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Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts.

Fukumoto S, Kiba T, Hall B, Iehara N, Nakamura T, Longenecker G, Krebsbach PH, Nanci A, Kulkarni AB, Yamada Y - J. Cell Biol. (2004)

Bottom Line: Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin.We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation.Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.

View Article: PubMed Central - PubMed

Affiliation: Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin- mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.

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Generation of ameloblastin- mice. (A) Structure of the Ambn gene, targeting vector, and targeted allele after homologous recombination. The location of the fragment used as a probe in Southern blotting is shown, as well as the sizes of the XbaI fragment detected for wild-type and targeted alleles. Exons are depicted as closed boxes. The PGK-neo and PGK-TK cassettes are depicted as open and oblique boxes. (B) Southern blot analysis of 6-wk-old mouse genomic DNA. Genomic DNA isolated from tails was digested with XbaI and hybridized with the probe containing exons 8–10. The wild-type and mutant alleles were detected as 4.5- and 2.3-kb fragments, respectively. (C) RT-PCR analysis of Ambn mRNA expression in wild-type, heterozygous, and homozygous mice. 2 μg of total RNA isolated from P3 mouse molars were reverse transcribed and amplified with primers for Ambn and GAPDH. (D) Western blot analysis of ameloblastin in wild-type, heterozygous, and homozygous mice. Two P3 maxillary first molars were dissected and lysed with 100 μl of lysis buffer, and 20 μl were separated by SDS-PAGE and immunoblotted with polyclonal anti-ameloblastin antibodies. (E) Incisors of 8-wk-old heterozygous and homozygous mice.
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fig1: Generation of ameloblastin- mice. (A) Structure of the Ambn gene, targeting vector, and targeted allele after homologous recombination. The location of the fragment used as a probe in Southern blotting is shown, as well as the sizes of the XbaI fragment detected for wild-type and targeted alleles. Exons are depicted as closed boxes. The PGK-neo and PGK-TK cassettes are depicted as open and oblique boxes. (B) Southern blot analysis of 6-wk-old mouse genomic DNA. Genomic DNA isolated from tails was digested with XbaI and hybridized with the probe containing exons 8–10. The wild-type and mutant alleles were detected as 4.5- and 2.3-kb fragments, respectively. (C) RT-PCR analysis of Ambn mRNA expression in wild-type, heterozygous, and homozygous mice. 2 μg of total RNA isolated from P3 mouse molars were reverse transcribed and amplified with primers for Ambn and GAPDH. (D) Western blot analysis of ameloblastin in wild-type, heterozygous, and homozygous mice. Two P3 maxillary first molars were dissected and lysed with 100 μl of lysis buffer, and 20 μl were separated by SDS-PAGE and immunoblotted with polyclonal anti-ameloblastin antibodies. (E) Incisors of 8-wk-old heterozygous and homozygous mice.

Mentions: To analyze the function of ameloblastin, we performed targeted disruption of the ameloblastin gene (Ambn) in mouse embryonic stem (ES) cells using homologous recombination. We deleted the sequence containing exons 5 and 6 of the gene and replaced it with the PGK-Neor cassette gene (Fig. 1 A). Chimeric mice were produced from ES cell clones with the modified Ambn locus as confirmed by genotyping with Southern blotting (Fig. 1 B). Neither Ambn transcript nor protein was detected in postnatal day (P) 3 maxillary first molar of homozygous (Ambn−/−) mice (Fig. 1, C and D). Heterozygous mice (Ambn+/−) showed similar expression levels of ameloblastin as wild-type mice.


Ameloblastin is a cell adhesion molecule required for maintaining the differentiation state of ameloblasts.

