Tooth morphogenesis is initiated by reciprocal interactions between the ectoderm and neural crest-derived mesenchyme. During tooth development, tooth cusps are regulated by precise control of proliferation of cell clusters, termed enamel knots, that are present among dental epithelial cells. The interaction of ectodysplasin-A (EDA) with its receptor, EDAR, plays a critical role in cusp formation by these enamel knots, and mutations of these genes is a cause of ectodermal dysplasia. It has also been reported that deficiency in , encoding a member of the NK2 homeobox family of transcription factors, leads to cusp absence in affected teeth. However, the molecular role of NKX2-3 in tooth morphogenesis is not clearly understood. Using gene microarray analysis in mouse embryos, we found that is highly expressed during tooth development and increased during the tooth morphogenesis, especially during cusp formation. We also demonstrate that NKX2-3 is a target molecule of EDA and critical for expression of the cell cycle regulator p21 in the enamel knot. Moreover, NKX2-3 activated the bone morphogenetic protein (BMP) signaling pathway by up-regulating expression levels of and in dental epithelium and decreased the expression of the dental epithelial stem cell marker SRY box 2 (SOX2). Together, our results indicate that EDA/NKX2-3 signaling is essential for enamel knot formation during tooth morphogenesis in mice.

Epithelial-mesenchymal interaction has critical roles for organ development including teeth, during which epithelial thickening and mesenchymal condensation are initiated by precise regulation of the signaling pathway. In teeth, neural crest-derived mesenchymal cells expressed PDGF receptors migrate and become condensed toward invaginated epithelium. To identify the molecular mechanism of this interaction, we explored the specific transcriptional start sites (TSSs) of tooth organs using cap analysis of gene expression (CAGE). We identified a tooth specific TSS detected in the chromosome 15qD1 region, which codes microRNA-875 (mir875). MiR875-5p is specifically expressed in dental mesenchyme during the early stage of tooth development. Furthermore, PRRX1/2 binds to the mir875 promoter region and enhances the expression of mir875. To assess the role of miR875-5p in dental mesenchyme, we transfected mimic miR875-5p into mouse dental pulp (mDP) cells, which showed that cell migration toward dental epithelial cells was significantly induced by miR875-5p via the PDGF signaling pathway. Those results also demonstrated that miR875-5p induces cell migration by inhibiting PTEN and STAT1, which are regulated by miR875-5p as part of post-transcriptional regulation. Together, our findings indicate that tooth specific miR875-5p has important roles in cell condensation of mesenchymal cells around invaginated dental epithelium and induction of epithelial-mesenchymal interaction. Tooth morphogenesis, initiated by reciprocal interactions between the ectoderm and neural crest-derived mesenchyme 1-3 , is a good model for understanding the molecular mechanism of epithelial-mesenchymal interaction, because of the well-defined developmental stages and distinctive cell types. In mice, that morphogenesis is initiated by thickening of dental epithelium to form the dental placode, followed by invagination into mesenchyme on embryonic day (E)11.5. In addition, neural crest-derived mesenchymal cells migrate and condense toward the dental epithelial placode. Thereafter, mesenchyme condenses around the epithelial tooth bud and gains an ability for tooth morphogenesis via expression of a particular set of transcription factors and signaling molecules 3,4. During tooth morphogenesis, invaginated epithelium and condensed mesenchymal tissues interact with each other (epithelial-mesenchymal interaction), and form the shape of the tooth. Finally, dental epithelium and mesenchyme differentiate into ameloblasts, which secretes enamel, and odontoblasts, secreting dentin. However, the molecular mechanism of mesenchymal cell migration and condensation have yet to be clearly elucidated. Cranial skeletal tissues including tooth, bone, nerves, and blood vessels are mainly derived from neural crest cells (NCCs) 5-7. The neural crest is composed of transient embryonic tissues present during neural tube formation, whose cells have a high potential for migration and differentiation 7-9. Disruption of cranial NCC migration during embryonic development can lead t...

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