Neuron type specific cell death and comparable regenerative time course in two distinct injury models. a-j Micrographs of retinal sections after mechanical (a, b, e-g) or metronidazole induced genetic ablation in Tg(ptf1a:Gal4 / UAS:nfsb-mCherry) (c, d, h-j, a, c) Retinal architecture of the uninjured retina at equivalent ages (b, d). Brackets indicate the amacrine neuron layer (weaker DAPI staining in the inner half of the INL) and arrows indicate the horizontal neuron layer (first row of flattened nuclei in the inner nuclear layer – INL). b, d Retinal architecture of injured retina revealed by DAPI staining shows disruption caused by the needle track immediately after ablation injury (0 dpi), affecting neurons types in each retinal layer (b), and loss of horizontal cells and amacrine cells (seen by the reduction in Ptf1a:GFP transgene expression, which specifically labels these two cell types) 4 days after injury, which is a timepoint following the main cell death phase (d). e-j TUNEL labelling at different days post-injury (dpi) in both injury models. TUNEL staining is observed in all retinal layers early after mechanical ablation (e-g) and more biased towards horizontal and amacrine cells (arrowheads in INL and displaced amacrine cells in GCL) layers among nitroreductase expressing (red) cells (h-j). k, l Quantification of TUNEL positive cells in the different retinal layers across days post-injury reveals a peak in cell death in the first two days distributed across all retinal layers in the mechanical ablation (k) and primarily confined to inhibitory neurons after genetic ablation (l) (n = 12 larvae per timepoint). Asterisks indicate timepoints at which TUNEL labelling was in a significantly higher proportion of inhibitory neurons in the genetic versus mechanical ablation (p-value <0.038). l Loss of nitroreductase-mCherry positive cells follows the cell death observed in genetic ablation (orange line, n = 12 larvae per timepoint). Results are mean ± SEM. ONL: outer nuclear layer; OPL: outer plexiform layer; IPL: inner plexiform layer; GCL: ganglion cell layer; nfsb: Nitroreductase. Scale bar in D (for a-d) = 50 μm, scale bar in J (for e-j) = 50 μm

Timing of PCNA labelled proliferation is comparable between injury models. a-c, e-g) Micrographs of retinal sections after mechanical injury (a-c) and genetic ablation injury (e-g). Retinal sections stained for PCNA (proliferating cell nuclear antigen, red) show cell clusters that span across multiple retinal layers in both injury models (b, f, g). d, h The graph shows the total number of PCNA cells after mechanical (d) and genetic ablation injury (h) model, suggesting that broadly, proliferation does not begin until 3–4 dpi and is active for at least three days (n = 12 larvae per timepoint per injury model). Results are mean ± SEM. INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar in G (for ac, e-g) = 50 μm

Progenitors and clones arise from Müller glia. a-g Micrographs of retinal sections of Tg(gfap:GFP) lines, with Müller glia cells (green) stained for PCNA (proliferating cell nuclear antigen red). As previously published, the needle stick injury causes proliferation in Müller glia (a). In our newly established genetic injury, PCNA labelled proliferation was also in Müller glia (b). c-h) A detailed time series and quantification (h) shows the peak proliferative stage during 5–7 days post-injury (dpi) (n = 12 larvae per timepoints 1–10 dpi, n = 8 larvae per timepoints 11–14, n = 6 larvae at 21 dpi). Proliferative cells in the first 5 dpi also almost exclusively co-labelled with progressively weaker GFAP:GFP, after which time there were also many proliferative cells that no longer expressed detectable GFAP:GFP. White insets (c’-g’) show higher power magnification of boxed region indicated in c-g. Results are mean ± SEM. ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar B (for a-b) = 50 μm, scale bar in G (for c-g) = 50 μm, scale bar G’ (for c’-g’) = 200 μm

