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. 2019 Mar 14;9:4420. doi: 10.1038/s41598-019-40734-z

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© The Author(s) 2019

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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Differentiation post-lesion in activated qRG of the qRG-layer. (a) Experimental design to study differentiation of proliferating radial-glia (pRG) at chase periods post-EdU injection. (b,c) Low and high magnification examples displaying the absence of co-labelling of EdU⁺ cells (pink) in the PGZ with the neuronal marker, HuC/D (green) at 2-wpi (b) and 4-wpi (c). (d–g) Representative images of co-labelling of EdU⁺/gfap:GFP⁺ cells in the qRG layer (qRG-L) of the PGZ at 2-wpi. White box in (d) shown at higher magnification and in separate channels confirming co-labelling of pRG with EdU (e–g; white arrows). (h) Total EdU⁺ cells in the upper neuronal layer and deeper qRG layer at consecutive chase periods (one-way ANOVA; F (3, 35) = 2.239, p = 0.1009; Tukey’s multiple comparisons test: 7-dpi vs 2-wpi, p = 0.2082; 7-dpi vs 4-wpi, p = 0.1072; 7-dpi vs 8 wpi, p = 0.3333; 2-wpi vs 4-wpi, p = 0.9863; 2-wpi vs 8-wpi, p = 0.9984; 4-wpi vs 8-wpi, p = 0.9621). Standard error of the mean for total EdU⁺ cells (neuronal layer + qRG layer): 7-dpi ± 2.4; 2-wpi ± 2.9; 4-wpi ± 2.7; 8-wpi ± 3.5. (i) Quantification of EdU⁺/GS⁺ cells at increasingly longer chase periods post tectal lesion in the qRG layer (one-way ANOVA; F (3, 17) = 0.7972, p = 0.5123; Tukey’s multiple comparisons test: 7-dpi vs 2-wpi, p = 0.5190; 7-dpi vs 4-wpi, p = 0.9994; 7-dpi vs 8- wpi, p = 0.9232; 2-wpi vs 4-wpi, p = 0.5845; 2-wpi vs 8-wpi, p = 0.9107; 4-wpi vs 8-wpi, p = 0.9533). (j) Fraction of EdU⁺/GS⁺ cells as a percentage of the total EdU⁺ population in the PGZ at four chase periods examined. (k) Experimental design to examine the contribution of macrophages, using the L-plastin antibody (green), to cell proliferation at the site of injury at 4-wpi. (l) The unlesioned hemisphere displays a small number of non-proliferative resident microglia in the TeO and PGZ. (m) A large population of amoeboid-like macrophages are observed in the TeO surrounding the tectal lesion site, with L-plastin staining in the PGZ resembling the unlesioned condition. Co-labelling with EdU (pink) reveals a population of L-plastin⁺ cells that are in a proliferative state (white arrows). White box is shown in higher magnification in panels o,p. (o,p) Merge and single channels showing a representative L-plastin⁺/EdU⁺ cell (white arrow) adjacent the lesion site at 4-wpi. Experimental replicates were combined for all statistical analyses. All data presented are mean ± S.E.M. Significance was accepted at *p < 0.05. Orange asterisk (*) denotes the lesion site in (b,d,m,n). In panels b–d, l–o, DAPI nuclear counterstaining (blue) was performed. In all cross-sectional images dorsal is oriented up. dpl, days post lesion; dpi, days post EdU injection; wpi, weeks post EdU injection.