Sample records for zebrafish

  1. Zebrafish Health Conditions in the China Zebrafish Resource Center and 20 Major Chinese Zebrafish Laboratories.

    PubMed

    Liu, Liyue; Pan, Luyuan; Li, Kuoyu; Zhang, Yun; Zhu, Zuoyan; Sun, Yonghua

    2016-07-01

    In China, the use of zebrafish as an experimental animal in the past 15 years has widely expanded. The China Zebrafish Resource Center (CZRC), which was established in 2012, is becoming one of the major resource centers in the global zebrafish community. Large-scale use and regular exchange of zebrafish resources have put forward higher requirements on zebrafish health issues in China. This article reports the current aquatic infrastructure design, animal husbandry, and health-monitoring programs in the CZRC. Meanwhile, through a survey of 20 Chinese zebrafish laboratories, we also describe the current health status of major zebrafish facilities in China. We conclude that it is of great importance to establish a widely accepted health standard and health-monitoring strategy in the Chinese zebrafish research community.

  2. Sprouting Buds of Zebrafish Research in Malaysia: First Malaysia Zebrafish Disease Model Workshop.

    PubMed

    Okuda, Kazuhide Shaun; Tan, Pei Jean; Patel, Vyomesh

    2016-04-01

    Zebrafish is gaining prominence as an important vertebrate model for investigating various human diseases. Zebrafish provides unique advantages such as optical clarity of embryos, high fecundity rate, and low cost of maintenance, making it a perfect complement to the murine model equivalent in biomedical research. Due to these advantages, researchers in Malaysia are starting to take notice and incorporate the zebrafish model into their research activities. However, zebrafish research in Malaysia is still in its infancy stage and many researchers still remain unaware of the full potential of the zebrafish model or have limited access to related tools and techniques that are widely utilized in many zebrafish laboratories worldwide. To overcome this, we organized the First Malaysia Zebrafish Disease Model Workshop in Malaysia that took place on 11th and 12th of November 2015. In this workshop, we showcased how the zebrafish model is being utilized in the biomedical field in international settings as well as in Malaysia. For this, notable international speakers and those from local universities known to be carrying out impactful research using zebrafish were invited to share some of the cutting edge techniques that are used in their laboratories that may one day be incorporated in the Malaysian scientific community.

  3. Automatic zebrafish heartbeat detection and analysis for zebrafish embryos.

    PubMed

    Pylatiuk, Christian; Sanchez, Daniela; Mikut, Ralf; Alshut, Rüdiger; Reischl, Markus; Hirth, Sofia; Rottbauer, Wolfgang; Just, Steffen

    2014-08-01

    A fully automatic detection and analysis method of heartbeats in videos of nonfixed and nonanesthetized zebrafish embryos is presented. This method reduces the manual workload and time needed for preparation and imaging of the zebrafish embryos, as well as for evaluating heartbeat parameters such as frequency, beat-to-beat intervals, and arrhythmicity. The method is validated by a comparison of the results from automatic and manual detection of the heart rates of wild-type zebrafish embryos 36-120 h postfertilization and of embryonic hearts with bradycardia and pauses in the cardiac contraction.

  4. Neurochemical measurements in the zebrafish brain

    PubMed Central

    Jones, Lauren J.; McCutcheon, James E.; Young, Andrew M. J.; Norton, William H. J.

    2015-01-01

    The zebrafish is an ideal model organism for behavioral genetics and neuroscience. The high conservation of genes and neurotransmitter pathways between zebrafish and other vertebrates permits the translation of research between species. Zebrafish behavior can be studied at both larval and adult stages and recent research has begun to establish zebrafish models for human disease. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique that permits the detection of neurotransmitter release and reuptake. In this study we have used in vitro FSCV to measure the release of analytes in the adult zebrafish telencephalon. We compare different stimulation methods and present a characterization of neurochemical changes in the wild-type zebrafish brain. This study represents the first FSCV recordings in zebrafish, thus paving the way for neurochemical analysis of the fish brain. PMID:26441575

  5. Zebrafish as tools for drug discovery.

    PubMed

    MacRae, Calum A; Peterson, Randall T

    2015-10-01

    The zebrafish has become a prominent vertebrate model for disease and has already contributed to several examples of successful phenotype-based drug discovery. For the zebrafish to become useful in drug development more broadly, key hurdles must be overcome, including a more comprehensive elucidation of the similarities and differences between human and zebrafish biology. Recent studies have begun to establish the capabilities and limitations of zebrafish for disease modelling, drug screening, target identification, pharmacology, and toxicology. As our understanding increases and as the technologies for manipulating zebrafish improve, it is hoped that the zebrafish will have a key role in accelerating the emergence of precision medicine.

  6. Zebrafish and Streptococcal Infections.

    PubMed

    Saralahti, A; Rämet, M

    2015-09-01

    Streptococcal bacteria are a versatile group of gram-positive bacteria capable of infecting several host organisms, including humans and fish. Streptococcal species are common colonizers of the human respiratory and gastrointestinal tract, but they also cause some of the most common life-threatening, invasive infections in humans and aquaculture. With its unique characteristics and efficient tools for genetic and imaging applications, the zebrafish (Danio rerio) has emerged as a powerful vertebrate model for infectious diseases. Several zebrafish models introduced so far have shown that zebrafish are suitable models for both zoonotic and human-specific infections. Recently, several zebrafish models mimicking human streptococcal infections have also been developed. These models show great potential in providing novel information about the pathogenic mechanisms and host responses associated with human streptococcal infections. Here, we review the zebrafish infection models for the most relevant streptococcal species: the human-specific Streptococcus pneumoniae and Streptococcus pyogenes, and the zoonotic Streptococcus iniae and Streptococcus agalactiae. The recent success and the future potential of these models for the study of host-pathogen interactions in streptococcal infections are also discussed. © 2015 The Foundation for the Scandinavian Journal of Immunology.

  7. Measuring zebrafish turning rate.

    PubMed

    Mwaffo, Violet; Butail, Sachit; di Bernardo, Mario; Porfiri, Maurizio

    2015-06-01

    Zebrafish is becoming a popular animal model in preclinical research, and zebrafish turning rate has been proposed for the analysis of activity in several domains. The turning rate is often estimated from the trajectory of the fish centroid that is output by commercial or custom-made target tracking software run on overhead videos of fish swimming. However, the accuracy of such indirect methods with respect to the turning rate associated with changes in heading during zebrafish locomotion is largely untested. Here, we compare two indirect methods for the turning rate estimation using the centroid velocity or position data, with full shape tracking for three different video sampling rates. We use tracking data from the overhead video recorded at 60, 30, and 15 frames per second of zebrafish swimming in a shallow water tank. Statistical comparisons of absolute turning rate across methods and sampling rates indicate that, while indirect methods are indistinguishable from full shape tracking, the video sampling rate significantly influences the turning rate measurement. The results of this study can aid in the selection of the video capture frame rate, an experimental design parameter in zebrafish behavioral experiments where activity is an important measure.

  8. INDUCED AND SPONTANEOUS NEOPLASIA IN ZEBRAFISH.

    EPA Science Inventory

    To address the potential of zebrafish as a cancer model, it is important to determine the susceptibility of zebrafish to tumors, and to compare zebrafish tumors with human tumors. To determine whether the commonly-used germ line mutagen, ethylnitrosourea (ENU) induces tumors, we ...

  9. Anesthesia and euthanasia in zebrafish.

    PubMed

    Matthews, Monte; Varga, Zoltán M

    2012-01-01

    Because of the relative ease of embryonic manipulation and observation, the ability to produce a great number of genetic mutations, efficient screening methods, and the continued advance of molecular genetic tools, such as the progress in sequencing and mapping of the zebrafish genome, the use of zebrafish (Danio rerio) as a biomedical model organism continues to expand. However, studies involving zebrafish husbandry and veterinary care struggle to keep pace with scientific progress. This article outlines some of the current, acceptable methods for providing anesthesia and euthanasia and provides some examples of how performance-based approaches can be used to advance the relatively limited number of anesthetic and euthanizing techniques available for zebrafish.

  10. Maintenance of Zebrafish Lines at the European Zebrafish Resource Center.

    PubMed

    Geisler, Robert; Borel, Nadine; Ferg, Marco; Maier, Jana Viktoria; Strähle, Uwe

    2016-07-01

    We have established a European Zebrafish Resource Center (EZRC) at the KIT. This center not only maintains and distributes a large number of existing mutant and transgenic zebrafish lines but also gives zebrafish researchers access to screening services and technologies such as imaging and high-throughput sequencing, provided by the Institute of Toxicology and Genetics (ITG). The EZRC maintains and distributes the stock collection of the Nüsslein-Volhard laboratory, comprising over 2000 publicly released mutations, as frozen sperm samples. Within the framework of the ZF-HEALTH EU project, the EZRC distributes over 10,000 knockout mutations from the Sanger Institute (United Kingdom), as well as over 100 mutant and transgenic lines from other sources. In this article, we detail the measures we have taken to ensure the health of our fish, including hygiene, quarantine, and veterinary inspections.

  11. Viral Diseases in Zebrafish: What Is Known and Unknown

    PubMed Central

    Crim, Marcus J.; Riley, Lela K.

    2013-01-01

    Naturally occurring viral infections have the potential to introduce confounding variability that leads to invalid and misinterpreted data. Whereas the viral diseases of research rodents are well characterized and closely monitored, no naturally occurring viral infections have been characterized for the laboratory zebrafish (Danio rerio), an increasingly important biomedical research model. Despite the ignorance about naturally occurring zebrafish viruses, zebrafish models are rapidly expanding in areas of biomedical research where the confounding effects of unknown infectious agents present a serious concern. In addition, many zebrafish research colonies remain linked to the ornamental (pet) zebrafish trade, which can contribute to the introduction of new pathogens into research colonies, whereas mice used for research are purpose bred, with no introduction of new mice from the pet industry. Identification, characterization, and monitoring of naturally occurring viruses in zebrafish are crucial to the improvement of zebrafish health, the reduction of unwanted variability, and the continued development of the zebrafish as a model organism. This article addresses the importance of identifying and characterizing the viral diseases of zebrafish as the scope of zebrafish models expands into new research areas and also briefly addresses zebrafish susceptibility to experimental viral infection and the utility of the zebrafish as an infection and immunology model. PMID:23382345

  12. Zebrafish Melanoma.

    PubMed

    Kaufman, Charles K

    2016-01-01

    Melanoma skin cancer is a potentially deadly disease in humans and has remained extremely difficult to treat once it has metastasized. In just the last 10 years, a number of models of melanoma have been developed in the zebrafish that are biologically faithful to the human disease and have already yielded important insights into the fundamental biology of melanoma and offered new potential avenues for treatment. With the diversity and breadth of the molecular genetic tools available in the zebrafish, these melanoma models will continue to be refined and expanded upon to keep pace with the rapidly evolving field of melanoma biology.

