Figure 6

EGFR signaling is necessary for Müller glia proliferation and ERK1/2 activity. (a) Potential EGFR activity in Müller glia (MG) in EGF-stimulated retinal explant culture upon indicated treatments and days ex vivo. For analysis, retinal sections were immunostained for the MG marker SOX2 and dual phosphorylation (P) of Tyr204 / Y187 of p44/42 mitogen-activated protein kinases 3 and 1 (MAPK3, MAPK1), also called P-ERK1/2 (extracellular signal-regulated kinases 1 and 2). EdU was given cumulatively throughout explant culture to monitor cell proliferation. EdU+ SOX2+ double-positive nuclei indicate MG proliferation. (a) Images and quantitative analysis showed that retinal explant culture induced a strong upregulation of P-ERK1/2 immunolabeling with radial morphology. Early on P-ERK1/2 overlaps with SOX2+ MG nuclei located in the INL, and later P-ERK1/2+ SOX2+ MG are also found in the ONL (arrow). Dashed white boxes show regions of interest at higher magnification (DEV 0 & 2, N = 7; DEV 0.75, N = 5; DEV 4, N = 3)). (b,c) Application of EGFR inhibitors PD153035 or PD158780 throughout retinal explant culture blocked (b) P-ERK1/2 upregulation, shown by IHC (N = 4) and Western blot analysis (N = 5), and (c) MG proliferation (N = 3) each in comparison to solvent control (CTRL). Bar graph color scheme indicates: GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer or cell across all layers (total cells). DEV, days ex vivo. Data are presented as mean ± SD (Western blot) and ± SEM all others; N ≥ 3 (see Table S4). *P < 0.05; **P < 0.01; ***P < 0.001 with Student’s t-test (unpaired, two-tailed). Scale bars: 50 µm. See Fig. S6.