Fig. 1

A shelf screen identifies JNK signaling as necessary for glial bridging. (A) A shelf screen of 11 compounds identifies Fgf, Tgf? and JNK as important signaling pathways for glial bridging in embryonic spinal cord regeneration. Control treatments are shown in blue and SP600125 treatment is shown in red for emphasis for panels A, B, D, E, G and H. The percentage of glial bridging is measured by dividing the width of the GFAP+ tissue bridge at 2 dpi by the width of the uninjured spinal cord anterior to the lesion site (n=20-25 in two experiments; Kruskal?Wallis with Dunn's multiple comparisons test). (B) Inhibition of Fgf, Tgf? or JNK signaling results in fewer fish able to form GFAP+ tissue bridges, defined as at least one process visually spanning the lesion site. n=20-25 in two experiments; Fischer's exact test. Five out of 20 animals did not form a bridge after JNK inhibition, compared with none of the 22 DMSO-treated controls. (C) GFAP+ glial bridges during spinal cord regeneration in DMSO-treated and SP600125-treated animals. Glial bridging percentage was calculated by taking the ratio of the width of the thinnest point of the glial bridge (yellow arrowhead) to the full width of the uninjured cord directly anterior to the lesion (white arrowhead). Scale bar: 100 µm. (D) JNK inhibition reduces the thickness of GFAP+ glial bridges at both 1 and 2 dpi. Glial bridges in SP600125-treated animals did not significantly change in width from 1 to 2 dpi (n=100 over three repeated experiments; two-tailed Mann?Whitney test). (E) JNK inhibition results in fewer fish forming GFAP+ tissue bridges at 1 and 2 dpi (n=100 over three repeated experiments; Fischer's exact test). At 1 dpi, 55 of 144 animals did not form a bridge after JNK inhibition at, compared with nine out of 100 in DMSO-treated controls. At 2 dpi, 33 of 110 did not form a bridge after JNK inhibition compared with none of the 100 DMSO-treated controls. (F) Neural bridging labeled by whole-mount immunofluorescence for acetylated ?-tubulin. Scale bar: 100 µm. (G) JNK inhibition does not measurably affect the thickness of neural bridges at 1 and 2 dpi (n=20-30 in two experiments; two-tailed Mann?Whitney test). (H) JNK inhibition results in slightly fewer fish able to form neural bridges at 2 dpi (n=20-30 in two experiments; Fischer's exact test).