Figure 7. Notch inhibition promotes the neurogenesis of reactive tectal RG.

(A) A schematic of Notch inhibition experiments shown in (B–D). In the control group, fish are administrated with DMSO from 1 to 6 dpi. In experimental groups, fish are administrated with LY411575 during either 1 to 3 dpi or 4 to 5 dpi. EdU is injected for six consecutive days after the injury. All the fish are sacrificed and analyzed at 7 dpi. (B–D) Representative images of HuC/D (green) and EdU (red) immunofluorescences of 7-dpi optic tecta treated with DMSO for 1–6 dpi, LY411575 for 1–3 dpi, or LY411575 for 4–5 dpi showing that significant more EdU⁺/HuC/D⁺ newborn neurons are only generated after the treatment of LY411575 during 4–5 dpi. White arrowheads indicate EdU⁺/HuC/D⁺ newborn neurons. (E–F) Quantification of EdU⁺ newborn cells (E) and EdU⁺/HuC/D⁺ newborn neurons (F) in (B–D). While Notch inhibition of 1–3 dpi or 4–5 dpi significantly increases the number of EdU⁺ newborn cells in the injured optic tectum, Notch inhibition during 4–5 dpi but not 1–3 dpi significantly increases the number of EdU⁺/HuC/D⁺ newborn neurons in the injured optic tectum. In the uninjured optic tecta, Notch inhibition during both 1–3 dpi and 4–5 dpi increases the number of EdU⁺ newborn cells, but not EdU⁺/HuC/D⁺ newborn neurons (mean ± SEM, ***p<0.001, **p<0.01, *p<0.05, ns, p>0.05; one-way ANOVA followed by Tukey’s HSD test). See also Figure 7—source datas 1 and 2 for quantification. (G) Proportion of EdU⁺/HuC/D⁺ newborn neurons to EdU⁺ newborn cells in (B–D). Notch inhibition during 4–5 dpi increases the proportion of the neuron production, whereas Notch inhibition during 1–3 dpi decreases the proportion (mean ± SEM, **p<0.01; ns, p>0.05; one-way ANOVA followed by Tukey’s HSD test). See also Figure 7—source data 3 for quantification. (H and I) Schematics of the experimental procedure for Notch inhibition experiments shown in (J-M). After the injury, fish are treated with either DMSO or LY411575 during 4–5 dpi and are injected with EdU for three consecutive days during 1–3 dpi (H) or 4–6 dpi (I). All the fish are sacrificed and analyzed at 7 dpi. (J–M) Representative images of HuC/D (green) and EdU (red) immunofluorescences of the 7-dpi optic tecta after the treatment in (H and I). With the treatment of LY411575 during 4–5 dpi, EdU pluses during 4–6 dpi (L and M) but not 1–3 dpi (J and K) label significant more newborn neurons. White arrowheads indicate EdU⁺/HuC/D⁺ newborn neurons. (N and O) Quantification of EdU⁺ newborn cells (N) and EdU⁺/HuC/D⁺ newborn neurons (O) in (J–M) (≥3 replicates for each group; mean ± SEM, ***p<0.001, ns, p>0.05; two-way ANOVA followed by Tukey’s HSD test). See also Figure 7—source datas 4 and 5 for quantification. (P) Proportion of EdU⁺/HuC/D⁺ newborn neurons to EdU⁺ newborn cells in (J–M). EdU pulses during 4–6 dpi significantly increase the proportion of neuron production (≥3 replicates for each group; mean ± SEM, ***p<0.001; ns, p>0.05; two-way ANOVA followed by Tukey’s HSD test). See also Figure 7—source data 6 for quantification. (Q) Schematic summary of the working model. Injury induces all RG underneath the injury site to become reactive. Only ~25% of reactive RG enter the cell cycle and become proliferative. The cell-cycle entry of reactive RG is regulated by Notch/Delta lateral inhibition. In the injury condition, proliferative RG largely undergo gliogenesis (~3–5% newborn neurons). The resulting newborn cells could survive up to 300 dpi. In the Notch inhibition condition, dormant RG can become proliferative but only generate ~1% of newborn neurons. However, Notch inhibition during 4–5 dpi drives reactive RG into the cell cycle, giving rise to significant more neurons (~12–20%). Interestingly, these over-produced neurons are largely diminished by 25 dpi. The numbers above the bars indicate the animals used. Yellow dashed lines represent the tectal ventricle boundary. RG, radial glia; TeO, tectum opticum; PGZ, periventricular gray zone; TS, torus semicircularis. Scale bars, 30 μm (B–D); 20 μm (J–M).

