Fig. 6.

Wnt3a protein acts as a proliferation stimulant on Axin2-positive tympanic border cells and their descendants. (A-E) When cultured with Wnt3a for 10 days, only Axin2^hi colonies significantly upregulated Ki67 expression (P<0.05). Conversely, the soluble Wnt antagonist Fz8CRD effectively suppressed Ki67-positive colonies in comparison with IgG-only control (P<0.005, n=3-5). (F,G) Wnt3a-treated Axin2^hi colonies robustly expressed Axin2 when compared with vehicle-only control. (H-J) Axin2^hi and Axin2^lo cells from Actin-DsRed-positive Axin2^lacZ/+ cochleae were cultured and DsRed-positive colonies were examined. Axin2^hi cells had higher colony forming capacity than Axin2^lo cells (P<0.01, one-way ANOVA). Wnt3a treatment significantly increased the Axin2^hi and not the Axin2^lo colonies (P<0.05, one-way ANOVA, n=3). (K-L″′) Third generation colonies from Wnt3a-treated Axin2^hi cells expressed myosin 7a and Sox2 (supplementary material Fig. S5F). Scale bars: 25 μm in A-D,F,G,K-L″′; 50 μm in H-I′. Data are mean±s.d. Asterisks indicate statistical significance.