Targeted p120-Catenin Ablation Disrupts Dental Enamel Development

Dental enamel development occurs in stages. The ameloblast cell layer is adjacent to, and is responsible for, enamel formation. When rodent pre-ameloblasts become tall columnar secretory-stage ameloblasts, they secrete enamel matrix proteins, and the ameloblasts start moving in rows that slide by one another. This movement is necessary to form the characteristic decussating enamel prism pattern. Thus, a dynamic system of intercellular interactions is required for proper enamel development. Cadherins are components of the adherens junction (AJ), and they span the cell membrane to mediate attachment to adjacent cells. p120 stabilizes cadherins by preventing their internalization and degradation. So, we asked if p120-mediated cadherin stability is important for dental enamel formation. Targeted p120 ablation in the mouse enamel organ had a striking effect. Secretory stage ameloblasts detached from surrounding tissues, lost polarity, flattened, and ameloblast E- and N-cadherin expression became undetectable by immunostaining. The enamel itself was poorly mineralized and appeared to be composed of a thin layer of merged spheres that abraded from the tooth. Significantly, p120 mosaic mouse teeth were capable of forming normal enamel demonstrating that the enamel defects were not a secondary effect of p120 ablation. Surprisingly, blood-filled sinusoids developed in random locations around the developing teeth. This has not been observed in other p120-ablated tissues and may be due to altered p120-mediated cell signaling. These data reveal a critical role for p120 in tooth and dental enamel development and are consistent with p120 directing the attachment and detachment of the secretory stage ameloblasts as they move in rows

Mosaic phenotype of the K14-Cre p120-cKO mouse incisor.

<p>A section through a mosaic cKO enamel organ (A) showing normal ameloblasts (Am) in the middle and malformed ameloblasts to the left (arrow). Es, enamel space. SEM analysis of the same cKO mosaic incisor (B). The right side of the bracket touches normal enamel and the left side touches an abnormal, enamel-free area. Between the two sides of the bracket is the malformed dysplastic enamel. Note that the cKO mouse is capable of forming normal enamel. Therefore, the observed enamel dysplasia is not a secondary effect of conditional p120 ablation.</p

Secretory and maturation stage enamel organ express E- and N-cadherins, p120 catenin and <i>Arvcf</i>.

<p>qPCR was performed to identify the expression levels of adherens junction proteins in enamel organs responsible for dental enamel development. Expression was assessed in first molar enamel organs from mice at the indicated age. At days 5–7, enamel organs are predominantly at the secretory stage and at days 9–11, enamel organs are predominantly at the maturation stage of enamel development. Note that p120 is expressed at constant levels across these development stages. Arvcf is highly homologous to p120 and this is the first demonstration of its expression in the mammalian enamel organ. Each time point was performed in duplicate with RNA from six different enamel organs. *, P<0.05; **, P<0.01; ***, P<0.001.</p

E-cadherin is only expressed in ameloblasts capable of expressing p120.

<p>In the same K14-Cre p120-cKO mosaic incisor shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0012703#pone-0012703-g008" target="_blank">Figure 8</a>, E-cadherin (top) and p120 catenin (bottom) was immunolocalized in adjacent cross-sections. E-cadherin is expressed exclusively in normal appearing ameloblasts (brackets) that also express p120. However, in flattened, malformed ameloblasts where p120 was ablated, immunostaining for E-cadherin was not detectable. EO, enamel organ; PO, pulp organ.</p

N-cadherin is expressed in wild-type secretory stage ameloblasts, but not in p120 ablated ameloblasts.

<p>In the less mature second molar (M2), N-cadherin was not expressed (A) in the enamel organ (EO) or pulp organ (PO) of three day-old mice. In the more mature first molar (M1), N-cadherin was expressed (B, C). After dentin matrix deposition, odontoblasts (Od) and ameloblasts (Am) showed lateral membrane immunostaining for N-cadherin, and the apical and basal terminal web apparatus of ameloblasts were also stained positively (B). After enamel matrix deposition, the odontoblasts stain intensely (C). A developing cusp tip from the first molar of a P3 K14-Cre p120-cKO mouse stained for N-cadherin (D). N-cadherin expression was detected in odontoblasts, but not in the ameloblasts from this molar. Note that the dentin appears rough and mildly dysplastic.</p

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

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

Enamel from K14-Cre p120-cKO mice does not mineralize properly.

<p>Faxitron analysis of wild-type and cKO mouse skulls reveals either absence of enamel or a thin layer of poorly mineralized enamel that is indistinguishable from the underlying dentin (Top). This assessment was corroborated by micro-CT analysis of molar and incisor teeth (Bottom). The wild-type molars had clearly defined opaque enamel layers whereas molars from the cKO mice did not (bracket identifies molars lacking a highly mineralized enamel layer). Note that small spots of more highly mineralized enamel are present on the cKO mouse molars. These data indicate that the globular material observed on the tooth surface by SEM is poorly mineralized. The cKO incisor tooth (bottom) had a thin layer of mineralized enamel covering a small portion of the labial tooth surface whereas the wild-type incisor had thick enamel covering the entire labial surface of the dentin.</p

Incisor tooth development in K14-p120-cKO mice appears normal until the secretory stage of development when the ameloblasts flatten and become dysmorphic.

<p>A wild-type mouse incisor with identified tissue structures is presented (A) as a comparison for the incisor from the cKO mice (B, C). The p120 ablated incisor ameloblasts separate from the dentin surface prior to mineral formation (B). Panel C is a higher magnification of the boxed area in panel B. The p120 null ameloblasts (Am) abruptly alter their morphology soon after they enter the secretory stage of enamel development and become short flattened cells (C). The odontoblast (Od) and pulp organ (PO) appear normal in these teeth (C).</p

Enamel from K14-Cre p120-cKO mice is dysplastic.

<p>Scanning electron microscopy (SEM) of molars and enamel from wild-type (A, D) and K14-Cre p120-cKO mice (B, C, E, F). Note that the general shape of the cusps and fissures in the cKO mice is not altered. The dysplastic enamel on the cKO mouse molars (B) does not protect the teeth from abrasion as does normal highly mineralized enamel. The first molar from a six week old mouse (panel C, left) shows pulp chamber exposure due to excessive attrition. The tooth surface of the K14-Cre p120-cKO (E) consists of an unusual alignment of modular structures into distinct rows. Higher magnification shows the rows are composed of spherical structures of about 0.1 to 0.2 µm in diameter. The wild-type surface enamel is very smooth (D).</p