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Critical roles for WDR72 in calcium transport and matrix protein removal during enamel maturation.

Wang SK, Hu Y, Yang J, Smith CE, Nunez SM, Richardson AS, Pal S, Samann AC, Hu JC, Simmer JP - Mol Genet Genomic Med (2015)

Bottom Line: The maturation stage mandibular incisor enamel did not stain with methyl red, indicating that the enamel did not acidify beneath ruffle-ended ameloblasts.Attachment of maturation ameloblasts to the enamel layer was weakened, and SLC24A4, a critical ameloblast calcium transporter, did not localize appropriately along the ameloblast distal membrane.We conclude that WDR72 serves critical functions specifically during the maturation stage of amelogenesis and is required for both protein removal and enamel mineralization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biologic and Materials Sciences, University of Michigan School of Dentistry 1210 Eisenhower Pl., Ann Arbor, Michigan, 48108.

ABSTRACT
Defects in WDR72 (WD repeat-containing protein 72) cause autosomal recessive hypomaturation amelogenesis imperfecta. We generated and characterized Wdr72-knockout/lacZ-knockin mice to investigate the role of WDR72 in enamel formation. In all analyses, enamel formed by Wdr72 heterozygous mice was indistinguishable from wild-type enamel. Without WDR72, enamel mineral density increased early during the maturation stage but soon arrested. The enamel layer was only a tenth as hard as wild-type enamel and underwent rapid attrition following eruption. Despite the failure to further mineralize enamel deposited during the secretory stage, ectopic mineral formed on the enamel surface and penetrated into the overlying soft tissue. While the proteins in the enamel matrix were successfully degraded, the digestion products remained inside the enamel. Interactome analysis of WDR72 protein revealed potential interactions with clathrin-associated proteins and involvement in ameloblastic endocytosis. The maturation stage mandibular incisor enamel did not stain with methyl red, indicating that the enamel did not acidify beneath ruffle-ended ameloblasts. Attachment of maturation ameloblasts to the enamel layer was weakened, and SLC24A4, a critical ameloblast calcium transporter, did not localize appropriately along the ameloblast distal membrane. Fewer blood vessels were observed in the papillary layer supporting ameloblasts. Specific WDR72 expression by maturation stage ameloblasts explained the observation that enamel thickness and rod decussation (established during the secretory stage) are normal in the Wdr72 mice. We conclude that WDR72 serves critical functions specifically during the maturation stage of amelogenesis and is required for both protein removal and enamel mineralization.

No MeSH data available.


Related in: MedlinePlus

Wdr72 knockout/NLS-lacZ knockin construct and expression. (A) Diagram of the wild-type Wdr72 gene. This diagram is based upon a comparison of the mouse Wdr72 NCBI cDNA and genomic reference sequences NM_001033500.3 and NC_000075.6, respectively. Exons are boxes with shaded areas corresponding to noncoding regions. (B) Knockin construct showing replacement of exon 2 starting at the translation initiation codon with NLS-lacZ. (C) Alternative splicing of transcripts from the Wdr72  allele that read through the transcription termination sequences introduced downstream of the NLS-lacZ sequence in intron 2. Arrows identify primer annealing sites for RT-PCR. (D) Ethidium bromide stained agarose gel showing RT-PCR products generated by amplification of RNA isolated from enamel organ epithelia (EOE). DNA sequencing of the bands showed that the modified exon 2 was sometimes deleted during RNA splicing. The higher bands marked by arrowheads are the wild-type amplification products; the lower bands marked by arrowheads are the same products lacking exon 2.
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fig01: Wdr72 knockout/NLS-lacZ knockin construct and expression. (A) Diagram of the wild-type Wdr72 gene. This diagram is based upon a comparison of the mouse Wdr72 NCBI cDNA and genomic reference sequences NM_001033500.3 and NC_000075.6, respectively. Exons are boxes with shaded areas corresponding to noncoding regions. (B) Knockin construct showing replacement of exon 2 starting at the translation initiation codon with NLS-lacZ. (C) Alternative splicing of transcripts from the Wdr72 allele that read through the transcription termination sequences introduced downstream of the NLS-lacZ sequence in intron 2. Arrows identify primer annealing sites for RT-PCR. (D) Ethidium bromide stained agarose gel showing RT-PCR products generated by amplification of RNA isolated from enamel organ epithelia (EOE). DNA sequencing of the bands showed that the modified exon 2 was sometimes deleted during RNA splicing. The higher bands marked by arrowheads are the wild-type amplification products; the lower bands marked by arrowheads are the same products lacking exon 2.

Mentions: To investigate the role of WDR72 in enamel formation in vivo, we used gene targeting to ablate Wdr72 in mouse strain C57Bl6 (Fig.1 and Fig. S1). Mouse Wdr72 has 20 exons, with the first exon being noncoding. We replaced the coding sequence of exon 2 and the 5′ end of intron 2 with a lacZ reporter gene encoding β-galactosidase fused to a mouse NLS. The translation initiation codon (ATG) of the inserted NLS-lacZ exactly replaced that of Wdr72, and thus precluded translation of native WDR72 and established a reporter gene in an optimal context to mimic Wdr72 transcription in vivo. The NLS-lacZ cassette included the coding sequence for bacterial β-galactosidase followed by a 3′-untranslated region (UTR) containing two polyadenylation signals to terminate transcription in intron 2.


