Abstract
Purpose :
We previously showed that loss of the small heat shock protein αA-crystallin leads to an increase in zebrafish lens developmental defects. However, loss of neither αB-crystallin altered lens development, and gene expression data suggest that αB-crystallin mRNA is not found in the lens through 5 days post fertilization (dpf). Therefore, we examined when αB-crystallin genes (cryaba and cryabb) become expressed in the zebrafish lens. We also assessed the impact of α-crystallin loss on lens clarity through 24mpf (months post fertilization).
Methods :
Zebrafish mutant lines were generated using CRISPR/Cas9 editing and loss of α-crystallins was confirmed by mass spectrometry. Reverse transcription qPCR included two reference genes (eef1a1l1 and rpl13a), negative RT controls and biological and technical triplicates. Lens clarity was assessed with DIC microscopy and ImageJ was used to quantify fish lengths and lens diameters. Statistical analyses were done in R and MedCalc.
Results :
Quantitative PCR showed cryaba mRNA in the lens by 10dpf and increasing at 19dpf. Lens cryabb mRNA was also present by 10dpf, but at lower levels than cryaba, and increased steadily into adulthood. Wild-type zebrafish showed cataract development with age, with 35% and 31% of individuals containing lens opacities at 18 and 24mpf, respectively. Loss of αA-crystallin did not increase the prevalence of general lens opacity through 18mpf, with a 24mpf timepoint still to be collected. However, loss of αBa-crystallin did increase cataract at 24mpf (p-value=0.021) and cryaba mutants were more likely to contain a unique localized anterior opacity (p-value=0.005). cryaba mutants also had smaller lenses than wild-type fish at 18 and 24mpf (p-values=0.012 and 0.003).
Conclusions :
The lack of a phenotype in our aging αA-crystallin null zebrafish lenses differs from findings in the mouse knockout model, which had smaller lenses and noticeable opacity by 10 weeks of age. Interestingly, it was loss of the lens-specific αBa-crystallin that led to increased general opacity, an anterior defect and smaller lenses. These results may suggest a switch in the relative importance of αA- and αBa-crystallin to maintenance of lens transparency during zebrafish aging. These findings impact the use of zebrafish in studying age-related cataract and our understanding of how α-crystallins have evolved to function in the vertebrate lens.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.