|
Fig. 1 Characterization of RGC:YFP-NTR2 paradigm. (A) Whole-fish view of RGC:YFP-NTR2 expression specificity (asterisk indicates zc65-associated myl7:TagRFP ‘tracer’ reporter expression in the heart, not YFP-NTR2 expression). (B) Plate reader-based quantification of YFP loss at 7 dpf (48 hpa) after a 5-6 dpf exposure to Mtz at the indicated concentrations. (C) Experimental setup for analyzing proliferative responses (PCNA immunolabeling) to RGC ablation. (D) Representative PCNA immunostaining (red cells) in the INL and ONL of RGC:YFP-NTR2 retinas treated with or without Mtz (asterisks indicate constitutive proliferation in CMZ). (E) Quantification of PCNA+ cells in the INL at 24, 48, 72 and 96 hpa. (F) Representative confocal images of RGC soma and axonal loss (48 hpa) and regeneration (168 hpa) in the retina (top) and tectum (bottom). (G) Imaris-based volumetric quantification of RGC axon loss and regeneration (YFP) from confocal z-stacks of the tectum. (H) Experimental setup of EdU-based lineage tracing of early retinal progenitors (24 h pulse, 96 hpa chase). (I) Representative images of EdU immunostaining in unablated (Cntr) and RGC-ablated (+Mtz) RGC:YFP-NTR2 larvae (asterisks indicate CMZ cells in the periphery and endothelial cells near the optic nerve; arrowhead marks EdU+ cells in the brain, none of which were included in the quantification). (J) Quantification of EdU+ cells in the GCL. (K) Representative swim traces of visual responses to a lights-off stimulus in control and RGC-ablated (+Mtz) larvae at 0, 48 and 96 hpa. (L) Quantification of visual responses in control and RGC-ablated larvae at 0, 48, 96 and 144 hpa. All white violin plots are unablated control larvae (Cntr), all orange violin plots are RGC-ablated larvae (+Mtz). Pair-wise and multiple comparisons are indicated by single lines and extended lines, respectively. **P≤0.01, ***P≤0.001, ****P≤0.0001; ns, not statistically significant.