Slc26a2-mediated sulfate metabolism is important in tooth development

Abstract

The sulfate transporter gene SLC26A2 is crucial for skeletal formation, as evidenced by its role in diastrophic dysplasia, a type of skeletal dysplasia in humans. Although SLC26A2-related chondrodysplasia also affects craniofacial and tooth development, its specific role in these processes remains unclear. In this study, we explored the pivotal roles of SLC26A2-mediated sulfate metabolism during tooth development. We found that Slc26a2 was predominantly expressed in dental tissues, including odontoblasts and ameloblasts. Slc26a2 knockout (Slc26a2-KO-Δexon2) mice exhibited distinct craniofacial abnormalities, such as a retrognathic upper jaw, small upper incisors and upper molar hypoplasia. These mice also showed flattened odontoblasts and loss of nuclear polarity in upper incisors and molars, with significant reductions in odontoblast differentiation markers Dspp and Dmp1. Ex vivo and in vitro studies further revealed dentin matrix hypoplasia, tooth root shortening and downregulation of Wnt signaling in Slc26a2-deficient cells. These findings highlight the crucial role of SLC26A2-mediated sulfate metabolism in tooth development and offer insights into the mechanisms underlying dental abnormalities in patients with SLC26A2-related chondrodysplasias.

Keywords: Extracellular matrix; Matrix biology; Odontoblasts; SLC26A2; Sulfate metabolism; Tooth development.

Fig. 1.

Pattern of Slc26a2 expression during tooth development in mice. (A) Re-analysis of a public single-cell RNA-sequencing dataset on isolated mouse incisors at postnatal day (P)0. Ten cell population clusters, including odontoblasts, pre-odontoblasts, ameloblasts and pre-ameloblasts, are identified in mice tooth germs at P0. IEE, inner enamel epithelium; OEE, outer enamel epithelium; SI, stratum intermedium; SR, stellate reticulum; UMAP, uniform manifold approximation and projection. (B) Dot plot showing the expression of Slc26 family members in various clusters. Dot size represents the percentage of cells expressing a specific gene. The intensity of color indicates the average expression level for a gene in the cluster. Slc26a2 is predominantly expressed in mouse tooth germs. (C,D) The expression of Slc26a2 during tooth development at embryonic day (E)18.5. Frontal sections through the upper (D, top row) and lower (D, bottom row) molar, and sagittal sections through the lower incisor (C), in wild type, processed by RNA in situ hybridization. Dashed red line boxes demarcate the magnified areas. Am, ameloblast; Od, odontoblast; P, dental pulp. Scale bars: 100 μm. All samples are wild-type C57B6/J.

Fig. 2.

Slc26a2-KO-Δexon2 mice show skeletal and dental abnormalities. (A-D) Gross phenotype of Slc26a2-KO-Δexon2 mice at E18.5. Whole-mount images of Slc26a2-KO-Δexon2 mice revealed hypoplasia of the maxilla and very short limbs (white arrowheads). Lateral (A,B) and frontal (C,D) views of the head are shown. Scale bars: 1 mm (A,B), 500 μm (C,D). (E-J) Whole-mount skeletal preparations of craniofacial bones (E,F), cranial base (G,H) and limbs (I,J) in control and Slc26a2-KO-Δexon2 mice at E18.5. Whole-mount skeletal preparations show that Slc26a2-KO-Δexon2 mice had chondrodysplasia and reduced Alcian Blue staining of the limbs and cranial cartilage. md, mandible; mx, maxilla; nc, nasal cavity. Scale bars: 2 mm. (K-R) Contrast-enhanced micro-computed tomography (CT) images of control and Slc26a2-KO-Δexon2 mouse tooth germs at E18.5. The upper molar and incisor widths of Slc26a2-KO-Δexon2 mice are smaller than those of the control. Scale bars: 50 μm. (S) Quantitative assessment of upper and lower tooth size in control and Slc26a2-KO-Δexon2 mice at E18.5. Slc26a2-KO-Δexon2 mice show hypoplasia of the upper and lower incisors and upper molars, but not of the lower molars, compared to those of the control mice. n=3; ***P<0.001, ****P<0.0001 (two-way ANOVA).

Fig. 3.

