Alpha crystallins are small heat shock proteins essential to normal ocular lens function. They also help maintain homeostasis in many non-ocular vertebrate tissues and their expression levels change in multiple diseases of the nervous and cardiovascular system and during cancer. The specific roles that α-crystallins may play in eye development are unclear. Studies with knockout mice suggested that only one of the two mammalian α-crystallins is required for normal early lens development. However, studies in two fish species suggested that reduction of αA-crystallin alone could inhibit normal fiber cell differentiation, cause cataract and contribute to lens degeneration. In this study we used synthetic antisense morpholino oligomers to suppress the expression of zebrafish αA-crystallin to directly test the hypothesis that, unlike mammals, the zebrafish requires αA-crystallin for normal early lens development. Despite the reduction of zebrafish αA-crystallin protein to undetectable levels by western analysis through 4 days of development we found no changes in fiber cell differentiation, lens morphology or transparency. In contrast, suppression of AQP0a expression, previously shown to cause lens cataract, produced irregularly shaped lenses, delay in fiber cell differentiation and lens opacities detectable by confocal microscopy. The normal development observed in αA-crystallin deficient zebrafish embryos may reflect similarly non-essential roles for this protein in the early stages of both zebrafish and mammalian lens development. This finding has ramifications for a growing number of researchers taking advantage of the zebrafish's transparent external embryos to study vertebrate eye development. Our demonstration that lens cataracts can be visualized in three-dimensions by confocal microscopy in a living zebrafish provides a new tool for studying the causes, development and prevention of lens opacities.
Bibliographical noteFunding Information:
This work was funded by an AREA grant from the National Institutes of Health / National Eye Institute to M.P. ( R15 EY13535 ). Two undergraduate student authors were provided summer room and board by Ashland University in support of this study. This study was initiated during a faculty sabbatical by M.P. supported by Ashland University and conducted in the Department of Biology at Case Western Reserve University. The AQP0a morpholino used in this study was provided by Daniel M. Clemens, UC Irvine and the crystallin antibodies were produced and provided by Thomas Vihtelic while at the University of Notre Dame. Neeley Meyers contributed to early experimental design for this study while an undergraduate student at Ashland University.
- Alpha crystallin
- Lens development
- Ocular lens
- Small heat shock protein