  13. Polygenic Sex Determination System in Zebrafish

    PubMed Central

    Liew, Woei Chang; Bartfai, Richard; Lim, Zijie; Sreenivasan, Rajini; Siegfried, Kellee R.; Orban, Laszlo

    2012-01-01

    Background Despite the popularity of zebrafish as a research model, its sex determination (SD) mechanism is still unknown. Most cytogenetic studies failed to find dimorphic sex chromosomes and no primary sex determining switch has been identified even though the assembly of zebrafish genome sequence is near to completion and a high resolution genetic map is available. Recent publications suggest that environmental factors within the natural range have minimal impact on sex ratios of zebrafish populations. The primary aim of this study is to find out more about how sex is determined in zebrafish. Methodology/Principal Findings Using classical breeding experiments, we found that sex ratios across families were wide ranging (4.8% to 97.3% males). On the other hand, repeated single pair crossings produced broods of very similar sex ratios, indicating that parental genotypes have a role in the sex ratio of the offspring. Variation among family sex ratios was reduced after selection for breeding pairs with predominantly male or female offspring, another indication that zebrafish sex is regulated genetically. Further examinations by a PCR-based “blind assay" and array comparative genomic hybridization both failed to find universal sex-linked differences between the male and female genomes. Together with the ability to increase the sex bias of lines by selective breeding, these data suggest that zebrafish is unlikely to utilize a chromosomal sex determination (CSD) system. Conclusions/Significance Taken together, our study suggests that zebrafish sex is genetically determined with limited, secondary influences from the environment. As we have not found any sign for CSD in the species, we propose that the zebrafish has a polygenic sex determination system. PMID:22506019

  14. What is the Thalamus in Zebrafish?

    PubMed Central

    Mueller, Thomas

    2012-01-01

    Current research on the thalamus and related structures in the zebrafish diencephalon identifies an increasing number of both neurological structures and ontogenetic processes as evolutionary conserved between teleosts and mammals. The patterning processes, for example, which during the embryonic development of zebrafish form the thalamus proper appear largely conserved. Yet also striking differences between zebrafish and other vertebrates have been observed, particularly when we look at mature and histologically differentiated brains. A case in point is the migrated preglomerular complex of zebrafish which evolved only within the lineage of ray-finned fish and has no counterpart in mammals or tetrapod vertebrates. Based on its function as a sensory relay station with projections to pallial zones, the preglomerular complex has been compared to specific thalamic nuclei in mammals. However, no thalamic projections to the zebrafish dorsal pallium, which corresponds topologically to the mammalian isocortex, have been identified. Merely one teleostean thalamic nucleus proper, the auditory nucleus, projects to a part of the dorsal telencephalon, the pallial amygdala. Studies on patterning mechanisms identify a rostral and caudal domain in the embryonic thalamus proper. In both, teleosts and mammals, the rostral domain gives rise to GABAergic neurons, whereas glutamatergic neurons originate in the caudal domain of the zebrafish thalamus. The distribution of GABAergic derivatives in the adult zebrafish brain, furthermore, revealed previously overlooked thalamic nuclei and redefined already established ones. These findings require some reconsideration regarding the topological origin of these adult structures. In what follows, I discuss how evolutionary conserved and newly acquired features of the developing and adult zebrafish thalamus can be compared to the mammalian situation. PMID:22586363

  15. Application of Zebrafish Model to Environmental Toxicology.

    PubMed

    Komoike, Yuta; Matsuoka, Masato

    2016-01-01

    Recently, a tropical freshwater fish, the zebrafish, has been generally used as a useful model organism in various fields of life science worldwide. The zebrafish model has also been applied to environmental toxicology; however, in Japan, it has not yet become widely used. In this review, we will introduce the biological and historical backgrounds of zebrafish as an animal model and their breeding. We then present the current status of toxicological experiments using zebrafish that were treated with some important environmental contaminants, including cadmium, organic mercury, 2,3,7,8-tetrachlorodibenzo-p-dioxin, and tributyltin. Finally, the future possible application of genetically modified zebrafish to the study of environmental toxicology is discussed.

  16. The importance of Zebrafish in biomedical research.

    PubMed

    Tavares, Bárbara; Santos Lopes, Susana

    2013-01-01

    Zebrafish (Danio rerio) is an ideal model organism for the study of vertebrate development. This is due to the large clutches that each couple produces, with up to 200 embryos every 7 days, and to the fact that the embryos and larvae are small, transparent and undergo rapid external development. Using scientific literature research tools available online and the keywords Zebrafish, biomedical research, human disease, and drug screening, we reviewed original studies and reviews indexed in PubMed. In this review we summarized work conducted with this model for the advancement of our knowledge related to several human diseases. We also focused on the biomedical research being performed in Portugal with the zebrafish model. Powerful live imaging and genetic tools are currently available for zebrafish making it a valuable model in biomedical research. The combination of these properties with the optimization of automated systems for drug screening has transformed the zebrafish into "a top model" in biomedical research, drug discovery and toxicity testing. Furthermore, with the optimization of xenografts technology it will be possible to use zebrafish to aide in the choice of the best therapy for each patient. Zebrafish is an excellent model organism in biomedical research, drug development and in clinical therapy.

  17. Zebrafish neurobehavioral phenomics for aquatic neuropharmacology and toxicology research.

    PubMed

    Kalueff, Allan V; Echevarria, David J; Homechaudhuri, Sumit; Stewart, Adam Michael; Collier, Adam D; Kaluyeva, Aleksandra A; Li, Shaomin; Liu, Yingcong; Chen, Peirong; Wang, JiaJia; Yang, Lei; Mitra, Anisa; Pal, Subharthi; Chaudhuri, Adwitiya; Roy, Anwesha; Biswas, Missidona; Roy, Dola; Podder, Anupam; Poudel, Manoj K; Katare, Deepshikha P; Mani, Ruchi J; Kyzar, Evan J; Gaikwad, Siddharth; Nguyen, Michael; Song, Cai

    2016-01-01

    Zebrafish (Danio rerio) are rapidly emerging as an important model organism for aquatic neuropharmacology and toxicology research. The behavioral/phenotypic complexity of zebrafish allows for thorough dissection of complex human brain disorders and drug-evoked pathological states. As numerous zebrafish models become available with a wide spectrum of behavioral, genetic, and environmental methods to test novel drugs, here we discuss recent zebrafish phenomics methods to facilitate drug discovery, particularly in the field of biological psychiatry. Additionally, behavioral, neurological, and endocrine endpoints are becoming increasingly well-characterized in zebrafish, making them an inexpensive, robust and effective model for toxicology research and pharmacological screening. We also discuss zebrafish behavioral phenotypes, experimental considerations, pharmacological candidates and relevance of zebrafish neurophenomics to other 'omics' (e.g., genomic, proteomic) approaches. Finally, we critically evaluate the limitations of utilizing this model organism, and outline future strategies of research in the field of zebrafish phenomics. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Mixtures, Metabolites, and Mechanisms: Understanding Toxicology Using Zebrafish.

    PubMed

    Gamse, Joshua T; Gorelick, Daniel A

    2016-10-01

    For more than 60 years, zebrafish have been used in toxicological studies. Due to their transparency, genetic tractability, and compatibility with high-throughput screens, zebrafish embryos are uniquely suited to study the effects of pharmaceuticals and environmental insults on embryonic development, organ formation and function, and reproductive success. This special issue of Zebrafish highlights the ways zebrafish are used to investigate the toxic effects of endocrine disruptors, pesticides, and heavy metals.

  19. Mycobacteriosis in zebrafish colonies.

    PubMed

    Whipps, Christopher M; Lieggi, Christine; Wagner, Robert

    2012-01-01

    Mycobacteriosis, a chronic bacterial infection, has been associated with severe losses in some zebrafish facilities and low-level mortalities and unknown impacts in others. The occurrence of at least six different described species (Mycobacterium abscessus, M. chelonae, M. fortuitum, M. haemophilum, M. marinum, M. peregrinum) from zebrafish complicates diagnosis and control because each species is unique. As a generalization, mycobacteria are often considered opportunists, but M. haemophilum and M. marinum appear to be more virulent. Background genetics of zebrafish and environmental conditions influence the susceptibility of fish and progression of disease, emphasizing the importance of regular monitoring and good husbandry practices. A combined approach to diagnostics is ultimately the most informative, with histology as a first-level screen, polymerase chain reaction for rapid detection and species identification, and culture for strain differentiation. Occurrence of identical strains of Mycobacterium in both fish and biofilms in zebrafish systems suggests transmission can occur when fish feed on infected tissues or tank detritus containing mycobacteria. Within a facility, good husbandry practices and sentinel programs are essential for minimizing the impacts of mycobacteria. In addition, quarantine and screening of animals coming into a facility is important for eliminating the introduction of the more severe pathogens. Elimination of mycobacteria from an aquatic system is likely not feasible because these species readily establish biofilms on surfaces even in extremely low nutrient conditions. Risks associated with each commonly encountered species need to be identified and informed management plans developed. Basic research on the growth characteristics, disinfection, and pathogenesis of zebrafish mycobacteria is critical moving forward.

  20. Episodic-like memory in zebrafish.

    PubMed

    Hamilton, Trevor J; Myggland, Allison; Duperreault, Erika; May, Zacnicte; Gallup, Joshua; Powell, Russell A; Schalomon, Melike; Digweed, Shannon M

    2016-11-01

    Episodic-like memory tests often aid in determining an animal's ability to recall the what, where, and which (context) of an event. To date, this type of memory has been demonstrated in humans, wild chacma baboons, corvids (Scrub jays), humming birds, mice, rats, Yucatan minipigs, and cuttlefish. The potential for this type of memory in zebrafish remains unexplored even though they are quickly becoming an essential model organism for the study of a variety of human cognitive and mental disorders. Here we explore the episodic-like capabilities of zebrafish (Danio rerio) in a previously established mammalian memory paradigm. We demonstrate that when zebrafish were presented with a familiar object in a familiar context but a novel location within that context, they spend more time in the novel quadrant. Thus, zebrafish display episodic-like memory as they remember what object they saw, where they saw it (quadrant location), and on which occasion (yellow or blue walls) it was presented.

  1. Mixtures, Metabolites, and Mechanisms: Understanding Toxicology Using Zebrafish

    PubMed Central

    Gamse, Joshua T.

    2016-01-01

    Abstract For more than 60 years, zebrafish have been used in toxicological studies. Due to their transparency, genetic tractability, and compatibility with high-throughput screens, zebrafish embryos are uniquely suited to study the effects of pharmaceuticals and environmental insults on embryonic development, organ formation and function, and reproductive success. This special issue of Zebrafish highlights the ways zebrafish are used to investigate the toxic effects of endocrine disruptors, pesticides, and heavy metals. PMID:27618129

  2. Zebrafish model systems for developmental neurobehavioral toxicology.

    PubMed

    Bailey, Jordan; Oliveri, Anthony; Levin, Edward D

    2013-03-01

    Zebrafish offer many advantages that complement classic mammalian models for the study of normal development as well as for the teratogenic effects of exposure to hazardous compounds. The clear chorion and embryo of the zebrafish allow for continuous visualization of the anatomical changes associated with development, which, along with short maturation times and the capability of complex behavior, makes this model particularly useful for measuring changes to the developing nervous system. Moreover, the rich array of developmental, behavioral, and molecular benefits offered by the zebrafish have contributed to an increasing demand for the use of zebrafish in behavioral teratology. Essential for this endeavor has been the development of a battery of tests to evaluate a spectrum of behavior in zebrafish. Measures of sensorimotor plasticity, emotional function, cognition and social interaction have been used to characterize the persisting adverse effects of developmental exposure to a variety of chemicals including therapeutic drugs, drugs of abuse and environmental toxicants. In this review, we present and discuss such tests and data from a range of developmental neurobehavioral toxicology studies using zebrafish as a model. Zebrafish provide a key intermediate model between high throughput in vitro screens and the classic mammalian models as they have the accessibility of in vitro models and the complex functional capabilities of mammalian models. Copyright © 2013 Wiley Periodicals, Inc.

