DOI: 10.5061/dryad.f4qrfj730
title: Loss of alpha Ba-crystallin, but not alpha A-crystallin, increases age-related cataract in the zebrafish lens

description: 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.
description: 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.
description: # Loss of alpha Ba-crystallin, but not alpha A-crystallin, increases
 age-related cataract in the zebrafish lens
 [https://doi.org/10.5061/dryad.f4qrfj730](https://doi.org/10.5061/dryad.f4qrfj730) # Description of the data and file structure This dataset contains images of excised zebrafish lenses from wild-type and mutant strains at 6, 12, 18, and 24 months of age. These images were used to analyze the effects of alpha crystallin loss on the development of eye lens cataract during aging. The primary data folder contains four folders labeled with each zebrafish strain. Three are mutants with CRISPR-damaged alpha crystallin genes and the fourth is a wild-type ZDR strain. • cryaa mutant • cryaba mutant • cryabb mutant • wild type Each of these folders contains subfolders with lens images from four different ages (6, 12, 18, and 24 months). Within each of these age folders are individual folders that list the date images were taken, the zebrafish strain, and its age. Individual files are labeled as follows: • A number that indicates an individual fish • A letter that indicates the lens from that fish (each numbered fish has images for two lenses: a and b) • A final number which is an image of that individual lens. Typically, three images were taken of each lens: ⁃ One image focused on the underlying micrometer to show lens clarity ⁃ Another on the circumference of the lens to measure lens diameter ⁃ A third image over the “mm” on the micrometer to also show clarity For example, the file name “4b-3” indicates the 4th fish imaged, its second lens and the third image of that lens. In our publication, all lens images were blind scored by two individuals by removing age and genotype information before scoring. Phenotype scores are presented in Figure 5 of the associated paper and all values included in Supplemental Tables 3 and 4. The diameters of excised lenses were measured using ImageJ, with the micrometer in each image used for calibration. These data are presented in Figure 6 of the associated paper and all values are included in Supplemental Table 6.