FIGURE 6

FIGURE 6

Generation of new sensory neurons from the neurod:GFP-positive progenitor pool upon lesion. (A) Short-term lineage tracing using the persistence of GFP in the SAG of neurod:GFP transgenic animals. Immunohistochemistry against the neuronal marker HuC/D and GFP label newborn neurons in sham-treated, and NaCl- and ouabain-injected animals as well as the unlesioned side (ULS) of either sham-treated, or NaCl- or ouabain-injected animals at 4, 8, and 57 days post lesion (dpl). Shown are representative overviews of the neurogenic niche of the SAG as well as close-ups to visualize co-staining. At 4 dpl, several neurod:GFP/HuC/D double-positive cells can be found in the lesioned side independent of the type of lesion, whereas neurogenesis is almost not existing in the corresponding unlesioned side (upper row). At 8 dpl, neurogenesis is abundant in the lesioned sides and increased as well in the respective unlesioned sides in sham-treated, and NaCl- and ouabain-injected animals (middle row). At 57 dpl, neurogenesis returned to low levels in the lesioned and unlesioned side with only a few newborn neurons present in the SAG (lower row). (B) Quantification of neurod:GFP/HuC/D double-positive cells per section at 2, 4, 8, 16, and 57 days post lesion. The number of neurod:GFP/HuC/D double-positive cells per section revealed a significant increase in neurogenesis in the lesioned side in comparison with the homeostatic baseline (0.56 neurod:GFP/Huc/D double-positive cells per section) calculated from Schwarzer et al., 2020 (gray box). The number of newborn neurons per section peaked at 4 dpl in ouabain-injected SAGs and at 8 dpl in sham-treated as well as in NaCl-injected SAGs, respectively. At 57 dpl, neurogenesis returned to homeostatic levels in all three lesion conditions. Interestingly, neurogenesis rates in the SAGs of the respective unlesioned sides were also increased over time and slightly above the homeostatic baseline at 8 dpl in sham-treated and NaCl-injected animals as well as at 16 dpl in ouabain-injected animals. Baseline levels in neurogenesis in the respective unlesioned sides was reached again at 16 and 57 dpl, respectively. (C) Time course for bromodeoxyuridine (BrdU) pulse chase experiment. Six-month-old Tg(neurod:GFP) zebrafish were lesioned, followed by two 24-h lasting BrdU treatments at 2 and 4 days post lesion (dpl). Animals were sacrificed at 19 dpl corresponding to 14 days after the second BrdU treatment. (D) Antibody staining against the thymidine analog BrdU, the neuronal marker HuC/D and GFP in the SAG of sham-treated, and NaCl- and ouabain-injected neurod:GFP animals at 19 dpl (14 days post second BrdU pulse). In the SAG of the unlesioned side, BrdU-positive cells can be found in close proximity to neurod:GFP-positive cells (gray arrow). In contrast, neurod:GFP/BrdU double-positive progenitors (arrow), neurod:GFP/BrdU/HuC/D triple-positive newborn neurons (arrowhead), and BrdU/HuC/D double-positive neurons (open arrowhead) can be found in the SAG of sham-treated, and NaCl- and ouabain-injected animals. Scale bars: 50 µm. For quantification sample size n = 3 (n = fish with three sections/SAG; exception: 4 dpl/NaCl: n = 2); data are presented as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons test; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05.