Loss of alpha Ba-crystallin, but not alpha A-crystallin, increases age-related cataract in the zebrafish lens

The vertebrate eye lens is an unusual organ in that most of its cells lack nuclei and the ability to replace aging protein. The small heat shock protein α-crystallins evolved to become key components of this lens, possibly because of their ability to prevent aggregation of aging protein that would otherwise lead to lens opacity. Most vertebrates express two α-crystallins, αA- and αB-crystallin, and mutations in each are linked to human cataract. In a mouse knockout model, only the loss of αA-crystallin led to early-stage lens cataract. We have used the zebrafish as a model system to investigate the role of α-crystallins during lens development. Interestingly, while zebrafish express one lens-specific αA-crystallin gene (cryaa), they express two αB-crystallin genes, with one evolving lens specificity (cryaba) and the other retaining the broad expression of its mammalian ortholog (cryabb). In this study, we used individual mutant zebrafish lines for all three α-crystallin genes to determine the impact of their loss on age-related cataract. Surprisingly, unlike mouse knockout models, we found that the loss of the αBa-crystallin gene cryaba led to an increase in lens opacity compared to cryaa null fish at 24 months of age. Loss of αA-crystallin did not increase the prevalence of cataract. We also used single-cell RNA-Seq and RT-qPCR data to show a shift in the lens expression of zebrafish α-crystallins between 5 and 10 days post fertilization (dpf), with 5 and 6 dpf lenses expressing cryaa almost exclusively, and expression of cryaba and cryabb becoming more prominent after 10 dpf. These data show that cryaa is the primary α-crystallin during early lens development, while the protective role for cryaba becomes more important during lens aging. This study is the first to quantify cataract prevalence in wild-type aging zebrafish, showing that lens opacities develop in approximately 25% of fish by 18 months of age. None of the three α-crystallin mutants showed a compensatory increase in the expression of the remaining two crystallins, or in the abundant βB1-crystallin. Overall, these findings indicate an ontogenetic shift in the functional importance of individual α-crystallins during zebrafish lens development. Our finding that the lens-specific zebrafish αBa-crystallin plays the leading role in preventing age-related cataract adds a new twist to our understanding of vertebrate lens evolution.

Methods
Lenses from adult wildtype fish and each of our three α-crystallin mutant lines were removed at 6, 12, 18, and 24 months to assess any abnormalities. Fish within one month of these ages were pooled together to represent that age. One fish from each tank was genotyped to confirm their identity and fish were not selected based on any apparent differences between individuals. We collected lenses from at least 10 fish of each age, except for 24 months, when at least 15 fish were used. The standard length of each fish was measured by ruler and any opacity of the lens or cornea from the intact eye was noted. Lenses were dissected from anesthetized individuals, placed in PBS, and imaged under a dissecting microscope at 40X total magnification over a stage micrometer. Lenses were imaged within 15 minutes of removal and placement in PBS as we often saw a visible optical separation between the lens nucleus and cortex after prolonged incubation in PBS. Images have not been processed in any way. Folders contain TIFF files produced with SPOT imaging software.