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The cell adhesion molecule nectin-1 is critical for normal enamel formation in mice.

Barron MJ, Brookes SJ, Draper CE, Garrod D, Kirkham J, Shore RC, Dixon MJ - Hum. Mol. Genet. (2008)

Bottom Line: While strong immunostaining for nectin-1 was observed at the interface between the maturation-stage ameloblasts and the underlying cells of the stratum intermedium (SI), its absence in nectin-1- mice correlated with separation of the cell layers at this interface.Nectins have been shown to regulate tight junction formation; however, this is the first report showing that they may also participate in the regulation of desmosome assembly.Importantly, our results show that integrity of the SI-ameloblast interface is essential for normal enamel mineralization.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, UK.

ABSTRACT
Nectin-1 is a member of a sub-family of immunoglobulin-like adhesion molecules and a component of adherens junctions. In the current study, we have shown that mice lacking nectin-1 exhibit defective enamel formation in their incisor teeth. Although the incisors of nectin-1- mice were hypomineralized, the protein composition of the enamel matrix was unaltered. While strong immunostaining for nectin-1 was observed at the interface between the maturation-stage ameloblasts and the underlying cells of the stratum intermedium (SI), its absence in nectin-1- mice correlated with separation of the cell layers at this interface. Numerous, large desmosomes were present at this interface in wild-type mice; however, where adhesion persisted in the mutant mice, the desmosomes were smaller and less numerous. Nectins have been shown to regulate tight junction formation; however, this is the first report showing that they may also participate in the regulation of desmosome assembly. Importantly, our results show that integrity of the SI-ameloblast interface is essential for normal enamel mineralization.

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Related in: MedlinePlus

Generation of Pvrl1−/− mice. (A) Structure of Pvrl1, gene targeting vector and targeted allele following homologous recombination. Exons are depicted as grey boxes, whereas the neomycin-resistance cassette (neo) is shown as a black box. The EcoR1 sites used for cloning, E, and the position of the probe used for Southern blot analysis are also shown. (B) Confirmation of the genotypes of wild-type (+/+), heterozygous (±) and homozygous mutant (−/−) mice by duplex PCR analysis in which the wild-type and targeted alleles are represented by 240 and 290 bp products, respectively. (C) Western blot analysis of protein extracts from wild-type (+/+), heterozygous (±) and homozygous mutant (−/−) mouse embryos. No nectin-1 protein was detected in samples obtained from Pvrl1−/− embryos. Actin-1 loading controls are also shown.
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DDN243F1: Generation of Pvrl1−/− mice. (A) Structure of Pvrl1, gene targeting vector and targeted allele following homologous recombination. Exons are depicted as grey boxes, whereas the neomycin-resistance cassette (neo) is shown as a black box. The EcoR1 sites used for cloning, E, and the position of the probe used for Southern blot analysis are also shown. (B) Confirmation of the genotypes of wild-type (+/+), heterozygous (±) and homozygous mutant (−/−) mice by duplex PCR analysis in which the wild-type and targeted alleles are represented by 240 and 290 bp products, respectively. (C) Western blot analysis of protein extracts from wild-type (+/+), heterozygous (±) and homozygous mutant (−/−) mouse embryos. No nectin-1 protein was detected in samples obtained from Pvrl1−/− embryos. Actin-1 loading controls are also shown.

Mentions: To investigate the function of nectin-1, we disrupted Pvrl1 in mouse embryonic stem cells by homologous recombination using a gene targeting construct in which exon 1, which contains the translation initiation codon, was replaced with a neomycin-resistance cassette (Fig. 1A). Three correctly targeted embryonic stem cell lines, as assessed by Southern blot analysis (data not shown), were used to generate chimeric male mice which were inter-crossed with C57BL/6 females to generate phenotypically normal Pvrl1± mice, the genotypes of which were confirmed by PCR analysis (Fig. 1B). Subsequently, Pvrl1± mice were inter-crossed to produce homozygous mutant animals. No nectin-1 protein was detected by western blot analysis in tissues extracted from mice homozygous for the targeted locus (Fig. 1C) while heterozygotes showed an intermediate level of protein (Fig. 1C). Pvrl1−/− mice were viable and fertile but manifested microphthalmia identical to that observed in the nectin-1- mice reported by Inagaki et al. (9) (data not shown).