Critical roles for WDR72 in calcium transport and matrix protein removal during enamel maturation.

Wang SK, Hu Y, Yang J, Smith CE, Nunez SM, Richardson AS, Pal S, Samann AC, Hu JC, Simmer JP - Mol Genet Genomic Med (2015)

Wdr72 knockout/NLS-lacZ knockin construct and expression. (A) Diagram of the wild-type Wdr72 gene. This diagram is based upon a comparison of the mouse Wdr72 NCBI cDNA and genomic reference sequences NM_001033500.3 and NC_000075.6, respectively. Exons are boxes with shaded areas corresponding to noncoding regions. (B) Knockin construct showing replacement of exon 2 starting at the translation initiation codon with NLS-lacZ. (C) Alternative splicing of transcripts from the Wdr72  allele that read through the transcription termination sequences introduced downstream of the NLS-lacZ sequence in intron 2. Arrows identify primer annealing sites for RT-PCR. (D) Ethidium bromide stained agarose gel showing RT-PCR products generated by amplification of RNA isolated from enamel organ epithelia (EOE). DNA sequencing of the bands showed that the modified exon 2 was sometimes deleted during RNA splicing. The higher bands marked by arrowheads are the wild-type amplification products; the lower bands marked by arrowheads are the same products lacking exon 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4521966&req=5

fig01: Wdr72 knockout/NLS-lacZ knockin construct and expression. (A) Diagram of the wild-type Wdr72 gene. This diagram is based upon a comparison of the mouse Wdr72 NCBI cDNA and genomic reference sequences NM_001033500.3 and NC_000075.6, respectively. Exons are boxes with shaded areas corresponding to noncoding regions. (B) Knockin construct showing replacement of exon 2 starting at the translation initiation codon with NLS-lacZ. (C) Alternative splicing of transcripts from the Wdr72 allele that read through the transcription termination sequences introduced downstream of the NLS-lacZ sequence in intron 2. Arrows identify primer annealing sites for RT-PCR. (D) Ethidium bromide stained agarose gel showing RT-PCR products generated by amplification of RNA isolated from enamel organ epithelia (EOE). DNA sequencing of the bands showed that the modified exon 2 was sometimes deleted during RNA splicing. The higher bands marked by arrowheads are the wild-type amplification products; the lower bands marked by arrowheads are the same products lacking exon 2.
Mentions: To investigate the role of WDR72 in enamel formation in vivo, we used gene targeting to ablate Wdr72 in mouse strain C57Bl6 (Fig.1 and Fig. S1). Mouse Wdr72 has 20 exons, with the first exon being noncoding. We replaced the coding sequence of exon 2 and the 5′ end of intron 2 with a lacZ reporter gene encoding β-galactosidase fused to a mouse NLS. The translation initiation codon (ATG) of the inserted NLS-lacZ exactly replaced that of Wdr72, and thus precluded translation of native WDR72 and established a reporter gene in an optimal context to mimic Wdr72 transcription in vivo. The NLS-lacZ cassette included the coding sequence for bacterial β-galactosidase followed by a 3′-untranslated region (UTR) containing two polyadenylation signals to terminate transcription in intron 2.

Bottom Line: The maturation stage mandibular incisor enamel did not stain with methyl red, indicating that the enamel did not acidify beneath ruffle-ended ameloblasts.Attachment of maturation ameloblasts to the enamel layer was weakened, and SLC24A4, a critical ameloblast calcium transporter, did not localize appropriately along the ameloblast distal membrane.We conclude that WDR72 serves critical functions specifically during the maturation stage of amelogenesis and is required for both protein removal and enamel mineralization.

View Article: PubMed Central - PubMed

Affiliation: Department of Biologic and Materials Sciences, University of Michigan School of Dentistry 1210 Eisenhower Pl., Ann Arbor, Michigan, 48108.

ABSTRACT
Defects in WDR72 (WD repeat-containing protein 72) cause autosomal recessive hypomaturation amelogenesis imperfecta. We generated and characterized Wdr72-knockout/lacZ-knockin mice to investigate the role of WDR72 in enamel formation. In all analyses, enamel formed by Wdr72 heterozygous mice was indistinguishable from wild-type enamel. Without WDR72, enamel mineral density increased early during the maturation stage but soon arrested. The enamel layer was only a tenth as hard as wild-type enamel and underwent rapid attrition following eruption. Despite the failure to further mineralize enamel deposited during the secretory stage, ectopic mineral formed on the enamel surface and penetrated into the overlying soft tissue. While the proteins in the enamel matrix were successfully degraded, the digestion products remained inside the enamel. Interactome analysis of WDR72 protein revealed potential interactions with clathrin-associated proteins and involvement in ameloblastic endocytosis. The maturation stage mandibular incisor enamel did not stain with methyl red, indicating that the enamel did not acidify beneath ruffle-ended ameloblasts. Attachment of maturation ameloblasts to the enamel layer was weakened, and SLC24A4, a critical ameloblast calcium transporter, did not localize appropriately along the ameloblast distal membrane. Fewer blood vessels were observed in the papillary layer supporting ameloblasts. Specific WDR72 expression by maturation stage ameloblasts explained the observation that enamel thickness and rod decussation (established during the secretory stage) are normal in the Wdr72 mice. We conclude that WDR72 serves critical functions specifically during the maturation stage of amelogenesis and is required for both protein removal and enamel mineralization.

No MeSH data available.


Related in: MedlinePlus