The tooth root is an important part of the tooth that works together with the surrounding periodontium to maintain the tooth in the alveolar socket. The root develops after crown morphogenesis. While the molecular and cellular mechanisms of early tooth development and crown morphogenesis have been extensively studied, little is known about the molecular mechanisms controlling tooth root formation. Here, we show that β-catenin is strongly expressed in odontoblast-lineage cells and is required for root formation. Tissue-specific inactivation of β-catenin in developing odontoblasts produced molars lacking roots and aberrantly thin incisors. At the beginning of root formation in the mutant molars, the cervical loop epithelium extended apically to form Hertwig's epithelial root sheath (HERS), but root odontoblast differentiation was disrupted and followed by the loss of some HERS inner layer cells. However, the outer layer of the HERS extended without the root, and the mutant molars finally erupted. The periodontal tissues extensively invaded the dental pulp. These results indicate that there is a cell-autonomous requirement for Wnt/β-catenin signaling in the dental mesenchyme for root formation.

Wnt signaling plays an essential role in the dental epithelium and mesenchyme during tooth morphogenesis. However, it remains unclear if Wnt ligands, produced from dental mesenchyme, are necessary for odontoblast differentiation and dentin formation. Here, we show that odontoblast-specific disruption of Wntless (Wls), a chaperon protein that regulates Wnt sorting and secretion, leads to severe defects in dentin formation and root elongation. Dentin thickness decreased remarkably and pulp chambers enlarged in the mandibular molars of OC-Cre;Wls CO/CO mice. Although the initial odontoblast differentiation was normal in the mutant crown, odontoblasts became cuboidal and dentin thickness was reduced. In immunohistochemistry, Wnt10a, β-catenin, type I collagen, and dentin sialoprotein were significantly down-regulated in the odontoblasts of mutant crown. In addition, roots were short and root canals were widened. Cell proliferation was reduced in the developing root apex of mutant molars. Furthermore, Wnt10a and Axin2 expression was remarkably decreased in the odontoblasts of mutant roots. Deletion of the Wls gene in odontoblasts appears to reduce canonical Wnt activity, leading to inhibition of odontoblast maturation and root elongation.

Bone and dentin share similar biochemical compositions and physiological properties. Dentin, a major tooth component, is formed by odontoblasts; in contrast, bone is produced by osteoblasts. Osterix (Osx), a zinc finger-containing transcription factor, has been identified as an essential regulator of osteoblast differentiation and bone formation. However, it has been difficult to establish whether Osx functions in odontoblast differentiation and dentin formation. To understand the role of Osx in dentin formation, we analyzed mice in which Osx was subjected to tissue-specific ablation under the control of either the Col1a1 or the OC promoter. Two independent Osx conditional knockout mice exhibited similar molar abnormalities. Although no phenotype was found in the crowns of these teeth, both mutant lines exhibited short molar roots due to impaired root elongation. Furthermore, the interradicular dentin in these mice showed severe hypoplastic features, which were likely caused by disruptions in odontoblast differentiation and dentin formation. These phenotypes were closely related to the temporospatial expression pattern of Osx during tooth development. These findings indicate that Osx is required for root formation by regulating odontoblast differentiation, maturation, and root elongation. Cumulatively, our data strongly indicate that Osx is a site-specific regulator in tooth root formation.

Transforming growth factor β (TGF-β) signaling has been implicated in dentin formation and repair; however, the molecular mechanisms underlying dentin formation remain unclear. To address the role of TGF-β signaling in dentin formation, we analyzed odontoblast-specific Tgfbr2 conditional knockout mice. The mutant mice had aberrant teeth with thin dysplastic dentin and pulpal obliteration, similar to teeth from human patients with dentinogenesis imperfecta type II and dentin dysplasia. In mutant, the odontoblasts lost their cellular polarity, and matrix secretion was disrupted after mantle dentin formation. As a consequence, the amount of predentin decreased significantly, and an ectopic fibrous matrix was formed below the odontoblast layer. This matrix gradually calcified and obliterated the pulp chamber with increasing age. Immunohistochemistry revealed decreased expression of alkaline phosphatase in mutant odontoblasts. In mutant dentin, Dsp expression was reduced, but Dmp1 expression increased significantly. Collagen type I, biglycan, and Dsp were expressed in the ectopic matrix. These results suggest that loss of responsiveness to TGF-β in odontoblasts results in impaired matrix formation and pulpal obliteration. Our study indicates that TGF-β signaling plays an important role in dentin formation and pulp protection. Furthermore, our findings may provide new insight into possible mechanisms underlying human hereditary dentin disorders and reparative dentin formation.

Feng et al. 2015) in response to our article titled "Osterix Regulates Tooth Root Formation in a Site-specific Manner" (Kim et al. 2015).A major issue is raised from our interpretation about the localization of Osterix and dentin phenotypes of 2 independent conditional Osterix knockout mouse lines (Kim et al. 2015). Dr. Feng's group recently reported that Osterix has an essential role in tooth root but not crown dentin formation (Zhang et al. 2015). Regardless of the difference in transactivators, dentin phenotype seems to be similar in Osterix mutants of 2 reports.In our observation, Osterix mutant mice did not show remarkable dentin phenotype in crown but did exhibit severe impairment of interradicular dentin in molar roots. In this region, both dental mesenchyme and HERS proliferation was relatively lower than in the root elongation region. This finding indicates that Osterix plays a critical role in odontoblast differentiation in a site-specific manner. In this context, it is notable that "rootless" teeth are produced without crown defect in the tissue-specific β-catenin inactivation mice (Kim et al. 2013).We also examined tooth phenotype of Osterix null newborn mice (Appendix Fig.). Although crown morphogenesis was normal, differentiation of both odontoblasts and ameloblasts was disrupted as reported previously (Clarke et al. 2014). In addition, Osterix is known to be short-lived protein, easily sumoylated and ubiquitinated (Hosoya et al. 2013;Peng et al. 2013). It could be associated with the discrepancies in Osterix expression between mRNA and protein level.We hope that our finding could contribute to further understanding the principles underlying tooth root formation.