  3. Zebrafish Model Systems for Developmental Neurobehavioral Toxicology

    PubMed Central

    Bailey, Jordan; Oliveri, Anthony; Levin, Edward D.

    2014-01-01

    Zebrafish offer many advantages that complement classic mammalian models for the study of normal development as well as for the teratogenic effects of exposure to hazardous compounds. The clear chorion and embryo of the zebrafish allow for continuous visualization of the anatomical changes associated with development, which, along with short maturation times and the capability of complex behavior, makes this model particularly useful for measuring changes to the developing nervous system. Moreover, the rich array of developmental, behavioral, and molecular benefits offered by the zebrafish have contributed to an increasing demand for the use of zebrafish in behavioral teratology. Essential for this endeavor has been the development of a battery of tests to evaluate a spectrum of behavior in zebrafish. Measures of sensorimotor plasticity, emotional function, cognition and social interaction have been used to characterize the persisting adverse effects of developmental exposure to a variety of chemicals including therapeutic drugs, drugs of abuse and environmental toxicants. In this review, we present and discuss such tests and data from a range of developmental neurobehavioral toxicology studies using zebrafish as a model. Zebrafish provide a key intermediate model between high throughput in vitro screens and the classic mammalian models as they have the accessibility of in vitro models and the complex functional capabilities of mammalian models. PMID:23723169

  4. Macrophage–Microbe Interactions: Lessons from the Zebrafish Model

    PubMed Central

    Yoshida, Nagisa; Frickel, Eva-Maria; Mostowy, Serge

    2017-01-01

    Macrophages provide front line defense against infections. The study of macrophage–microbe interplay is thus crucial for understanding pathogenesis and infection control. Zebrafish (Danio rerio) larvae provide a unique platform to study macrophage–microbe interactions in vivo, from the level of the single cell to the whole organism. Studies using zebrafish allow non-invasive, real-time visualization of macrophage recruitment and phagocytosis. Furthermore, the chemical and genetic tractability of zebrafish has been central to decipher the complex role of macrophages during infection. Here, we discuss the latest developments using zebrafish models of bacterial and fungal infection. We also review novel aspects of macrophage biology revealed by zebrafish, which can potentiate development of new therapeutic strategies for humans. PMID:29250076

  5. Contextual Fear Conditioning in Zebrafish

    ERIC Educational Resources Information Center

    Kenney, Justin W.; Scott, Ian C.; Josselyn, Sheena A.; Frankland, Paul W.

    2017-01-01

    Zebrafish are a genetically tractable vertebrate that hold considerable promise for elucidating the molecular basis of behavior. Although numerous recent advances have been made in the ability to precisely manipulate the zebrafish genome, much less is known about many aspects of learning and memory in adult fish. Here, we describe the development…

  6. Knockdown of prothrombin in zebrafish.

    PubMed

    Day, Kenneth; Krishnegowda, Naveen; Jagadeeswaran, Pudur

    2004-01-01

    Thrombin is a serine protease generated from its zymogen, prothrombin, and plays a central role in the coagulation cascade. It is also important for mammalian development. The zebrafish has now been established as an excellent genetic model for studies on mammalian hemostasis and development. In this report, we used prothrombin-specific antisense morpholinos to knock down the levels of prothrombin to characterize the effects of prothrombin deficiency in the zebrafish embryo. Prothrombin morpholino-injected zebrafish embryos yielded an early phenotype exhibiting severe abnormalities that later showed occasional bleeding. In a second late phenotype, the embryos had no observable morphological abnormalities in early stages, but showed occasional bleeding at later stages. These phenotypes resembled characteristics shown by prothrombin knockout mice. Laser-induced vascular injury on some of the normal appearing phenotypic larvae showed a prolonged time to occlusion, and recombinant zebrafish prothrombin injected into these larvae restored a normal time to occlusion thus showing the specificity of the morpholino effect. The system developed here should be useful for investigation of the role of thrombin in vertebrate development.

  7. A Zebrafish Heart Failure Model for Assessing Therapeutic Agents.

    PubMed

    Zhu, Xiao-Yu; Wu, Si-Qi; Guo, Sheng-Ya; Yang, Hua; Xia, Bo; Li, Ping; Li, Chun-Qi

    2018-03-20

    Heart failure is a leading cause of death and the development of effective and safe therapeutic agents for heart failure has been proven challenging. In this study, taking advantage of larval zebrafish, we developed a zebrafish heart failure model for drug screening and efficacy assessment. Zebrafish at 2 dpf (days postfertilization) were treated with verapamil at a concentration of 200 μM for 30 min, which were determined as optimum conditions for model development. Tested drugs were administered into zebrafish either by direct soaking or circulation microinjection. After treatment, zebrafish were randomly selected and subjected to either visual observation and image acquisition or record videos under a Zebralab Blood Flow System. The therapeutic effects of drugs on zebrafish heart failure were quantified by calculating the efficiency of heart dilatation, venous congestion, cardiac output, and blood flow dynamics. All 8 human heart failure therapeutic drugs (LCZ696, digoxin, irbesartan, metoprolol, qiliqiangxin capsule, enalapril, shenmai injection, and hydrochlorothiazide) showed significant preventive and therapeutic effects on zebrafish heart failure (p < 0.05, p < 0.01, and p < 0.001) in the zebrafish model. The larval zebrafish heart failure model developed and validated in this study could be used for in vivo heart failure studies and for rapid screening and efficacy assessment of preventive and therapeutic drugs.

  8. Zebrafish: an animal model for research in veterinary medicine.

    PubMed

    Nowik, N; Podlasz, P; Jakimiuk, A; Kasica, N; Sienkiewicz, W; Kaleczyc, J

    2015-01-01

    The zebrafish (Danio rerio) has become known as an excellent model organism for studies of vertebrate biology, vertebrate genetics, embryonal development, diseases and drug screening. Nevertheless, there is still lack of detailed reports about usage of the zebrafish as a model in veterinary medicine. Comparing to other vertebrates, they can lay hundreds of eggs at weekly intervals, externally fertilized zebrafish embryos are accessible to observation and manipulation at all stages of their development, which makes possible to simplify the research techniques such as fate mapping, fluorescent tracer time-lapse lineage analysis and single cell transplantation. Although zebrafish are only 2.5 cm long, they are easy to maintain. Intraperitoneal and intracerebroventricular injections, blood sampling and measurement of food intake are possible to be carry out in adult zebrafish. Danio rerio is a useful animal model for neurobiology, developmental biology, drug research, virology, microbiology and genetics. A lot of diseases, for which the zebrafish is a perfect model organism, affect aquatic animals. For a part of them, like those caused by Mycobacterium marinum or Pseudoloma neutrophila, Danio rerio is a natural host, but the zebrafish is also susceptible to the most of fish diseases including Itch, Spring viraemia of carp and Infectious spleen and kidney necrosis. The zebrafish is commonly used in research of bacterial virulence. The zebrafish embryo allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host. Plenty of common pathogens can be examined using zebrafish model: Streptococcus iniae, Vibrio anguillarum or Listeria monocytogenes. The steps are taken to use the zebrafish also in fungal research, especially that dealing with Candida albicans and Cryptococcus neoformans. Although, the zebrafish is used commonly as an animal model to study diseases caused by external agents, it is also useful in studies of metabolic

  9. Zebrafish: A Versatile Animal Model for Fertility Research.

    PubMed

    Hoo, Jing Ying; Kumari, Yatinesh; Shaikh, Mohd Farooq; Hue, Seow Mun; Goh, Bey Hing

    2016-01-01

    The utilization of zebrafish in biomedical research is very common in the research world nowadays. Today, it has emerged as a favored vertebrate organism for the research in science of reproduction. There is a significant growth in amount numbers of scientific literature pertaining to research discoveries in reproductive sciences in zebrafish. It has implied the importance of zebrafish in this particular field of research. In essence, the current available literature has covered from the very specific brain region or neurons of zebrafish, which are responsible for reproductive regulation, until the gonadal level of the animal. The discoveries and findings have proven that this small animal is sharing a very close/similar reproductive system with mammals. More interestingly, the behavioral characteristics and along with the establishment of animal courtship behavior categorization in zebrafish have laid an even stronger foundation and firmer reason on the suitability of zebrafish utilization in research of reproductive sciences. In view of the immense importance of this small animal for the development of reproductive sciences, this review aimed at compiling and describing the proximate close similarity of reproductive regulation on zebrafish and human along with factors contributing to the infertility, showing its versatility and its potential usage for fertility research.

  10. Zebrafish: A Versatile Animal Model for Fertility Research

    PubMed Central

    Hoo, Jing Ying; Kumari, Yatinesh; Shaikh, Mohd Farooq; Hue, Seow Mun

    2016-01-01

    The utilization of zebrafish in biomedical research is very common in the research world nowadays. Today, it has emerged as a favored vertebrate organism for the research in science of reproduction. There is a significant growth in amount numbers of scientific literature pertaining to research discoveries in reproductive sciences in zebrafish. It has implied the importance of zebrafish in this particular field of research. In essence, the current available literature has covered from the very specific brain region or neurons of zebrafish, which are responsible for reproductive regulation, until the gonadal level of the animal. The discoveries and findings have proven that this small animal is sharing a very close/similar reproductive system with mammals. More interestingly, the behavioral characteristics and along with the establishment of animal courtship behavior categorization in zebrafish have laid an even stronger foundation and firmer reason on the suitability of zebrafish utilization in research of reproductive sciences. In view of the immense importance of this small animal for the development of reproductive sciences, this review aimed at compiling and describing the proximate close similarity of reproductive regulation on zebrafish and human along with factors contributing to the infertility, showing its versatility and its potential usage for fertility research. PMID:27556045

  11. Physical exercise improves learning in zebrafish, Danio rerio.

    PubMed

    Luchiari, Ana Carolina; Chacon, Diana Marques Martins

    2013-11-01

    Zebrafish is an ideal vertebrate model for neuroscience studies focusing on learning and memory. Although genetic manipulation of zebrafish is available, behavioral protocols are often lacking. In this study we tested whether physical activity can facilitate zebrafish's learning process in an associative conditioning task. Learning was inferred by the approach of the feeding area just after the conditioned stimulus (light). Unexercised zebrafish showed conditioning response from the 5th testing day while fish previously submitted to swim against the water current showed learning by the 3rd day of testing. It seems that physical activity may accelerate associative learning response in zebrafish, indicating the benefits of exercise for cognitive processes. We suggest that this preliminary work could be useful for high throughput screening. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Normal anatomy and histology of the adult zebrafish.

