Induction of RG proliferation during the early stages after stab wound injury. (A–D) Representative images of proliferative RG (BLBP+ PCNA+) in intact fish (A) and injured fish during the early stages of regeneration, 6, 12, and 24 hpi (B–D). (A’–D’) Magnified images of the boxed area in each image. White arrowheads indicate BLBP+ PCNA+ cells and dashed lines (B–D) indicate area injured by needle insertion. Scale bar: 100 μm in (A–D), 50 μm in (A’–D’). (E) Quantification of proliferative RG in intact fish (n = 4) and on the uninjured and injured hemispheres of injured fish at 6, 12, and 24 hpi. Statistical analyses between uninjured and injured hemispheres at each time point were evaluated using paired Student’s t-tests. (F) Quantification of proliferative cells except RG (BLBP-PCNA+) in intact fish (n = 4) and the uninjured and injured hemispheres of injured fish at 6, 12, and 24 hpi. Statistical analyses between uninjured and injured hemispheres at each time point were evaluated using paired Student’s t-tests.

Induction of IB4+ macrophage migration during the early stages after stab wound injury. (A–D) Representative images of IB4-positive macrophages in intact fish (A) and the injured hemisphere of injured fish during the early stages of regeneration, 6, 12, and 24 hpi (B–D). (A’–D’) Magnified images of the boxed area in each image. White arrowheads indicate BLBP+ PCNA+ cells and dashed lines (B–D) indicate the area injured by needle insertion. Scale bar: 100 μm in (A–D), 50 μm in (A’–D’). (E) Quantification of IB4-positive cells in intact fish (n = 4) and the uninjured and injured hemispheres of injured fish at 6, 12, and 24 hpi. Statistical analyses between uninjured and injured hemispheres at each time point were evaluated using paired Student’s t-tests.

Transcriptomic and enrichment analyses during the early stages of optic tectum regeneration. (A,B) Venn diagram indicating upregulated (A) and downregulated (B) differentially expressed genes (DEGs) identified by DEseq2 between injured (6, 12, and 24 hpi) and intact optic tectum. Fold change >2 in both directions and FDR < 0.05. (C–E) Heatmaps indicating clusters of significantly enriched GO biological process (BP) (C), KEGG pathway (D), and Reactome pathway (E) terms based on DEGs between the injured and intact tectum (6, 12, and 24 hpi) using Metascape enrichment analysis. UP and DOWN indicate enrichment analyses based on up- and downregulated DEGs, respectively.

Activation of IL6/Stat3 signaling during the early stages of optic tectum regeneration. (A–F) Quantitative real-time (qRT-) PCR analyses of IL6 superfamily cytokines il6 (A), il11a (B), and il11b (C), and Jak/Stat signaling components stat3 (D), jak1 (E), and gfap (F). Each graph indicates relative gene expression in the injured tectum from 1 to 7 dpi compared to intact optic tectum (n = 4 per gene per time point). Statistical analyses between intact and injured hemispheres at each time point were evaluated using unpaired Student’s t-tests. (G) Schematic diagram of the cell sorting workflow using the RG-specific reporter line, Tg(gfap:GFP). (H) Representative images of FACS plots for GFP-positive cells from the optic tectum of wild-type (left) or Tg(gfap:GFP) (right) fish. (I) Quantification of stat3 expression in GFPhigh-positive cells from uninjured and injured optic tectum at 24 hpi using qPCR. The graph shows relative stat3 expression in GFP^high-positive cells from injured tectum at 24 hpi compared to uninjured tectum (n = 11). Statistical analyses between uninjured and injured hemispheres was evaluated using unpaired Student’s t-tests.

Stat3 inhibitor S3I-201 suppresses RG proliferation after stab wound injury in adult optic tectum. (A–C) Representative images of proliferative RG (BLBP+ PCNA+) in injured optic tectum treated with 1% DMSO (A), 10 μM S3I-201 (B), or 100 μM S3I-201 (C) at 1 dpi. (A’–C’) Magnified images of the boxed area in each image. White arrowheads indicate BLBP+ PCNA+ cells and dashed lines indicate the area injured by needle insertion. Scale bar: 50 μm in (A–C) and (A’–C’). (D) Schematic diagram of drug administration. (E) Quantification of proliferative RG (BLBP+ PCNA+) treated with 1% DMSO, 10 μM S3I-201 (n = 4), or 100 μM S3I-201 (n = 5) at 1 dpi. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test. (F) Quantification of proliferative cells except RG (BLBP-PCNA+) treated with 1% DMSO, 10 μM S3I-201 (n = 4), or 100 μM S3I-201 (n = 5) at 1 dpi. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test.

IL6 induces RG proliferation without stab wound injury in adult optic tectum. (A,B) Representative images of proliferative RG (BLBP+ PCNA+) in the contralateral side with PBS or 100 ng/μL IL6 at 1 dpi. Allow heads indicate BLBP+ PCNA+ cells and arrows indicate BLBP- PCNA+ cells. Scale bar: 100 μm in (A–D) and (A’,B’). (C) Schematic diagram of cerebroventricular microinjection. A small hole was made in the center of the skull above the right hemisphere of the optic tectum using a 30G needle. The injected solution spread through the corticospinal fluid. (D) Quantification of proliferative RG (BLBP+ PCNA+) in the injected and contralateral hemisphere with PBS (n = 4) or 100 ng/μL IL6 (n = 3) at 1 dpi. (E) Quantification of BLBP- PCNA+ cells in the injected and contralateral hemisphere with PBS or IL6 at 1 dpi. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test.