No abstract

The a-crystallins are evolutionarily related members of the small heat shock protein (sHSP) superfamily which are taxonomically ubiquitous components of the vertebrate eye lens [1]. The aA-crystallin and aB-crystallin genes arose through a gene duplication event that occurred early in vertebrate history and are most closely related to sHsp20 [2]. Mammalian aA-crystallin is primarily lens specific and has lost the stress induction response that characterizes most sHsps, although some metals induce its expression [3]. In contrast, multiple cellular stresses induce mammalian aB-crystallin expression in a variety of tissues [4]. The mammalian a-crystallins act as chaperone-like molecules by binding to and preventing the aggregation of non-native We previously reported that zebrafish aB-crystallin is not constitutively expressed in nervous or muscular tissue and has reduced chaperone-like activity compared with its human ortholog. Here we characterize the tissue expression pattern and chaperone-like activity of a second zebrafish aBcrystallin. Expressed sequence tag analysis of adult zebrafish lens revealed the presence of a novel a-crystallin transcript designated cryab2 and the resulting protein aB2-crystallin. The deduced protein sequence was 58.2% and 50.3% identical with human aB-crystallin and zebrafish aB1-crystallin, respectively. RT-PCR showed that aB2-crystallin is expressed predominantly in lens but, reminiscent of mammalian aB-crystallin, also has lower constitutive expression in heart, brain, skeletal muscle and liver. The chaperone-like activity of purified recombinant aB2 protein was assayed by measuring its ability to prevent the chemically induced aggregation of a-lactalbumin and lysozyme. At 25°C and 30°C, zebrafish aB2 showed greater chaperone-like activity than human aB-crystallin, and at 35°C and 40°C, the human protein provided greater protection against aggregation. 2D gel electrophoresis indicated that aB2-crystallin makes up 0.16% of total zebrafish lens protein. Zebrafish is the first species known to express two different aB-crystallins. Differences in primary structure, expression and chaperone-like activity suggest that the two zebrafish aB-crystallins perform divergent physiological roles. After gene duplication, zebrafish aB2 maintained the widespread protective role also found in mammalian aB-crystallin, while zebrafish aB1 adopted a more restricted, nonchaperone role in the lens. Gene duplication may have allowed these functions to separate, providing a unique model for studying structure-function relationships and the regulation of tissue-specific expression patterns.Abbreviations sHSP, small heat shock protein.

The vertebrate lens is a valuable model system for investigating the gene expression changes that coordinate tissue differentiation due to its inclusion of two spatially separated cell types, the outer epithelial cells and the deeper denucleated fiber cells that they support. Zebrafish are a useful model system for studying lens development given the organ's rapid development in the first several days of life in an accessible, transparent embryo. While we have strong foundational knowledge of the diverse lens crystallin proteins and the basic gene regulatory networks controlling lens development, no study has detailed gene expression in a vertebrate lens at single cell resolution. Here we report an atlas of lens gene expression in zebrafish embryos at single cell resolution through five days of development, identifying a number of novel regulators of lens development as potential targets for future functional studies. Our temporospatial expression data address open questions about the function of α-crystallins during lens development and provides the first detailed view of β-and γ-crystallin expression in and outside the lens. We describe subfunctionalization in transcription factor genes that occur as paralog pairs in the zebrafish. Finally, we examine the expression dynamics of cytoskeletal, RNA-binding, and transcription factors genes, identifying a number of novel patterns. Overall these data provide a foundation for identifying and characterizing lens developmental regulatory mechanisms and revealing targets for future functional studies with potential therapeutic impact.

Gallic acid has been reported to be responsible for the invasive success of nonnative genotypes of Phragmites australis in North America. We have been unable to confirm previous reports of persistent high concentrations of gallic acid in the rhizosphere of invasive P. australis, and of high concentrations of gallic acid and gallotannins in P. australis rhizomes. The half-life of gallic acid in nonsterile P. australis soil was measured by aqueous extraction of soils and found to be less than 1 day at added concentrations up to 10,000 μg g(-1). Furthermore, extraction of P. australis soil collected in North Carolina showed no evidence of gallic acid, and extractions of both rhizomes and leaves of samples of four P. australis populations confirmed to be of invasive genotype show only trace amounts of gallic acid and/or gallotannins. The detection limits were less than 20 μg gallic acid g(-1) FW in the rhizome samples tested, which is approximately 0.015 % of the minimum amount of gallic acid expected based on previous reports. While the occurrence of high concentrations of gallic acid and gallotannins in some local populations of P. australis cannot be ruled out, our results indicate that exudation of gallic acid by P. australis cannot be a primary, general explanation for the invasive success of this species in North America.

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.