Liver development in zebrafish （Danio rerio）

Liver is one of the largest internal organs in the body and its importance for metabolism, detoxification and homeostasis has been well established. In this review, we summarized recent progresses in studying liver initiation and development during embryogenesis using zebrafish as a model system. We mainly focused on topics related to the specification of hepatoblasts from endoderm, the formation and growth of liver bud, the differentiation of hepatocytes and bile duct cells from hepatoblasts, and finally the role of mesodermal signals in controlling liver development in zebrafish.     

Overexpression of gankyrin induces liver steatosis in zebrafish (Danio rerio)

Gankyrin is a small ankyrin-repeat protein that previous research has confirmed to be overexpressed in hepatocellular carcinoma (HCC). Although relevant literature has reported on gankyrin functions in cellular proliferation and tumorigenesis, the exact role of gankyrin is poorly understood in animal model systems. This study analyzed hepatic lipid accumulation in gankyrin transgenic (GK) zebrafish. Bromodeoxyuridine (BrdU)-positive cells were predominantly increased in the liver bud of GK larvae, indicating that gankyrin functionally promoted cell proliferation at the larval stage in GK fish. However, over 90% of the viable GK adults showed an increased lipid content, leading in turn to liver steatosis. Liver histology and oil red O staining also indicated the accumulation of fatty droplets in GK fish, consistent with the specific pathological features of severe steatosis. Molecular analysis revealed that gankyrin overexpression induced hepatic steatosis and modulated the expression profiles of four hepatic microRNAs, miR-16, miR-27b, miR-122, and miR-126, and 22 genes involved in lipid metabolism. Moreover, significantly increased hepatic cell apoptosis resulted in liver damage in GK adults, leading to liver failure and death after approximately 10months. This study is the first to report gankyrin as a potential link between microRNAs and liver steatosis in zebrafish.     

Acridine mutagenesis of zebrafish (Danio rerio)

Mutagenesis screening, in which heritable traits are isolated following damage to the genome, is a powerful approach for investigating gene function. Among vertebrate model organisms, the zebrafish (Danio rerio) is ideally suited to mutagenesis screens. The success of large-scale screens is dependent on the way in which changes are identified. The type of damage induced is also pivotal. Single base coding region deletions and insertions are suited to abolition of gene function whilst inducing a small physical alteration to the genome. Such mutations are not commonly found following mutagenesis schemes reported to date. Here, we show that an acridine mutagen, ICR191, which in other model organisms frequently induces single base deletions and insertions, is mutagenic in zebrafish. ICR191 induces hallmark phenotypes associated with genetic damage in treated embryos. Alterations are heritable. Offspring of mutagenised fish had mutations in a marker gene and were found to produce offspring with abnormal development. Using an adaptation of a molecular mutation detection method, fluorescent arbitrary primed PCR, we identified an induced alteration directly. The estimated frequency of induced mutations was sufficiently high to make it feasible to employ this approach for mutagenesis screening.     

Biophysics of zebrafish (Danio rerio) sperm

In the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37 ± 0.02 (SEM), an isosmotic cell volume (V_o) of 12.1 ± 0.2 μm³ (SEM), a water permeability (L_p) in 10% dimethyl sulfoxide of 0.021 ± 0.001(SEM) μm/min/atm, and a cryoprotectant permeability (P_s) of 0.10 ± 0.01 (SEM) × 10⁻³ cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24 h at 0 °C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques.     

Locomotor behaviors in zebrafish (Danio rerio) larvae

Locomotor behaviors were examined in two experiments using zebrafish (Danio rerio) larvae at 4, 5, 6 and 7 days post fertilization (dpf). Larvae were observed in individual wells of a 12-well plate for 1 h a day. In Experiment 1, the same larvae were observed for four consecutive days beginning on post-fertilization day 4; in Experiment 2, different groups of larvae from the same egg collection were observed at 4, 5, 6 and 7 dpf. Automated images collected every 6 s were analyzed for information about larval location, orientation and general activity. In both experiments, 4 dpf larvae rested significantly more, used a smaller area of the well more frequently, and were generally less active than older larvae. All larvae exhibited a preference for facing away from the center of the well and for the edge of the well. However, prolonged exposure to the well influenced overall activity, orientation, and preference for the edge region. The implications of these results for understanding the development of larval behavior and for the design of procedures to measure the effects of experience in zebrafish larvae are discussed.     

