The transcription factor NKX2-3 mediates p21 expression and ectodysplasin-A signaling in the enamel knot for cusp formation in tooth development

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 Nkx2-3, 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 Nkx2-3 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 Bmp2 and Bmpr2 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.