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Mason Posner, Baley Bernthisel, Joshua Allman, Cassie Craig, Kelly Murray; The zebrafish cloche mutant can be used as an in vivo model to test the ability of α-crystallins to prevent lens cataract. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5584.
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© ARVO (1962-2015); The Authors (2016-present)
The zebrafish cloche mutant develops lens cataracts that can be prevented by expression of endogenous α-crystallin. In this study we characterize the early developmental defects in the cloche lens and show the effects of both increasing and decreasing αA-crystallin expression levels.
Live embryos from crossed heterozygous cloche zebrafish were examined by Nomarski microscopy to assess the prevalence of lens cataract. Fixed embryos were also cryosectioned and stained with hematoxylin and eosin or DAPI to examine defects in lens development. Total RNA collected from 3 dpf embryos was used to reverse transcribe cDNA for qPCR experiments to compare the expression levels of αA- and αBb-crystallin between homozygous cloche embryos and wildtype zebrafish. Linearized plasmid (50-100 picograms) containing the zebrafish αA-crystallin gene driven by the human βB1-crystallin promoter were injected into cloche zygotes and the resulting embryos were examined at 3 dpf to assess the effect on lens cataract development. Translation-blocking morpholinos were injected into the yolks of 1-4 cell stage cloche embryos to determine the effects of reduced αA-crystallin levels on non-cloche phenotype embryos.
By 3 dpf cloche phenotype zebrafish embryos showed irregularly shaped lenses with cataract and retention of fiber cell nuclei. Five dpf cloche phenotype lenses were highly reduced in size compared to non-cloche siblings. Quantitative PCR analysis showed only a small decrease in αA-crystallin expression (RQ 0.7) in cloche-phenotype embryos compared to wildtype embryos. There was no difference in αBb-crystallin expression. Expression of exogenous αA-crystallin reduced the proportion of 3 dpf cloche embryos with cataract from 96% to 44%. Preliminary experiments suggest that morpholino-induced suppression of αA-crystallin translation in non-cloche phenotype siblings increased the proportion of embryos with cataract, while having no effect on wildtype zebrafish embryos.
Our results confirm that lens cataract and abnormalities in fiber cell differentiation are readily visualized in cloche embryos and demonstrate that cloche lens phenotypes can be altered by increasing or decreasing αA-crystallin expression levels. The cloche mutant zebrafish is promising as an in vivo model for studying the ability of various α-crystallins to prevent lens cataract.
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