Loss of αBa-crystallin, but not αA-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 ßB1-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.

Loss of αBa-crystallin, but not αA-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 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 ßB1-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.

Impact of α-crystallin protein loss on zebrafish lens development.

The α-crystallin small heat shock proteins contribute to the transparency and refractive properties of the vertebrate eye lens and prevent the protein aggregation that would otherwise produce lens cataracts, the leading cause of human blindness. There are conflicting data in the literature as to what role the α-crystallins may play in early lens development. In this study, we used CRISPR gene editing to produce zebrafish lines with mutations in each of the three α-crystallin genes (cryaa, cryaba and cryabb) to prevent protein production. The absence of each α-crystallin protein was analyzed by mass spectrometry, and lens phenotypes were assessed with differential interference contrast microscopy and histology. Loss of αA-crystallin produced a variety of lens defects with varying severity in larvae at 3 and 4 dpf but little substantial change in normal fiber cell denucleation. Loss of αBa-crystallin produced no substantial lens defects. Our cryabb mutant produced a truncated αBb-crystallin protein and showed no substantial change in lens development. Mutation of each α-crystallin gene did not alter the mRNA levels of the remaining two, suggesting a lack of genetic compensation. These data suggest that αA-crystallin plays some role in lens development, but the range of phenotype severity in null mutants indicates its loss simply increases the chance for defects and that the protein is not essential. Our finding that cryaba and cryabb mutants lack noticeable lens defects is congruent with insubstantial transcript levels for these genes in lens epithelial and fiber cells through five days of development. Future experiments can explore the molecular mechanisms leading to lens defects in cryaa null mutants and the impact of αA-crystallin loss during zebrafish lens aging.