Dose-Dependent Rescue of KO Amelogenin Enamel by Transgenes in Vivo

Mice lacking amelogenin (KO) have hypoplastic enamel. Overexpression of the most abundant amelogenin splice variant M180 and LRAP transgenes can substantially improve KO enamel, but only ~40% of the incisor thickness is recovered and the prisms are not as tightly woven as in WT enamel. This implies that the compositional complexity of the enamel matrix is required for different aspects of enamel formation, such as organizational structure and thickness. The question arises, therefore, how important the ratio of different matrix components, and in particular amelogenin splice products, is in enamel formation. Can optimal expression levels of amelogenin transgenes representing both the most abundant splice variants and cleavage product at protein levels similar to that of WT improve the enamel phenotype of KO mice? Addressing this question, our objective was here to understand dosage effects of amelogenin transgenes (Tg) representing the major splice variants M180 and LRAP and cleavage product CTRNC on enamel properties. Amelogenin KO mice were mated with M180Tg, CTRNCTg and LRAPTg mice to generate M180Tg and CTRNCTg double transgene and M180Tg, CTRNCTg, LRAPTg triple transgene mice with transgene hemizygosity (on one allelle) or homozygosity (on both alleles). Transgene homo- vs. hemizygosity was determined by qPCR and relative transgene expression confirmed by Western blot. Enamel volume and mineral density were analyzed by microCT, thickness and structure by SEM, and mechanical properties by Vickers microhardness testing. There were no differences in incisor enamel thickness between amelogenin KO mice with three or two different transgenes, but mice homozygous for a given transgene had significantly thinner enamel than mice hemizygous for the transgene (p < 0.05). The presence of the LRAPTg did not improve the phenotype of M180Tg/CTRNCTg/KO enamel. In the absence of endogenous amelogenin, the addition of amelogenin transgenes representing the most abundant splice variants and cleavage product can rescue abnormal enamel properties and structure, but only up to a maximum of ~80% that of molar and ~40% that of incisor wild-type enamel

Helium ion microscopy of enamel crystallites and extracellular tooth enamel matrix

An unresolved problem in tooth enamel studies has been to analyze simultaneously and with sufficient spatial resolution both mineral and organic phases in their three dimensional (3D) organization in a given specimen. This study aims to address this need using high-resolution imaging to analyze the 3D structural organization of the enamel matrix, especially amelogenin, in relation to forming enamel crystals. Chemically fixed hemi-mandibles from wild type mice were embedded in LR White acrylic resin, polished and briefly etched to expose the organic matrix in developing tooth enamel. Full-length amelogenin was labeled with specific antibodies and 10 nm immuno-gold. This allowed us to use and compare two different high-resolution imaging techniques for the analysis of uncoated samples. Helium ion microscopy (HIM) was applied to study the spatial organization of organic and mineral structures, while field emission scanning electron microscopy (FE-SEM) in various modes, including backscattered electron detection, allowed us to discern the gold-labeled proteins. Wild type enamel in late secretory to early maturation stage reveals adjacent to ameloblasts a lengthwise parallel alignment of the enamel matrix proteins, including full-length amelogenin proteins, which then transitions into a more heterogeneous appearance with increasing distance from the mineralization front. The matrix adjacent to crystal bundles forms a smooth and lacey sheath, whereas between enamel prisms it is organized into spherical components that are interspersed with rod-shaped protein. These findings highlight first, that the heterogeneous organization of the enamel matrix can be visualized in mineralized en bloc samples. Second, our results illustrate that the combination of these techniques is a powerful approach to elucidate the 3D structural organization of organic matrix molecules in mineralizing tissue in nanometer resolution

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

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.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.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

Gene expression analysis in WT and N-cadherin cKO mouse enamel organs.

<p>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).</p

Primers used for quantitative real-time PCR (qPCR).

<p>Primers used for quantitative real-time PCR (qPCR).</p

No difference in Enamel hardness between WT and N-cadherin cKO mice.

<p>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.</p

BMP2 signaling may be responsible for maintaining E-cadherin expression during the secretory stage in N-cadherin cKO enamel organs.

<p>qPCR was performed on WT and N-cadherin cKO postnatal day-5 enamel organs. <i>Bmp2</i> expression increased approximately 1.7 fold over WT levels in the N-cadherin ablated enamel organs (**, p<0.005). Conversely, the BMP signaling antagonist <i>Nog</i> decreased by approximately 80% of the WT expression level (*, p<0.05). Results were obtained from 4 mice per genotype.</p

E-Cadherin Can Replace N-Cadherin during Secretory-Stage Enamel Development - Figure 1

<p>(<b>a</b>) 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-<i>Cre</i>-N-cadherin-<i>LoxP</i>+/+) had 0.19 fold of the WT N-cadherin expression level (***, p<0.001) and the heterozygous ablated mice (K14-<i>Cre</i>-N-cadherin-<i>LoxP</i>+/−) had 0.75 fold of the WT expression level (*, p<0.05). (<b>b</b>) N-cadherin protein expression was ablated in the ameloblast layer of K14-<i>Cre</i>-N-cadherin-<i>LoxP</i>+/+ 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.</p

Synchrotron imaging and Markov Chain Monte Carlo reveal tooth mineralization patterns

The progressive character of tooth formation records aspects of mammalian life history, diet, seasonal behavior and climate. Tooth mineralization occurs in two stages: secretion and maturation, which overlap to some degree. Despite decades of study, the spatial and temporal pattern of elemental incorporation during enamel mineralization remains poorly characterized. Here we use synchrotron X-ray microtomography and Markov Chain Monte Carlo sampling to estimate mineralization patterns from an ontogenetic series of sheep molars (n = 45 M1s, 18 M2s). We adopt a Bayesian approach that posits a general pattern of maturation estimated from individual- and population-level mineral density variation over time. This approach converts static images of mineral density into a dynamic model of mineralization, and demonstrates that enamel secretion and maturation waves advance at nonlinear rates with distinct geometries. While enamel secretion is ordered, maturation geometry varies within a population and appears to be driven by diffusive processes. Our model yields concrete expectations for the integration of physiological and environmental signals, which is of particular significance for paleoseasonality research. This study also provides an avenue for characterizing mineralization patterns in other taxa. Our synchrotron imaging data and model are available for application to multiple disciplines, including health, material science, and paleontological research