Müller glia in the mdkami5001 mutant arrest in the G1 phase of the cell cycle. A–C, qPCR assay for cell cycle regulator cyclins, ccnd1, ccne1, and ccna2 following photolytic lesion. Both WT and mdkami5001 upregulate G1 cyclins, ccnd1 (A; WT: 30 hpl, p = 0.0107, 36 hpl, p = 0.0013; mdkami5001: 30 hpl, p = 0.0048, 36 hpl, p = 0.0213; F-ratio = 12.0576) and ccne1 (B; WT: 30 hpl, p < 0.0001, 36 hpl, p < 0.0001; mdkami5001: 30 hpl, p = 0.0012, 36 hpl, p = 0.0001; F-ratio = 36.5808), following lesion (A,B). The mdkami5001 mutant fails to upregulate S phase cyclin, ccna2 (C; WT: 36 hpl, p = 0.0011; F- ratio = 12.5433). D, S phase assay with BrdU labeling (green) between 24 and 30 hpl. Müller glia in the mdkami5001 mutants did not incorporate BrdU following lesion. E–H, qPCR of additional cell cycle regulators. Expression of cyclin-dependent kinases, cdk4 (E; WT: 36 hpl, p < 0.0001; F-ratio = 15.8783) and cdk6 (F; WT: 36 hpl, p = 0.0087; F- ratio = 8.3300), is dysregulated in the mdkami5001 retinas (E,F). The WT transiently upregulates id2a at 30 hpl (p = 0.0003; F- ratio = 7.2578), whereas expression levels did not change in the mdkami5001 (G). Expression of the cell cycle inhibitor, p130, decreases in the WT after lesion, whereas mdkami5001 maintains steady levels of expression (H; p = 0.002, relative to WT; F- ratio = 6.3823). ANOVA with post hoc Tukey, relative to unlesioned. Scale bar: D, 30 μm. onl, Outer nuclear layer; inl, inner nuclear layer; gcl, ganglion cell layer. *p < 0.03, #p < 0.01.
|
