Figure 6. Injury-induced RG are largely undergoing gliogenesis.

(A–B₃) Images of a representative 2 cells clone at 77 dpi. Both cells are EdU⁺/BLBP⁺/HuC/D⁻ RG (white arrowheads, white dashed circles). The white asterisk in (A) indicates the blood vessel. Yellow asterisks in (B₁–B₃) indicate other EdU⁺/BLBP⁺/HuC/D⁻ RG underneath the injury site at 77 dpi. (B–B₃) The high-magnification images of the boxed area in (A) (C) A schematic showing the procedures used for 7 days EdU pulse-and-stain assay. Fish are injected with EdU for six consecutive days after the injury, the injured and uninjured optic tecta are analyzed at 7 dpi. (D–G₃) Representative images of EdU (red), BLBP (gray) and HuC/D (green) immunofluorescences showing that most of the newborn cells are EdU⁺/BLBP⁺ RG, while a few newborn cells are EdU⁺/HuC/D⁺ neurons (white arrowheads). The newborn RG forms a bulge underneath the injury site. (E-E₃) The high-magnification images of the boxed area in (D). (F) The representative image of uninjured optic tectum. (G–G₃) The high-magnification images of the boxed area in (F). (H) Quantification of EdU⁺ newborn cells, EdU⁺/BLBP⁺/HuC/D⁻ newborn RG and EdU⁺/BLBP⁻/HuC/D⁺ newborn neurons at 7 dpi. The number of EdU⁺/BLBP⁻/HuC/D⁺ newborn neurons on the injured side is significantly increased compared with that on the uninjured side (mean ± SEM; ***p<0.001, **p<0.01, Wilcoxon test). See also Figure 6—source data 1 for quantification. (I) A schematic showing the procedure of EdU pulse-and-staining assay for 300 days long-term tracing. Fish are injected with EdU for six consecutive days after the injury, the injured and uninjured optic tecta are analyzed at 300 dpi. (J–M₃) Representative images of EdU (red), BLBP (gray) and HuC/D (green) immunofluorescences showing that EdU⁺ newborn cells that survive up to 300 dpi are largely EdU⁺/BLBP⁺ newborn RG, but a few cells are EdU⁺/HuC/D⁺ newborn neurons (white arrowheads). (K–K₃) The high-magnification representative images of the boxed area in (J). (L) The representative image of uninjured optic tectum. (M–M₃) The high-magnification representative images of the boxed area in (L). (N) Quantification of EdU⁺ newborn cells, EdU⁺/BLBP⁺/HuC/D⁻ newborn RG and EdU⁺/BLBP⁻/HuC/D⁺ newborn neurons at 300 dpi (mean ± SEM, ***p<0.001, *p<0.05, Wilcoxon test). See also Figure 6—source data 2 for quantification. The numbers above the bars indicate the animals used. White dashed lines represent the tectal ventricle boundary. Yellow dashed lines indicate the boundary of bulges. RG, radial glia; TeO, tectum opticum; PGZ, periventricular gray zone; TS, torus semicircularis. Scale bars, 20 μm (A-B₃, E-E₃, G-G₃, K-K₃, and M-M₃); 50 μm (D, F, J and L).

Figure 6—figure supplement 1. The injury wounds are failed to be restored.

(A–B₃) Representative images of EdU (red), HuC/D (green) and BLBP (gray) immunofluorescences showing a EdU⁺/HuC/D⁺ neuron migrates to the upper region of injured optic tectum at 300 dpi. (B–B₃) The high-magnification images of boxed area in (A). White arrowheads indicate the EdU⁺/HuC/D⁺ neuron. Yellow dashed lines indicate the RG layer. White dashed lines and white asterisks indicate the stab wound that is not restored at 300 dpi. (C–D₃) Representative images of EdU (red), HuC/D (green) and BLBP (gray) immunofluorescences showing the processes terminal of RG near the injury site are hypertrophic with high level of BLBP expression at 7 dpi. (D–D₃) The representative images of uninjured optic tectum. (E) Quantification of the area of stab wounds at 1–4 (1343 ± 315.7 μm²) and 300–400 dpi (1339 ± 768.6 μm²) showing that the stab wounds are not completely restored (mean ± SEM, ns, p>0.05; Wilcoxon test). (F–I₃) Representative images of EdU (red), HuC/D (green) and BLBP (gray) immunofluorescences showing stab wound that is surrounded by hypertrophic RG processes terminal (high level of BLBP expression, gray signals) is remained at 300 dpi. (H–H₃) are the representative images of uninjured optic tectum. (G–G₃) and (I–I₃) The high-magnification images of boxed areas in (F) and (H), respectively. (J and K) t-SNE and violin plots showing that hmgb2a and hmgb2b are enriched in proliferative-S and -G2 RG. (L–M₂) In situ hybridization showing PCNA⁺ RG (green cells) express hmgb2a (red signals) at 3 dpi. (M–M2) are the representative images of uninjured optic tectum. Yellow arrow heads indicate the injury site. RG, radial glia; TeO, tectal opticum; PGZ, periventricular gray zone; TS, torus semicircularis; Val, valvula cerebelli. Scale bars, 50 μm (A, F and H); 30 μm (B–B₃, C–C₃, D–D₃, G–G₃, I–I₃ and L–M₂).