doi: 10.1159/000272318. Epub 2009 Dec 24.
Unique modulation of cadherin expression pattern during posterior frontal cranial suture development and closure
Affiliations
- PMID:20051668
- PMCID: PMC2859230
- DOI: 10.1159/000272318
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Unique modulation of cadherin expression pattern during posterior frontal cranial suture development and closure
David E Sahar et al. Cells Tissues Organs.2010.
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Abstract
Cranial suture development involves coordinated expression of multiple genes and tissue contribution from neural crest cells and paraxial mesoderm for timely sutural morphogenesis. Transcription factors, growth factors, and neural crest determinant genes play critical roles in calvarial growth ensuring normal development of the underlying brain. In vitro studies have implicated cell-cell adhesion molecules as a driving force behind suture closure. We performed cDNA microarray to study differential expression of adhesion molecules during the timing of suture closure in a mouse model where only the posterior frontal (PF) suture closes. Our results indicate increased expression of E-cadherin during the period of PF suture closure. Quantitative RT-PCR analysis of E- and N-cadherin in PF closing suture revealed a biphasic expression of N-cadherin, the first phase coinciding with cellular condensation preceding chondrogenesis followed by a second phase coinciding with E-cadherin co-expression and suture closure. Furthermore, expression analysis of the N-cadherin and E-cadherin transcriptional repressors Wnt7a and Snail indicate a specific temporal regulation of these genes, suggesting their potential role as regulators of both E- and N-cadherin during the PF suture development and closure. Finally, given the in vitro evidence of fibroblast growth factor (FGF)-2 as a potential regulator of E- and N-cadherin we investigated the expression of E-cadherin during PF suture closure in Fgf-2 deficient mice. In contrast to in vitrodata previously reported, E-cadherin expression is normal in these animals, and PF suture closure occurs properly, probably due to potential redundancy of FGF ligands ensuring normal temporal expression of E-cadherin and PF suture closure.
Figures
DifferentialE-cadherin expression in cranial sutures.a Microarray profile ofE-cadherin gene on SAG and PF suture shows that the gene is up-regulated exclusively in the PF suture at p15. Green = Lower than mean expression; red = higher than mean expression.b Quantitative real-time RT-PCR confirmed the up-regulation ofE-cadherin gene expression in PF suture at p15. Quantified mRNA values were normalized by the amounts ofGapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments.
E-cadherin expression in posterior frontal suture during the first month of life.a Quantitative real-time PCR forE-cadherin on PF suture reveals up-regulation ofE-cadherin gene starting at day p9 with maximal expression at days p13–p15, a period coinciding with active bone deposition in the PF suture and suture closure. Quantified mRNA values were normalized by the amounts ofGapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments from 3 independent litters. * p < 0.05.b Immunohistochemistry analysis of E-cadherin protein performed using a specific E-cadherin antibody, shows at day p5 absence of positive cells in the suture mesenchyme PF suture (boxed area), while positive cells are detected in the surrounding osteogenic fronts. At day p7, a faint E-cadherin staining of few cells is observed, E-cadherin staining is also detected in pericranium and dura mater cells. Starting from day p9, more E-cadherin positive cells are detected in differentiating mesenchyme of the endocranial layer of PF suture. At day p15 an intense E-cadherin staining is observed in the bony bridge of closed endocranial layer of PF suture (boxed area). Boxed areas corresponding to the suture mesenchyme undergoing to endochondral ossification are magnified in panels on the right column. Dashed lines mark the osteogenic front of approaching bone plates Note, immunostaining is also observed in the area of suture populated by red blood cells, vessels and lymphocytes (asterisk). Pr = Pericranium; DM = dura mater; PF- endo = PF endocranial suture. Scale bar: 200 μm (at ×20) and 50 μm (at ×40).c Quantitative real-time PCR forE-cadherin performed on SAG suture and its comparison with expression profile observed in PF suture. Expression ofE-cadherin gene in SAG suture remains steady during the first month of life. The results are presented as the mean ± SD of 3 independent experiments from 3 independent litters.