Oxidative stress in zebrafish (Danio rerio) sperm

Laboratories around the world have produced tens of thousands of mutant and transgenic zebrafish lines. As with mice, maintaining all of these valuable zebrafish genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Because reducing oxidative stress has become an important aspect of reducing the variability in mouse sperm cryopreservation, we examined whether antioxidants might improve cryopreservation of zebrafish sperm. Four experiments were conducted in this study. First, we used the xanthine-xanthine oxidase (X-XO) system to generate reactive oxygen species (ROS). The X-XO system was capable of producing a stress reaction in zebrafish sperm reducing its sperm motility in a concentration dependent manner (P<0.05). Second, we examined X-XO and the impact of antioxidants on sperm viability, ROS and motility. Catalase (CAT) mitigated stress and maintained viability and sperm motility (P>0.05), whereas superoxide dismutase (SOD) and vitamin E did not (P<0.05). Third, we evaluated ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility. Methanol (8%) reduced viability and sperm motility (P<0.05), but the addition of CAT mitigated these effects (P>0.05), producing a mean 2.0 to 2.9-fold increase in post-thaw motility. Fourth, we examined the effect of additional cryoprotectants and CAT on fresh sperm motility. Cryoprotectants, 8% methanol and 10% dimethylacetamide (DMA), reduced the motility over the control value (P<0.5), whereas 10% dimethylformamide (DMF) with or without CAT did not (P>0.05). Zebrafish sperm protocols should be modified to improve the reliability of the cryopreservation process, perhaps using a different cryoprotectant. Regardless, the simple addition of CAT to present-day procedures will significantly improve this process, assuring increased and less variable fertilization success and allowing resource managers to dependably plan how many straws are needed to safely cryopreserve a genetic line.     

Sex-specific recombination rates in zebrafish (Danio rerio)

In many organisms, the rate of genetic recombination is not uniform along the length of chromosomes or between sexes. To compare the relative recombination rates during meiosis in male and female zebrafish, we constructed a genetic map based on male meiosis. We developed a meiotic mapping panel of 94 androgenetic haploid embryos that were scored for genetic polymorphisms. The resulting male map was compared to female and sex-average maps. We found that the recombination rate in male meiosis is dramatically suppressed relative to that of female meiosis, especially near the centromere. These findings have practical applications for experimental design. The use of exclusively female meiosis in a positional cloning project maximizes the ratio of genetic map distance to physical distance. Alternatively, the use of exclusively male meiosis to localize a mutation initially to a linkage group or to maintain relationships of linked alleles minimizes recombination, thereby facilitating some types of analysis.     

Visual discrimination learning in zebrafish (Danio rerio)

Three experiments demonstrated visual discrimination learning in zebrafish (Danio rerio). In each experiment, zebrafish were given a choice between two visually distinct arms of a T-maze. Choice of one stimulus was always followed by a food reward, but choice of the other stimulus was not rewarded. Different colored sleeves fitted around the arms of the T-maze were used in Experiments 1 (green and purple) and 2 (red and blue). The stimuli used in Experiment 3 were white sleeves lined with horizontal or vertical black stripes. In all three experiments, zebrafish acquired a significant preference for the stimulus that led to a food reward. Experiments 1 and 2 also showed that zebrafish could learn a reversal of the discrimination. Finally, the effect of discontinuing food rewards was examined after reversal training in Experiment 2 and after initial discrimination training in Experiments 1 and 3. Non-reinforcement led to a decrease in correct responding in Experiments 2 and 3 independent of stimulus identity, but to an asymmetrical pattern of responding in Experiment 1. The median latency to make a choice response decreased over the course of acquisition in all three experiments; during extinction, median response times did not change at all in Experiment 1 and increased only very slightly in Experiment 2, but showed a substantial increase in Experiment 3. The implications of these results for the zebrafish as a model system for genetic studies of learning and memory are discussed.     

