Barrie D. Robison

Status and opportunities for genomics research with rainbow trout

The rainbow trout (Oncorhynchus mykiss) is one of the most widely studied of model fish species. Extensive basic biological information has been collected for this species, which because of their large size relative to other model fish species are particularly suitable for studies requiring ample quantities of specific cells and tissue types. Rainbow trout have been widely utilized for research in carcinogenesis, toxicology, comparative immunology, disease ecology, physiology and nutrition. They are distinctive in having evolved from a relatively recent tetraploid event, resulting in a high incidence of duplicated genes. Natural populations are available and have been well characterized for chromosomal, protein, molecular and quantitative genetic variation. Their ease of culture, and experimental and aquacultural significance has led to the development of clonal lines and the widespread application of transgenic technology to this species. Numerous microsatellites have been isolated and two relatively detailed genetic maps have been developed. Extensive sequencing of expressed sequence tags has begun and four BAC libraries have been developed. The development and analysis of additional genomic sequence data will provide distinctive opportunities to address problems in areas such as evolution of the immune system and duplicate genes.

Effect of starvation on transcriptomes of brain and liver in adult female zebrafish (Danio rerio)

We used microarray and quantitative real-time PCR (qRT-PCR) analyses in adult female zebrafish (Danio rerio) to identify metabolic pathways regulated by starvation in the liver and brain. The transcriptome of whole zebrafish brain showed little response to 21 days of starvation. Only agouti-related protein 1 (agrp1) significantly responded, with increased expression in brains of starved fish. In contrast, a 21-day period of starvation significantly downregulated 466 and upregulated 108 transcripts in the liver, indicating an overall decrease in metabolic activity, reduced lipid metabolism, protein biosynthesis, proteolysis, and cellular respiration, and increased gluconeogenesis. Starvation also regulated expression of many components of the unfolded protein response, the first such report in a species other than yeast (Saccharomyces cerevisiae) and mice (Mus musculus). The response of the zebrafish hepatic transcriptome to starvation was strikingly similar to that of rainbow trout (Oncorhynchus mykiss) and less similar to mouse, while the response of common carp (Cyprinus carpio) differed considerably from the other three species.

The effects of early and adult social environment on zebrafish (Danio rerio) behavior

Because early social experience can have a profound effect on later mate and social choices, the availability of options and decisions made early in development can have major effects on adult behavior. Herein, we use strain differences among zebrafish, Danio rerio, as an experimental tool to test the effects of social experience on behavior. By manipulating the strain composition of groups in which the subject fish are housed at different ages, we tested (1) whether mixing with dissimilar individuals influenced subsequent behavior, (2) whether prolonged mixing during the juvenile stage had a more pronounced effect than a shorter period of mixing during adulthood and (3) whether mixing had a lasting effect after animals were resorted into groups of same strain animals. We found that social experience had a profound impact on social behavior. Both Nadia and TM1 individuals engaged in more frequent biting after having been in mixed strain groups compared to pure strain groups. This was true of groups mixed as juveniles, as well as adults, indicating that this response was not dependent on exposure during a critical developmental period. Also, TM1 fish (but not Nadia) having recently been housed in mixed-strain groups were more willing to leave the immediate vicinity of a shoal than were TM1 fish raised in pure strain groups. This change was more pronounced in groups mixed as juveniles than as adults. In addition, the observed changes persisted after mixed groups were separated into pure strain groups for a month. Other behavioral measures including Activity Level, Predator Response and Stress Recovery were unaffected by previous social experience.

Sexual dimorphism in hepatic gene expression and the response to dietary carbohydrate manipulation in the zebrafish (Danio rerio)

