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M. Posner, N. Meyers, J. O. Liang; Morpholino Knockdown of -Crystallin in the Zebrafish (Danio rerio). Invest. Ophthalmol. Vis. Sci. 2009;50(13):4361.
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To determine the importance of A-crystallin in the development of the zebrafish lens.
Microinjection of antisense morpholinos was used to block the translation of A-crystallin mRNA in developing zebrafish. Two non-overlapping 25-basepair morpholinos designed to bind to either the start codon region or 5’ untranslated region of A-crystallin mRNA were injected into the 1-2 cell stage of zebrafish embryos. Control zebrafish were left uninjected or injected with a control morpholino that does not recognize any zebrafish mRNA sequence. Injected and uninjected embryos were cryosectioned and stained to assess the histology of the developing lens. Heads from other sacrificed embryos were homogenized and the resulting protein was probed by western blot to confirm A-crystallin knockdown.
Western blotting confirmed A-crystallin protein expression in control zebrafish at 2 and 4 days post ferilization (dpf), consistent with previous reports. However, A-crystallin was not detectable by western blot in morpholino injected zebrafish through 4 days (dpf). There were no detectable differences in either the gross anatomy or lens histology between A-crystallin depleted and control embryos. Cell nuclei were observable in the central region of lenses from all treatments at 2 dpf, and had disappeared in all treatments by 3 dpf.
Knockdown of A-crystallin to undetectable levels did not affect the normal histological development of the zebrafish lens. Previous studies of a fish lens mutant and a natural blind cavefish population suggested that reduced levels of A-crystallin might inhibit fiber cell differentiation, possibly by interfering with the regulation of apoptosis and leading to lens regression. The data presented here suggest that lack of A-crystallin alone does not interfere with fiber cell differentiation. This result is similar to the knockout of mouse A-crystallin, in which lenses initially develop normally but are smaller and produce early cataract.
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