Figure 5. Notch inhibition mediates the proliferation of reactive tectal RG.

(A) Experimental time course of Notch inhibition experiments shown in (B–I). Fish are administrated with LY411575, a Notch inhibitor, or DMSO for two consecutive and are analyzed at 2 dpi. (B–I) LY411575 administration increases the number of proliferative RG (PCNA⁺, green cells) underneath the injury site in the optic tectum with (B and C) or without injury (D and E). (F–I) The high-magnification images of boxed areas in (B–E). (J) Quantification of PCNA⁺ cell in (B–E). LY411575 administration significantly increases the number of proliferative RG (PCNA⁺ green cells) in the optic tectum with or without the injury. Very few RG is proliferative in the uninjured DMSO-treated control optic tectum (mean ± SEM; ***p<0.001, ns, p>0.05; two-way ANOVA followed by Tukey’s HSD test). See also Figure 5—source data 1 for quantification. (K) Experimental time course of heat shock-induced Notch over-activation experiments shown in (L–M₃). Tg(hsp70l:gal4 ×UAS:NICD-Myc) fish are injured in the optic tecta and followed by heat shocks for three consecutive days (1 hr per day) and are analyzed at 3 dpi. (L–M₃) NICD-overexpressed RG (open white arrowheads, red cells) underneath the injury site are not proliferative after the stab injury whereas RG (white arrowheads, green cells) become proliferative in the control optic tectum with the injury. The expression of NICD-Myc is controlled by the gal4-UAS system. It is a mosaic labeling genetic system so that only a subset of cells could be induced to express NICD-Myc. To avoid obscure the signal, only two representative cells were indicated by arrowheads in (L–M₃). See also Figure 5—figure supplement 1C-C₃. (N) Quantification of NICD-Myc⁺/PCNA⁻ and NICD-Myc⁺/PCNA⁺ RG in (L–M₃) showing ~94% NICD-Myc-overexpressed RG are PCNA⁻. (31/33 cells in 15 sections of 6 fish). The numbers above the bars indicate the animals used. White dashed lines represent the tectal ventricle boundary. RG, radial glia; TeO, tectum opticum; PGZ, periventricular gray zone; TS, torus semicircularis. Scale bars, 100 μm (B–E); 50 μm (F–I); and 30 μm (L–M₃).

Figure 5—figure supplement 1. Notch signaling regulates the proliferation of RG.

(A–D) Representative images of PCNA (red) immunofluorescence showing 2 days’ treatment of RO4929097, a putative Notch inhibitor, results in more RG (PCNA⁺, white arrowheads) proliferation in injured (B) and uninjured (D) optic tecta. (A and C) The representative images of injured and uninjured optic tecta treated with DMSO. (E–E₃) Representative images of Myc (red) and PCNA (green) immunofluorescences showing a single RG with NICD-Myc overexpression (red cell, white arrowheads) is lack of PCNA expression (green signals) while many neighboring cells (green cells, open white arrowheads) without NICD-Myc expression are proliferative. This is the most common NICD-Myc and PCNA expression pattern in the heat shock-induced Notch over-activation experiments. (F–F₄) Representative images of her4.1:dRFP (red), PCNA (green) and DAPI (gray) immunofluorescences showing that the boundary (white dashed lines and yellow arrowheads) of PGZ and TS could be unambiguously defined by the DAPI signal. The cells under the boundary are her4.1:dRFP⁺, indicating their glial identity. The lack of PCNA expression in them indicates they are largely dormant. (G–G₃) Representative images of gfap:GFP (green) and DAPI (magenta) immunofluorescences showing that the boundary (white dashed lines and yellow arrowheads) of PGZ and TS could be unambiguously defined by the DAPI signal. White dashed lines represent the tectal ventricle boundary. RG, radial glia; TeO, tectal opticum; PGZ, periventricular gray zone; TS, torus semicircularis, Val, valvula cerebelli. Scale bars, 100 μm (A–D, F and G); and 20 μm (E–E₃, F₁–F₄ and G₁–G₃).