In this study, we tested for the presence of sexual dimorphism in the hepatic transcriptome of the adult zebrafish and examined the effect of long term manipulation of dietary carbohydrate on gene expression in both sexes. Zebrafish were fed diets comprised of 0%, 15%, 25%, or 35% carbohydrate from the larval stage through sexual maturity, then sampled for hepatic tissue, growth, proximate body composition, and retention efficiencies. Using Affymetrix microarrays and qRT-PCR, we observed substantial sexual dimorphism in the hepatic transcriptome. Males up-regulated genes associated with oxidative metabolism, carbohydrate metabolism, energy production, and amelioration of oxidative stress, while females had higher expression levels of genes associated with translation. Restriction of dietary carbohydrate (0% diet) significantly affected hepatic gene expression, growth performance, retention efficiencies of protein and energy, and percentages of moisture, lipid, and ash. The response of some genes to dietary manipulation varied by sex; with increased dietary carbohydrate, males up-regulated genes associated with oxidative metabolism (e.g. hadhbeta) while females up-regulated genes associated with glucose phosphorylation (e.g. glucokinase). Our data support the use of the zebrafish model for the study of fish nutritional genomics, but highlight the importance of accounting for sexual dimorphism in these studies.

Assessing Social Behavior Phenotypes in Adult Zebrafish: Shoaling, Social Preference, and Mirror Biting Tests

Zebrafish are a popular model organism in neuroscience research, recently emerging as an excellent species to study complex social phenotypes. For example, zebrafish actively form shoals, which can be used to quantify their shoaling behaviors. Zebrafish also display strong social preference when placed in a tank with conspecific fish, a trait that can easily be quantified in the two-compartment preference test. The mirror biting test, based on mirror image stimulation, is another well-established method for studying zebrafish boldness and sociability. This chapter will describe three simple and efficient paradigms—shoaling, social preference, and mirror biting tests—for quantifying social behaviors in adult zebrafish. Reflecting different aspects of zebrafish social phenotypes, these models can be used individually or within a test battery.

Brain transcriptome variation among behaviorally distinct strains of zebrafish (Danio rerio).

BackgroundDomesticated animal populations often show profound reductions in predator avoidance and fear-related behavior compared to wild populations. These reductions are remarkably consistent and have been observed in a diverse array of taxa including fish, birds, and mammals. Experiments conducted in common environments indicate that these behavioral differences have a genetic basis. In this study, we quantified differences in fear-related behavior between wild and domesticated zebrafish strains and used microarray analysis to identify genes that may be associated with this variation.ResultsCompared to wild zebrafish, domesticated zebrafish spent more time near the water surface and were more likely to occupy the front of the aquarium nearest a human observer. Microarray analysis of the brain transcriptome identified high levels of population variation in gene expression, with 1,749 genes significantly differentially expressed among populations. Genes that varied among populations belonged to functional categories that included DNA repair, DNA photolyase activity, response to light stimulus, neuron development and axon guidance, cell death, iron-binding, chromatin reorganization, and homeobox genes. Comparatively fewer genes (112) differed between domesticated and wild strains with notable genes including gpr177 (wntless), selenoprotein P1a, synaptophysin and synaptoporin, and acyl-CoA binding domain containing proteins (acbd3 and acbd4).ConclusionsMicroarray analysis identified a large number of genes that differed among zebrafish populations and may underlie behavioral domestication. Comparisons with similar microarray studies of domestication in rainbow trout and canids identified sixteen evolutionarily or functionally related genes that may represent components of shared molecular mechanisms underlying convergent behavioral evolution during vertebrate domestication. However, this conclusion must be tempered by limitations associated with comparisons among microarray studies and the low level of population-level replication inherent to these studies.

Is Behavioral Variation along the Bold-Shy Continuum Associated with Variation in the Stress Axis in Zebrafish?*

We tested whether boldness is associated with attenuation of the physiological stress response in behaviorally selected lines of zebrafish Danio rerio. We measured three component behaviors of boldness: cortisol levels under control and stressed conditions, growth rate, and expression of key genes linked to the hypothalamic-pituitary-interrenal axis in the brain. Surprisingly, bold animals did not differ from shy animals with respect to cortisol levels. However, significant differences between these animals in the expression of glucocorticoid receptors and genes that regulate production of stress hormones indicate that there may still be a relationship between bold behavior and the stress axis. Perhaps the most surprising result of this study was the degree of sexual dimorphism: female zebrafish were bolder than male zebrafish, had significantly lower levels of cortisol, and differed significantly in the expression of several genes in the brain. Our data indicate that a bold behavioral type is associated with transcriptional attenuation of stress axis genes, but we do not yet know whether evolution along the bold-shy continuum is attributable to genetic changes in the stress axis. The bold and shy zebrafish lines will be valuable tools for additional research into the relationship between stress and behavior and the mechanisms regulating sexual dimorphism in these traits.

