Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. However, little is known about zebrafish α-crystallin promoter function, how it compares to that of mammals, or whether mammalian α-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of α-crystallin promoter fragments from each species combined with the coding sequence for green fluorescent protein (GFP) into zebrafish zygotes to determine the resulting spatiotemporal expression patterns in the developing embryo. We also measured mRNA levels and protein abundance for all three zebrafish α-crystallins. Our data showed that mouse and zebrafish αA-crystallin promoters generated similar GFP expression in the lens, but with earlier onset when using mouse promoters. Expression was also found in notochord and skeletal muscle in a smaller percentage of embryos. Mouse αB-crystallin promoter fragments drove GFP expression primarily in zebrafish skeletal muscle, with less common expression in notochord, lens, heart and in extraocular regions of the eye. A short fragment containing only a lens-specific enhancer region increased lens and notochord GFP expression while decreasing muscle expression, suggesting that the influence of mouse promoter control regions carries over into zebrafish embryos. The two paralogous zebrafish αB-crystallin promoters produced subtly different expression profiles, with the aBa promoter driving expression equally in notochord and skeletal muscle while the αBb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish αA increased between 1 and 2 days post fertilization (dpf), αBa increased between 4 and 5 dpf, but αBb remained at baseline levels through 5 dpf. Parallel reaction monitoring (PRM) mass spectrometry was used to detect αA, aBa, and αBb peptides in digests of zebrafish embryos. In whole embryos, αA-crystallin was first detected by 2 dpf, peaked in abundance by 4–5 dpf, and was localized to the eye. αBa was detected in whole embryo at nearly constant levels from 1–6 dpf, was also localized primarily to the eye, and its abundance in extraocular tissues decreased from 4–7 dpf. In contrast, due to its low abundance, no αBb protein could be detected in whole embryo, or dissected eye and extraocular tissues. Our results show that mammalian α-crystallin promoters can be efficiently screened in zebrafish embryos and that their controlling regions are well conserved. An ontogenetic shift in zebrafish aBa-crystallin promoter activity provides an interesting system for examining the evolution and control of tissue specificity. Future studies that combine these promoter based approaches with the expanding ability to engineer the zebrafish genome via techniques such as CRISPR/Cas9 will allow the manipulation of protein expression to test hypotheses about lens crystallin function and its relation to lens biology and disease.

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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 cataract, 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 null mutations for each of the three α-crystallin genes (cryaa, cryaba and cryabb). Absence of protein was confirmed 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 larval lenses at 3 and 4 dpf, but little significant change in normal fiber cell denucleation. Loss of either αBa- or αBb-crystallin produced no significant lens defects. Mutation of each α-crystallin gene did not alter the expression levels of the remaining two, suggesting a lack of genetic compensation. These data confirm a developmental role for αA-crystallin in lens development, but the range of phenotype severity suggests its loss simply increases the chance for defect, and that the protein is not essential. Our finding that cryaba and cryabb null mutants lack noticeable lens defects is congruent with insignificant transcript levels in lens epithelial and fiber cells. Future experiments can explore the molecular consequences of cryaa mutation and causes of lens defects in this null mutant, as well as the roles of other genes in lens development and function.