Figure 7—figure supplement 1. Late Notch inhibition-induced over-produced neurons are short-lived.

(A–F) Representative images of EdU (gray) and HuC/D (red) immunofluorescences showing the over-produced neurons in the optic tecta of the fish treated with LY411575 during 4–5 dpi (D–F) always shown as a cell cluster. (A–C) are the representative images of DMSO-treated optic tecta. Yellow dashed circles indicate the newborn neurons. (G) A schematic of the experimental procedure for Notch inhibition experiments shown in (H–K). Fish are injected with EdU for three consecutive days (4–6 dpi) and administrated with LY411575 or DMSO for two consecutive days (4–5 dpi). Fish are analyzed at 7 dpi or 25 dpi. (H–K) Representative images of HuC/D (green), BLBP (blue) and EdU (red) immunofluorescences showing that many newborn neurons (white arrowheads in (I)) are existed in Notch-inhibited (4–5 dpi) optic tectum at 7 dpi, but only a few newborn neurons (white arrowheads in (K)) are still remained at 25 dpi. (H and J) The representative images of DMSO-treated control optic tecta at 7 and 25 dpi, respectively. (L–N) Quantification of EdU⁺ newborn cells (L), EdU⁺/BLBP⁺ newborn RG (M), and EdU⁺/HuC/D⁺ newborn neuron (N) in (H–K) (≥3 replicates for each group; mean ± SEM,***p<0.001; *p<0.05; ns, p>0.05; Wilcoxon test in (L and M), two-way ANOVA followed by Tukey’s HSD test in (N)). (O–O₃) Representative images of Tg(1016tuba1α:GFP) (green), EdU (gray) and HuC/D (red) immunofluorescences showing the remaining neuron (white arrowheads) in the optic tectum of the fish treated with LY411575 during 4–5 dpi can survive up to 86 dpi if it is not eliminated by 25 dpi. (P) Quantification of the EdU⁺ newborn cells, EdU⁺/HuC/D⁺ newborn neurons and EdU⁺/BLBP⁺ RG in (O–O₃) (mean ± SEM, n = 4). (Q and R) Schematic diagrams of the distribution of newborn neurons in the injured 25-dpi optic tecta of the fish treated with DMSO or LY411575 during 4–5 dpi. Circles in different colors represent the newborn neurons from different individuals. (S) Proportion of the newborn neurons in TeO and PGZ of the optic tectum shown in (Q and R). Most of the neurons are existed in the TeO. (T) Representative images of EdU (gray) and HuC/D (red) immunofluorescences showing Notch inhibition during 4–5 dpi does not help to complete restoration of stab wound of the injured optic tectum. Yellow dashed circle indicate the stab wound. (U) Quantification of the area of stab wounds from the ~85 dpi fish treated with LY411575 during 4–5 dpi (1670 ± 704 μm²; mean ± SEM, n = 3). The numbers above the bars indicate the animals used. Yellow arrow heads indicate the injury sites. White dashed lines represent the tectal ventricle boundary. RG, radial glia; PGZ, periventricular gray zone; TeO, tectal opticum; TS, torus semicircularis. Scale bars, 50 μm (O–O₃ and T); 20 μm (H–K); 5 μm (A–F).