Fig. 7.
RPE-specific knockout of REV-ERBα in mice shows similar ocular pathologies as Rev-erbα−/−mice. (A–B) Protein levels of REV-ERBα and NRF2 were assayed (A) and quantified (B) in Rev-erbαfl/fl, BEST1-Cre, and BEST1-cre: Rev-erbαfl/fl RPEs by Western blotting. The BEST1-cre: Rev-erbαfl/fl RPE showed ∼75% efficiency of REV-ERBα knockout at protein levels, and NRF2 protein levels were downregulated by ∼40% in BEST1-cre: Rev-erbαfl/fl RPE. n = 3–4 mice/group. (C–D) Immunohistochemistry confirmed absence of REV-ERBα (magenta) in RPE/choroid flat mounts (C) counterstained with β-catenin (green) and DAPI (blue) and cross-sections (D) in 3-month-old BEST1-cre: Rev-erbαfl/fl vs. Rev-erbαfl/fl mice. Scale bars, C: 20 μm; D: 20 μm. (E) Rev-erbαfl/fl, BEST1-Cre, and BEST1-cre: Rev-erbαfl/fl mice were examined by fundus imaging at 3, 6, and 9 months old. BEST1-cre: Rev-erbαfl/fl retinas showed whitish lesions starting from 6 months old. (F) Quantification of lesion numbers in fundus images of Rev-erbαfl/fl, BEST1-Cre, and BEST1-cre: Rev-erbαfl/fl eyes shows significant difference in lesion count in BEST1-cre: Rev-erbαfl/fl eyes vs. both BEST1-Cre, and Rev-erbαfl/fl control groups. n = 10–16 eyes/group, mixed genders. (G) Immunohistochemistry of β-catenin (green) and DAPI (blue) in BEST1-cre: Rev-erbαfl/fl RPE/choroid flat mounts showed more severe patchy, distorted RPE cells than BEST1-cre mice. Scale bar, 10 μm. (H) mRNA expression of Nr1d1 (REV-ERBα), Clock (positive control), Nfe2l2 (NRF2), Sod1, Sod2 and catalase was examined in the RPE of 6-month-old Rev-erbαfl/fl and BEST1-cre: Rev-erbαfl/fl mice. qPCR results were normalized to 18S and then normalized again to the expression levels in Rev-erbαfl/fl RPE cells. n = 6/group. (I) Representative images of immunostaining of 8-OHdG in 6-month-old BEST1-cre and BEST1-cre:Rev-erbαfl/fl retinas. Scale bar, 10 μm. (J) Representative images of H&E staining showed discontinued RPE layer in 9-month-old BEST1-cre: Rev-erbαfl/fl vs. BEST1-cre eye cross-sections. Scale bar, 10 μm **: P < 0.01, ***: P < 0.001****: P < 0.0001. Error bars indicate SD. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)