Figure 6.

Wnt/β-catenin signaling modulates reparative dentinogenesis. Pulp cells rapidly proliferate in response to tooth damage shown by PCNA staining, with a significant peak in proliferation occurring 3 days post-damage and returning to baseline 14 days post-damage (Fig. 2). New odontoblast-like cells are detected by DSPP expression as early as 5 days post-damage and a dentine bridge is seen 14 days post-damage (Fig. 1). Our data shows that pulp exposure first triggers proliferation, followed by odontoblast differentiation and secretion of reparative dentine to form a dentine bridge. Wnt reporter mice (TCF/Lef:H2B-GFP) demonstrated proliferating cells are Wnt responsive 3 days post-damage (Fig. 3). Real-time qPCR analysis of Axin2 expression demonstrated that Axin2 is significantly elevated 3 days post-damage, indicating that the Wnt responsive cells are Axin2 positive ((Fig. 3C), Supplementary Figure 1). Lineage tracing of Axin2 cells in Axin2^{CreERT2, Rosa26 mTmG fl/+} mice demonstrated that these cells undergo a proliferative expansion and differentiate into odontoblast-like cells indicated by their co-expression of Dspp 5 days post-damage, characteristic odontoblast morphology and close association with the dentine bridge 14 days post-damage (Fig. 4). Loss of Wnt signaling in Wls^fl/fl mice demonstrated that damaged teeth no longer repair as a dentine bridge is absent 14 days post-damage compared to WT (Fig. 5B). Moreover, specifically deleting Wls in Axin2-expressing cells in Axin2^CreERT2; Wls^fl/fl mice severely impaired dentine bridge formation 14 days post-damage compared to WT (Fig. 5D). This suggests that Axin2 cells are producing their own source of Wnt to modulate their fate in an autocrine manner. Additionally, Wnt signaling is important for damage induced proliferation as the number of proliferating cells are significantly reduced in Axin2^CreERT2; Wls^fl/fl compared to WT at 3 and 5 days post-injury (Fig. 5E).