Fig. 5

pSmad3 expression is present in perineurial glia early after injury. (a–e) Representative images of 5 or 6 dpf larvae with motor axons (magenta), nkx2.2a⁺ nuclei (green, white open arrows), and anti-pSmad3 (cyan) in uninjured (a), 2 (b), 3 (c), 6 (d) hpi larvae, and larvae treated with SB431542 at 3 hpi (e). Anti-pSmad3 labeling is observed in nkx2.2a⁺ nuclei (open white arrows) and along the motor nerve (open yellow arrows) at 2, 3, and 6 hpi. 6 dpf larvae treated with 10 μM SB431542 show loss of anti-pSmad3 staining at 3 hpi (bottom panels). 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. (f,g) Quantification of the percentage of nerves that had anti-pSmad3⁺ labeling (black) co-localized with nkx2.2a⁺ nuclei (f) or motor nerves (g). Chi-square tests were performed to determine significance. (f) 0% of uninjured nerves, 46% of 2 hpi nerves, 71% of 3 hpi nerves, and 22% of 6 hpi nerves had anti-pSmad3⁺ labeling in nkx2.2a⁺ nuclei (n = 13 nerves in 9 larvae, 20 nerves in 4 larvae, 21 nerves in 6 larvae, 17 nerves in 4 larvae, respectively) (Chi-square: 137.7, df: 3, p < .0001). (g) 0% of uninjured nerves, 31% of 2 hpi nerves, 43% of 3 hpi nerves, and 55% of 6 hpi had anti-pSmad3⁺ labeling along the nerve (n = 13 nerves in 9 larvae, 20 nerves in 4 larvae, 21 nerves in 6 larvae, 17 nerves in 4 larvae, respectively) (Chi-square: 63.92, df: 3, p < .0001). Scale bars, 25 μm; magnified insets, 10 μm; 3D renderings, 5 μm.