AJ components fail to accumulate at the apical domains of epithelial cells in nok mutants. (A) Nok protein was not expressed in lens epidermal cells at 24 hpf. a (anterior) and p (posterior) show the lens orientation. (B) Nok started to be expressed in lens epithelial cells at 28 hpf when epidermal-to-epithelial transition occurred. (C,D) Apical enriched β-catenin colocalized with Nok in WT lens epithelial cells at 36 hpf. β-catenin lost its apical enrichment in nok mutants at 36 hpf. C′-C‴ and D′-D‴ are higher magnifications of the white boxed regions in C and D, respectively. (E) Apical zonula adherens disappeared in nok mutants at 36 hpf. a (apical) and b (basal) show the orientation of lens epithelial cells. (F) Quantification of the number of adhesion plaques in lens epithelial cells in WT and nok mutants at 36 hpf. In WT lens, the apical AJs between epithelia (AJs_EE, 1 per epithelium) and the apical AJs between epithelia and fiber cells (AJs_EF, 1.68 per epithelium) were the major types of adhesion plaques. In nok mutants, AJs_EE number significantly decreased (0.45 per epithelium) and the number of other types of adhesion plaques, including the basal AJs between epithelia (BJs_EE), the lateral AJs between epithelia (LJs_EE) and AJs_EF, significantly increased. (G) Quantification of the length of adhesion plaques in epithelial cells in WT and nok mutants at 36 hpf. The length of AJs_EE (0.41±0.03 μm) was significantly longer than LJs_EE (0.15±0.01 μm) and AJs_EF (0.18±0.01 μm) in WT. The length of AJs_EE in nok mutants (0.19±0.02 μm) was significantly reduced and comparable with LJs_EE and AJs_EF (n=49 epithelial cells for WT and 59 for nok mutants). (H,I) Compared with WT (H), a large number of Zl1-immunoreactive negative cells (arrows) accumulated in the inner lens in nok mutants at 28 hpf (I). Scale bars: 10 μm (A-D,H,I), 400 nm (E). ***P<0.001, n.s>0.05.
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