The Role Of Hippo Signaling Pathway In Mouse Tooth Development

Background and ObjectiveActivation of the Hippo pathway tumor suppressor genes Mstl/2-Savl and Latsl/2-Mob lead to phosphorylation of YAP/TAZ and induce its degradation. YAP/TAZ is also regulated by cell-cell contact via tight junction and adheres junction. Inhibition of Hippo signaling pathway relieves YAP/TAZ, resulting in its nuclear translocation. The Hippo signaling pathway controls organ size in animals through the regulation of cell proliferation and apoptosis. Yap is inactivated during mouse embryonic stem cell differentiation and activated in induced pluripotent stem (iPS) cells. Yap knockdown in mouse embryonic stem cells leads to loss of pluripotency, whereas ectopic expression of Yap prevents embryonic stem cell differentiation Additionally, the Hippo pathway also regulates tissue-specific progenitor cells. Yap expression is generally restricted to the progenitor cells in normal mouse intestines, and transgenic expression of Yap in mouse intestines causes a marked expansion of the progenitor cell. Yap induced loss of differentiation. Yap expression expands basal epidermal progenitors in mouse skin and inhibits their terminal differentiation. Yap activation reversibly increases liver size more than4-fold and also dramatic lead myocardial overgrowth causing near chamber obliteration. In mammals, there is also evidence for a role of Yap in tissue regeneration. In wild-type crypts, Yap was detected in the entire crypt epithelium. Intestinal damage markedly induces Yap expression, and loss of Yap severely impairs dextran sodium sulfate-induced intestinal regeneration. In the mouse liver, previous results found that loss of Yap in the liver not only results in defects in hepatocyte survival, but also leads to a profound defect in bile duct development.The growth of mouse incisors continues throughout life and is supported by dental epithelial stem cells localized within the cervical loop of the incisor tooth organ. The dental epithelium gives rise to the inner enamel epithelium (IEE), stratum intermedium, stellate reticulum (SR), and outer enamel epithelium (OEE). The IEE was previously proposed to represent transit-amplifying (TA) cells and will differentiate into ameloblasts. In the labial cervical loop, the epithelial stem cells proliferate actively and migrate along the labial surface, differentiating into enamel-forming ameloblasts. In contrast, the lingual cervical loop contains fewer proliferating stem cells, and the lingual surface of the incisor lacks ameloblasts and enamel The Pitx2gene encodes three isoforms, Pitx2a, Pitx2b and Pitx2c in mice and a fourth Pitx2isoform, Pitx2d, has been identified in humans. Pitx2a, Pitx2b and the Pitx2c isoforms are co-expressed in oral ectoderm and, at later stages, within tooth epithelial structures.MethodsThe embryo from different mice, including Pitx2-Cre; TomatoAi14and C57, were sampled at different embryonic stages, and fluorescent tracer method was used to detect the Pitx2gene expression pattern at different stages. The in situ was used to determine the Yap gene expression manner during the mouse tooth development. Pitx2-Cre recombinase was used to generate the Yap conditional knockout and transgenic mouse, to determine the role of Yap in the development of dental epithelium. ResultsPart11. Pitx-Cre were highly expressed in molar, incisor, inner and outer epithelium and cervical loop at different states, including cape, bell and secretary stages.2. Yapl were expressed in both molar and incisor, and it main distributes in lingual side cervical loop region, and lower expression in dental mesenchyme.Part21. Pitx2-Cre; Yapflox/flox had the similar body size and normal facial development as wild distribution. The surface is smoothness compared with wild type. The u-CT scan showed that the enamel of molar exhibited low density. The occlusal surfaces showed enamel defect, and the mesio-distal side showed serious defect; however, the buccal and lingual side was normal. The outline of the high point lines changed dramatically, the tooth root showed bigger size and the periodontal ligament is not obvious. Incisors showed high-density in lingual side was the ectopic expression of enamel, while the buccal side enamel coverage decreased, only a small amount of enamel covering near the midline.2. The SEM results showed cut like shaped incisor, and small amount of prism wwrapped by matrix. The eruption time was delayed, and the cusp development was abnormal, with a large number of enamel beads surrounded it.3. H&E staining results showed normal morphology in the first molar at E14.5; at E16.5/E18.5molar size was reduced, and the dental papilla and dental follicle seemed normal. The inner and outer enamel epithelial layer was break; a large blood vessel was found in the stellate reticulum and showed a trend of invasive to dental papilla layer. The size of cervical loop was decreased at E16.5/E18.5; the dental papilla and dental follicle was normal compared with wild type.4. In situ results showed fgf3,Fgf9were found in the cervical loop region. Msxl expression was decreased; Amelogenin expression was finding at lingual side enamal region. The Dspp expression was also increased at E18.5in the odontoblast. The p21 expression was normal in the incisor at E18.5.Part31. When the Yap was knockout driven by Pitx2-Cre, the mouse body size became smaller than wild type mouse. On the3weeks old, the defect on the enamel appeared, with a low density appearance. The root was shorter than normal and the periodontal ligaments were not as obvious as wild type. Enamel was disappeared in the occlusal surface of molar; the enamel cover area in the incisor reduced, with only a small amount in the midline.2. HE staining results showed that the appearance molar dental papilla and dental follicle were normal at E14.5, E16.5, and E18.5. At the same time, the development of outer epithelium layer and SR were not affected. On E16.5, inner epithelium expanded slightly, and on E18.5, cervical loop area increased. On E16.5and E18.5, the dental follicle and dental papilla was normal; the cervical loop area expands, and the outer epithelium showed hyperplasia.3. Brdu staining showed no obvious changes in molar dental papilla and dental follicle. In enamel organ region, the proliferation was normal. In incisor, the dental papilla, dental follicle and inner epithelium was normal, but outer epithelium proliferation increased.4. In situ results showed that no difference in Fgf3expression; Fgf9expression was increased in labial side cervical loop; Msx1expression density was not changed, but localization was changed, with expression on outer epithelium region. For Amelogenin, there was no significant difference found. For Dspp, its expression was increased in the incisor tip, and the p21was also expressed on this region.Conclusion1. Yap consistently expresses in the normal mouse tooth development but the location and intensity are different; Pitx2-Cre can drive gene expression in specific area.2. Pitx2-Cre mediated specific knockout in dental epithelial cells, inhibit the labial cervical loop stem cell proliferation, resulting in the labial side enamel development disorders and lead to label side cervical loop size reduced; lingual ameloblast differentiation increase and lead to ectopic enamel.3. Pitx2-Cre mediated of dental epithelial cells over expression inhibit ameloblast differentiation and minimization.a large emale defect apaears in the molar and incisor,the lable side of cervical loop become expansion.