1State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China 2The Brain Science Center, Beijing Institute of Basic Medical Sciences, Beijing 100850, China 3College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China 4Beijing Institute of Pharmacology and Toxicology, Beijing 100850, China 5State Key Laboratory of Toxicology and Medical Countermeasures, Beijing 100850, China
Cataract is a major cause of blindness worldwide, its complicated and unclear etiopathogenesis limit effective therapy. Here, we found that Yap, a downstream effector of the Hippo pathway, is specifically expressed in lens epithelial cells and Yap conditional knockout (cKO) in the lens leads to cataract. Histologically, Yap deficient lens show fewer epithelial cells, retention of nuclei and accumulation of morgagnian globules in the transitional zone and the posterior area. Mechanistically, GFAP-mediated Yap cKO leads to the reduced proliferation of epithelial cells, delayed fiber cell denucleation and increased cellular senescence in lens. Further RNA profiling analysis reveals Yap cKO results in a significant alteration in gene transcription that is involved in eye development, lens structure, inflammation, cellular proliferation and polarity. Collectively, our data reveal a novel function of Yap in the lens and links Yap deficiency with the development of cataract, making Yap a promising target for cataract therapy.
Figure 1. The expression patterns of Yap and GFAP-Cre recombinase in postnatal mouse eyes. (A) Schematic of a transverse section of mouse eye. (B-C) Immunostaining with anti-Yap antibody (green) on frozen eye sections at different ages. Nuclei were counterstained with DAPI (blue). Yap staining was detected in scattered cells within the INL (arrowheads) and GCL of the retina and the lens epithelium (arrows). (D) Cre recombinase (red) was expressed in the lens epithelium and INL, GCL of retina in frozen eye sections of Tomatof/+; GFAP-Cre mice at P14. Nuclei were counterstained with DAPI (blue). LE, lens epithelium; TZ, transitional zone; RPE, retinal pigment epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Scale bars: 25 μm (B-C), 100 μm (D).
Figure 2. Cataract occurs in 1.5-month-old Yap-deficient mice. (A) 1.5-month-old Yapf/f; GFAP-Cre mouse demonstrating severe nuclear cataract, with opaque cloudiness (arrowheads) in lens. (B) Incidence and latency of cataract formation in Yapf/f; GFAP-Cre mice.
Figure 3. Abnormal lens structure in Yap-deficient mice at different stages. (A-H) H&E staining of lens from Yapf/f and Yapf/f; GFAP-Cre mice at P21, 1-month, 2-month and 3-month, respectively. (A1-H1) Selected high magnification views of lens epithelium (A-H), showed the disorganization of anterior lens epithelium (arrows indicate lens epithelial cells) of Yapf/f; GFAP-Cre lens. (A2-H2) High magnification views of transitional zone regions (A-H), showed the accumulation of ectopic cells and morgagnian globules at the transitional zone and posterior region of Yapf/f; GFAP-Cre lens (asterisks indicate morgagnian globules). Arrowheads indicate hematoxylin-positive cells. (I-J) Immunofluorescent staining revealed the abnormal distribution of AQP0 in Yap-deficient lens compared with wildtype (1.5-month). Asterisks indicate morgagnian globules and arrowheads indicate displaced cell nuclei. Scale bars: 100 μm.
Figure 4. Yap deficiency suppresses cell proliferation in vivo and in vitro. (A-F) The Ki67 positive ratio of lens epithelial cells decreased in Yap-deficient mice at different stages (arrowheads indicate Ki67 positive cells). (G) The relative number of Ki67 positive lens epithelial cells (number of Ki67 positive lens epithelial cells / lens epithelium area). The data are shown as mean ± S.E.M. (Student’s t-test, *P<0.05, **P<0.01, n=10). (H-I) Knockdown efficiency of Yap in αTN4 cell using siRNA. (J-K) Cell viability and growth assay revealed that proliferation was downregulated in Yap knockdown αTN4 cells. The data are shown as mean ± S.E.M. (Two-way RM ANOVA, **P<0.01, n=5). Scale bars: 50 μm.
Figure 5. Yap deficiency results in cellular senescence in the lens. (A) Frozen eye sections from 1.5-month-old Yapf/f and Yapf/f; GFAP-Cre littermate mice were stained for SA β-galactosidase activity (arrowheads). (B and C) qRT-PCR analysis of p21 and p53 relative mRNA levels in the lens from Yapf/f and Yapf/f; GFAP-Cre littermate mice at different stages. The data are shown as mean ± S.E.M. (Student’s t-test, *P<0.05, **P<0.01, n=6). Scale bars: 50 μm.
Figure 6. Analysis of differential gene expression in Yap-deficient mice. (A) Volcano map of significantly expressed genes in the lens from 1.5-month-old Yapf/f and Yapf/f; GFAP-Cre littermate mice. Red dots and green dots represent significantly up-regulated and down-regulated genes, respectively (P<0.01 and fold change >2). (B) The distributions of biological processes (BP). The curve indicates p-value of BP terms and the bar graph indicates the gene number distribution in BP terms. (C) The list of 24 significant transcripts that are regulated by Yap deficiency were classified into five categories including eye development, lens structure, inflammation and cell proliferation as well as polarity determination. (D-E) Secondary validation of the differentially expressed genes with qRT-PCR analysis. The data are expressed as mean ± S.E.M. (Student’s t-test, **P<0.01, n=6).
Figure 7. Secondary validation of differentially expressed genes in Yap-deficient lenses at different stages. (A-G) qRT-PCR analysis of relative Sox2, Pax6, Dnase2b, Crygc, Crygd, Il6 and Tnfα mRNA levels in lens from Yapf/f and Yapf/f; GFAP-Cre littermate mice at different stages. The data are shown as mean ± S.E.M. (Student’s t-test, **P<0.01, n=6).
Figure 8. Proposed model of cataract formation in Yap conditional knockout mice. The model proposes that Yap deficiency in the lens led to a cascade of events, including: 1) reduced cell proliferation; 2) abnormal denucleation of fiber cells; 3) cellular senescence; 4) increased inflammation; 5) and consequently, cataract with cloudy and dense organelle-free-zone.
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