Fig. 7

Tracing and CRISPR/Cas9 mutagenesis of iNeuron marker genes in zebrafish.A?C Rostral-to-caudal neuronal tracing at 1 wpi. Biocytin was applied 4 mm rostral to the lesion and co-labeled with syt11b and HuC/D. SC cross sections 750 ?m caudal to the lesion are shown. Dotted lines delineate central canal edges. Biocytin-labeled neurons were normalized to total neurons in (B). Biocytin-labeled iNeurons were normalized to traced neurons in (C). D Pipeline for CRISPR/Cas9 mutagenesis of iNeuron marker genes and regeneration assessment. E Swim endurance in CRISPR/Cas9 targeted animals at 4 wpi. Uninjected control siblings were used (ANOVA p < 0.0001). F Swim behavior in targeted animals at 4 wpi (ANOVA p < 0.0001). Swim distance was tracked under water current velocities of 0, 10 and 20 cm/s. G Glial bridging in targeted animals at 4 wpi. Gfap+ bridges are shown at the lesion site of atf3, vamp4 and syt11a/b crispants relative to controls. Percent bridging represents the cross-sectional area of the glial bridge at the lesion (0 µm) relative to the intact SC 750 µm rostral to the lesion (ANOVA p: 0.0049). H Anterograde axon tracing in targeted animals at 4 wpi. Biocytin was applied rostrally and analyzed 600 ?m (proximal) (ANOVA p < 0.0001) and 1500 ?m (distal) (ANOVA p < 0.0001) caudal to the lesion. Representative biocytin traces are shown at the proximal level. Axon growth was first normalized to biocytin 450 and 750 µm rostral to the lesion, and then to labeling in wild-type controls. Data points in all bar charts indicate individual animal and sample sizes are indicated in parentheses. Brown-Forsythe and Welch ANOVA were performed in (G) with Dunnett?s T3 multiple comparisons performed across different time points with 95% CI. Ordinary one-way ANOVA was performed in (E) with Dunnett?s multiple comparison with 95% CI. Two-way ANOVA with Tukey?s multiple comparisons (95% CI) was performed in (F, H). In all bar charts, data are presented as mean values +/- SEM. All statistical tests are two-sided. *p ? 0.05, **p ? 0.01, ***p ? 0.001. Scale bars, 50 µm. Source data are provided as a Source Data file.