MIPfun water permeability regulation via R33L and less via N40H is required for lens clarity, optics, and lens nucleus centralization. Representative dissected lenses from adult aqp0a−/− (A) (SL = 23.8 mm), aqp0a−/−MIPfun (B) (SL 24.7 mm), aqp0a−/−MIPfunN40H (C) (SL = 20 mm) or aqp0a−/−MIPfunR33L (D) SL = 21.5 mm) were imaged through the optical axis under bright field (BF) or dark field (DF) illumination. The ability to focus was tested by focusing through the lens onto a grid. Whereas most aqp0a−/− mutant lenses misexpressing N40H were transparent, some as shown had cataract and inability to focus a grid clearly, as did the R33L expressing lenses. (E) In adult zebrafish of SL > 20 mm, the frequency of optical defects was similar between aqp0a−/− and aqp0a−/−MIPfunN40H, while aqp0a−/−MIPfunR33L had higher frequency of cataract than aqp0a−/−. Refer to Supplementary Figure S1 for phenotype frequency by developmental stage. The same lenses from A to D were imaged perpendicular to the optical axis displayed the relative localization of the lens nucleus (F–I). (J) Overexpression of R33L in aqp0a−/− did not rescue the failure to centralize the lens nucleus, whereas overexpression of MIPfunN40H lead to a more centralized lens nucleus. Mean cross-sectional lens nucleus localization between aqp0a−/− and mutants overexpressing MIPfunN40H were different at mid and longer SL (P = 0.000 and P = 0.001). Scale bars = 500 µm.
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