Loss of alpha Ba-crystallin, but not alpha A-crystallin, increases age-related cataract in the zebrafish lens
Data files
May 16, 2024 version files 4.45 GB
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Posner_EER_Lens_Images_2024.zip
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README.md
Abstract
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.
README: 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
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.
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.