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. 2018 Jun 4;9(6):669. doi: 10.1038/s41419-018-0578-x

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — 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