Fig 1.
Reference lines for animal dissection.
A. In 4-week-old rats, the secretory- (S) and maturation-stage (M) enamel organs along the enamel surface of mandibular incisor are separated by the reference line between the first and the second molar [3,43]. B. Two reference lines were used to partition secretory-stage (S) from maturation-stage (M) enamel organ. The first reference line, vertical to the inferior border of mandibular cortical bone, divided the mesial-distal width of the first molar into halves. The second reference line, also vertical to the inferior to the bony border of the mandible, was located between the second and the third molar.
Table 1.
Antibodies used for western blot, immunoperoxidase immunostaining, immunofluorescence and co-immunoprecipitation analyses.
Fig 2.
Real-time PCR analysis of Slc26a1, Slc26a6 and Slc26a7 expression during amelogenesis.
The expression levels of Slc26a1, Slc26a6 and Slc26a7 were normalized to those of Beta-Actin and are presented in fold changes. The expression levels of Slc26a1, Slc26a6 and Slc26a7 were up-regulated by ~10.0, ~4.1 and ~15.3 fold, respectively, at maturation stage relative to secretory stage.
Fig 3.
Western blot analysis of Slc26a1, Slc26a6 and Slc26a7.
A1-C1. Protein-level expression of Slc26a1, Slc26a6 and Slc26a7 was detected by western blot analysis using samples obtained from both secretory- and maturation-stage enamel organs (4-week-old rat incisors). Protein samples extracted from kidney (4-week-old rat) were used as reference controls. The molecular weights for Slc26a1, Slc26a6 and Slc26a7 are 75kDa, 90kDa and 72kDa, respectively. Beta-Actin served as the control for sample loading. A2-C2. The intensities of the bands (relative to Beta-Actin) were measured using ImageJ. The average fold changes of Slc26a1, Slc26a6 and Slc26a7 at the protein level were ~1.6, ~6.1, ~4.2, respectively.
Table 2.
Average fold changes of Slc26 gene family members during maturation stage relative to secretory stage based on genome-wide mRNA transcriptome analysis.
(N/A, expression not detected; α = 0.05).
Fig 4.
Immunoperoxidase immunostaining of Slc26a1, Slc26a6 and Slc26a7 in secretory- and maturation-stage enamel organ.
Immunostaining procedures were applied to the sagittal sections prepared from paraffin-embedded 4-week-old rat mandibles. A. Slc26a1 in secretory-stage ameloblasts (S); B. Slc26a1 in smooth-ended ameloblasts at maturation stage (M-SA); C. Slc26a1 in ruffle-ended ameloblasts at maturation stage (M-RA); D. Slc26a6 in secretory-stage ameloblasts (S); E. Slc26a6 in smooth-ended ameloblasts at maturation stage (M-SA); F. Slc26a6 in ruffle-ended ameloblasts at maturation stage (M-RA); G. Slc26a7 in secretory-stage ameloblasts (S); H. Slc26a7 in smooth-ended ameloblasts at maturation stage (M-SA); I. Slc26a7 in ruffle-ended ameloblasts at maturation stage (M-RA); J-L. The sections that were incubated without antibodies served as negative controls for immunostaining. All images were collected under 20x magnification. Scale bar shown in Panel J (50μm). Slc26a1, Slc26a6 and Slc26a7 all showed expression on the apical membrane and/or within subapical cytoplasmic region (double black arrows). Positive staining in other regions was indicated by double black asterisks. SI—Stratum intermedium; Am—Ameloblast; ES—Enamel space; CT—Connective tissue; PL—Papillary layer.
Fig 5.
Co-localization analysis of Slc26a1, Slc26a6, Slc26a7 with Ae2, Lamp1 and Cftr.
(A-B) Co-localization of Slc26a1 with Ae2 and Lamp1 in maturation-stage ameloblasts by confocal microscopy at 63x magnification. The signals of Slc26a1 were mainly seen on the apical membrane of maturation-stage ameloblasts (Panels A and B; Green). In contrast, Ae2 was localized to the basolateral membrane (Panel A; Red) and the basal pole (Panel C; Red), while Lamp1 showed a cytoplasmic and/or peri-nuclear distribution pattern in ameloblasts (Panel B; Red). The sections were stained with DAPI to highlight the nuclei (Panels A and B; Blue). (C-D) Co-localization of Slc26a6 with Ae2 and Lamp1 in maturation-stage ameloblasts. Slc26a6 exhibited a similar expression pattern to that of Slc26a1—on the apical membrane of maturation-stage ameloblasts (Panels C and D; Green). The fluorescence signals of Ae2 (Panel C; Red) and Lamp1 (Panel D; Red) were used as references. The sections were stained with DAPI to highlight the nuclei (Panels C and D; Blue). (E-G) Co-localization of Slc26a7 with Ae2, Lamp1 and Cftr in maturation-stage ameloblasts. The expression of Slc26a7 was found both on the apical membrane and within the cytoplasmic region (Panels E-G; Green). The fluorescence signals of Slc26a7 partially overlapped with those of Lamp1 (Panel F; Red) and Cftr (Panel G; Red), rather than Ae2 (Panel E; Red). The sections were stained with DAPI to highlight the nuclei (Panels E-G; Blue). The images were collected under confocal microscopy (63x magnification). Scale bar shown in Panel G (10μm).
