Figure 1. Injury reactivates dormant RG to proliferate and divide.

(A–B₃) Tg(gfap:GFP) (green), GS (red) and PCNA (gray) immunofluorescences show that PCNA⁺ proliferative cells (gray cells) are restricted to the TPZ (white arrow in (A)) and very few radial glia (RG) (1.4 ± 0.2%, n = 5, mean ± SEM, gray cells, white arrowheads in (B–B₃)) is PCNA⁺. (B–B₃) The high-magnification images of the boxed area (white box) in (A). (C) Schematic representation of stab injury assay. A 30G needle is stabbed into the central-dorsal region of the right hemisphere of zebrafish optic tectum. The red asterisk and yellow arrowhead indicates the injury site. RG (green cells) at the bottom of PGZ underneath the injury site are analyzed. (D–G) Tg(gfap:GFP) (green) and PCNA (red) immunofluorescences show that injury induces the proliferation of RG (GFP⁺/PCNA⁺, yellow cells) underneath the injury site at 3 days post-injury (dpi). (F and G) The high-magnification images of boxed areas in (D) and (E), respectively. (H) The design of Cre-loxP transgenic fish lines used for clonal analysis of individual tectal RG. Fish expressing mCherryT2ACreER^T2 controlled by the her4.1-promotor are crossed to red-to-green reporter fish controlled by the hsp70l promoter. In Tg(her4.1:mCherry-CreER^T2×hsp70l:DsRed2(floxed)EGFP) double transgenic fish, EGFP expression is specifically induced in her4.1-expressing RG and their progeny by TAM applications and heat shocks. (I) Experimental time course of Cre-loxP-based clonal analysis experiments shown in (J–O₁). Double transgenic fish are administrated with TAM for three consecutive days (black dots) before the injury. EdU is injected to the injured fish to label the newborn cells for six consecutive days (red dots). Fish (21 to 24 dpi) are heat-shocked to induce EGFP expression in recombined cells and their progeny. (J–K₃) Representative RG-derived clone (EGFP⁺/EdU⁺, white arrows) underneath the injury site at 8 dpi. (K–K₃) The high-magnification images of the boxed area in (J). Two EGFP⁺/EdU⁺ (white arrowheads) cells and an EGFP⁺ radial process (open white arrowhead in (J)) are found underneath the injury site in this clone. (L–N₂) Representative 1 cell (L–L₂), 2 cells (M–M₂) and 3 cells clones (N–N₂) derived from single RG in response to the stab injury. In these clones, cells are EGFP⁺/EdU⁺ newborn cells (white arrowheads). (O and O₁) The size distribution of collected 29 clones. 2-cells clones (20/29) are the most abundant clones. White dashed lines represent the tectal ventricle boundary. A, anterior; P, posterior; D, dorsal; V, ventral; L, lateral; Tel, telencephalon; OT, optic tectum; Ce, cerebellum; SC, spinal cord; TAM, tamoxifen; RG, radial glia; TeO, tectum opticum; TPZ, tectal proliferation zone; PGZ, periventricular gray zone; TS, torus semicircularis; Val, valvula cerebelli. Scale bars, 100 μm (A); 50 μm (B-B₃, D-G, and J); 20 μm (K–K₃); and 10 μm (L–N₂).

Figure 1—figure supplement 1. Tectal RG are largely dormant under physiological conditions.

(A–C₂) Representative images of Tg(gfap:GFP) (A-A₂, green), Tg(her4.1:dRFP) (B-B₂, red) and GS (C-C₂, red) immunofluorescences showing the RG extend their processes into the superficial neuropils of the optic tectum. The yellow arrowheads indicate the boundary of PGZ and TS. (D–F₁) Tg(her4.1:dRFP) (red) and BrdU (green) immunofluorescences show that BrdU signal is restricted to tectal proliferation zone (TPZ) (yellow arrows in (D–E₁)) and is absent from the RG layer, very few RG (white arrows in F-F₁) is labeled. The fish are administrated with BrdU for one day before analysis. (E–F₁) The high-magnification images of the boxed areas shown in (D). White dashed lines represent the boundary of tectal ventricle. RG, radial glia; TeO, tectal opticum; TPZ, tectal proliferation zone; PGZ, periventricular gray zone; Val, valvula cerebelli. Scale bars, 100 μm (D); 50 μm (A–C₂, E–F₁).

Figure 1—figure supplement 2. Injury responses of RG in different geographical regions in the optic tectum.

(A–B₂) Representative images of Tg(gfap:GFP) (green) and BrdU (red) immunofluorescences showing RG underneath the injury site enter the S phase at 3 dpi. After the injury, the fish are administrated with BrdU for 1 day (2-3dpi) and analyzed at 3 dpi. (B–B₂) The high-magnification images of boxed area shown in (A). White arrowheads (in B-B₂) indicate the BrdU⁺/GFP⁺ RG underneath the injury site. (C) Quantification of BrdU⁺ RG in (A–B₃) showing 30 ± 4 RG enter the S phase. (mean ± SEM, ***p<0.001; Wilcoxon test). (D) Experimental time courses of Cre-loxP-based single RG genetic labeling shown in (E). Double transgenic fish are administrated with tamoxifen (TAM) for three consecutive days and analyzed at day 8, before sacrifice the fish are heat shocked for 3 times (one hour per day) to induce EGFP expression in recombined cells. (E) Representative image of sparsely labeled tectal RG (EGFP⁺ green cells, white arrows) at day 8. (F–O) Representative images of PCNA (red) immunofluorescence showing injury induces proliferation of RG underneath the injury site at 3 dpi in different regions of optic tecta. (K–O) The high-magnification images of boxed area in (F–J). (P) Quantification of PCNA⁺ cells in (F–O). Across the five regions, only the RG in the medial-dorsal region shows low proliferation capacity after injury (mean ± SEM; **p<0.01; ns, p>0.05; one-way ANOVA followed by Tukey’s HSD test). (Q–S) Representative images of PCNA (red) and GS (green) immunofluorescences showing that injury induces less proliferation of RG (white arrowhead in (R)) in the medial-dorsal region compared with the RG (white arrowheads in (S)) in the central-dorsal region of a two-sites injured optic tectum. (R and S) The high-magnification images of boxed areas in (Q). (T) Quantification of PCNA⁺/GS⁺ RG in (R and S). Injury induces significant more RG proliferation in the central-dorsal region than in the medial-dorsal region (mean ± SEM, **p<0.01; Wilcoxon test). The numbers above the bars indicate the animals used. White dashed lines indicate the boundary of the tectal ventricle. Yellow arrowheads indicate the injury sites. A, anterior; P, posterior; D, dorsal; V, ventral; L, lateral; Tel, telencephalon; OT, optic tectum; Ce, cerebellum; SC, spinal cord; RG, radial glia; TeO, tectal opticum; PGZ, periventricular gray zone; TS, torus semicircularis; Val, valvula cerebelli. Scale bars, 100 μm (F–J and Q); 50 μm (A and E); 30 μm (F–J); 20 μm (K–O); and 10 μm (R and S).