Abstract
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.
Highlights
The mouse tooth is a good model for the study of regulatory pathways involved in cell differentiation, proliferation and organogenesis
To clarify the mechanism by which Wingless integration site (Wnt)/β-catenin-mediated hyperdontia occurs, we studied the expression of selected genes related to the initiation of odontogenesis during the early stages of dental development, after overactivating the canonical Wnt pathway by bromoindirubin oxime reagent (BIO) in isolated mouse branchial arches
When we studied the apoptotic effect of 20 μM BIO in E14.5 molars cultured for 6 days, Caspase3 was detected in cells of the oral epithelium of control and BIO-treated molars
Summary
The mouse tooth is a good model for the study of regulatory pathways involved in cell differentiation, proliferation and organogenesis. Mouse tooth development begins around embryonic day 10.5 (E10,5) with a local thickening and oral epithelial invagination. Continuation of this invagination process results in the formation of epithelial tooth buds at E12.5–13.5. At approximately E12.5, the potential to induce tooth formation is transferred from the dental epithelium to the dental mesenchyme (Mina and Collar, 1987; Lumsden, 1988). 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
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