Fig 6.
Co-immunoprecipitation (Co-IP) assay of Cftr with Slc26a1, Slc26a6 and Slc26a7.
Co-IP was conducted using protein samples extracted from the maturation-stage enamel organs of 4-week-old rat incisors (50~100 μg initial input). The interaction complexes were pulled down by anti-Cftr antibody (Table 3). The subsequent western blot analyses were performed using primary antibodies to Slc26a1, Slc26a6 and Slc26a7 (Table 3). The positive control was the pre-cleared total protein without the following immunoprecipitation procedures, and the negative control was prepared by skipping the step of applying the antibody against Cftr.
Fig 7.
Micro-CT analysis of Slc26a1-/- and Slc26a7-/- mandibles.
The mandibles from wild-type, Slc26a1 null and Slc26a7 null animals (at 8 weeks of age) were subject to micro-CT analysis (n = 3). The relative density and thickness of enamel on the labial incisor where the cortical bone enclosing just begins (A1-C2) were measured. There was no statistical difference between mutant and wild-type animals with respect to these two parameters (D-E). (Enamel Em, Dentin De)
Fig 8.
SEM images of mature enamel in Slc26a1-/- and Slc26a7-/- animals.
The surface of the enamel of incisor and molars in Slc26a1-/- and Slc26a7-/- animals were similar to those of wild-type animals (A1-B3, magnification 35x). When the internal structures of enamel (incisor) were observed in coronal section (C1-C3, magnification 5000x), the enamel from mutant animals showed a mild disruption in rod density and diameter (C2-C3) compared with wild-type enamel (C1). The arrangement of the enamel rod and inter-rod structures (incisor) did not seem to be impacted significantly by the deletion of Slc26a1 or Slc26a7 (C1-C3).
Fig 9.
EDS analysis of mature enamel Slc26a1-/- and Slc26a7-/- animals.
(A-C) EDS spectrum of mature enamel in wild-type, Slc26a1-/- and Slc26a7-/- animals (n = 6 per group). (D) No statistically significant differences were detected in the At% of Ca, P and O between mutant and wild-type enamel. (E) The At% of Cl increased significantly by ~34% in both Slc26a1-/- (P = 0.012) and Slc26a7-/- (P = 0.035) enamel. There was a significant decrease in the At% of C in Slc26a1-/- enamel (P = 0.028). A similar difference in the At% of C was detected between Slc26a7-/- and wild-type enamel, but was only marginally significant (P = 0.078). The At% of Na in Slc26a7-/- enamel also significantly decreased (P = 0.028).
Table 3.
Enamel and dentin Vickers microhardness of mutant animals.
Fig 10.
Up-regulated genes in Slc26a1-/- and Slc26a7-/- animals compared with wild types.
Most genes that showed significant changes in expression were up-regulated (Panels A-S), indicating a compensatory effect induced by the deletion of Slc26a1 or Slc26a7. The expression values of differentially expressed genes were normalized to those of Beta-Actin. For all the two-tailed t tests used, the significance level was 0.05. * <0.05; ** <0.01.
Fig 11.
Downregulated genes in Slc26a1-/- and Slc26a7-/- animals compared with wild types.
The expression values of differentially expressed genes were normalized to those of Beta-Actin. For all the two-tailed t tests used, the significance level was 0.05. * <0.05; ** <0.01.
Fig 12.
Schematic diagram depicting the distribution of major pH regulators in maturation-stage ameloblasts.
Cftr, Slc26a1, Slc26a4, Slc26a6 and Slc26a7 are localized to the apical membrane and Slc26a1, Slc26a6 and Slc26a7 physically interact with Cftr to form regulation complexes. Slc26a7 is also found on the endo-lysosomal membrane. Ae2, Nhe1 and NBCe1 are localized basolaterally. Clcn7 is expressed on the endo-lysosomal membrane. CA2 exhibits intracellular distribution whereas CA6 functions in extracellular enamel matrix. Lamp1 in this image was used as a marker of late lysosomes.