Mason Posner

Gene duplication and separation of functions in αB-crystallin from zebrafish (Danio rerio)

We previously reported that zebrafish αB‐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 αB‐crystallin. Expressed sequence tag analysis of adult zebrafish lens revealed the presence of a novel α‐crystallin transcript designated cryab2 and the resulting protein αB2‐crystallin. The deduced protein sequence was 58.2% and 50.3% identical with human αB‐crystallin and zebrafish αB1‐crystallin, respectively. RT‐PCR showed that αB2‐crystallin is expressed predominantly in lens but, reminiscent of mammalian αB‐crystallin, also has lower constitutive expression in heart, brain, skeletal muscle and liver. The chaperone‐like activity of purified recombinant αB2 protein was assayed by measuring its ability to prevent the chemically induced aggregation of α‐lactalbumin and lysozyme. At 25 °C and 30 °C, zebrafish αB2 showed greater chaperone‐like activity than human αB‐crystallin, and at 35 °C and 40 °C, the human protein provided greater protection against aggregation. 2D gel electrophoresis indicated that αB2‐crystallin makes up ≈ 0.16% of total zebrafish lens protein. Zebrafish is the first species known to express two different αB‐crystallins. Differences in primary structure, expression and chaperone‐like activity suggest that the two zebrafish αB‐crystallins perform divergent physiological roles. After gene duplication, zebrafish αB2 maintained the widespread protective role also found in mammalian αB‐crystallin, while zebrafish αB1 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.

Functional validation of hydrophobic adaptation to physiological temperature in the small heat shock protein αA-crystallin.

Small heat shock proteins (sHsps) maintain cellular homeostasis by preventing stress and disease-induced protein aggregation. While it is known that hydrophobicity impacts the ability of sHsps to bind aggregation-prone denaturing proteins, the complex quaternary structure of globular sHsps has made understanding the significance of specific changes in hydrophobicity difficult. Here we used recombinant protein of the lenticular sHsp α A-crystallin from six teleost fishes environmentally adapted to temperatures ranging from -2°C to 40°C to identify correlations between physiological temperature, protein stability and chaperone-like activity. Using sequence and structural modeling analysis we identified specific amino acid differences between the warm adapted zebrafish and cold adapted Antarctic toothfish that could contribute to these correlations and validated the functional consequences of three specific hydrophobicity-altering amino acid substitutions in αA-crystallin. Site directed mutagenesis of three residues in the zebrafish (V62T, C143S, T147V) confirmed that each impacts either protein stability or chaperone-like activity or both, with the V62T substitution having the greatest impact. Our results indicate a role for changing hydrophobicity in the thermal adaptation of α A-crystallin and suggest ways to produce sHsp variants with altered chaperone-like activity. These data also demonstrate that a comparative approach can provide new information about sHsp function and evolution.

Loss of the small heat shock protein αA-crystallin does not lead to detectable defects in early zebrafish lens development

Alpha crystallins are small heat shock proteins essential to normal ocular lens function. They also help maintain homeostasis in many non-ocular vertebrate tissues and their expression levels change in multiple diseases of the nervous and cardiovascular system and during cancer. The specific roles that α-crystallins may play in eye development are unclear. Studies with knockout mice suggested that only one of the two mammalian α-crystallins is required for normal early lens development. However, studies in two fish species suggested that reduction of αA-crystallin alone could inhibit normal fiber cell differentiation, cause cataract and contribute to lens degeneration. In this study we used synthetic antisense morpholino oligomers to suppress the expression of zebrafish αA-crystallin to directly test the hypothesis that, unlike mammals, the zebrafish requires αA-crystallin for normal early lens development. Despite the reduction of zebrafish αA-crystallin protein to undetectable levels by western analysis through 4 days of development we found no changes in fiber cell differentiation, lens morphology or transparency. In contrast, suppression of AQP0a expression, previously shown to cause lens cataract, produced irregularly shaped lenses, delay in fiber cell differentiation and lens opacities detectable by confocal microscopy. The normal development observed in αA-crystallin deficient zebrafish embryos may reflect similarly non-essential roles for this protein in the early stages of both zebrafish and mammalian lens development. This finding has ramifications for a growing number of researchers taking advantage of the zebrafishs transparent external embryos to study vertebrate eye development. Our demonstration that lens cataracts can be visualized in three-dimensions by confocal microscopy in a living zebrafish provides a new tool for studying the causes, development and prevention of lens opacities.

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.

Piccolo paralogs and orthologs display conserved patterns of alternative splicing within the C2A and C2B domains

Piccolo is an organizational component of the presynaptic active zone, a specialized region of nerve terminals where synaptic vesicles fuse and release their neurotransmitter contents. Alternative splicing (AS) of the mouse Piccolo gene (PCLO) produces two primary splice isoforms: isoform-1 that includes two C2 domains (C2A and C2B) and isoform-2 with only C2A. Genome-wide association studies have identified variations located in or near the C2A domain of human Piccolo that predispose individuals to affective disorders and in rare cases leads to altered brain development. In zebrafish a genome duplication event led to the generation of PCLO-a and PCLO-b: gene paralogs that display strikingly similar genomic organization with other PCLO orthologs. Given this conservation in genomic structure, it is likely that AS patterns of zebrafish PCLO paralogs are similar to mammalian PCLO. We used a RT-PCR strategy to identify four zebrafish isoforms generated from zebrafish PCLO-a and PCLO-b that are equivalent to mouse Piccolo isoform-1 and isoform-2. Additionally, we identified an exon skipping event that leads to exclusion of a 27 nucleotide exon in both zebrafish Piccolo-a and Piccolo-b. Elimination of this exon in mammalian Piccolo alters the calcium binding property of the C2A domain. We also measured transcriptional levels of mouse and zebrafish Piccolo splice variants and demonstrate that despite similarities in AS, there are quantitative differences in gene expression. Our results indicate that AS of Piccolo is similar across diverse taxa and further support the use of zebrafish to study the role of Piccolo in neurodevelopment and synaptic signaling.