    PubMed

    Menke, Aswin L; Spitsbergen, Jan M; Wolterbeek, Andre P M; Woutersen, Ruud A

    2011-08-01

    The zebrafish has been shown to be an excellent vertebrate model for studying the roles of specific genes and signaling pathways. The sequencing of its genome and the relative ease with which gene modifications can be performed have led to the creation of numerous human disease models that can be used for testing the potential and the toxicity of new pharmaceutical compounds. Many pharmaceutical companies already use the zebrafish for prescreening purposes. So far, the focus has been on ecotoxicity and the effects on embryonic development, but there is a trend to expand the use of the zebrafish with acute, subchronic, and chronic toxicity studies that are currently still carried out with the more conventional test animals such as rodents. However, before we can fully realize the potential of the zebrafish as an animal model for understanding human development, disease, and toxicology, we must first greatly advance our knowledge of normal zebrafish physiology, anatomy, and histology. To further this knowledge, we describe, in the present article, location and histology of the major zebrafish organ systems with a brief description of their function.

  13. Making Waves: New Developments in Toxicology With the Zebrafish.

    PubMed

    Horzmann, Katharine A; Freeman, Jennifer L

    2018-05-01

    The laboratory zebrafish (Danio rerio) is now an accepted model in toxicologic research. The zebrafish model fills a niche between in vitro models and mammalian biomedical models. The developmental characteristics of the small fish are strategically being used by scientists to study topics ranging from high-throughput toxicity screens to toxicity in multi- and transgenerational studies. High-throughput technology has increased the utility of zebrafish embryonic toxicity assays in screening of chemicals and drugs for toxicity or effect. Additionally, advances in behavioral characterization and experimental methodology allow for observation of recognizable phenotypic changes after xenobiotic exposure. Future directions in zebrafish research are predicted to take advantage of CRISPR-Cas9 genome editing methods in creating models of disease and interrogating mechanisms of action with fluorescent reporters or tagged proteins. Zebrafish can also model developmental origins of health and disease and multi- and transgenerational toxicity. The zebrafish has many advantages as a toxicologic model and new methodologies and areas of study continue to expand the usefulness and application of the zebrafish.

  14. Identification and characterization of the zebrafish glutathione S-transferase Pi-1.

    PubMed

    Abunnaja, Maryam S; Kurogi, Katsuhisa; Mohammed, Yasir I; Sakakibara, Yoichi; Suiko, Masahito; Hassoun, Ezdihar A; Liu, Ming-Cheh

    2017-10-01

    Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S-transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi-1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3-month-old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione-conjugating activity toward 1-chloro-2,4-dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH-dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined. © 2017 Wiley Periodicals, Inc.

  15. Automated measurement of zebrafish larval movement

    PubMed Central

    Cario, Clinton L; Farrell, Thomas C; Milanese, Chiara; Burton, Edward A

    2011-01-01

    Abstract The zebrafish is a powerful vertebrate model that is readily amenable to genetic, pharmacological and environmental manipulations to elucidate the molecular and cellular basis of movement and behaviour. We report software enabling automated analysis of zebrafish movement from video recordings captured with cameras ranging from a basic camcorder to more specialized equipment. The software, which is provided as open-source MATLAB functions, can be freely modified and distributed, and is compatible with multiwell plates under a wide range of experimental conditions. Automated measurement of zebrafish movement using this technique will be useful for multiple applications in neuroscience, pharmacology and neuropsychiatry. PMID:21646414

  16. Automated measurement of zebrafish larval movement.

    PubMed

    Cario, Clinton L; Farrell, Thomas C; Milanese, Chiara; Burton, Edward A

    2011-08-01

    The zebrafish is a powerful vertebrate model that is readily amenable to genetic, pharmacological and environmental manipulations to elucidate the molecular and cellular basis of movement and behaviour. We report software enabling automated analysis of zebrafish movement from video recordings captured with cameras ranging from a basic camcorder to more specialized equipment. The software, which is provided as open-source MATLAB functions, can be freely modified and distributed, and is compatible with multiwell plates under a wide range of experimental conditions. Automated measurement of zebrafish movement using this technique will be useful for multiple applications in neuroscience, pharmacology and neuropsychiatry.

  17. Learning and memory in zebrafish larvae

    PubMed Central

    Roberts, Adam C.; Bill, Brent R.; Glanzman, David L.

    2013-01-01

    Larval zebrafish possess several experimental advantages for investigating the molecular and neural bases of learning and memory. Despite this, neuroscientists have only recently begun to use these animals to study memory. However, in a relatively short period of time a number of forms of learning have been described in zebrafish larvae, and significant progress has been made toward their understanding. Here we provide a comprehensive review of this progress; we also describe several promising new experimental technologies currently being used in larval zebrafish that are likely to contribute major insights into the processes that underlie learning and memory. PMID:23935566

  18. Production of Androgenetic Zebrafish (Danio Rerio)

    PubMed Central

    Corley-Smith, G. E.; Lim, C. J.; Brandhorst, B. P.

    1996-01-01

    To help investigate the evolutionary origin of the imprinting (parent-of-origin mono-allelic expression) of paternal genes observed in mammals, we constructed haploid and diploid androgenetic zebrafish (Danio rerio). Haploid androgenotes were produced by fertilizing eggs that had been X-ray irradiated to eliminate the maternal genome. Subsequent inhibition of the first mitotic division of haploid androgenotes by heat shock produced diploid androgenotes. The lack of inheritance of maternal-specific DNA markers (RAPD and SSR) by putative diploid and haploid androgenotes confirmed the androgenetic origin of their genomes. Marker analysis was performed on 18 putative androgenotes (five diploids and 13 haploids) from six families. None of 157 maternal-specific RAPD markers analyzed, some of which were apparently homozygous, were passed on to any of these putative androgenotes. A mean of 7.7 maternal-specific markers were assessed per family. The survival of androgenetic zebrafish suggests that if paternal imprinting occurs in zebrafish, it does not result in essential genes being inactivated when their expression is required for development. Production of haploid androgenotes can be used to determine the meiotic recombination rate in male zebrafish. Androgenesis may also provide useful information about the mechanism of sex determination in zebrafish. PMID:8846903

  19. Development of sensory systems in zebrafish (Danio rerio)

    NASA Technical Reports Server (NTRS)

    Moorman, S. J.

    2001-01-01

    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.

  20. UNUSUAL FINDINGS IN ZEBRAFISH, DANIO RERIO, FROM TOXICOLOGICAL STUDIES AND THE ZEBRAFISH INTERNATIONAL RESOURCE CENTER DIAGNOSTIC SERVICE

    EPA Science Inventory

    A number of interesting and unusual lesions have been diagnosed in zebrafish that have been evaluated from toxicological studies or submitted as cases to the Diagnostic Service at Oregon State University. Lesions were observed in various wild-type and mutant lines of zebrafish an...

  1. Contextual fear conditioning in zebrafish.

    PubMed

    Kenney, Justin W; Scott, Ian C; Josselyn, Sheena A; Frankland, Paul W

    2017-10-01

    Zebrafish are a genetically tractable vertebrate that hold considerable promise for elucidating the molecular basis of behavior. Although numerous recent advances have been made in the ability to precisely manipulate the zebrafish genome, much less is known about many aspects of learning and memory in adult fish. Here, we describe the development of a contextual fear conditioning paradigm using an electric shock as the aversive stimulus. We find that contextual fear conditioning is modulated by shock intensity, prevented by an established amnestic agent (MK-801), lasts at least 14 d, and exhibits extinction. Furthermore, fish of various background strains (AB, Tu, and TL) are able to acquire fear conditioning, but differ in fear extinction rates. Taken together, we find that contextual fear conditioning in zebrafish shares many similarities with the widely used contextual fear conditioning paradigm in rodents. Combined with the amenability of genetic manipulation in zebrafish, we anticipate that our paradigm will prove to be a useful complementary system in which to examine the molecular basis of vertebrate learning and memory. © 2017 Kenney et al.; Published by Cold Spring Harbor Laboratory Press.

  2. Automatic multiple zebrafish larvae tracking in unconstrained microscopic video conditions.

    PubMed

    Wang, Xiaoying; Cheng, Eva; Burnett, Ian S; Huang, Yushi; Wlodkowic, Donald

    2017-12-14

    The accurate tracking of zebrafish larvae movement is fundamental to research in many biomedical, pharmaceutical, and behavioral science applications. However, the locomotive characteristics of zebrafish larvae are significantly different from adult zebrafish, where existing adult zebrafish tracking systems cannot reliably track zebrafish larvae. Further, the far smaller size differentiation between larvae and the container render the detection of water impurities inevitable, which further affects the tracking of zebrafish larvae or require very strict video imaging conditions that typically result in unreliable tracking results for realistic experimental conditions. This paper investigates the adaptation of advanced computer vision segmentation techniques and multiple object tracking algorithms to develop an accurate, efficient and reliable multiple zebrafish larvae tracking system. The proposed system has been tested on a set of single and multiple adult and larvae zebrafish videos in a wide variety of (complex) video conditions, including shadowing, labels, water bubbles and background artifacts. Compared with existing state-of-the-art and commercial multiple organism tracking systems, the proposed system improves the tracking accuracy by up to 31.57% in unconstrained video imaging conditions. To facilitate the evaluation on zebrafish segmentation and tracking research, a dataset with annotated ground truth is also presented. The software is also publicly accessible.

  3. Computerized image analysis for quantitative neuronal phenotyping in zebrafish.

    PubMed

    Liu, Tianming; Lu, Jianfeng; Wang, Ye; Campbell, William A; Huang, Ling; Zhu, Jinmin; Xia, Weiming; Wong, Stephen T C

    2006-06-15

    An integrated microscope image analysis pipeline is developed for automatic analysis and quantification of phenotypes in zebrafish with altered expression of Alzheimer's disease (AD)-linked genes. We hypothesize that a slight impairment of neuronal integrity in a large number of zebrafish carrying the mutant genotype can be detected through the computerized image analysis method. Key functionalities of our zebrafish image processing pipeline include quantification of neuron loss in zebrafish embryos due to knockdown of AD-linked genes, automatic detection of defective somites, and quantitative measurement of gene expression levels in zebrafish with altered expression of AD-linked genes or treatment with a chemical compound. These quantitative measurements enable the archival of analyzed results and relevant meta-data. The structured database is organized for statistical analysis and data modeling to better understand neuronal integrity and phenotypic changes of zebrafish under different perturbations. Our results show that the computerized analysis is comparable to manual counting with equivalent accuracy and improved efficacy and consistency. Development of such an automated data analysis pipeline represents a significant step forward to achieve accurate and reproducible quantification of neuronal phenotypes in large scale or high-throughput zebrafish imaging studies.

  4. Designing and Testing of Self-Cleaning Recirculating Zebrafish Tanks.

    PubMed

    Nema, Shubham; Bhargava, Yogesh

    2016-08-01

    Maintenance of large number of zebrafish in captive conditions is a daunting task. This can be eased by the use of recirculating racks with self-cleaning zebrafish tanks. Commercially available systems are costly, and compatibility of intercompany products has never been investigated. Although various cost-effective designs and methods of construction of custom-made recirculating zebrafish racks are available in literature, the design of self-cleaning zebrafish tanks is still not available. In this study, we report the design and method of construction of the self-cleaning unit, which can be fitted in any zebrafish tank. We validated the design by investigating sediment cleaning process in rectangular and cylindrical tank geometries using time lapse imaging. Our results suggest that for both tank geometries, the tanks fitted with self-cleaning unit provided superior sediment cleaning than the tanks fitted with overflow-drain unit. Although the self-cleaning unit could clean the sediment completely from both geometries over prolonged period, the cleaning of sediments was faster in the cylindrical tank than the rectangular tank. In conclusion, cost and efforts of zebrafish maintenance could be significantly reduced through the installation of our self-cleaning unit in any custom-made zebrafish tank.

