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Mason Posner, Heather Prior, Neeley Meyers, Jackie Skiba, Jennifer O. Liang; Alpha A-crystallin is not Required for Normal Development of the Zebrafish (Danio rerio) Lens. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2275.
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© ARVO (1962-2015); The Authors (2016-present)
While genetic knockout of the mouse alpha A-crystallin gene produces minimal to no effects on lens development, recent studies using the zebrafish and a closely related species suggest that alpha A-crystallin is needed in these fish species for fiber cell differentiation and continued lens viability. We directly tested this hypothesis by chemically inhibiting zebrafish alpha A-crystallin translation and assessing possible resulting phenotypes. Considering the possibility of alpha crystallin functional redundancy, we also initiated experiments to inhibit translation of zebrafish alpha Bb-crystallin.
Zebrafish embryos at 1-8 cell stage were injected with 4.5 ng of a 1:1 mixture of two non-overlapping antisense morpholinos that block translation of alpha A-crystallin mRNA into protein. Control zebrafish were left uninjected or injected with 4.5 ng of a control morpholino that does not recognize any zebrafish mRNA sequence. Western blots of protein homogenates from injected and uninjected embryos were used to confirm specificity of alpha A-crystallin knockdown. Cryosectioning and subsequent hematoxylin and eosin staining as well as measurements of eye diameter and body lengths were used to identify possible phenotypes. Preliminary injections with various amounts of a single translation blocking morpholino to zebrafish alpha Bb-crystallin were used to identify an appropriate dose for knockdown of this second alpha crystallin.
Western blotting confirmed that our morpholino injections suppressed alpha A-crystallin protein expression to undetectable levels through 4 days post fertilization (dpf), beyond the point at which the lens becomes optically functional. Injections had no effect on beta B1-crystallin expression. There were no detectable changes in the gross anatomy of injected fish, and histology showed normal lens development. Neither body length nor eye diameter differed significantly between alpha A-crystallin depleted and control injected embryos. A 4.5 ng dose of a single alpha Bb-crystallin morpholino produced defects in gross anatomy, suggesting non-specific toxicity, which disappeared at a lower dose of 2.25 ng.
These data indicate that alpha A-crystallin is not necessary for normal larval development of the zebrafish lens, similar to results from gene knockout studies in the mouse. This result suggests that reduction of alpha A-crystallin protein levels alone may not be a significant cause of cataract, inhibition of fiber cell differentiation and lens regression observed in previous studies on the fish lens.
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