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. 2017 May 7;77(9):1114–1129. doi: 10.1002/dneu.22499

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© 2017 The Authors. Developmental Neurobiology Published by Wiley Periodicals, Inc.

This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

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Coinjection of 5′UTR MO and zf pgrn‐a, zf pgrn‐b, or hGRN mRNA rescues most aspects of knockdown retinal phenotype. (A–O) Cross‐sections through central retina of 72 hpf uninjected (UI, A–C), 5′UTR MO‐injected (D–F), 5′UTR MO and zf pgrn‐a mRNA coinjected (G–I), 5′UTR MO and zf pgrn‐b mRNA coinjected (J–L), and 5′UTR MO and hGRN mRNA coinjected (M–O) embryos at 72 hpf. Sections are immunolabeled (cyan) with markers for ganglion cells (Zn‐5, left column), red‐green double cone photoreceptors (Zpr1, middle column), and amacrine cells (HPC1, right column), EdU (fusia), and DAPI (gray). (P) Histogram showing relative retinal area of UI (17437.4 ± 2286.1 μm²; n = 23; 95% CI: 16970 (14744–19196)), 5′UTR MO‐injected (12961.2 ± 2251.8 μm²; n = 21; 95% CI: 11329 (10561–12097)), 5′UTR MO and eGFP mRNA co‐injected (10809 ± 2282.5 μm²; n = 8; 95% CI: 14374 (12599–16149)), 5′UTR MO and zf pgrn‐a mRNA co‐injected (16435.7 ± 2882.2 μm²; n = 12; 95% CI: 16860 (15697–18023)), 5′UTR MO and zf pgrn‐b mRNA co‐injected (15357.8 ± 2750.2 μm²; n = 13; 95% CI: 14834 (13529–16139)), and 5′UTR MO and hGRN mRNA co‐injected (17146.1 ± 2930.7 μm²; n = 12; 95% CI: 18579 (17174–19984)) embryos at 72 hpf; ***p<0.001. (Q) Histogram showing the percent of the retina labeled with EdU in UI (10.5 ± 3.9%; n = 7; 95% CI: 11.0 (7–15)), 5′UTR MO‐injected (45 ± 7.9%; n = 18; 95% CI: 45 (41–49)), 5′UTR MO and eGFP mRNA co‐injected (41.1 ± 4.5%; n = 6; 95% CI: 41.0 (36–46)), 5′UTR MO and zf pgrn‐a mRNA co‐injected (15.2 ± 6.8%; n = 22; 95% CI: 15.0 (12–18)), 5′UTR MO and zf pgrn‐b mRNA co‐injected (20.8 ± 9.6%; n = 18; 95% CI: 21 (16‐26)), and 5′UTR MO and hGRN mRNA co‐injected (21.8 ± 8.5%; n = 12; 95% CI: 15 (9–21)) embryos at 72 hpf; ***p<0.001. (R) Histogram showing the number of retinal microglia (4C4+ cells) at 72 hpf in UI (95 ± 30.5 cells; n = 23; 95% CI: 95.0 (82.5–107.5)), 5′UTR MO‐injected (8.4 ± 6.8 cells; n = 21; 95% CI: 8.4 (5.5–11.3)), 5′UTR MO and eGFP mRNA co‐injected (5.4 ± 4.8 cells; n = 8; 95% CI: 5.4 (2.1–8.7)), 5′UTR MO and zf pgrn‐a mRNA co‐injected (17.7 ± 10.2 cells; n = 12; 95% CI: 17.7 (11.9–23.5)), 5′UTR MO and zf pgrn‐b mRNA co‐injected (28.9 ± 14.7 cells; n = 13; 95% CI: 28.9 (20.9′36.9)), and 5′UTR MO and hGRN mRNA co‐injected (20.6 ± 10.8; n = 12; 95% CI: 20.6 (14.5′26.7)) embryos; ***p<0.001. Quantitative data are represented as mean; error bars represent the standard deviation. Outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL); ciliary marginal zone (CMZ, brackets). Scale bar equals 50 µm.