  5. Genomic Approaches to Zebrafish Cancer

    PubMed Central

    2017-01-01

    The zebrafish has emerged as an important model for studying cancer biology. Identification of DNA, RNA and chromatin abnormalities can give profound insight into the mechanisms of tumorigenesis and the there are many techniques for analyzing the genomes of these tumors. Here, I present an overview of the available technologies for analyzing tumor genomes in the zebrafish, including array based methods as well as next-generation sequencing technologies. I also discuss the ways in which zebrafish tumor genomes can be compared to human genomes using cross-species oncogenomics, which act to filter genomic noise and ultimately uncover central drivers of malignancy. Finally, I discuss downstream analytic tools, including network analysis, that can help to organize the alterations into coherent biological frameworks that can then be investigated further. PMID:27165352

  6. Zebrafish tracking using convolutional neural networks.

    PubMed

    Xu, Zhiping; Cheng, Xi En

    2017-02-17

    Keeping identity for a long term after occlusion is still an open problem in the video tracking of zebrafish-like model animals, and accurate animal trajectories are the foundation of behaviour analysis. We utilize the highly accurate object recognition capability of a convolutional neural network (CNN) to distinguish fish of the same congener, even though these animals are indistinguishable to the human eye. We used data augmentation and an iterative CNN training method to optimize the accuracy for our classification task, achieving surprisingly accurate trajectories of zebrafish of different size and age zebrafish groups over different time spans. This work will make further behaviour analysis more reliable.

  7. Zebrafish tracking using convolutional neural networks

    NASA Astrophysics Data System (ADS)

    Xu, Zhiping; Cheng, Xi En

    2017-02-01

    Keeping identity for a long term after occlusion is still an open problem in the video tracking of zebrafish-like model animals, and accurate animal trajectories are the foundation of behaviour analysis. We utilize the highly accurate object recognition capability of a convolutional neural network (CNN) to distinguish fish of the same congener, even though these animals are indistinguishable to the human eye. We used data augmentation and an iterative CNN training method to optimize the accuracy for our classification task, achieving surprisingly accurate trajectories of zebrafish of different size and age zebrafish groups over different time spans. This work will make further behaviour analysis more reliable.

  8. Zebrafish as model organisms for studying drug-induced liver injury

    PubMed Central

    Vliegenthart, A D Bastiaan; Tucker, Carl S; Del Pozo, Jorge; Dear, James W

    2014-01-01

    Drug-induced liver injury (DILI) is a major challenge in clinical medicine and drug development. New models are needed for predicting which potential therapeutic compounds will cause DILI in humans, and new markers and mediators of DILI still need to be identified. This review highlights the strengths and weaknesses of using zebrafish as a high-throughput in vivo model for studying DILI. Although the zebrafish liver architecture is different from that of the mammalian liver, the main physiological processes remain similar. Zebrafish metabolize drugs using similar pathways to those in humans; they possess a wide range of cytochrome P450 enzymes that enable metabolic reactions including hydroxylation, conjugation, oxidation, demethylation and de-ethylation. Following exposure to a range of hepatotoxic drugs, the zebrafish liver develops histological patterns of injury comparable to those of mammalian liver, and biomarkers for liver injury can be quantified in the zebrafish circulation. The zebrafish immune system is similar to that of mammals, but the zebrafish inflammatory response to DILI is not yet defined. In order to quantify DILI in zebrafish, a wide variety of methods can be used, including visual assessment, quantification of serum enzymes and experimental serum biomarkers and scoring of histopathology. With further development, the zebrafish may be a model that complements rodents and may have value for the discovery of new disease pathways and translational biomarkers. PMID:24773296

  9. Heart Repair and Regeneration: Recent Insights from Zebrafish Studies

    PubMed Central

    Lien, Ching-Ling; Harrison, Michael R.; Tuan, Tai-Lan; Starnes, Vaughn A

    2012-01-01

    Cardiovascular disease is the leading cause of death in United States and worldwide. Failure to properly repair or regenerate damaged cardiac tissues after myocardial infarction is a major cause of heart failure. In contrast to humans and other mammals, zebrafish hearts regenerate after substantial injury or tissue damage. Here, we review recent progress in studying zebrafish heart regeneration, addressing the molecular and cellular responses in the three tissue layers of the heart: myocardium, epicardium, and endocardium. We also compare different injury models utilized to study zebrafish heart regeneration, and discuss the differences in responses to injury between mammalian and zebrafish hearts. By learning how zebrafish hearts regenerate naturally, we can better design therapeutic strategies for repairing human hearts after myocardial infarction. PMID:22818295

  10. The zebrafish genome: a review and msx gene case study.

    PubMed

    Postlethwait, J H

    2006-01-01

    Zebrafish is one of several important teleost models for understanding principles of vertebrate developmental, molecular, organismal, genetic, evolutionary, and genomic biology. Efficient investigation of the molecular genetic basis of induced mutations depends on knowledge of the zebrafish genome. Principles of zebrafish genomic analysis, including gene mapping, ortholog identification, conservation of syntenies, genome duplication, and evolution of duplicate gene function are discussed here using as a case study the zebrafish msxa, msxb, msxc, msxd, and msxe genes, which together constitute zebrafish orthologs of tetrapod Msx1, Msx2, and Msx3. Genomic analysis suggests orthologs for this difficult to understand group of paralogs.

  11. Biosecurity and Health Monitoring at the Zebrafish International Resource Center

    PubMed Central

    Varga, Zoltán M.; Kent, Michael L.

    2016-01-01

    Abstract The Zebrafish International Resource Center (ZIRC) is a repository and distribution center for mutant, transgenic, and wild-type zebrafish. In recent years annual imports of new zebrafish lines to ZIRC have increased tremendously. In addition, after 15 years of research, we have identified some of the most virulent pathogens affecting zebrafish that should be avoided in large production facilities, such as ZIRC. Therefore, while importing a high volume of new lines we prioritize safeguarding the health of our in-house fish colony. Here, we describe the biosecurity and health-monitoring program implemented at ZIRC. This strategy was designed to prevent introduction of new zebrafish pathogens, minimize pathogens already present in the facility, and ensure a healthy zebrafish colony for in-house uses and shipment to customers. PMID:27031282

  12. Advancing epilepsy treatment through personalized genetic zebrafish models.

    PubMed

    Griffin, A; Krasniak, C; Baraban, S C

    2016-01-01

    With an increase in the number of disease causing genetic mutations identified from epilepsy cohorts, zebrafish are proving to be an attractive vertebrate model for functional analysis of these allele variants. Not only do zebrafish have conserved gene functions, but larvae harboring mutations in identified human epileptic genes show spontaneous seizure activity and mimic the convulsive behavioral movements observed in humans. With zebrafish being compatible with medium to high-throughput screening, they are also proving to be a unique and powerful system for early preclinical drug screening, including novel target identification, pharmacology, and toxicology. Additionally, with recent advances in genomic engineering technologies, it is now possible to study the precise pathophysiology of patient-specific gene mutations in zebrafish. The following sections highlight how the unique attributes of zebrafish, in combination with genetic modifications, are continuing to transform our understanding of epilepsy and help identify personalized therapeutics for specific patient cohorts. © 2016 Elsevier B.V. All rights reserved.

  13. Examination of a Palatogenic Gene Program in Zebrafish

    PubMed Central

    Swartz, Mary E.; Sheehan-Rooney, Kelly; Dixon, Michael J.; Eberhart, Johann K.

    2011-01-01

    Human palatal clefting is debilitating and difficult to rectify surgically. Animal models enhance our understanding of palatogenesis and are essential in strategies designed to ameliorate palatal malformations in humans. Recent studies have shown that the zebrafish palate, or anterior neurocranium, is under similar genetic control to the amniote palatal skeleton. We extensively analyzed palatogenesis in zebrafish to determine the similarity of gene expression and function across vertebrates. By 36 hpf palatogenic cranial neural crest cells reside in homologous regions of the developing face compared to amniote species. Transcription factors and signaling molecules regulating mouse palatogenesis are expressed in similar domains during palatogenesis in zebrafish. Functional investigation of a subset of these genes, fgf10a, tgfb2, pax9 and smad5 revealed their necessity in zebrafish palatogenesis. Collectively, these results suggest that the gene regulatory networks regulating palatogenesis may be conserved across vertebrate species, demonstrating the utility of zebrafish as a model for palatogenesis. PMID:22016187

  14. Systematic approaches to toxicology in the zebrafish.

    PubMed

    Peterson, Randall T; Macrae, Calum A

    2012-01-01

    As the current paradigms of drug discovery evolve, it has become clear that a more comprehensive understanding of the interactions between small molecules and organismal biology will be vital. The zebrafish is emerging as a complement to existing in vitro technologies and established preclinical in vivo models that can be scaled for high-throughput. In this review, we highlight the current status of zebrafish toxicology studies, identify potential future niches for the model in the drug development pipeline, and define the hurdles that must be overcome as zebrafish technologies are refined for systematic toxicology.

  15. Zebrafish models in neuropsychopharmacology and CNS drug discovery.

    PubMed

    Khan, Kanza M; Collier, Adam D; Meshalkina, Darya A; Kysil, Elana V; Khatsko, Sergey L; Kolesnikova, Tatyana; Morzherin, Yury Yu; Warnick, Jason E; Kalueff, Allan V; Echevarria, David J

    2017-07-01

    Despite the high prevalence of neuropsychiatric disorders, their aetiology and molecular mechanisms remain poorly understood. The zebrafish (Danio rerio) is increasingly utilized as a powerful animal model in neuropharmacology research and in vivo drug screening. Collectively, this makes zebrafish a useful tool for drug discovery and the identification of disordered molecular pathways. Here, we discuss zebrafish models of selected human neuropsychiatric disorders and drug-induced phenotypes. As well as covering a broad range of brain disorders (from anxiety and psychoses to neurodegeneration), we also summarize recent developments in zebrafish genetics and small molecule screening, which markedly enhance the disease modelling and the discovery of novel drug targets. © 2017 The British Pharmacological Society.

  16. Incorporating zebrafish omics into chemical biology and toxicology.

    PubMed

    Sukardi, Hendrian; Ung, Choong Yong; Gong, Zhiyuan; Lam, Siew Hong

    2010-03-01

    In this communication, we describe the general aspects of omics approaches for analyses of transcriptome, proteome, and metabolome, and how they can be strategically incorporated into chemical screening and perturbation studies using the zebrafish system. Pharmacological efficacy and selectivity of chemicals can be evaluated based on chemical-induced phenotypic effects; however, phenotypic observation has limitations in identifying mechanistic action of chemicals. We suggest adapting gene-expression-based high-throughput screening as a complementary strategy to zebrafish-phenotype-based screening for mechanistic insights about the mode of action and toxicity of a chemical, large-scale predictive applications and comparative analysis of chemical-induced omics signatures, which are useful to identify conserved biological responses, signaling pathways, and biomarkers. The potential mechanistic, predictive, and comparative applications of omics approaches can be implemented in the zebrafish system. Examples of these using the omics approaches in zebrafish, including data of ours and others, are presented and discussed. Omics also facilitates the translatability of zebrafish studies across species through comparison of conserved chemical-induced responses. This review is intended to update interested readers with the current omics approaches that have been applied in chemical studies on zebrafish and their potential in enhancing discovery in chemical biology.

