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Fig. 4
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 ?m2; n?=?23; 95% CI: 16970 (14744?19196)), 5?UTR MO-injected (12961.2?±?2251.8 ?m2; n?=?21; 95% CI: 11329 (10561?12097)), 5?UTR MO and eGFP mRNA co-injected (10809?±?2282.5 ?m2; n?=?8; 95% CI: 14374 (12599?16149)), 5?UTR MO and zf pgrn-a mRNA co-injected (16435.7?±?2882.2 ?m2; n?=?12; 95% CI: 16860 (15697?18023)), 5?UTR MO and zf pgrn-b mRNA co-injected (15357.8?±?2750.2 ?m2; n?=?13; 95% CI: 14834 (13529?16139)), and 5?UTR MO and hGRN mRNA co-injected (17146.1?±?2930.7 ?m2; 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.