Fig. 10

pSmad3 expression is absent in nkx2.2a⁺ nuclei in ctgfa mutants. (a–c) Representative images of 5 or 6 dpf larvae with motor axons (magenta), nkx2.2a⁺ nuclei (green), and anti-pSmad3 (cyan) in ctgfa^+/+ (a), ctgfa^+/− (b), and ctgfa^−/− (c) larvae at 3 hpi. Anti-pSmad3 labeling is observed in nkx2.2a⁺ nuclei (open white arrows) and along the motor nerve (open yellow arrows) in ctgfa^+/+ and some ctgfa^+/− larvae. ctgfa^−/− larvae show loss of anti-pSmad3 staining in nkx2.2a⁺ nuclei at 3 hpi. Dashed circles indicate injury sites. Solid-line boxes indicate the area represented in enhanced single z planes and nuclei in 3D rendered images (Imaris). White dotted-lines outline nkx2.2a⁺ nuclei and anti-pSmad3 labeling. (d,e) Quantification of the percentage of nerves that had anti-pSmad3⁺ labeling (black) co-localized with nkx2.2a⁺ nuclei (d) or motor nerves (e). Chi-square tests were performed to determine significance (ctgfa^+/+: n = 52 nerves in 10 larvae, ctgfa^+/−: n = 20 nerves in 7 larvae, ctgfa^−/−: n = 23 nerves in 6 larvae). (d) 64% of ctgfa^+/+ nerves, 55% of ctgfa^+/− nerves, and 9% of ctgfa^−/− nerves had anti-pSmad3⁺ labeling in nkx2.2a⁺ nuclei (Chi-square: 71.16, df: 2, p < .0001). (e) 24% of ctgfa^+/+ nerves, 15% of ctgfa^+/− nerves, and 0% of ctgfa^−/− nerves had anti-pSmad3⁺ labeling in nkx2.2a⁺ nuclei (Chi-square: 25.99, df: 2, p < .0001). Scale bars, 25 μm; magnified insets, 10 μm; 3D renderings, 5 μm.