The cell adhesion molecule nectin-1 is critical for normal enamel formation in mice.

Barron MJ, Brookes SJ, Draper CE, Garrod D, Kirkham J, Shore RC, Dixon MJ - Hum. Mol. Genet. (2008)

Generation of Pvrl1−/− mice. (A) Structure of Pvrl1, gene targeting vector and targeted allele following homologous recombination. Exons are depicted as grey boxes, whereas the neomycin-resistance cassette (neo) is shown as a black box. The EcoR1 sites used for cloning, E, and the position of the probe used for Southern blot analysis are also shown. (B) Confirmation of the genotypes of wild-type (+/+), heterozygous (±) and homozygous mutant (−/−) mice by duplex PCR analysis in which the wild-type and targeted alleles are represented by 240 and 290 bp products, respectively. (C) Western blot analysis of protein extracts from wild-type (+/+), heterozygous (±) and homozygous mutant (−/−) mouse embryos. No nectin-1 protein was detected in samples obtained from Pvrl1−/− embryos. Actin-1 loading controls are also shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2572697&req=5

DDN243F1: Generation of Pvrl1−/− mice. (A) Structure of Pvrl1, gene targeting vector and targeted allele following homologous recombination. Exons are depicted as grey boxes, whereas the neomycin-resistance cassette (neo) is shown as a black box. The EcoR1 sites used for cloning, E, and the position of the probe used for Southern blot analysis are also shown. (B) Confirmation of the genotypes of wild-type (+/+), heterozygous (±) and homozygous mutant (−/−) mice by duplex PCR analysis in which the wild-type and targeted alleles are represented by 240 and 290 bp products, respectively. (C) Western blot analysis of protein extracts from wild-type (+/+), heterozygous (±) and homozygous mutant (−/−) mouse embryos. No nectin-1 protein was detected in samples obtained from Pvrl1−/− embryos. Actin-1 loading controls are also shown.
Mentions: To investigate the function of nectin-1, we disrupted Pvrl1 in mouse embryonic stem cells by homologous recombination using a gene targeting construct in which exon 1, which contains the translation initiation codon, was replaced with a neomycin-resistance cassette (Fig. 1A). Three correctly targeted embryonic stem cell lines, as assessed by Southern blot analysis (data not shown), were used to generate chimeric male mice which were inter-crossed with C57BL/6 females to generate phenotypically normal Pvrl1± mice, the genotypes of which were confirmed by PCR analysis (Fig. 1B). Subsequently, Pvrl1± mice were inter-crossed to produce homozygous mutant animals. No nectin-1 protein was detected by western blot analysis in tissues extracted from mice homozygous for the targeted locus (Fig. 1C) while heterozygotes showed an intermediate level of protein (Fig. 1C). Pvrl1−/− mice were viable and fertile but manifested microphthalmia identical to that observed in the nectin-1- mice reported by Inagaki et al. (9) (data not shown).

Bottom Line: While strong immunostaining for nectin-1 was observed at the interface between the maturation-stage ameloblasts and the underlying cells of the stratum intermedium (SI), its absence in nectin-1- mice correlated with separation of the cell layers at this interface.Nectins have been shown to regulate tight junction formation; however, this is the first report showing that they may also participate in the regulation of desmosome assembly.Importantly, our results show that integrity of the SI-ameloblast interface is essential for normal enamel mineralization.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, University of Manchester, Manchester, UK.

ABSTRACT
Nectin-1 is a member of a sub-family of immunoglobulin-like adhesion molecules and a component of adherens junctions. In the current study, we have shown that mice lacking nectin-1 exhibit defective enamel formation in their incisor teeth. Although the incisors of nectin-1- mice were hypomineralized, the protein composition of the enamel matrix was unaltered. While strong immunostaining for nectin-1 was observed at the interface between the maturation-stage ameloblasts and the underlying cells of the stratum intermedium (SI), its absence in nectin-1- mice correlated with separation of the cell layers at this interface. Numerous, large desmosomes were present at this interface in wild-type mice; however, where adhesion persisted in the mutant mice, the desmosomes were smaller and less numerous. Nectins have been shown to regulate tight junction formation; however, this is the first report showing that they may also participate in the regulation of desmosome assembly. Importantly, our results show that integrity of the SI-ameloblast interface is essential for normal enamel mineralization.

Show MeSH
Related in: MedlinePlus