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Fig. 5 Ca2+ regulation of MIPfun water permeability is required for lens transparency, optics, and lens nucleus centralization. (A) Representative lenses from adult aqp0a−/− mutants (A) (SL = 23.8 mm), aqp0a−/− mutants overexpressing MIPfun (B) (SL = 24.7 mm), aqp0a−/− mutants overexpressing MIPfunY75G (C) (SL = 24.8 mm) or MIPfunR153A (D) (SL = 25.1 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. The lenses misexpressing MIPfunY75G revealed severe nuclear cataract and poor focusing through a grid, whereas there are no obvious transparency defects in the lenses with R153A mutation. (E) The frequency of severe cataract in adult zebrafish SL > 20 mm was higher in +MIPfunY75G compared to aqp0a−/− mutants, whereas mutants overexpressing MIPfunR153A only had transparency defects at the same frequency as MIPfun. 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 MIPfunY75G in aqp0a−/− resulted in failure of the lens nucleus centralizing at all developmental stages with the relative nucleus localization similar to aqp0a−/− at all stages. Overexpression of MIPfunR153A resulted in more centralized lens nuclei in mid and longer SLs compared to aqp0a−/− (P = 0.001 and P = 0.000) indicating partial rescue of centralization. At shorter SLs, MIPfunR153A overexpressing lenses had more anteriorly placed lens nuclei, like aqp0a−/−. Scale bars = 500 µm.