Impact of α-crystallin protein loss on zebrafish lens development.

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 cataracts, 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 mutations in each of the three α-crystallin genes (cryaa, cryaba and cryabb) to prevent protein production. The absence of each α-crystallin protein was analyzed 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 larvae at 3 and 4 dpf but little substantial change in normal fiber cell denucleation. Loss of αBa-crystallin produced no substantial lens defects. Our cryabb mutant produced a truncated αBb-crystallin protein and showed no substantial change in lens development. Mutation of each α-crystallin gene did not alter the mRNA levels of the remaining two, suggesting a lack of genetic compensation. These data suggest that αA-crystallin plays some role in lens development, but the range of phenotype severity in null mutants indicates its loss simply increases the chance for defects and that the protein is not essential. Our finding that cryaba and cryabb mutants lack noticeable lens defects is congruent with insubstantial transcript levels for these genes in lens epithelial and fiber cells through five days of development. Future experiments can explore the molecular mechanisms leading to lens defects in cryaa null mutants and the impact of αA-crystallin loss during zebrafish lens aging.

Archaeal viruses, not archaeal phages: an archaeological dig.

Viruses infect members of domains Bacteria, Eukarya, and Archaea. While those infecting domain Eukarya are nearly universally described as "Viruses", those of domain Bacteria, to a substantial extent, instead are called "Bacteriophages," or "Phages." Should the viruses of domain Archaea therefore be dubbed "Archaeal phages," "Archaeal viruses," or some other construct? Here we provide documentation of published, general descriptors of the viruses of domain Archaea. Though at first the term "Phage" or equivalent was used almost exclusively in the archaeal virus literature, there has been a nearly 30-year trend away from this usage, with some persistence of "Phage" to describe "Head-and-tail" archaeal viruses, "Halophage" to describe viruses of halophilic Archaea, use of "Prophage" rather than "Provirus," and so forth. We speculate on the root of the early 1980's transition from "Phage" to "Virus" to describe these infectious agents, consider the timing of introduction of "Archaeal virus" (which can be viewed as analogous to "Bacterial virus"), identify numerous proposed alternatives to "Archaeal virus," and also provide discussion of the general merits of the term, "Phage." Altogether we identify in excess of one dozen variations on how the viruses of domain Archaea are described, and document the timing of both their introduction and use.

Why does the zebrafish cloche mutant develop lens cataract?

The zebrafish has become a valuable model for examining ocular lens development, physiology and disease. The zebrafish cloche mutant, first described for its loss of hematopoiesis, also shows reduced eye and lens size, interruption in lens cell differentiation and a cataract likely caused by abnormal protein aggregation. To facilitate the use of the cloche mutant for studies on cataract development and prevention we characterized variation in the lens phenotype, quantified changes in gene expression by qRT-PCR and RNA-Seq and compared the ability of two promoters to drive expression of introduced proteins into the cloche lens. We found that the severity of cloche embryo lens cataract varied, while the decrease in lens diameter and retention of nuclei in differentiating lens fiber cells was constant. We found very low expression of both αB-crystallin genes (cryaba and cryabb) at 4 days post fertilization (dpf) by both qRT-PCR and RNA-Seq in cloche, cloche sibling and wildtype embryos and no significant difference in αA-crystallin (cryaa) expression. RNA-Seq analysis of 4 dpf embryos identified transcripts from 25,281 genes, with 1,329 showing statistically significantly different expression between cloche and wildtype samples. Downregulation of eight lens ß- and γM-crystallin genes and 22 retinal related genes may reflect a general reduction in eye development and growth. Six stress response genes were upregulated. We did not find misregulation of any known components of lens development gene regulatory networks. These results suggest that the cloche lens cataract is not caused by loss of αA-crystallin or changes to lens gene regulatory networks. Instead, we propose that the cataract results from general physiological stress related to loss of hematopoiesis. Our finding that the zebrafish αA-crystallin promoter drove strong GFP expression in the cloche lens demonstrates its use as a tool for examining the effects of introduced proteins on lens crystallin aggregation and cataract prevention.

The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function.

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.