Fukumoto S, Kiba T, Hall B, Iehara N, Nakamura T, Longenecker G, Krebsbach PH, Nanci A, Kulkarni AB, Yamada Y - J. Cell Biol. (2004)

Generation of ameloblastin- mice. (A) Structure of the Ambn gene, targeting vector, and targeted allele after homologous recombination. The location of the fragment used as a probe in Southern blotting is shown, as well as the sizes of the XbaI fragment detected for wild-type and targeted alleles. Exons are depicted as closed boxes. The PGK-neo and PGK-TK cassettes are depicted as open and oblique boxes. (B) Southern blot analysis of 6-wk-old mouse genomic DNA. Genomic DNA isolated from tails was digested with XbaI and hybridized with the probe containing exons 8–10. The wild-type and mutant alleles were detected as 4.5- and 2.3-kb fragments, respectively. (C) RT-PCR analysis of Ambn mRNA expression in wild-type, heterozygous, and homozygous mice. 2 μg of total RNA isolated from P3 mouse molars were reverse transcribed and amplified with primers for Ambn and GAPDH. (D) Western blot analysis of ameloblastin in wild-type, heterozygous, and homozygous mice. Two P3 maxillary first molars were dissected and lysed with 100 μl of lysis buffer, and 20 μl were separated by SDS-PAGE and immunoblotted with polyclonal anti-ameloblastin antibodies. (E) Incisors of 8-wk-old heterozygous and homozygous mice.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172447&req=5

fig1: Generation of ameloblastin- mice. (A) Structure of the Ambn gene, targeting vector, and targeted allele after homologous recombination. The location of the fragment used as a probe in Southern blotting is shown, as well as the sizes of the XbaI fragment detected for wild-type and targeted alleles. Exons are depicted as closed boxes. The PGK-neo and PGK-TK cassettes are depicted as open and oblique boxes. (B) Southern blot analysis of 6-wk-old mouse genomic DNA. Genomic DNA isolated from tails was digested with XbaI and hybridized with the probe containing exons 8–10. The wild-type and mutant alleles were detected as 4.5- and 2.3-kb fragments, respectively. (C) RT-PCR analysis of Ambn mRNA expression in wild-type, heterozygous, and homozygous mice. 2 μg of total RNA isolated from P3 mouse molars were reverse transcribed and amplified with primers for Ambn and GAPDH. (D) Western blot analysis of ameloblastin in wild-type, heterozygous, and homozygous mice. Two P3 maxillary first molars were dissected and lysed with 100 μl of lysis buffer, and 20 μl were separated by SDS-PAGE and immunoblotted with polyclonal anti-ameloblastin antibodies. (E) Incisors of 8-wk-old heterozygous and homozygous mice.
Mentions: To analyze the function of ameloblastin, we performed targeted disruption of the ameloblastin gene (Ambn) in mouse embryonic stem (ES) cells using homologous recombination. We deleted the sequence containing exons 5 and 6 of the gene and replaced it with the PGK-Neor cassette gene (Fig. 1 A). Chimeric mice were produced from ES cell clones with the modified Ambn locus as confirmed by genotyping with Southern blotting (Fig. 1 B). Neither Ambn transcript nor protein was detected in postnatal day (P) 3 maxillary first molar of homozygous (Ambn−/−) mice (Fig. 1, C and D). Heterozygous mice (Ambn+/−) showed similar expression levels of ameloblastin as wild-type mice.

Bottom Line: Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin.We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation.Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.

View Article: PubMed Central - PubMed

Affiliation: Craniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
Tooth morphogenesis results from reciprocal interactions between oral epithelium and ectomesenchyme culminating in the formation of mineralized tissues, enamel, and dentin. During this process, epithelial cells differentiate into enamel-secreting ameloblasts. Ameloblastin, an enamel matrix protein, is expressed by differentiating ameloblasts. Here, we report the creation of ameloblastin- mice, which developed severe enamel hypoplasia. In mutant tooth, the dental epithelium differentiated into enamel-secreting ameloblasts, but the cells were detached from the matrix and subsequently lost cell polarity, resumed proliferation, and formed multicell layers. Expression of Msx2, p27, and p75 were deregulated in mutant ameloblasts, the phenotypes of which were reversed to undifferentiated epithelium. We found that recombinant ameloblastin adhered specifically to ameloblasts and inhibited cell proliferation. The mutant mice developed an odontogenic tumor of dental epithelium origin. Thus, ameloblastin is a cell adhesion molecule essential for amelogenesis, and it plays a role in maintaining the differentiation state of secretory stage ameloblasts by binding to ameloblasts and inhibiting proliferation.

Show MeSH
Related in: MedlinePlus