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Background:  We used an  in vitro  tooth development model to investigate the effects of overactivation of the Wnt/β-catenin pathway during odontogenesis by bromoindirubin oxime reagent (BIO), a specific inhibitor of GSK-3 activity.   Results:  Overactivating the Wnt/β-catenin pathway at tooth initiation upregulated and ectopically expressed the epithelial markers  Sonic Hedgehog (Shh), Epiprofin (Epfn), and Fibroblast growth factor8 (Fgf8) , which are involved in the delimitation of odontogenic fields in the oral ectoderm. This result indicated an ectopic extension of the odontogenic potential. During tooth morphogenesis,  Fibroblast growth factor4 (Fgf4), Fibroblast growth factor10 (Fgf10), Muscle segment homeobox 1 (Msx-1), Bone Morphogenetic protein 4 (Bmp4), and Dickkopf WNT signaling pathway inhibitor 1 (Dkk-1)  were overexpressed in first molars cultured with BIO. Conversely, the expression levels of  Wingless integration site 10b (Wnt-10b)  and  Shh  were reduced. Additionally, the odontoblast differentiation markers Nestin and  Epfn  showed ectopic overexpression in the dental mesenchyme of BIO-treated molars. Moreover, alkaline phosphatase activity increased in the dental mesenchyme, again suggesting aberrant, ectopic mesenchymal cell differentiation. Finally, Bmp4 downregulated  Epfn  expression during dental morphogenesis.   Conclusions:  We suggest the presence of a positive feedback loop wherein  Epfn  and β -catenin  activate each other. The balance of the expression of these two molecules is essential for proper tooth development. We propose a possible link between Wnt, Bmp, and Epfn that would critically determine the correct patterning of dental cusps and the differentiation of odontoblasts and ameloblasts.

Wnt/β-Catenin Regulates the Activity of Epiprofin/Sp6, SHH, FGF, and BMP to Coordinate the Stages of Odontogenesis