The rw341 mutant gene encodes VPS45. (A) Genomic and cDNA organization of vps45 gene in rw341 mutants. (B) 5 dpf lenses of rw341 mutants (left) and rw341 mutants injected with wild-type (middle) or mutant (right) vps45 mRNA. Wild-type vps45 mRNA rescues multilayer phenotypes (middle, arrowheads). (C) Percentage of transparent lens fiber area in wild type, rw341 mutants and in rw341 mutants injected with wild-type or mutant vps45 mRNA. Wild-type vps45 mRNA significantly increases transparent area size in rw341 mutants. (D) 5 dpf lenses of wild type (left), rw341 mutants (middle) and rw341 mutants expressing VPS45-GFP under control of the foxe3 promoter (right). foxe3 promoter-mediated VPS45-GFP rescues multilayer phenotypes (right, arrowheads). (E) Percentage of transparent lens fiber area in wild type, rw341 mutants and rw341 mutants expressing VPS45-GFP under control of the foxe3 promoter. foxe3 promoter-mediated VPS45-GFP fully recovers transparent area size in rw341 mutants. (F) 5 dpf lenses of wild-type (left), rw341 mutants (middle) and rw341 mutants expressing VPS45-GFP under control of the αA-crystallin promoter (right). Multilayer phenotypes of lens epithelium become milder, but there is still cell aggregation in the posterior lens area in rw341 mutants overexpressing VPS45-GFP under the αA-crystallin promoter (right, asterisk). (G) Percentage of transparent lens fiber area in wild type, rw341 mutants and rw341 mutants expressing VPS45-GFP under control of the αA-crystallin promoter. αA-crystallin promoter-mediated VPS45-GFP partially recovers transparent area size in rw341 mutants, but the recovery does not reach the wild-type level. (H) Prox1 expression (green) in wild type, rw341 mutants and rw341 mutants expressing VPS45-GFP under control of foxe3 promoter or αA-crystallin promoter. Bottom panels indicate higher magnification of outlined areas. Ectopic Prox1 expression in rw341 mutants is inhibited by foxe3 promoter-induced VPS45-GFP (arrowheads), but not by αA-crystallin promoter-induced VPS45-GFP (asterisks). Fluorescent signals of VPS45-GFP were inactivated by the paraffin sectioning process. (C,E,G) Data are mean±s.d. Two-way (C) and one-way (E,G) ANOVA: *P<0.05, ***P<0.005. Scale bars: 20 µm.
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