction in the GFP expression in the mutants as well as shortened OFT and hypoplastic RV. Scale bar, 200 μm. (D) GFP sorted cells isolated from control and mutant embryos. Cdh2 mutants show reduced number of AHF-GFP+ cells (∼60%) compared to the wild-type control. Error bars indicate SD, n = 4 experiments, **P = 0.003, evaluated by Student's t-test. (E) Representative image of day 6 embryoid bodies (EB) differentiated from transgenic AHF-Cre+/−; AHF-GFP+; Cdh2fl/+(top panel) and AHF-Cre+/−; AHF-GFP+; Cdh2fl/fl(bottom panel) ES cells. Note the reduction in GFP expression in the mutant EBs. Scale bar, 200 μm. (F) Quantification of GFP+ cells isolated from control and mutant EBs. Error bars indicate SD, n = 3 experiments, **P = 0.01, evaluated by Student's t-test.
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fig2: Impaired development of AHF derivatives in AHF-specific Cdh2 mutant. (A) The percentage of surviving embryos dissected at various embryonic stages compared to expected Mendelian ratio. Note that mutant embryos are dying between E10.5 and E11.5. Viability was measured by the presence of a heartbeat. (B) AHF-specific Cdh2 mutant embryos exhibit hypoplastic cardiac outflow tract (OFT) compared to controls at E9.5. H&E staining of the sagittal sections revealed smaller OFT in the mutant at E9.5 (enlarged image). Cardiomyocytes are stained dark red. Scale bar, 200 μm. (C) Whole embryo images of AHF-GFP expression in control and mutant embryos. Note reduction in the GFP expression in the mutants as well as shortened OFT and hypoplastic RV. Scale bar, 200 μm. (D) GFP sorted cells isolated from control and mutant embryos. Cdh2 mutants show reduced number of AHF-GFP+ cells (∼60%) compared to the wild-type control. Error bars indicate SD, n = 4 experiments, **P = 0.003, evaluated by Student's t-test. (E) Representative image of day 6 embryoid bodies (EB) differentiated from transgenic AHF-Cre+/−; AHF-GFP+; Cdh2fl/+(top panel) and AHF-Cre+/−; AHF-GFP+; Cdh2fl/fl(bottom panel) ES cells. Note the reduction in GFP expression in the mutant EBs. Scale bar, 200 μm. (F) Quantification of GFP+ cells isolated from control and mutant EBs. Error bars indicate SD, n = 3 experiments, **P = 0.01, evaluated by Student's t-test.

Mentions: To study the function of N-cadherin in Isl1+ CPCs, we performed conditional gene knockout using the Cre-loxP system. Because the Cre is expressed under the control of AHF-specific enhancer, its expression is restricted to the CPCs and their progenies22. In N-cadherin conditional mutant mouse line, the first exon containing the start codon is flanked by loxP sites23, resulting in a Cdh2 floxed allele (Cdh2fl). To delete N-cadherin specifically in AHF-CPCs, we crossed double-heterozygous (AHF-Cre; Cdh2fl/+) male mice with females homozygous for the Cdh2fl allele (Cdh2fl/fl). The disruption of N-cadherin expression in the AHF-CPCs caused embryonic lethality around E9.5-E10.5, with no viable embryos retrieved at E11.5 (Figure 2A). Examination of the mutant embryos at E9.0-E9.5 revealed that all of them contained defects in the cardiac OFT and right ventricle. The cardiac OFT in the mutants was found to be shorter compared to that in the control embryos (Figure 2B and 2C). The looping of the developing heart is also abnormal, with the mutant cardiac OFT and RV protruding forward instead of turning toward the body (Figure 2B and 2C). This phenotype suggests disrupted growth of the cardiac OFT and RV. The disruption in growth could be attributed to defects in CPCs that contribute to the OFT and RV.


