FIGURE 6

Apoptosis level was restored by combinatorial treatment with teratogen and Wnt agonists. (A) Immunofluorescence images of apoptotic cells in the palate at 96 hpf. Experimental time course is the same as in Figure 4A. WAF (30 μM) exposure induced cleft palate and increased the number of active Cas3-positive cells in the palate. The number of active Cas3-positive cells was restored to normal by combinatorial treatment with BIO (100 nM), CHIR99021 (300 nM) or WAY-262611 (250 nM). White indicates cartilage cells double stained with anti-coll2 antibody and lectin PNA. Green dotted lines trace the shape of the palate. Yellow indicates apoptotic cells stained with anti-caspase3 antibody. (B) Quantification of the number of active Cas3-positive cells in the palate. Numerical value is normalized to 10⁴ μm². (C) VPA (20 μM) exposure induced cleft palate and increased the number of active Cas3-positive cells. The number of active Cas3-positive cells was restored to normal by combinatorial treatment with BIO (100 nM), CHIR99021 (300 nM) or WAY-262611 (250 nM). (D) Quantification of the number of active Cas3-positive cells in the palate. Numerical value is normalized to 10⁴ μm². n = 15 (Control), 15 (WAF), 14 (WAF + BIO), 14 (WAF + CHIR), 14 (WAF + WAY) in (B), n = 15 (Control), 15 (VPA), 14 (VPA + BIO), 13 (VPA + CHIR), 11 (VPA + WAY) in (D), *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA followed by Dunnett’s multiple comparison test). Scale bar, 50 μm.