  17. Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing

    PubMed Central

    Chatterjee, Aniruddha; Ozaki, Yuichi; Stockwell, Peter A; Horsfield, Julia A; Morison, Ian M; Nakagawa, Shinichi

    2013-01-01

    Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish. PMID:23975027

  18. Zebrafish models for the functional genomics of neurogenetic disorders.

    PubMed

    Kabashi, Edor; Brustein, Edna; Champagne, Nathalie; Drapeau, Pierre

    2011-03-01

    In this review, we consider recent work using zebrafish to validate and study the functional consequences of mutations of human genes implicated in a broad range of degenerative and developmental disorders of the brain and spinal cord. Also we present technical considerations for those wishing to study their own genes of interest by taking advantage of this easily manipulated and clinically relevant model organism. Zebrafish permit mutational analyses of genetic function (gain or loss of function) and the rapid validation of human variants as pathological mutations. In particular, neural degeneration can be characterized at genetic, cellular, functional, and behavioral levels. Zebrafish have been used to knock down or express mutations in zebrafish homologs of human genes and to directly express human genes bearing mutations related to neurodegenerative disorders such as spinal muscular atrophy, ataxia, hereditary spastic paraplegia, amyotrophic lateral sclerosis (ALS), epilepsy, Huntington's disease, Parkinson's disease, fronto-temporal dementia, and Alzheimer's disease. More recently, we have been using zebrafish to validate mutations of synaptic genes discovered by large-scale genomic approaches in developmental disorders such as autism, schizophrenia, and non-syndromic mental retardation. Advances in zebrafish genetics such as multigenic analyses and chemical genetics now offer a unique potential for disease research. Thus, zebrafish hold much promise for advancing the functional genomics of human diseases, the understanding of the genetics and cell biology of degenerative and developmental disorders, and the discovery of therapeutics. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

  19. Morphologic analysis of the zebrafish digestive system.

    PubMed

    Trotter, Andrew J; Parslow, Adam C; Heath, Joan K

    2009-01-01

    The zebrafish provides an ideal model for the study of vertebrate organogenesis, including the formation of the digestive tract and its associated organs. Despite optical transparency of embryos, the internal position of the developing digestive system and its close juxtaposition with the yolk initially made morphological analysis relatively challenging, particularly during the first 3 d of development. However, methodologies have been successfully developed to address these problems and comprehensive morphologic analysis of the developing digestive system has now been achieved using a combination of light and fluorescence microscope approaches-including confocal analysis-to visualize wholemount and histological preparations of zebrafish embryos. Furthermore, the expanding number of antibodies that cross-react with zebrafish proteins and the generation of tissue-specific transgenic green fluorescent protein reporter lines that mark specific cell and tissue compartments have greatly enhanced our ability to successfully image the developing zebrafish digestive system.

  20. Open-RAC: Open-Design, Recirculating and Auto-Cleaning Zebrafish Maintenance System.

    PubMed

    Nema, Shubham; Bhargava, Yogesh

    2017-08-01

    Zebrafish is a vertebrate animal model. Their maintenance in large number under laboratory conditions is a daunting task. Commercially available recirculating zebrafish maintenance systems are used to efficiently handle the tasks of automatic sediment cleaning from zebrafish tanks with minimal waste of water. Due to their compact nature, they also ensure the maximal use of available lab space. However, the high costs of commercial systems present a limitation to researchers with limited funds. A cost-effective zebrafish maintenance system with major features offered by commercially available systems is highly desirable. Here, we describe a compact and recirculating zebrafish maintenance system. Our system is composed of cost-effective components, which are available in local markets and/or can be procured via online vendors. Depending on the expertise of end users, the system can be assembled in 2 days. The system is completely customizable as it offers geometry independent zebrafish tanks that are capable of auto-cleaning the sediments. Due to these features, we called our setup as Open-RAC (Open-design, Recirculating and Auto-Cleaning zebrafish maintenance system). Open-RAC is a cost-effective and viable alternative to the currently available zebrafish maintenance systems. Thus, we believe that the use of Open-RAC could promote the zebrafish research by removing the cost barrier for researchers.

  1. Transvection Arising from Transgene Interactions in Zebrafish.

    PubMed

    Keefe, Matthew D; Bonkowsky, Joshua L

    2017-02-01

    There has been a rapid expansion in use of transgenic technologies in zebrafish. We report a novel example of transinteractions of genetic elements, or transvection. This interaction led to a novel expression pattern and illustrates a precautionary example regarding use of transgenes in zebrafish.

  2. Behavioural fever in zebrafish larvae.

    PubMed

    Rey, Sonia; Moiche, Visila; Boltaña, Sebastian; Teles, Mariana; MacKenzie, Simon

    2017-02-01

    Behavioural fever has been reported in different species of mobile ectotherms including the zebrafish, Danio rerio, in response to exogenous pyrogens. In this study we report, to our knowledge for the first time, upon the ontogenic onset of behavioural fever in zebrafish (Danio rerio) larvae. For this, zebrafish larvae (from first feeding to juveniles) were placed in a continuous thermal gradient providing the opportunity to select their preferred temperature. The novel thermal preference aquarium was based upon a continuous vertical column system and allows for non-invasive observation of larvae vertical distribution under isothermal (T R at 28 °C) and thermal gradient conditions (T CH : 28-32 °C). Larval thermal preference was assessed under both conditions with or without an immersion challenge, in order to detect the onset of the behavioural fever response. Our results defined the onset of the dsRNA induced behavioural fever at 18-20 days post fertilization (dpf). Significant differences were observed in dsRNA challenged larvae, which prefer higher temperatures (1-4 °C increase) throughout the experimental period as compared to non-challenged larvae. In parallel we measured the abundance of antiviral transcripts; viperin, gig2, irf7, trim25 and Mxb mRNAs in dsRNA challenged larvae under both thermal regimes: T R and T Ch . Significant increases in the abundance of all measured transcripts were recorded under thermal choice conditions signifying that thermo-coupling and the resultant enhancement of the immune response to dsRNA challenge occurs from 18 dpf onwards in the zebrafish. The results are of importance as they identify a key developmental stage where the neuro-immune interface matures in the zebrafish likely providing increased resistance to viral infection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Zebrafish models for translational neuroscience research: from tank to bedside

    PubMed Central

    Stewart, Adam Michael; Braubach, Oliver; Spitsbergen, Jan; Gerlai, Robert; Kalueff, Allan V.

    2014-01-01

    The zebrafish (Danio rerio) is emerging as a new important species for studying mechanisms of brain function and dysfunction. Focusing on selected central nervous system (CNS) disorders (brain cancer, epilepsy, and anxiety) and using them as examples, we discuss the value of zebrafish models in translational neuroscience. We further evaluate the contribution of zebrafish to neuroimaging, circuit level, and drug discovery research. Outlining the role of zebrafish in modeling a wide range of human brain disorders, we also summarize recent applications and existing challenges in this field. Finally, we emphasize the potential of zebrafish models in behavioral phenomics and high-throughput genetic/small molecule screening, which is critical for CNS drug discovery and identifying novel candidate genes. PMID:24726051

  4. Characterization of behavioral and endocrine effects of LSD on zebrafish.

    PubMed

    Grossman, Leah; Utterback, Eli; Stewart, Adam; Gaikwad, Siddharth; Chung, Kyung Min; Suciu, Christopher; Wong, Keith; Elegante, Marco; Elkhayat, Salem; Tan, Julia; Gilder, Thomas; Wu, Nadine; Dileo, John; Cachat, Jonathan; Kalueff, Allan V

    2010-12-25

    Lysergic acid diethylamide (LSD) is a potent hallucinogenic drug that strongly affects animal and human behavior. Although adult zebrafish (Danio rerio) are emerging as a promising neurobehavioral model, the effects of LSD on zebrafish have not been investigated previously. Several behavioral paradigms (the novel tank, observation cylinder, light-dark box, open field, T-maze, social preference and shoaling tests), as well as modern video-tracking tools and whole-body cortisol assay were used to characterize the effects of acute LSD in zebrafish. While lower doses (5-100 microg/L) did not affect zebrafish behavior, 250 microg/L LSD increased top dwelling and reduced freezing in the novel tank and observation cylinder tests, also affecting spatiotemporal patterns of activity (as assessed by 3D reconstruction of zebrafish traces and ethograms). LSD evoked mild thigmotaxis in the open field test, increased light behavior in the light-dark test, reduced the number of arm entries and freezing in the T-maze and social preference test, without affecting social preference. In contrast, LSD affected zebrafish shoaling (increasing the inter-fish distance in a group), and elevated whole-body cortisol levels. Overall, our findings show sensitivity of zebrafish to LSD action, and support the use of zebrafish models to study hallucinogenic drugs of abuse. Copyright (c) 2010 Elsevier B.V. All rights reserved.

  5. 15 years of zebrafish chemical screening

    PubMed Central

    Rennekamp, Andrew J.; Peterson, Randall T.

    2015-01-01

    In 2000, the first chemical screen using living zebrafish in a multi-well plate was reported. Since then, more than 60 additional screens have been published describing whole-organism drug and pathway discovery projects in zebrafish. To investigate the scope of the work reported in the last 14 years and to identify trends in the field, we analyzed the discovery strategies of 64 primary research articles from the literature. We found that zebrafish screens have expanded beyond the use of developmental phenotypes to include behavioral, cardiac, metabolic, proliferative and regenerative endpoints. Additionally, many creative strategies have been used to uncover the mechanisms of action of new small molecules including chemical phenocopy, genetic phenocopy, mutant rescue, and spatial localization strategies. PMID:25461724

  6. Differences in Acute Alcohol-Induced Behavioral Responses Among Zebrafish Populations

    PubMed Central

    Gerlai, Robert; Ahmad, Fahad; Prajapati, Sonal

    2009-01-01

    Background With the arsenal of genetic tools available for zebrafish, this species has been successfully used to investigate the genetic aspects of human diseases from developmental disorders to cancer. Interest in the behavior and brain function of zebrafish is also increasing as CNS disorders may be modeled and studied with this species. Alcoholism and alcohol abuse are among the most devastating and costliest diseases. However, the mechanisms of these diseases are not fully understood. Zebrafish has been proposed as a model organism to study such mechanisms. Characterization of alcohol’s effects on zebrafish is a necessary step in this research. Methods Here, we compare the effects of acute alcohol (EtOH) administration on the behavior of zebrafish from 4 distinct laboratory-bred populations using automated as well as observation based behavioral quantification methods. Results Alcohol treatment resulted in significant dose-dependent behavioral changes but the dose–response trajectories differed among zebrafish populations. Conclusions The results demonstrate for the first time a genetic component in alcohol responses in adult zebrafish and also show the feasibility of high throughput behavioral screening. We discuss the exploration and exploitation of the genetic differences found. PMID:18652595