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)

Impaired development of AHF derivatives in AHF-specific Cdh2 mutant. (A) The percentage of surviving embryos dissected at various embryonic stages compared to expected Mendelian ratio. Note that mutant embryos are dying between E10.5 and E11.5. Viability was measured by the presence of a heartbeat. (B) AHF-specific Cdh2 mutant embryos exhibit hypoplastic cardiac outflow tract (OFT) compared to controls at E9.5. H&E staining of the sagittal sections revealed smaller OFT in the mutant at E9.5 (enlarged image). Cardiomyocytes are stained dark red. Scale bar, 200 μm. (C) Whole embryo images of AHF-GFP expression in control and mutant embryos. Note reduction in the GFP expression in the mutants as well as shortened OFT and hypoplastic RV. Scale bar, 200 μm. (D) GFP sorted cells isolated from control and mutant embryos. Cdh2 mutants show reduced number of AHF-GFP+ cells (∼60%) compared to the wild-type control. Error bars indicate SD, n = 4 experiments, **P = 0.003, evaluated by Student's t-test. (E) Representative image of day 6 embryoid bodies (EB) differentiated from transgenic AHF-Cre+/−; AHF-GFP+; Cdh2fl/+(top panel) and AHF-Cre+/−; AHF-GFP+; Cdh2fl/fl(bottom panel) ES cells. Note the reduction in GFP expression in the mutant EBs. Scale bar, 200 μm. (F) Quantification of GFP+ cells isolated from control and mutant EBs. Error bars indicate SD, n = 3 experiments, **P = 0.01, evaluated by Student's t-test.
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fig2: Impaired development of AHF derivatives in AHF-specific Cdh2 mutant. (A) The percentage of surviving embryos dissected at various embryonic stages compared to expected Mendelian ratio. Note that mutant embryos are dying between E10.5 and E11.5. Viability was measured by the presence of a heartbeat. (B) AHF-specific Cdh2 mutant embryos exhibit hypoplastic cardiac outflow tract (OFT) compared to controls at E9.5. H&E staining of the sagittal sections revealed smaller OFT in the mutant at E9.5 (enlarged image). Cardiomyocytes are stained dark red. Scale bar, 200 μm. (C) Whole embryo images of AHF-GFP expression in control and mutant embryos. Note reduction in the GFP expression in the mutants as well as shortened OFT and hypoplastic RV. Scale bar, 200 μm. (D) GFP sorted cells isolated from control and mutant embryos. Cdh2 mutants show reduced number of AHF-GFP+ cells (∼60%) compared to the wild-type control. Error bars indicate SD, n = 4 experiments, **P = 0.003, evaluated by Student's t-test. (E) Representative image of day 6 embryoid bodies (EB) differentiated from transgenic AHF-Cre+/−; AHF-GFP+; Cdh2fl/+(top panel) and AHF-Cre+/−; AHF-GFP+; Cdh2fl/fl(bottom panel) ES cells. Note the reduction in GFP expression in the mutant EBs. Scale bar, 200 μm. (F) Quantification of GFP+ cells isolated from control and mutant EBs. Error bars indicate SD, n = 3 experiments, **P = 0.01, evaluated by Student's t-test.
Mentions: To study the function of N-cadherin in Isl1+ CPCs, we performed conditional gene knockout using the Cre-loxP system. Because the Cre is expressed under the control of AHF-specific enhancer, its expression is restricted to the CPCs and their progenies22. In N-cadherin conditional mutant mouse line, the first exon containing the start codon is flanked by loxP sites23, resulting in a Cdh2 floxed allele (Cdh2fl). To delete N-cadherin specifically in AHF-CPCs, we crossed double-heterozygous (AHF-Cre; Cdh2fl/+) male mice with females homozygous for the Cdh2fl allele (Cdh2fl/fl). The disruption of N-cadherin expression in the AHF-CPCs caused embryonic lethality around E9.5-E10.5, with no viable embryos retrieved at E11.5 (Figure 2A). Examination of the mutant embryos at E9.0-E9.5 revealed that all of them contained defects in the cardiac OFT and right ventricle. The cardiac OFT in the mutants was found to be shorter compared to that in the control embryos (Figure 2B and 2C). The looping of the developing heart is also abnormal, with the mutant cardiac OFT and RV protruding forward instead of turning toward the body (Figure 2B and 2C). This phenotype suggests disrupted growth of the cardiac OFT and RV. The disruption in growth could be attributed to defects in CPCs that contribute to the OFT and RV.

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