Development of sensory systems in zebrafish (Danio rerio)

Zebrafish possess all of the classic sensory modalities: taste, tactile, smell, balance, vision, and hearing. For each sensory system, this article provides a brief overview of the system in the adult zebrafish followed by a more detailed overview of the development of the system. By far the majority of studies performed in each of the sensory systems of the zebrafish have involved some aspect of molecular biology or genetics. Although molecular biology and genetics are not major foci of the paper, brief discussions of some of the mutant strains of zebrafish that have developmental defects in each specific sensory system are included. The development of the sensory systems is only a small sampling of the work being done using zebrafish and provides a mere glimpse of the potential of this model for the study of vertebrate development, physiology, and human disease.     

Boldness predicts social status in zebrafish (Danio rerio)

This study explored if boldness could be used to predict social status. First, boldness was assessed by monitoring individual zebrafish behaviour in (1) an unfamiliar barren environment with no shelter (open field), (2) the same environment when a roof was introduced as a shelter, and (3) when the roof was removed and an unfamiliar object (Lego® brick) was introduced. Next, after a resting period of minimum one week, social status of the fish was determined in a dyadic contest and dominant/subordinate individuals were determined as the winner/loser of two consecutive contests. Multivariate data analyses showed that males were bolder than females and that the behaviours expressed by the fish during the boldness tests could be used to predict which fish would later become dominant and subordinate in the ensuing dyadic contest. We conclude that bold behaviour is positively correlated to dominance in zebrafish and that boldness is not solely a consequence of social dominance.     

Dietary fatty acid composition influences the fertilization rate of zebrafish(Danio rerio Hamilton-Buchanan)

Reports in the literature show that the dietary fatty acid (FA) composition is reflected in fish muscle, their internal organs and eggs. Fish eggs are used in toxicity tests to determine the subchronic toxicity of various substances. In toxicity tests, physico-chemical test parameters have been standardized. However, parental age and diet may also influence results of toxicity tests and if so would also require standardization. Our investigations hitherto indicate that the FA composition of eggs and testes in zebrafish is caused by the FA composition of fish diet. Diets with different ratios of n-3/n-6 polyunsaturated fatty acids led to different contents of these FA in the reproductive organs and finally to variations in the fertilization rates. This rate is elevated in the eggs of zebrafish ( Danio rerio Hamilton-Buchanan) if the quotient of n-3/n-6 is low.     

In vivo studies of liver-type fatty acid binding protein (L-FABP) gene expression in liver of transgenic zebrafish (Danio rerio)

Mammalian liver fatty acid binding protein (L-FABP) is a small cytosolic protein in various tissues including liver, small intestine and kidney and is thought to play a crucial role in intracellular fatty acid trafficking and metabolism. To better understand its tissue-specific regulation during zebrafish hepatogenesis, we isolated 5'-flanking sequences of the zebrafish L-FABP gene and used a green fluorescent protein (GFP) transgenic strategy to generate liver-specific transgenic zebrafish. The 2.8-kb 5'-flanking sequence of zebrafish L-FABP gene was sufficient to direct GFP expression in liver primordia, first observed in 2 dpf embryos and then continuously to the adult stage. This pattern of transgenic expression is consistent with the expression pattern of the endogenous gene. F2 inheritance rates of 42-51% in all the seven transgenic lines were consistent with the ratio of Mendelian segregation. Further, hhex and zXbp-1 morphants displayed a visible liver defect, which suggests that it is possible to establish an in vivo system for screening genes required for liver development.     