  7. The Effect of Chronic Arsenic Exposure in Zebrafish

    PubMed Central

    Hallauer, Janell; Geng, Xiangrong; Yang, Hung-Chi; Shen, Jian; Tsai, Kan-Jen

    2016-01-01

    Abstract Arsenic is a prevalent environmental toxin and a Group one human carcinogenic agent. Chronic arsenic exposure has been associated with many human diseases. The aim of this study is to evaluate zebrafish as an animal model to assess arsenic toxicity in elevated long-term arsenic exposure. With prolonged exposure (6 months) to various concentrations of arsenic from 50 ppb to 300 ppb, effects of arsenic accumulation in zebrafish tissues, and phenotypes were investigated. Results showed that there are no significant changes of arsenic retention in zebrafish tissues, and zebrafish did not exhibit any visible tumor formation under arsenic exposure conditions. However, the zebrafish demonstrate a dysfunction in their neurological system, which is reflected by a reduction of locomotive activity. Moreover, elevated levels of the superoxide dismutase (SOD2) protein were detected in the eye and liver, suggesting increased oxidative stress. In addition, the progenies of arsenic-treated parents displayed a smaller biomass (four-fold reduction in body weight) compared with those from their parental controls. This result indicates that arsenic may induce genetic or epigenetic changes that are then passed on to the next generation. Overall, this study demonstrates that zebrafish is a convenient vertebrate model with advantages in the evaluation of arsenic-associated neurological disorders as well as its influences on the offspring. PMID:27140519

  8. Zebrafish sex: a complicated affair

    PubMed Central

    Liew, Woei Chang

    2014-01-01

    In this review, we provide a detailed overview of studies on the elusive sex determination (SD) and gonad differentiation mechanisms of zebrafish (Danio rerio). We show that the data obtained from most studies are compatible with polygenic sex determination (PSD), where the decision is made by the allelic combinations of several loci. These loci are typically dispersed throughout the genome, but in some teleost species a few of them might be located on a preferential pair of (sex) chromosomes. The PSD system has a much higher level of variation of SD genotypes both at the level of gametes and the sexual genotype of individuals, than that of the chromosomal sex determination systems. The early sexual development of zebrafish males is a complicated process, as they first develop a ‘juvenile ovary’, that later undergoes a transformation to give way to a testis. To date, three major developmental pathways were shown to be involved with gonad differentiation through the modulation of programmed cell death. In our opinion, there are more pathways participating in the regulation of zebrafish gonad differentiation/transformation. Introduction of additional powerful large-scale genomic approaches into the analysis of zebrafish reproduction will result in further deepening of our knowledge as well as identification of additional pathways and genes associated with these processes in the near future. PMID:24148942

  9. Zebrafish Models for Human Acute Organophosphorus Poisoning.

    PubMed

    Faria, Melissa; Garcia-Reyero, Natàlia; Padrós, Francesc; Babin, Patrick J; Sebastián, David; Cachot, Jérôme; Prats, Eva; Arick Ii, Mark; Rial, Eduardo; Knoll-Gellida, Anja; Mathieu, Guilaine; Le Bihanic, Florane; Escalon, B Lynn; Zorzano, Antonio; Soares, Amadeu M V M; Raldúa, Demetrio

    2015-10-22

    Terrorist use of organophosphorus-based nerve agents and toxic industrial chemicals against civilian populations constitutes a real threat, as demonstrated by the terrorist attacks in Japan in the 1990 s or, even more recently, in the Syrian civil war. Thus, development of more effective countermeasures against acute organophosphorus poisoning is urgently needed. Here, we have generated and validated zebrafish models for mild, moderate and severe acute organophosphorus poisoning by exposing zebrafish larvae to different concentrations of the prototypic organophosphorus compound chlorpyrifos-oxon. Our results show that zebrafish models mimic most of the pathophysiological mechanisms behind this toxidrome in humans, including acetylcholinesterase inhibition, N-methyl-D-aspartate receptor activation, and calcium dysregulation as well as inflammatory and immune responses. The suitability of the zebrafish larvae to in vivo high-throughput screenings of small molecule libraries makes these models a valuable tool for identifying new drugs for multifunctional drug therapy against acute organophosphorus poisoning.

  10. Live imaging of apoptotic cells in zebrafish

    PubMed Central

    van Ham, Tjakko J.; Mapes, James; Kokel, David; Peterson, Randall T.

    2010-01-01

    Many debilitating diseases, including neurodegenerative diseases, involve apoptosis. Several methods have been developed for visualizing apoptotic cells in vitro or in fixed tissues, but few tools are available for visualizing apoptotic cells in live animals. Here we describe a genetically encoded fluorescent reporter protein that labels apoptotic cells in live zebrafish embryos. During apoptosis, the phospholipid phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane. The calcium-dependent protein Annexin V (A5) binds PS with high affinity, and biochemically purified, fluorescently labeled A5 probes have been widely used to detect apoptosis in vitro. Here we show that secreted A5 fused to yellow fluorescent protein specifically labels apoptotic cells in living zebrafish. We use this fluorescent probe to characterize patterns of apoptosis in living zebrafish larvae and to visualize neuronal cell death at single-cell resolution in vivo.—Van Ham, T. J., Mapes, J., Kokel, D., Peterson, R. T. Live imaging of apoptotic cells in zebrafish. PMID:20601526

  11. HCV IRES-Mediated Core Expression in Zebrafish

    PubMed Central

    Zhang, Jing-Pu; Hu, Zhan-Ying; Tong, Jun-Wei; Ding, Cun-Bao; Peng, Zong-Gen; Zhao, Li-Xun; Song, Dan-Qing; Jiang, Jian-Dong

    2013-01-01

    The lack of small animal models for hepatitis C virus has impeded the discovery and development of anti-HCV drugs. HCV-IRES plays an important role in HCV gene expression, and is an attractive target for antiviral therapy. In this study, we report a zebrafish model with a biscistron expression construct that can co-transcribe GFP and HCV-core genes by human hepatic lipase promoter and zebrafish liver fatty acid binding protein enhancer. HCV core translation was designed mediated by HCV-IRES sequence and gfp was by a canonical cap-dependent mechanism. Results of fluorescence image and in situ hybridization indicate that expression of HCV core and GFP is liver-specific; RT-PCR and Western blotting show that both core and gfp expression are elevated in a time-dependent manner for both transcription and translation. It means that the HCV-IRES exerted its role in this zebrafish model. Furthermore, the liver-pathological impact associated with HCV-infection was detected by examination of gene markers and some of them were elevated, such as adiponectin receptor, heparanase, TGF-β, PDGF-α, etc. The model was used to evaluate three clinical drugs, ribavirin, IFNα-2b and vitamin B12. The results show that vitamin B12 inhibited core expression in mRNA and protein levels in dose-dependent manner, but failed to impact gfp expression. Also VB12 down-regulated some gene transcriptions involved in fat liver, liver fibrosis and HCV-associated pathological process in the larvae. It reveals that HCV-IRES responds to vitamin B12 sensitively in the zebrafish model. Ribavirin did not disturb core expression, hinting that HCV-IRES is not a target site of ribavirin. IFNα-2b was not active, which maybe resulted from its degradation in vivo for the long time. These findings demonstrate the feasibility of the zebrafish model for screening of anti-HCV drugs targeting to HCV-IRES. The zebrafish system provides a novel evidence of using zebrafish as a HCV model organism. PMID:23469178

  12. Analysis of Lethality and Malformations During Zebrafish (Danio rerio) Development.

    PubMed

    Raghunath, Azhwar; Perumal, Ekambaram

    2018-01-01

    The versatility offered by zebrafish (Danio rerio) makes it a powerful and an attractive vertebrate model in developmental toxicity and teratogenicity assays. Apart from the newly introduced chemicals as drugs, xenobiotics also induce abnormal developmental abnormalities and congenital malformations in living organisms. Over the recent decades, zebrafish embryo/larva has emerged as a potential tool to test teratogenicity potential of these chemicals. Zebrafish responds to compounds as mammals do as they share similarities in their development, metabolism, physiology, and signaling pathways with that of mammals. The methodology used by the different scientists varies enormously in the zebrafish embryotoxicity test. In this chapter, we present methods to assess lethality and malformations during zebrafish development. We propose two major malformations scoring systems: binomial and relative morphological scoring systems to assess the malformations in zebrafish embryos/larvae. Based on the scoring of the malformations, the test compound can be classified as a teratogen or a nonteratogen and its teratogenic potential is evaluated.

  13. Conserved gene regulation during acute inflammation between zebrafish and mammals

    PubMed Central

    Forn-Cuní, G.; Varela, M.; Pereiro, P.; Novoa, B.; Figueras, A.

    2017-01-01

    Zebrafish (Danio rerio), largely used as a model for studying developmental processes, has also emerged as a valuable system for modelling human inflammatory diseases. However, in a context where even mice have been questioned as a valid model for these analysis, a systematic study evaluating the reproducibility of human and mammalian inflammatory diseases in zebrafish is still lacking. In this report, we characterize the transcriptomic regulation to lipopolysaccharide in adult zebrafish kidney, liver, and muscle tissues using microarrays and demonstrate how the zebrafish genomic responses can effectively reproduce the mammalian inflammatory process induced by acute endotoxin stress. We provide evidence that immune signaling pathways and single gene expression is well conserved throughout evolution and that the zebrafish and mammal acute genomic responses after lipopolysaccharide stimulation are highly correlated despite the differential susceptibility between species to that compound. Therefore, we formally confirm that zebrafish inflammatory models are suited to study the basic mechanisms of inflammation in human inflammatory diseases, with great translational impact potential. PMID:28157230

  14. Zebrafish as a model system to study toxicology.

    PubMed

    Dai, Yu-Jie; Jia, Yong-Fang; Chen, Na; Bian, Wan-Ping; Li, Qin-Kai; Ma, Yan-Bo; Chen, Yan-Ling; Pei, De-Sheng

    2014-01-01

    Monitoring and assessing the effects of contaminants in the aquatic eco-environment is critical in protecting human health and the environment. The zebrafish has been widely used as a prominent model organism in different fields because of its small size, low cost, diverse adaptability, short breeding cycle, high fecundity, and transparent embryos. Recent studies have demonstrated that zebrafish sensitivity can aid in monitoring environmental contaminants, especially with the application of transgenic technology in this area. The present review provides a brief overview of recent studies on wild-type and transgenic zebrafish as a model system to monitor toxic heavy metals, endocrine disruptors, and organic pollutants for toxicology. The authors address the new direction of developing high-throughput detection of genetically modified transparent zebrafish to open a new window for monitoring environmental pollutants. © 2013 SETAC.

