Abstract
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.
Highlights
Tooth morphogenesis is initiated by reciprocal interactions between the ectoderm and neural crest– derived mesenchyme
To confirm whether Nkx2-3 is expressed in dental epithelium or mesenchyme, embryonic day 14 (E14) dental epithelium was separated from mesenchyme under a microscope after treatment with dispase, and expression levels were determined by qRT-PCR
The dental epithelium-specific gene Krt14 was shown to be highly expressed in epithelium, whereas Vim, which encodes the mesenchyme-specific protein vimentin, was highly expressed in mesenchyme, and Nkx2-3 was mainly expressed in dental epithelium (Fig. 1D)
Summary
Microarray analysis was performed to identify expression of tooth-specific genes in the tooth morphogenesis stage. Compared with the mock group, the Nkx2-3– overexpressed group had a 2-fold increase in transcriptional activity of the WT sequence Cdkn1a promoter, whereas the use of a triple-base mutation of the Cdkn1a promoter region resulted in a decrease of transcriptional activity as compared with the WT (Fig. 4F) These findings indicated that NKX2-3 directly binds to the promoter region of Cdkn1a and regulates its gene expression in dental epithelial cells. QRT-PCR results revealed that Nkx as well as Eda and Edar were expressed at higher levels in molars as compared with incisors (Fig. 5A), suggesting that NKX2-3 is a key regulator for enamel knot formation. We added Eda-a1 to organ culture medium used for culturing teeth transfected with Nkx siRNA, which apparently abrogated the decrease in cusp height (Fig. 5C), suggesting that NKX2-3 is a target transcription factor regulated by the EDA/EDAR signaling pathway in the enamel knot (Fig. 5I). These results suggest that NKX2-3 contributes to cell fate determination of the enamel knot for cusp formation (Fig. 5I)
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