Proliferation time course measured with 24 h pulse BrdU incorporation is comparable to PCNA time course. a-g) Micrographs of retinal sections after mechanical injury stained with DAPI (blue) and for BrdU (green). a-g) BrdU positive cell clusters were observed between 3 to 7 days post-injury (dpi) with cells across multiple retinal layers. h The graph shows that BrdU positive cells were most abundant within a 2–3 day time period (n = 12 larvae). Results are mean ± SEM. Scale bar G (for a-g) = 50 μm

Prolonged BrdU exposure reveals cell type specific replacement. a, b Micrographs of mechanical and genetic ablated juveniles in prolonged BrdU exposure between 3 and 7 dpi. Retinal lamination has recovered by this timepoint with horizontal cells (arrows) and amacrine cell layer (brackets) re-establishing after genetic ablation. c, d Graphs indicating the total number of BrdU cells in each retinal layer across 5 time points observed in the mechanical (c) and genetic (d) ablation injury models. Statistics indicate comparison of the proportion of inhibitory neurons compared to age-matched uninjured control composition. After genetic ablation the vast majority of proliferative cells at 7 dpi are confined to the inhibitory layers, most notably the amacrine layer (*** p-value = 2.2 × 10−7 compared to WT proportion). In both injuries, the total number of cells per layer increases after 7 dpi and decreases by 14 dpi (genetic) and 17 dpi (mechanical) (n = 12 larvae at 7 and 10 dpi, 8 larvae at 14 dpi and 6 larvae at 17 and 21 dpi). Ns: not significant (p-value >0.05), * p-value = 0.004. Results are mean ± SEM. ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer; Scale bars = 50 μm

Fate determinant expression during regeneration does not recapitulate developmental sequence after different injuries. a, b) In uninjured control, a prolonged BrdU pulse labels neurons in the peripheral ciliary margin zone, which results in a stripe of BrdU positive cells after BrdU withdrawal, as BrdU negative cells continue to be added from the ciliary margin. This BrdU stripe is observed in micrographs from control (b). There are no BrdU cells in the mature retina found more centrally. c-h) Using prolonged exposure, BrdU labelled cells observed in this central mature retina region reflects regeneration. Micrographs show retinal sections from 14 days post-injury (dpi). The proportion of BrdU positive GCL cells after mechanical injury (n = 17 larvae - 10 dpi, 9 larvae −14 dpi, 12 larvae - 17 dpi) is higher compared to genetic injury (n = 8 larvae - 10 dpi, 15 larvae - 14 dpi, 19 larvae - 17 dpi) at 10 and 14 dpi. The firstborn ganglion cell marker Tg(atoh7:GFP) shows more co-labelling after mechanical injury. A large proportion of BrdU positive labelled cells in the bipolar layer (outer half of INL) show high expression of Tg(vsx1:GFP) indicative of bipolar differentiation (last born during development) after both injuries, starting earlier after mechanical (n = 13 larvae - 10 dpi, 24 larvae - 14 dpi, 21 larvae - 17 dpi) than genetic (n = 14 larvae - 10 dpi, 21 larvae - 14 dpi, 11 larvae - 17 dpi) injury. For both injuries, strongly labelled Vsx1 cells are observed prior to strongly labelled Atoh7 GCL cells. Results are mean ± SEM ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar B = 100 μm, scale bar C (for c, d, f, g) = 50 μm, scale bar in insets C (for insets in c, d, f, g) = 20 μm

Following genetic ablation, new horizontal and amacrine cells can be observed prior to the proliferative wave. a-c) Micrographs of retinal sections in Tg(ptf1a:GFP) larvae at different days post injury (dpi). d Quantification shows an initial reduction and subsequent increase in the number of Ptf1a:GFP labelled inhibitory neurons. At 3 and 4 dpi, the number is significantly lower (* p-value = 0.018, ** p-value ≤0.01) compared to 1 dpi (baseline) or 5 dpi (regenerated), which are not significantly different from each other (p-value = 0.50). Ns: not significant (p-value >0.05). Results are mean ± SEM. INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar C = 50 μm

Acknowledgments
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