Figure 8. p27Kip1 is part of a gene regulatory network promoting pigmentation in the R227W retina.

(A) p27 mRNA was reduced by approximately half in R227W; Mitf^mi/+ and R227W; p27^+/− compound mutant retinas compared to R227W at E12.5. (B–D) Phenotypes of R227W, p27Kip1^+/− compound mutant eyes at P0. (B) Retinal tissue (blue) was restored along with a concomitant loss of pigmentation (white) in the retina. The peripheral retina was not rescued to the same extent as the central region. Eye size was also enhanced in the compound mutant (inset). POU4F2 (C) and the amacrine cell marker SOX2 (D) were expressed in the compound mutant. SOX2 is also expressed in RPCs. (E) Expression of Mitf and Otx1 mRNAs were reduced by approximately half in compound mutant retinas (black bars) compared to R227W (white bars) at E12.5. (F) The expression level of D-Mitf mRNA was reduced in compound mutant retinas, whereas H-Mitf mRNA levels were were not significantly different. (G) p27 overexpression in HEK293 cells increased luciferase activity from pGL3B-DMitf, but to a much lesser extent from pGL3B-HMitf. (H) ChIP assays of E12.5 R227W retinal lysates probed with p27 antibody. ChIP panel on left shows products obtained with primer set 1; panel on right shows products obtained with primer set 13. Sequence-verified products denoted by arrowheads. * P≤0.05; ** P≤0.01; *** P≤0.001 Scale bars: 100 µm (B); 1 mm (inset); 50 µm(C,D).