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

The p120 null ameloblasts detach from the incisor dentin surface, the stratum intermedium, and from each other.

<p>Secretory ameloblasts (Am) are identified by positive immunostaining for amelogenin. Panels A–D represent sequential sections of an incisor from a cKO mouse. Pre-secretory ameloblasts do not express amelogenin (A) whereas secretory stage ameloblasts do express amelogenin (B–D). Note the vacuoles above the ameloblasts in the stratum intermedium (SI) that grow progressively larger as development progresses. The right side of panel D shows ameloblasts that have completely lost contact with the stratum intermedium. However, the ameloblasts also lose contact with the dentin surface and with each other (E, F). The ameloblasts in panels E and F are disorganized and appear to have lost most but not all cell adhesion properties so they appear stretched between the stratum intermedium and dentin. Od, odontoblast; SR, stellate reticulum.</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

Molar enamel organs from K14-Cre p120-cKO mice are dysplastic and are adjacent to blood filled sinusoids.

<p>Wild-type mouse molars with identified tissue structures are presented (A) as a comparison to the molars from the cKO mice (B–D). Note that the enamel space (resulting from the demineralization of enamel) in the cKO mouse molars was greatly reduced in width (B) when compared to wild-type molars. Unusual large, blood-filled sinusoids were present, such as the one between the first (M1) and second (M2) molars in this section. The enamel organ morphology became dysplastic in the p120 ablated mice (C) and a high magnification (D) of an enamel organ between two cusp tips (CT) revealed the presence of clear vacuoles (Vac) where tissues were separated. With few exceptions, the ameloblasts, stratum intermedium, and stellate reticulum layers could not be definitively identified. A thick normal appearing layer of dentin was present in the cKO mouse molar teeth. 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

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

Assessment of control and <i>Mmp20</i> transgenic enamel by light microscopy.

<p>Wild-type control (Tg– <i>Mmp20^+/+</i>) incisors had a sharp incisal tip and the characteristic yellow-brown coloration. The molars contained a fully thick enamel layer and the cusp tips were well defined. In contrast, <i>Mmp20</i> null (Tg– <i>Mmp20^−/−</i>) incisors had a blunted tip and showed little or no enamel and no yellow-brown color. The molars were worn and the remaining cusp tips appeared thin from abrasion and absence of a full thickness enamel layer. For the transgenes in the <i>Mmp20</i> null background (Tg+ <i>Mmp20^−/−</i>): The Tg6 transgenic enamel had a sharp incisal tip, but the yellow-brown color was mostly missing while the molars appeared fully recovered from the null phenotype. The Tg24 transgenic animals had incisors that appeared sharp and somewhat yellow-brown in color, but the enamel surface was rough and appeared slightly chalky rather than translucent. The molar cusp tips were worn and the enamel layer appeared hypoplastic. The Tg42 transgenic mice had incisors that were blunted with a rough enamel surface that was mildly yellow-brown color with a chalky appearance like the Tg24 transgenic incisors. The molars appeared well formed and fully recovered from the null phenotype. For the transgenes in the <i>Mmp20</i> wild-type background (Tg+ <i>Mmp20^+/+</i>): The Tg6 transgenic mice had well pigmented but blunted incisors with a chalky-white appearance and rough enamel surface. The molars were severely compromised having abraded cusp tips with pitted enamel surfaces. The Tg24 transgenic enamel appeared almost identical to the Tg6 enamel in the wild-type background. In contrast, Tg42 transgenic incisors had a sharp tip and smooth enamel, but with almost no yellow-brown color. The Tg42 transgenic molars appeared no different than the wild-type molars. The observed Tg6 transgenic enamel was the best example of an overall recovery (both incisors and molars) from the <i>Mmp20</i> null phenotype.</p