E-cadherin expression in posterior frontal suture during the first month of life.a Quantitative real-time PCR forE-cadherin on PF suture reveals up-regulation ofE-cadherin gene starting at day p9 with maximal expression at days p13–p15, a period coinciding with active bone deposition in the PF suture and suture closure. Quantified mRNA values were normalized by the amounts ofGapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments from 3 independent litters. * p < 0.05.b Immunohistochemistry analysis of E-cadherin protein performed using a specific E-cadherin antibody, shows at day p5 absence of positive cells in the suture mesenchyme PF suture (boxed area), while positive cells are detected in the surrounding osteogenic fronts. At day p7, a faint E-cadherin staining of few cells is observed, E-cadherin staining is also detected in pericranium and dura mater cells. Starting from day p9, more E-cadherin positive cells are detected in differentiating mesenchyme of the endocranial layer of PF suture. At day p15 an intense E-cadherin staining is observed in the bony bridge of closed endocranial layer of PF suture (boxed area). Boxed areas corresponding to the suture mesenchyme undergoing to endochondral ossification are magnified in panels on the right column. Dashed lines mark the osteogenic front of approaching bone plates Note, immunostaining is also observed in the area of suture populated by red blood cells, vessels and lymphocytes (asterisk). Pr = Pericranium; DM = dura mater; PF- endo = PF endocranial suture. Scale bar: 200 μm (at ×20) and 50 μm (at ×40).c Quantitative real-time PCR forE-cadherin performed on SAG suture and its comparison with expression profile observed in PF suture. Expression ofE-cadherin gene in SAG suture remains steady during the first month of life. The results are presented as the mean ± SD of 3 independent experiments from 3 independent litters.
E-cadherin expression in posterior frontal suture during the first month of life.a Quantitative real-time PCR forE-cadherin on PF suture reveals up-regulation ofE-cadherin gene starting at day p9 with maximal expression at days p13–p15, a period coinciding with active bone deposition in the PF suture and suture closure. Quantified mRNA values were normalized by the amounts ofGapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments from 3 independent litters. * p < 0.05.b Immunohistochemistry analysis of E-cadherin protein performed using a specific E-cadherin antibody, shows at day p5 absence of positive cells in the suture mesenchyme PF suture (boxed area), while positive cells are detected in the surrounding osteogenic fronts. At day p7, a faint E-cadherin staining of few cells is observed, E-cadherin staining is also detected in pericranium and dura mater cells. Starting from day p9, more E-cadherin positive cells are detected in differentiating mesenchyme of the endocranial layer of PF suture. At day p15 an intense E-cadherin staining is observed in the bony bridge of closed endocranial layer of PF suture (boxed area). Boxed areas corresponding to the suture mesenchyme undergoing to endochondral ossification are magnified in panels on the right column. Dashed lines mark the osteogenic front of approaching bone plates Note, immunostaining is also observed in the area of suture populated by red blood cells, vessels and lymphocytes (asterisk). Pr = Pericranium; DM = dura mater; PF- endo = PF endocranial suture. Scale bar: 200 μm (at ×20) and 50 μm (at ×40).c Quantitative real-time PCR forE-cadherin performed on SAG suture and its comparison with expression profile observed in PF suture. Expression ofE-cadherin gene in SAG suture remains steady during the first month of life. The results are presented as the mean ± SD of 3 independent experiments from 3 independent litters.
