E-cadherin can replace N-cadherin during secretory-stage enamel development

Abstract

Background: N-cadherin is a cell-cell adhesion molecule and deletion of N-cadherin in mice is embryonic lethal. During the secretory stage of enamel development, E-cadherin is down-regulated and N-cadherin is specifically up-regulated in ameloblasts when groups of ameloblasts slide by one another to form the rodent decussating enamel rod pattern. Since N-cadherin promotes cell migration, we asked if N-cadherin is essential for ameloblast cell movement during enamel development.

Methodology/principal findings: The enamel organ, including its ameloblasts, is an epithelial tissue and for this study a mouse strain with N-cadherin ablated from epithelium was generated. Enamel from wild-type (WT) and N-cadherin conditional knockout (cKO) mice was analyzed. μCT and scanning electron microscopy showed that thickness, surface structure, and prism pattern of the cKO enamel looked identical to WT. No significant difference in hardness was observed between WT and cKO enamel. Interestingly, immunohistochemistry revealed the WT and N-cadherin cKO secretory stage ameloblasts expressed approximately equal amounts of total cadherins. Strikingly, E-cadherin was not normally down-regulated during the secretory stage in the cKO mice suggesting that E-cadherin can compensate for the loss of N-cadherin. Previously it was demonstrated that bone morphogenetic protein-2 (BMP2) induces E- and N-cadherin expression in human calvaria osteoblasts and we show that the N-cadherin cKO enamel organ expressed significantly more BMP2 and significantly less of the BMP antagonist Noggin than did WT enamel organ.

Conclusions/significance: The E- to N-cadherin switch at the secretory stage is not essential for enamel development or for forming the decussating enamel rod pattern. E-cadherin can substitute for N-cadherin during these developmental processes. Bmp2 expression may compensate for the loss of N-cadherin by inducing or maintaining E-cadherin expression when E-cadherin is normally down-regulated. Notably, this is the first demonstration of a natural endogenous increase in E-cadherin expression due to N-cadherin ablation in a healthy developing tissue.

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.