Zebrafish (Danio rerio) vary by strain and sex in their behavioral and transcriptional responses to selenium supplementation

We used the Nadia, Gaighatta, Scientific Hatcheries, and TM1 zebrafish (Danio rerio) strains to test the hypothesis that variation among populations influences the behavioral and transcriptional responses to selenium supplementation. When fed a diet with control levels of selenium, zebrafish strains differed significantly in behavior, characterized as their mean horizontal and vertical swimming positions within the tank. The four strains also differed in brain expression of selenoprotein P1a (sepp1a), glutathione peroxidase 3 (gpx3), thioredoxin reductase 1 (txnrd1), and tRNA selenocysteine associated protein 1 (secp43). Iodothyronine deiodinase 2 (dio2) did not differ among strains but showed a sex-specific expression pattern. When supplemented with selenium, all strains spent a greater proportion of time near the front of the tank, but the response of vertical swimming depth varied by strain. Selenium supplementation also caused changes in selenoprotein expression in the brain that varied by strain for sepp1a, secp43, and dio2, and varied by strain and sex for txnrd1. Expression of gpx3 was unaffected by selenium. Our data indicate that selenium homeostasis in the brain may be a regulator of behavior in zebrafish, and the strain-specific effects of selenium supplementation suggest that genetic heterogeneity among populations can influence the results of selenium supplementation studies.

Retinal regeneration is facilitated by the presence of surviving neurons

Teleost fish regenerate their retinas after damage, in contrast to mammals. In zebrafish subjected to an extensive ouabain‐induced lesion that destroys all neurons and spares Müller glia, functional recovery and restoration of normal optic nerve head (ONH) diameter take place at 100 days postinjury. Subsequently, regenerated retinas overproduce cells in the retinal ganglion cell (RGC) layer, and the ONH becomes enlarged. Here, we test the hypothesis that a selective injury, which spares photoreceptors and Müller glia, results in faster functional recovery and fewer long‐term histological abnormalities. Following this selective retinal damage, recovery of visual function required 60 days, consistent with this hypothesis. In contrast to extensively damaged retinas, selectively damaged retinas showed fewer histological errors and did not overproduce neurons. Extensively damaged retinas had RGC axons that were delayed in pathfinding to the ONH, and showed misrouted axons within the ONH, suggesting that delayed functional recovery following an extensive lesion is related to defects in RGC axons exiting the eye and/or reaching their central targets. The atoh7, fgf8a, Sonic hedgehog (shha), and netrin‐1 genes were differentially expressed, and the distribution of hedgehog protein was disrupted after extensive damage as compared with selective damage. Confirming a role for Shh signaling in supporting rapid regeneration, shhat4+/‐ zebrafish showed delayed functional recovery after selective damage. We suggest that surviving retinal neurons provide structural/molecular information to regenerating neurons, and that this patterning mechanism regulates factors such as Shh. These factors in turn control neuronal number, retinal lamination, and RGC axon pathfinding during retinal regeneration.

Genetic backgrounds, standard lines, and husbandry of zebrafish.

Publisher Summary This chapter outlines the current issues surrounding the construction, maintenance, and use of genetically defined lines of zebrafish. The increasing number of zebrafish strains used for research raises a number of significant issues. Careful identification of zebrafish lines is critical in establishing the generality of research results. Each line of zebrafish is usually derived from a separate founder stock, and therefore each might harbor a unique genetic background. Through epistatic gene action, the genetic background of a given line can have marked effects on experimental results. Differences among the backgrounds of different wild-type lines could conceivably result in a given mutation displaying different phenotypes, which could have a variety of effects. This is particularly important in developmental biology because of the extensive use of forward genetic mutagenesis screens. The goals of a breeding program can be organized into three broad categories: genetic uniformity, maintenance of genetic variation, and selection for desirable phenotypes. Genetic monitoring of standard lines has made use of morphological, behavioral, immunological, and general line performance traits. Distribution of a line should provide the lines entire genetic background.