MIPfun partially rescues lens transparency, optics and lens nucleus centralization in aqp0a?/?/aqp0b?/? double mutants. Representative juvenile lenses dissected from wild type (A, D) (standard length (SL) 9.2 mm), aqp0a?/?/aqp0b?/? (B, E) (SL 11 mm) or transgenic aqp0a?/?/aqp0b?/? zebrafish stably overexpressing MIPfun (C, F) (SL 11.2 mm) are shown. Wild type lens imaged through the optical axis under brightfield (BF) or darkfield (DF) illumination are clear (A), whereas aqp0a?/?/aqp0b?/? double mutants lens (B) reveal severe nuclear cataract with cortical transparency defects and inability to focus a microgrid which is reduced with overexpression of MIPfun due improved refraction and focal ability (C). The same lenses from A to C were imaged perpendicular to the optical axis and used to display the relative localization of the lens nucleus (D?F). The measurement of the localization of the lens nucleus center in relation to the lens center is shown (E), where * = lens nucleus center, + = lens center, a = lens radius, and r = distance of center of lens nucleus from lens center. (G) Overexpression of MIPfun in aqp0a?/?/aqp0b?/? resulted in a more centrally localized lens nucleus compared with aqp0a?/?/aqp0b?/? at mid-age (SL = 9?17 mm), with no differences at shorter SL (<9 mm) or longer SL (>17 mm) stages. The mean axial nucleus localization at a mean SL = 12.87 mm ± 1.5 SD of normalized lens nucleus was statistically significantly different (P = 0.001) between the 2 groups, whereas there was no difference in the 2 slopes (P = 0.589). Scale bars = 250 µm.

MIPfun partially restores lens transparency, optics, and nucleus centralization due to loss of Aqp0a. Representative lenses dissected from adult wild type (A) (SL = 20.1 mm), aqp0a?/? (B) (SL = 23.8 mm), and aqp0a?/?MIPfun transgenic zebrafish (C) (SL = 24.7 mm) imaged in equatorial orientation (through the optical axis) under brightfield (BF) or darkfield (DF) illumination reveal improved transparency in lenses expressing MIPfun (C) compared to aqp0a?/? (B). Similar improvement was seen in the ability of lenses to focus a grid. (D) Frequency of abnormal phenotypes in lenses from adult zebrafish (SL > 20 mm) was reduced in aqp0a?/? overexpressed MIPfun, with a complete loss of cataract. Refer to Supplementary Figure S1 for phenotype frequency by developmental stage. The same lenses from A to C were imaged perpendicular to the optical axis displayed the relative localization of the lens nucleus (E?G). (H) Overexpression of MIPfun results in the lens nucleus centralizing (closer to 0.0) more similar to wild type, compared to aqp0a?/? mutants, where the lens nucleus fails to centralize during development. The mean crossectional lens nucleus localization is statistically different at longer SLs between aqp0a?/? and transgenics overexpressing MIPfun (P = 0.000) as well as a difference in slopes (0.003). There is also a statistically significant difference between wild type and aqp0a?/?MIPfun slopes (P = 0.001) and at the shortest (P = 0.003) and longest (P = 0.002) SL cross-sectional means indicating partial rescue. Scale bars = 500 µm.

Auto-adhesion by MIPfun is not required for lens transparency, optics, and lens nucleus centralization. Representative lenses from adult aqp0a?/? mutants (A) (SL = 23.8 mm), aqp0a?/? mutants overexpressing MIPfun (B), (SL = 24.7 mm) or aqp0a?/? mutants overexpressing an auto-adhesive defective MIPfunN110T (C) (SL = 23.9 mm) were imaged through the optical axis under bright field (BF) or dark field (DF) illumination. Lens defects evident in aqp0a?/? mutants (A), as well as the optical defects when focusing a grid. The optical properties were improved by overexpression of MIPfun (B) or MIPfunN110T (C). (D) Both MIPfun or MIPfunN110T increased the frequency of normal phenotypes in adult fish SL > 20 mm, with no cataract observed. Refer to Supplementary Figure S1 for phenotype frequency by developmental stage. The same lenses from A to C were imaged perpendicular to the optical axis displayed the relative localization of the lens nucleus (E?G). (H) Overexpression of MIPfunN110T results in the lens nucleus centralizing closer to 0.0) like seen in MIPfun, compared to aqp0a?/? mutants, where the lens nucleus fails to centralize. The mean crossectional lens nucleus localization is statistically different at mid and longer SL between aqp0a?/? and in transgenics overexpressing MIPfunN110T (P = 0.000), as is the slope of the trends (P = 0.001). At shorter SL, the mean nucleus localization is similar between aqp0a?/? and MIPfunN110T overexpressing mutants. Scale bars = 500 µm.

Water transport of MIPfun is required for lens transparency, optics, and lens nucleus centralization. Representative lenses from adult aqp0a?/? mutants (A) (SL = 23.8 mm), aqp0a?/? mutants overexpressing MIPfun (B) (SL = 24.7 mm) or aqp0a?/? mutants overexpressing a water transport defective MIPfunN68Q (C) (SL = 25.9 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. A dense nuclear cataract, poor transparency in the center of the lenses, and inability to focus a grid clearly were observed in aqp0a-/-MIPfunN68Q lenses. (D) The frequency of cataract in adult fish SL > 20 mm was higher in +MIPfunN68Q compared to aqp0a?/? mutants. Refer to Supplementary Figure S1 for phenotype frequency by developmental stage. The same lenses from A to C were imaged perpendicular to the optical axis displayed the relative localization of the lens nucleus (E?G). (H) Overexpression of MIPfunN68Q in aqp0a?/? resulted in failure of the lens nucleus centralization, and the nucleus is more anteriorly localized than in aqp0a?/? at shorter SLs (P = 007 at cross sectional mean nucleus r/a at SL = 1.96 mm ± 1.5 SD) and mid SLs (P = 0.000 at cross sectional mean nucleus r/a at SL = 12.87 mm ± 1.5 SD. The slope of graph is statistically different between aqp0a?/? and mutants expressing MIPfunN68Q (P = 0.003). Scale bars = 500 µm.

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.

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.