Vision is enabled by a properly shaped and transparent lens – a deeply conserved structure found within the camera-eye of groups otherwise highly divergent such as cephalopods and vertebrates. The vertebrate eye lens is primarily composed of proteins from three families: the α, β and γ-crystallins, which contribute to the transparency and refractive power required to focus light onto the retina (Bloemendal & Cate, 1959; Harding & Dilley, 1976; Wistow & Piatigorsky, 1988; Bloemendal & Jong, 1991; Andley, 2007). Various vertebrates also contain what have been termed taxon-specific crystallins, such as the δ-crystallins found in birds and other reptiles (Zwaan, 1968; Jong, Stapel & Zweers, 1981). Surprisingly, α-crystallin gene sequences were found to be similar to the small heat shock protein genes in Drosophila, suggesting that these abundant lens materials were evolutionarily coopted from stress-induced protective proteins (Ingolia & Craig, 1982). Vertebrate lenses have two distinct cell-types: fiber and epithelial cells. In fiber cells, the mammalian α-crystallin has been shown to function as a molecular chaperone, preventing the aggregation of denatured protein that could otherwise lead to lens cataract (Horwitz, 1992). This chaperone function is critical for lens homeostasis as lens fiber cells lose their nuclei during development and are not able to replace aging protein (Kuwabara & Imaizumi, 1974; PIATIGORSKY, 1981; Bassnett, 2002).

Mammals and birds produce two α-crystallin proteins due to a gene duplication event early in vertebrate evolution (Wistow & Piatigorsky, 1988). One of these, αA-crystallin, is primarily expressed in the lens, but its paralog αB-crystallin is widely expressed in lens, nervous and muscular tissue (Bhat & Nagineni, 1989; Dubin, Wawrousek & Piatigorsky, 1989). Mutations in the cryaa gene for αA-crystallin can lead to congenital cataract, while those in the cryab gene for αB-crystallin can produce both cataract and cardiomyopathies (Litt et al., 1998; Vicart et al., 1998; Berry et al., 2001). A variety of α-crystallin mutants and genetic modifications have been used to explore the structure/function relationships of these proteins (Shiels & Hejtmancik, 2021). In mouse knockout models, loss of αA-crystallin led to lens cataract while loss of αB-crystallin did not, suggesting at least in this species that αA-crystallin is more protective against the loss of lens transparency (Brady et al., 1997, 2001). However, expression of a human cataract-linked αB-crystallin mutation, the R120G mutant, also produces cataract when expressed in the mouse lens, suggesting its importance to lens function (Andley et al., 2011).

The zebrafish lens proteome shares many similarities with mammals (Posner et al., 2008; Greiling, Houck & Clark, 2009). However, the genome duplication that occurred at the base of teleost evolution led to the presence of two αB-crystallin paralogs (Postlethwait et al., 1998; Smith et al., 2006). Interestingly, one of these paralogs, αBa-crystallin, has evolved lens-preferred expression while the other, αBb-crystallin, maintains the broad expression found in the single-copy mammalian protein (Posner, Kantorow & Horwitz, 1999; Smith et al., 2006). The protective chaperone activities of the two αB-crystallin paralogs have also diverged when measured in vitro, although there are conflicting data on which is the stronger chaperone (Smith et al., 2006; Koteiche et al., 2015).

Zebrafish are an excellent model system for studying the genetic and molecular processes that guide lens development (Vihtelic, 2008). Their external development, and our ability to perform mutant-analysis, make zebrafish embryos and larvae ideally suited to investigating early events in lens cell-type specification. Mutations of each of the three αB-crystallin genes have been used to examine their impact on early lens development. Zebrafish mutants that do not express αA-crystallin show subtle defects, such as roughness and irregular borders between fiber cells, that were observable using differential interference contrast microscopy (Zou et al., 2015; Posner et al., 2023). Results with αB-crystallin mutants were mixed, with one study showing lens developmental defects while another showed no effect (Mishra et al., 2018; Posner et al., 2023). It is not known how the loss of α-crystallins might affect cataract development as fish age. In fact, little is known about the prevalence of lens cataract in lab-raised zebrafish. Most work on fish lens cataract has focused on aquacultured species, such as salmon and lumpfish (Richardson et al., 1985; Jonassen et al., 2017). Considering the presence of two α-crystallins with lens-preferred expression in zebrafish, it is unclear which of these proteins performs the protective role played by αA-crystallin in the mammalian lens.

The goal of this study was to investigate the role of α-crystallin during lens aging. Zebrafish raised in the lab can live for 3 to 4 years. In this study we examined fish at 6-month intervals through 24 months of age. We hypothesized that the presence of two lens-preferred αB-crystallins in zebrafish might have led to evolutionary changes in their protective roles against cataract formation. The data generated address fundamental questions about the role played by a group of proteins central to the evolutionary success of the vertebrate lens.