a TUNEL assay (A, B) and HuC/D (C, D) immunostaining of PTU-treated normal Sb and pnt^tq209 mutant zebrafish at 72 hpf to detect apoptotic cells and differentiated neurons, respectively. The merge between the two fluorescent signals is shown in E and F. b Quantification of apoptotic bodies, detected with the TUNEL assay, in Sb and pnt^tq209 zebrafish, showing a significant increase in the total number of apoptotic bodies in the brain and retina of mutant larvae (number of apoptotic bodies (TUNEL-positive puncta) in the brain, Sb: 31.10 ± 2.35, pnt^tq209: 57.53 ± 3.12; in the retina, Sb: 5.33 ± 0.85, pnt^tq209: 21.80 ± 1.52; results are reported as mean ± S.E.M. (n = 15–20)). c apoE in situ hybridization (A, B) and NR staining (C–F) were carried out in PTU-treated, 72 hpf zebrafish to visualise microglia. d, e Quantification of apoE-positive (d) and NR-positive (e) cells in the whole CNS. A significant increase in the number of microglial cells characterizes pnt^tq209 zebrafish ((number of apoE-positive puncta, Sb: 13.10 ± 1.28, pnt^tq209: 25.20 ± 1.38; number of NR-positive puncta, Sb: 17.50 ± 1.18, pnt^tq209: 23.13 ± 1.35; results are reported as mean ± S.E.M. (apoE: n = 10; NR: n = 24)). f Analysis of the size of NR puncta. pnt^tq209 zebrafish showed a significant increase in the size of microglia when compared to Sb (microglial area (NR-positive pixels), Sb: 210.45 ± 15.65, pnt^tq209: 379.35 ± 26.73; results are reported as mean ± S.E.M. (n = 15)). g, h Evaluation of the number of cells stained with apoE (g) and NR (h) in the brain and retina, separately. The results demonstrate that, in pnt^tq209 larvae, the increase in microglial cell population is also extended to the retina ((number of apoE-positive puncta in the brain, Sb: 4.40 ± 0.56, pnt^tq209: 9.60 ± 1.38; in the retina, Sb: 8.70 ± 1.58, pnt^tq209: 15.60 ± 1.73; number of NR-positive puncta in the brain, Sb: 11.19 ± 1.10, pnt^tq209: 16.50 ± 1.21; in the retina, Sb: 5.44 ± 0.93, pnt^tq209: 7.94 ± 1.23; results are reported as mean ± S.E.M. (apoE: n = 10; NR: n = 15)). FB forebrain, HB hindbrain, MB midbrain, R retina

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a, b Fluorescent IHC of whole-mount 72 hpf zebrafish, stained with anti-OPA1 antibody. Representative images (a) and quantification of OPA1 fluorescence intensity (b) are reported, showing a reduction in the OPA1 expression levels in the brain and retina of pnt^tq209 larvae (OPA1 fluorescence (A.U.), Sb: 1.00 ± 0.01, pnt^tq209: 0.70 ± 0.09; results are presented as mean ± S.E.M. (n = 3)). c, d Analysis of eye morphology in Sb and pnt^tq209 zebrafish obtained through anti-acetylated α-tubulin IHC of whole-mount 72 hpf zebrafish. No major morphological differences were observed between normal and mutant larvae, as shown in the prototypical images (c) and relative quantification of eye size (d) (eye size (pixels), Sb: 2.58 ± 0.10, pnt^tq209: 2.53 ± 0.06; results are presented as mean ± S.E.M. (n = 10)). e, f Quantification of OPA1 levels via western blotting analysis in 72 hpf wild type and pnt^tq209 larvae. The representative membrane blotted for both OPA1 isoform (e) and the bar chart (f) show a significant decrease in the levels of the short isoform of the protein in pnt^tq209 larvae. g–j (OPA1 band density relative to ACTB, OPA1 long isoform Sb: 1.00 ± 0.01, pnt^tq209:0.97 ± 0.01; OPA1 short isoform Sb: 1.00 ± 0.01, pnt^tq209:0.84 ± 0.01; results are presented as mean ± S.E.M. (n = 3), Quantitative western blot analysis of OPA1 levels in the brain (frontal cortex/hippocampus) (j, k) and isolated optic nerve (l, m) of WT and Atpif1^−/− mice. Representative blots (j, l) and quantitated band densities relative to GAPDH (k, m) are reported. Significantly lower levels of short and long OPA1 isoforms expression were found in the brain and, specifically, in the optic nerve of mutant mice (OPA1 band density relative to GAPDH, OPA1 long isoform WT: 1.00 ± 0.01, Atpif1^−/−: 0.52 ± 0.01 in the frontal cortex/hippocampus; in the optic nerve, WT: 1.00 ± 0.05, Atpif1^−/−: 0.59 ± 0.09; in the frontal cortex/hippocampus OPA1 short isoform WT: 1.00 ± 0.01, Atpif^1−/−: 0.68 ± 0.01; in the optic nerve, WT: 1.00 ± 0.05, Atpif1^−/−: 0.548 ± 0.01; results are presented as mean ± S.E.M. (n = 3). FB forebrain, HB hindbrain, MB midbrain, NR neural retina, L lens

a Anti-acetylated α-tubulin IHC of whole-mount 72-hpf zebrafish. No marked differences in axonal growth and morphology were observed between Sb and pnt^tq209 larvae. Images were acquired at three different focal planes to visualize the lateral line (F1) and the ventral and dorsal motor axons (F2 and F3, respectively). b–d Locomotor activity assay in 72-hpf zebrafish. Total distance moved (b), mean velocity (c) and maximum velocity (d) were measured (average total distance moved (mm), Sb: 18.39 ± 8.60, pnt^tq209: 21.22 ± 8.46; average mean velocity (mm/s), Sb: 0.02 ± 0.01, pnt^tq209: 0.05 ± 0.03; average maximum velocity (mm/s), Sb: 41.57 ± 15.74, pnt^tq209: 42.47 ± 12.62; results are reported as mean ± S.E.M. (n = 9)). No variances were observed between Sb and pnt^tq209 larvae