  15. Silver nanoparticles induce endoplasmatic reticulum stress response in zebrafish

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Christen, Verena; Capelle, Martinus; Fent, Karl, E-mail: karl.fent@fhnw.ch

    2013-10-15

    Silver nanoparticles (AgNPs) find increasing applications, and therefore humans and the environment are increasingly exposed to them. However, potential toxicological implications are not sufficiently known. Here we investigate effects of AgNPs (average size 120 nm) on zebrafish in vitro and in vivo, and compare them to human hepatoma cells (Huh7). AgNPs are incorporated in zebrafish liver cells (ZFL) and Huh7, and in zebrafish embryos. In ZFL cells AgNPs lead to induction of reactive oxygen species (ROS), endoplasmatic reticulum (ER) stress response, and TNF-α. Transcriptional alterations also occur in pro-apoptotic genes p53 and Bax. The transcriptional profile differed in ZFL andmore » Huh7 cells. In ZFL cells, the ER stress marker BiP is induced, concomitant with the ER stress marker ATF-6 and spliced XBP-1 after 6 h and 24 h exposure to 0.5 g/L and 0.05 g/L AgNPs, respectively. This indicates the induction of different pathways of the ER stress response. Moreover, AgNPs induce TNF-α. In zebrafish embryos exposed to 0.01, 0.1, 1 and 5 mg/L AgNPs hatching was affected and morphological defects occurred at high concentrations. ER stress related gene transcripts BiP and Synv are significantly up-regulated after 24 h at 0.1 and 5 mg/L AgNPs. Furthermore, transcriptional alterations occurred in the pro-apoptotic genes Noxa and p21. The ER stress response was strong in ZFL cells and occurred in zebrafish embryos as well. Our data demonstrate for the first time that AgNPs lead to induction of ER stress in zebrafish. The induction of ER stress can have several consequences including the activation of apoptotic and inflammatory pathways. - Highlights: • Effects of silver nanoparticles (120 nm AgNPs) are investigated in zebrafish. • AgNPs induce all ER stress reponses in vitro in zebrafish liver cells. • AgNPs induce weak ER stress in zebrafish embryos. • AgNPs induce oxidative stress and transcripts of pro-apoptosis genes.« less

  16. New frontiers for zebrafish management.

    PubMed

    Lawrence, C

    2016-01-01

    The zebrafish (Danio rerio) is a preeminent model organism with a wide and expanding utility for numerous scientific disciplines. The same features that once endeared this small freshwater minnow to developmental biologists combined with its relatively high genetic similarity to mammals and the advent of new, more efficient methods for genome editing are now helping to spur expanded growth in its usage in various fields, including toxicology, drug discovery, transplant biology, disease modeling, and even aquaculture. Continued maturation and adoption of the zebrafish model system in these and other fields of science will require that methods and approaches for husbandry and management of these fish in controlled settings be refined and improved to the extent that, ultimately, zebrafish research becomes more reproducible, defined, cost-effective, and accessible to the masses. Knowledge and technology transfer from laboratory animal science and commercial aquaculture will be a necessary part of this development. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Mayo Clinic Zebrafish Facility Overview.

    PubMed

    Leveque, Ryan E; Clark, Karl J; Ekker, Stephen C

    2016-07-01

    The zebrafish (Danio rerio) is a premier nonmammalian vertebrate model organism. This small aquatic fish is utilized in multiple disciplines in the Mayo Clinic community and by many laboratories around the world because of its biological similarity to humans, its advanced molecular genetics, the elucidation of its genome sequence, and the ever-expanding and outstanding new biological tools now available to the zebrafish researcher. The Mayo Clinic Zebrafish Facility (MCZF) houses ∼2,000 tanks annotated using an in-house, Internet cloud-based bar-coding system tied to our established zfishbook.org web infrastructure. Paramecia are the primary food source for larval fish rearing, using a simplified culture protocol described herein. The MCZF supports the specific ongoing research in a variety of laboratories, while also serving as a local hub for new scientists as they learn to tap into the potential of this model system for understanding normal development, disease, and as models of health.

  18. Culturable Gut Microbiota Diversity in Zebrafish

    PubMed Central

    Sørby, Jan Roger Torp; Aleström, Peter; Sørum, Henning

    2012-01-01

    Abstract The zebrafish (Danio rerio) is an increasingly used laboratory animal model in basic biology and biomedicine, novel drug development, and toxicology. The wide use has increased the demand for optimized husbandry protocols to ensure animal health care and welfare. The knowledge about the correlation between culturable zebrafish intestinal microbiota and health in relation to environmental factors and management procedures is very limited. A semi-quantitative level of growth of individual types of bacteria was determined and associated with sampling points. A total of 72 TAB line zebrafish from four laboratories (Labs A–D) in the Zebrafish Network Norway were used. Diagnostic was based on traditional bacterial culture methods and biochemical characterization using commercial kits, followed by 16S rDNA gene sequencing from pure subcultures. Also selected Gram-negative isolates were analyzed for antibiotic susceptibility to 8 different antibiotics. A total of 13 morphologically different bacterial species were the most prevalent: Aeromonas hydrophila, Aeromonas sobria, Vibrio parahaemolyticus, Photobacterium damselae, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas luteola, Comamonas testosteroni, Ochrobactrum anthropi, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus warneri. Only Lab B had significantly higher levels of total bacterial growth (OR=2.03), whereas numbers from Lab C (OR=1.01) and Lab D (OR=1.12) were found to be similar to the baseline Lab A. Sexually immature individuals had a significantly higher level of harvested total bacterial growth than mature fish (OR=0.82), no statistically significant differences were found between male and female fish (OR=1.01), and the posterior intestinal segment demonstrated a higher degree of culturable bacteria than the anterior segment (OR=4.1). Multiple antibiotic (>3) resistance was observed in 17% of the strains. We propose that a rapid

  19. Culturable gut microbiota diversity in zebrafish.

    PubMed

    Cantas, Leon; Sørby, Jan Roger Torp; Aleström, Peter; Sørum, Henning

    2012-03-01

    The zebrafish (Danio rerio) is an increasingly used laboratory animal model in basic biology and biomedicine, novel drug development, and toxicology. The wide use has increased the demand for optimized husbandry protocols to ensure animal health care and welfare. The knowledge about the correlation between culturable zebrafish intestinal microbiota and health in relation to environmental factors and management procedures is very limited. A semi-quantitative level of growth of individual types of bacteria was determined and associated with sampling points. A total of 72 TAB line zebrafish from four laboratories (Labs A-D) in the Zebrafish Network Norway were used. Diagnostic was based on traditional bacterial culture methods and biochemical characterization using commercial kits, followed by 16S rDNA gene sequencing from pure subcultures. Also selected Gram-negative isolates were analyzed for antibiotic susceptibility to 8 different antibiotics. A total of 13 morphologically different bacterial species were the most prevalent: Aeromonas hydrophila, Aeromonas sobria, Vibrio parahaemolyticus, Photobacterium damselae, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas luteola, Comamonas testosteroni, Ochrobactrum anthropi, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus warneri. Only Lab B had significantly higher levels of total bacterial growth (OR=2.03), whereas numbers from Lab C (OR=1.01) and Lab D (OR=1.12) were found to be similar to the baseline Lab A. Sexually immature individuals had a significantly higher level of harvested total bacterial growth than mature fish (OR=0.82), no statistically significant differences were found between male and female fish (OR=1.01), and the posterior intestinal segment demonstrated a higher degree of culturable bacteria than the anterior segment (OR=4.1). Multiple antibiotic (>3) resistance was observed in 17% of the strains. We propose that a rapid conventional

  20. An assay for lateral line regeneration in adult zebrafish.

    PubMed

    Pisano, Gina C; Mason, Samantha M; Dhliwayo, Nyembezi; Intine, Robert V; Sarras, Michael P

    2014-04-08

    Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al. that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

  1. UPLC/MS MS data of testosterone metabolites in human and zebrafish liver microsomes and whole zebrafish larval microsomes.

    PubMed

    Saad, Moayad; Bijttebier, Sebastiaan; Matheeussen, An; Verbueken, Evy; Pype, Casper; Casteleyn, Christophe; Van Ginneken, Chris; Maes, Louis; Cos, Paul; Van Cruchten, Steven

    2018-02-01

    This article represents data regarding a study published in Toxicology in vitro entitled " in vitro CYP-mediated drug metabolism in the zebrafish (embryo) using human reference compounds" (Saad et al., 2017) [1]. Data were acquired with ultra-performance liquid chromatography - accurate mass mass spectrometry (UPLC-amMS). A full spectrum scan was conducted for the testosterone (TST) metabolites from the microsomal stability assay in zebrafish and humans. The microsomal proteins were extracted from adult zebrafish male (MLM) and female (FLM) livers, whole body homogenates of 96 h post fertilization larvae (EM) and a pool of human liver microsomes from 50 donors (HLM). Data are expressed as the abundance from the extracted ion chromatogram of the metabolites.

  2. Fishing anti(lymph)angiogenic drugs with zebrafish.

    PubMed

    García-Caballero, Melissa; Quesada, Ana R; Medina, Miguel A; Marí-Beffa, Manuel

    2018-02-01

    Zebrafish, an amenable small teleost fish with a complex mammal-like circulatory system, is being increasingly used for drug screening and toxicity studies. It combines the biological complexity of in vivo models with a higher-throughput screening capability compared with other available animal models. Externally growing, transparent embryos, displaying well-defined blood and lymphatic vessels, allow the inexpensive, rapid, and automatable evaluation of drug candidates that are able to inhibit neovascularisation. Here, we briefly review zebrafish as a model for the screening of anti(lymph)angiogenic drugs, with emphasis on the advantages and limitations of the different zebrafish-based in vivo assays. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Effectiveness of recommended euthanasia methods in larval zebrafish (Danio rerio).

    PubMed

    Strykowski, Jennifer L; Schech, Joseph M

    2015-01-01

    The popularity of zebrafish and its use as a model organism in biomedical research including genetics, development, and toxicology, has increased over the past 20 y and continues to grow. However, guidelines for euthanasia remain vague, and the responsibility of creating appropriate euthanasia protocols essentially falls on individual facilities. To reduce variation in experimental results among labs, a standard method of euthanasia for zebrafish would be useful. Although various euthanasia methods have been compared, few studies focus on the effectiveness of euthanasia methods for larval zebrafish. In this study, we exposed larval zebrafish to each of 3 euthanasia agents (MS222, eugenol, and hypothermic shock) and assessed the recovery rate. Hypothermic shock appeared to be the most effective method for euthanizing zebrafish at 14 d after fertilization; however, this method may not be considered an efficient method for large numbers of larval zebrafish. Exposure to chemicals, such as MS222 and eugenol, were ineffective methods for euthanasia at this stage of development. When these agents are used, secondary measures should be taken to ensure death. Choosing a euthanasia method that is effective, efficient, and humane can be challenging. Determining a method of euthanasia that is suitable for fish of all stages will bring the zebrafish community closer to meeting this challenge.

  4. Effectiveness of Recommended Euthanasia Methods in Larval Zebrafish (Danio rerio)

    PubMed Central

    Strykowski, Jennifer L; Schech, Joseph M

    2015-01-01

    The popularity of zebrafish and its use as a model organism in biomedical research including genetics, development, and toxicology, has increased over the past 20 y and continues to grow. However, guidelines for euthanasia remain vague, and the responsibility of creating appropriate euthanasia protocols essentially falls on individual facilities. To reduce variation in experimental results among labs, a standard method of euthanasia for zebrafish would be useful. Although various euthanasia methods have been compared, few studies focus on the effectiveness of euthanasia methods for larval zebrafish. In this study, we exposed larval zebrafish to each of 3 euthanasia agents (MS222, eugenol, and hypothermic shock) and assessed the recovery rate. Hypothermic shock appeared to be the most effective method for euthanizing zebrafish at 14 d after fertilization; however, this method may not be considered an efficient method for large numbers of larval zebrafish. Exposure to chemicals, such as MS222 and eugenol, were ineffective methods for euthanasia at this stage of development. When these agents are used, secondary measures should be taken to ensure death. Choosing a euthanasia method that is effective, efficient, and humane can be challenging. Determining a method of euthanasia that is suitable for fish of all stages will bring the zebrafish community closer to meeting this challenge. PMID:25651096

  5. The zebrafish world of colors and shapes: preference and discrimination.

    PubMed

    Oliveira, Jessica; Silveira, