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

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

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

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

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

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

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

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

Assessment of incisor microhardness on incisor longitudinal sections.

<p>Approximately 20 indentations throughout the enamel layer were obtained per incisor and the results were average to generate one data point for the graph. Measurements from at least 4 incisors from each genotype were used to generate a bar on the graph. Whiskers denote the data range and the horizontal line within the box represents the median microhardness value. When present in the <i>Mmp20</i> null background (A), the Tg6i (M) <i>Mmp20^−/−</i> (N = 4) enamel was slightly softer (P<0.05) than wild-type mouse enamel (Tg– <i>Mmp20^+/+</i>, N = 8), but the Tg24i (H) <i>Mmp20^−/−</i> (N = 4) enamel had a wider range of microhardness values and was therefore not significantly different from wild-type. The Tg42i (L) <i>Mmp20^−/−</i> (N = 7) enamel was much softer than enamel from wild-type mice and this difference was highly significant (P<0.0001). In contrast, each of the transgenic mice had enamel that was harder than the enamel from the <i>Mmp20</i> null mouse incisors (Tg– <i>Mmp20^−/−</i>, N = 11) and these differences were all highly significant (P<0.0001). Enamel hardness values positively correlated to the level of transgene expression in mouse incisors when the transgenes were in the <i>Mmp20</i> null background. When present in the <i>Mmp20</i> wild-type background (B), the Tg6i (M) <i>Mmp20^+/+</i> (N = 4) incisor enamel was softer (P<0.01) than enamel from wild-type mice (Tg– <i>Mmp20^+/+</i>, N = 8) and the Tg24i (H) <i>Mmp20^+/+</i> (N = 4) enamel was much softer than enamel from wild-type mice (P<0.0001). However, no difference in enamel hardness was observed between wild-type and Tg42i (L) <i>Mmp20^+/+</i> (N = 4) enamel. Enamel hardness values negatively correlated to the level of transgene expression in mouse incisors when the transgenes were in the wild-type background.</p

µCT analyses of incisor enamel from <i>Mmp20</i> transgenics and controls.

<p>Enamel on rodent incisors is present only on the labial side (arrows). The presented longitudinally oriented incisors were reconstructed from µCT images. The incisors protrude from bone (arrowheads) and are arranged so that the labial side is to the left. The wild-type incisors (Tg– MMP20^+/+, top right panel) have a bright line of mineralized enamel that extends from the apical region (bottom arrow) to the labial incisal tip (top arrow). This mineralized enamel was mostly missing from the <i>Mmp20</i> null incisors (Tg– <i>Mmp20^−/−</i>, top left panel). The transgenic incisors in the null background (Tg+ <i>Mmp20^−/−</i>) recovered some or most of the enamel layer along the labial surface. When the transgenes were present in the wild-type background (Tg+ Mmp20^+/+), the enamel layer seemed relatively normal on incisors from mice transgenic for Tg6i (M) or Tg42i (L), but it was severely disrupted on the Tg24i (H) <i>Mmp20^+/+</i> incisors. For incisors, Tg24 was the highest, Tg6 the middle and Tg42 the lowest expressing transgene.</p