Table 1.
Primers used for quantitative real-time PCR (qPCR).
Figure 1.
(a) qPCR analysis of N-cadherin gene expression in wild-type (WT) and N-cadherin conditional knockout (cKO) mouse enamel organs. mRNA was extracted from postnatal day-5 enamel organs from 3 mice per genotype for qPCR analysis. Results are presented as expression ratios relative to the WT levels. The ablated N-cadherin cKO enamel organs (K14-Cre-N-cadherin-LoxP+/+) had 0.19 fold of the WT N-cadherin expression level (***, p<0.001) and the heterozygous ablated mice (K14-Cre-N-cadherin-LoxP+/−) had 0.75 fold of the WT expression level (*, p<0.05). (b) N-cadherin protein expression was ablated in the ameloblast layer of K14-Cre-N-cadherin-LoxP+/+ mice. Immunohistochemical staining of N-cadherin was performed on paraffin-imbedded incisor sections from both WT and N-cadherin cKO mice. In WT mice N-cadherin was not expressed highly in pre-secretory stage ameloblasts, but was strongly up-regulated during the secretory stage and was later down-regulated when the ameloblasts progressed into the maturation stage. In contrast, regardless of developmental stage, N-cadherin expression was not observed in the N-cadherin cKO ameloblasts demonstrating that N-cadherin expression was successfully deleted in these mice. A, ameloblast layer; O, odontoblast layer.
Figure 2.
(a) N-cadherin cKO mice have normal enamel mineral levels. WT and N-cadherin cKO hemi-mandibles were subjected to micro-computed tomography (μCT). The same arbitrary threshold was selected for both genotypes and enamel 3D structures were generated. Results were repeated with 5 mice per genotype. (b) Enamel defects were not observed in the N-cadherin cKO mice. Scanning electron microscopy (SEM) analysis was performed on WT and N-cadherin cKO incisors. The cKO incisors had a smooth enamel surface without abnormalities. Fractured incisors revealed that both WT and cKO samples had similar enamel thicknesses and both had the characteristic decussating prism pattern. With higher magnification (right side) normal enamel rods were clearly observed for both samples.
Figure 3.
No difference in Enamel hardness between WT and N-cadherin cKO mice.
Adult mouse incisors were harvested and indented for Vickers microhardness measurements. Enamel hardness from 4 mice per genotype was measured and results were averaged. WT samples have an averaged Vickers hardness number of 219.6±24.5, while N-cadherin cKO samples possess a slightly higher value of 235.1±23.8.
Figure 4.
Gene expression analysis in WT and N-cadherin cKO mouse enamel organs.
qPCR was performed on WT and N-cadherin cKO postnatal day-5 enamel organs with 7 mice assessed per genotype. No significant difference was observed in expression levels of p120 and β-catenin between WT and N-cadherin ablated enamel organs. Various cadherins were assessed for expression in secretory stage enamel organ and for those that were expressed, expression levels were assessed. A comparison of expression levels between WT and N-cadherin cKO enamel organs revealed that VE-cadherin expression was slightly but significantly reduced compared to WT and that E-cadherin expression was significantly increased by approximately 1.5 fold compared to WT. No significant differences by genotype were observed for P-cadherin or cadherin-11 (*, p<0.05).
Figure 5.
E-cadherin expression is abnormally elevated in secretory stage N-cadherin cKO ameloblasts.
Immunohistochemical staining with pan-cadherin (total cadherin) or E-cadherin antibodies was performed on sectioned incisors from WT and N-cadherin cKO mice. For the pan-cadherin antibody, both genotypes stained pre-secretory and secretory stage ameloblasts with no apparent difference in staining intensity observed between WT and N-cadherin cKO ameloblasts. This indicates that total cadherin levels were similar between genotypes. E-cadherin stained well in the WT pre-secretory stage ameloblasts and faded as the ameloblasts entered the secretory stage. In contrast, E-cadherin staining in the N-cadherin cKO sample remained strong from the pre-secretory stage through the secretory stage.
Figure 6.
BMP2 signaling may be responsible for maintaining E-cadherin expression during the secretory stage in N-cadherin cKO enamel organs.
qPCR was performed on WT and N-cadherin cKO postnatal day-5 enamel organs. Bmp2 expression increased approximately 1.7 fold over WT levels in the N-cadherin ablated enamel organs (**, p<0.005). Conversely, the BMP signaling antagonist Nog decreased by approximately 80% of the WT expression level (*, p<0.05). Results were obtained from 4 mice per genotype.