Zinc transporter expression in zebrafish (Danio rerio) during development

Zinc is a micronutrient important in several biological processes including growth and development. We have limited knowledge on the impact of maternal zinc deficiency on zinc and zinc regulatory mechanisms in the developing embryo due to a lack of in vivo experimental models that allow us to directly study the effects of maternal zinc on embryonic development following implantation. To overcome this barrier, we have proposed to use zebrafish as a model organism to study the impact of zinc during development. The goal of the current study was to profile the mRNA expression of all the known zinc transporter genes in the zebrafish across embryonic and larval development and to quantify the embryonic zinc concentrations at these corresponding developmental time points. The SLC30A zinc transporter family (ZnT) and SLC39A family, Zir-,Irt-like protein (ZIP) zinc transporter proteins were profiled in zebrafish embryos at 0, 2, 6, 12, 24, 48 and 120 h post fertilization to capture expression patterns from a single cell through full development. We observed consistent embryonic zinc levels, but differential expression of several zinc transporters across development. These results suggest that zebrafish is an effective model organism to study the effects of zinc deficiency and further investigation is underway to identify possible molecular pathways that are dysregulated with maternal zinc deficiency.     

Life-stage-dependent sensitivity of zebrafish (Danio rerio) to estrogen exposure

The aim of this study was to identify periods in zebrafish (Danio rerio) development when estrogen exposure has long-term consequences on reproductive capabilities at the adult stage. To this end, zebrafish were exposed to 10 ng/L ethynylestradiol (EE(2)) during three stages of gonadal differentiation: (i) the juvenile hermaphroditic stage when gonads display the morphology of an immature ovary (in our zebrafish colony this lasted from 15 to 42 days post-fertilization [dpf]), (ii) the gonad transition stage when the hermaphroditic gonad differentiates into either testes or ovary (from 43 to day 71 dpf), and (iii) the premature stage of testicular and ovarian development (from 72 to 99 dpf). The consequences of stage-specific exposure to EE(2) were assessed by determining time to first spawning, fecundity (number of eggs per female per day), fertilization success (percentage of fertilized eggs) and sex ratio of the adults. Exposure during the gonad transition period induced a delay in the onset of spawning and a significant reduction of fecundity and fertilization success, whereas exposure during the hermaphroditic stage or during the premature stage had no significant impact on the reproductive parameters of adult fish. The results from this experiment pointed to the gonad transition stage as being most susceptible to persistent effects of developmental estrogen exposure. In a second experiment, the concentration dependency of the EE(2)effects was evaluated by exposing zebrafish during the gonad transition stage (43-71 dpf) to 1.67, 3 or 10 ng EE(2)/L. Significant effects of EE(2) on adult reproduction were found with 3 and 10 ng EE(2)/L, but not with 1.67 ng/L. Histological examination of the gonads revealed that at termination of EE(2) exposure (71 dpf), all individuals in the 3 and 10 ng EE(2)/L treatment possessed ovaries. However, this feminising effect appeared to be reversible since at the adult stage (190 dpf), both fish with ovaries and with testes were found. Thus, EE(2) exposure during the gonad transition stage seems to have no persistent effect on gonad histology but on reproductive capabilities.     

Mycobacterium haemophilum infections of zebrafish (Danio rerio) in research facilities

In May 2005, a disease outbreak was investigated at a zebrafish (Danio rerio) research facility experiencing severe losses. Mycobacterium haemophilum was isolated from these fish and the disease was subsequently recreated in experimentally infected zebrafish. Fish exhibited signs characteristic of mycobacteriosis, including granuloma formation and severe, diffuse, chronic inflammation. Bacteria were observed in multiple tissues, including the central nervous system. Biofilm samples from the outbreak facility were PCR positive for M. haemophilum, suggesting biofilms might act as a reservoir for infection. Zebrafish appear to be particularly vulnerable to M. haemophilum, and measures such as quarantine and treatment of incoming water should be implemented to minimize the likelihood of introduction of this bacterium to zebrafish research facilities. Zebrafish are already a well-established laboratory animal model for genetics, toxicology and disease, their susceptibility to M. haemophilum may make them useful for the study of this bacterium in the future.