aN-cadherin expression in the posterior frontal suture during the first month of life. Top panel: Quantitative real-time PCR forN-cadherin on PF suture reveals a biphasic expression profile ofN-cadherin gene.N-cadherin is first up-regulated on p7, a period coinciding with a cellular condensation in the endocranial plate of the PF suture. A second phase ofN-cadherin gene up-regulation is observed on p13–p15, a period marked by osteogenesis and closure of PF suture. Bottom panel: Quantitative real-time PCR forE-cadherin on SAG suture shows that the expression is steady overtime. Quantified mRNA values were normalized by the amounts ofGapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments from 3 independent litters. * p < 0.05.bImmunolocalization of N-cadherin protein on PF suture using a specific anti-N-cadherin antibody mirrors the biphasic gene expression profile and localizes N-cadherin protein in the suture mesenchyme of the endocranial layer of PF suture during cellular precondensation at day p5 and p7. By day p9 only a few condroblasts present in the differentiating suture mesenchyme stain positively. At day p11 none of the cells present in the suture stain positive. In contrast, by day p13 a strong N-cadherin staining is observed in the endocranial layer of PF suture. Day p15, which marks the closure of PF suture, is also characterized by strong N-cadherin staining in the area of endochondral ossification (boxed area). Pericranium, osteoblasts and dura mater, also stain positively for N-cadherin. Boxed areas are magnified in panels on the right column. Pr = pericranium; DM = dura mater; PF-endo = PF endocranial suture. Scale bar: 200 μm (at ×20), 50 μm (at ×40).
aN-cadherin expression in the posterior frontal suture during the first month of life. Top panel: Quantitative real-time PCR forN-cadherin on PF suture reveals a biphasic expression profile ofN-cadherin gene.N-cadherin is first up-regulated on p7, a period coinciding with a cellular condensation in the endocranial plate of the PF suture. A second phase ofN-cadherin gene up-regulation is observed on p13–p15, a period marked by osteogenesis and closure of PF suture. Bottom panel: Quantitative real-time PCR forE-cadherin on SAG suture shows that the expression is steady overtime. Quantified mRNA values were normalized by the amounts ofGapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments from 3 independent litters. * p < 0.05.bImmunolocalization of N-cadherin protein on PF suture using a specific anti-N-cadherin antibody mirrors the biphasic gene expression profile and localizes N-cadherin protein in the suture mesenchyme of the endocranial layer of PF suture during cellular precondensation at day p5 and p7. By day p9 only a few condroblasts present in the differentiating suture mesenchyme stain positively. At day p11 none of the cells present in the suture stain positive. In contrast, by day p13 a strong N-cadherin staining is observed in the endocranial layer of PF suture. Day p15, which marks the closure of PF suture, is also characterized by strong N-cadherin staining in the area of endochondral ossification (boxed area). Pericranium, osteoblasts and dura mater, also stain positively for N-cadherin. Boxed areas are magnified in panels on the right column. Pr = pericranium; DM = dura mater; PF-endo = PF endocranial suture. Scale bar: 200 μm (at ×20), 50 μm (at ×40).
Expression ofN- andE-cadherin transcriptional repressorsWnt7a andSnail.a Quantitative real-time PCR forWnt7a on PF suture reveals up-regulation ofWnt7a geneas early as p7, with maximal expression on p11 and return to baseline by p13.b The inverse gene expression correlation betweenWnt7a andN-cadherin. Note thatWnt7a gene expression is specifically up-regulated during the down-regulation ofN-cadherin gene.c Quantitative real-time PCR forSnail on PF suture reveals a moderate up-regulation ofSnail genestarting on p5, with a decrease on p9 and a return to baseline by p15. * p < 0.01).d Graph showing the inverse gene expression profile ofSnail andE-cadherin during the PF suture patterning and closure. Quantified mRNA values were normalized by the amounts ofGapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments. * p < 0.05.
E-cadherin expression is not down-regulated in the PF suture ofFgf-2 null mice.aE-cadherin expression in PF suture ofFgf-2 null mice during the first month of life was assessed by quantitative real-time PCR. Up-regulation ofE-cadherin gene is observed starting on p5 with maximal level on p9 and return to baseline by p11. Note that up-regulation ofE-cadherin gene inFgf-2 null mice occurs earlier than in CD-1 wild-type mice.bN-cadherin expression in PF suture ofFgf-2 null mice also shows a pattern similar to that of wild-type CD-1 mice. Quantified mRNA values were normalized by the amounts ofGapdh mRNA, and results are given as fold induction. The results are presented as the mean ± SD of 3 independent experiments. * p < 0.05.
Temporal expression ofN- andE- cadherins and their regulators,Wnt7a andSnail. The diagram summarizes the temporal gene expression profiles and the key differentiation events occurring during PF suture closing.
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