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Hassane S Mchaourab, Ping Zou, Hanane A Koteiche, Sanjay Mishra, Poonam Rathore, Wenbiao Chen; Expression of cataract-linked mutants of γ-crystallin in the Zebrafish lens and effects of αA-crystallin expression. Invest. Ophthalmol. Vis. Sci. 2014;55(13):4053.
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A number of mutations in γ-crystallins have been associated with congenital cataracts in humans and mouse models. Some of these mutants are associated with changes in stability, i.e. shifts in the unfolding equilibrium. Studies of two such mutants, I4F and V76D, in mouse models suggest multiple mechanistic origins of cataract. However, the interaction of these mutants with the resident chaperone, α-crystallin, in the context of a lens fiber cell has not been investigated. To address this question, we have generated transgenic zebrafish expressing γ-crystallin mutants. The transgenic fish were then crossed with either a transgenic line overexpressing mouse αA-crystallin or an αA-crystallin knowckout strain.
Embryos were screened for lens defects by bright light microscope at 3 dpf, followed by differential interference contrast (DIC) and reflectance analysis. DIC images were used as a general locator for reflectance analysis and reflected light was analyzed by digital image analysis program Image J.
Expression of γ-crystallin mutants lead to abnormalities in the zebrafish lens. We observed clear evidence of increased scattering that were more frequent and severe in the strain expressing the double mutant γ-crystallin I4F/V76D relative to the mutant I4F. Crossing the I4F expressing zebrasifh with an αA-crystallin knockout strain accentuated the severity of the phenotype relative to either strain alone. Crossing the I4F/V76D line with transgenic fish expressing mouse αA-crystallin did not attenuate the phenotype.
The results establish the use of zebrafish lens to study the mechanistic basis of cataracts. The failure of αA-crystallin to rescue the phenotype suggests that its chaperone activity may not be sufficient to overcome the deleterious effects of the γ-crystallin mutants.
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