Slc26a2 deficiency leads to impaired differentiation of odontoblasts in the upper tooth. (A,B,C,D) Hematoxylin and Eosin (H&E) staining of frontal sections of upper incisor and molar tooth germ. Upper incisor and upper molar tooth germ in Slc26a2-KO-Δexon2 mice show flattened odontoblasts cell body and nuclei, and loss of intracellular polarity. (A′,B′,C′,D′) Magnified views of the boxed areas in A, B, C and D, respectively. (E,F) Quantitative PCR (qPCR) analysis of Dspp and Dmp1 expression in odontoblasts of upper tooth germ at E18.5. In Slc26a2-KO-Δexon2 mice, the expression of Dspp and Dmp1 in upper molar tooth germ was significantly decreased compared to that in control mice. n=3; **P<0.01, ***P<0.001 (unpaired two-tailed Student's t-test). (G-J′) H&E staining of sagittal sections of lower incisor (G-H3) and lower molar (I-J′) tooth germ. The overall structure of lower molar tooth germ is comparable in control and Slc26a2-KO-Δexon2 mice (I,J). In the high-magnification images of the differentiation zone (G2,H2), the pre-secretary ameloblast layer is larger in Slc26a2-KO-Δexon2 mice than that in control mice. The heights of secretary and mature ameloblasts are lower in Slc26a2-KO-Δexon2 mice than those in control mice (G3,H3). Reduction in enamel and dentin thickness is observed in the incisor tooth germ in Slc26a2-KO-Δexon2 mice (G3,H3). The tooth phenotype in lower molar tooth germ in Slc26a2-KO-Δexon2 mice is much milder than that in upper molar tooth germ (I-J′). Am, ameloblast; Od, odontoblasts; P, dental pulp. Scale bars: 200 μm (A,B); 100 μm (C,D,G,H,I,J); 50 μm (A′,B′,C′,D′,G2,G3,H2,H3,I′,J′); 20 μm (G1,H1).

Fig. 4.

Analysis of the effect of Slc26a2 deletion on dentinogenesis. (A) SLC26A2-depleted and control human dental pulp stem cells (hDPSCs) were cultured for 7 days in odontoblast differentiation medium. The expression of SLC26A2, DSPP and DMP1 was evaluated by qPCR. Note that SLC26A2, DSPP and DMP1 expression was significantly decreased in SLC26A2-depleted cells compared to that in the control cells. GAPDH was used as an internal control for normalization. Means±s.d. (n=3) are shown. **P<0.01 (two-way ANOVA). (B) Ex vivo organ culture of tooth germ by implantation of the tooth germ under the kidney capsule of nude mice. After 4 weeks of organ culture, the implanted tooth germs were collected and analyzed by micro-CT. Sagittal sections of control and Slc26a2-KO-Δexon2 mice tooth germ micro-CT images show hypoplasia of the tooth crown, consisting of enamel and dentin. Tooth root shortening was also observed in Slc26a2-deficient tooth germ. Scale bars: 450 μm. (C) Quantitative assessment of the dentin volume demonstrated a significant reduction in Slc26a2-KO-Δexon2 mouse tooth germ compared to that in control tooth germ. n=5; *P<0.05 (two-way ANOVA).

Fig. 5.

Slc26a2 deficiency leads to impaired Wnt signaling in mouse dental papilla mesenchymal cells. (A) Hierarchical clustering heatmap displaying standardized gene expression values ranging from −1.0 to 1.0, with a mean of 0 for sh-Slc26a2 and sh-control mouse dental papilla mesenchymal cells (mDPCs). Red represents genes with high expression levels; blue indicates genes with low expression levels. (B) Volcano plot visualization of genes upregulated or downregulated in Slc26a2-silenced mDPCs. Gene values with |log2FC|>1 were considered differentially expressed genes (DEGs). Red dots represent upregulated genes; blue dots represent downregulated genes; gray dots represent nonsignificantly DEGs (non-DEGs). CPM, counts per million; FC, fold change. (C) Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs. BP, biological process. (D) Expression of Axin2, Wnt10a, Dmp1, Dspp and Slc26a2 in sh-Slc26a2 and sh-control mDPCs after 3 days of differentiation. n=3; *P<0.05, **P<0.01 (unpaired two-tailed Student's t-test).

Fig. 6.

The sulfate transporter defect resulting from Slc26a2 deficiency is partly compensated in lower tooth germ. (A) The total amount of sulfated glycosaminoglycans in upper molar tooth germs was significantly decreased by Slc26a2 deficiency. n=3; n.s., not significant; **P<0.01, ***P<0.001, ****P<0.0001 (two-way ANOVA). (B) The odontoblast layer was extracted by micro-dissection from the sagittal section of upper and lower molars. Absolute quantification was performed to evaluate Slc26a1 and Slc26a2 expression. The expression of Slc26a2 was significantly higher than that of Slc26a1 in odontoblast of upper tooth germs. Conversely, the expression of Slc26a1 was higher than that of Slc26a2 in odontoblasts of lower tooth germs. n=3; *P<0.05, **P<0.01 (two-way ANOVA).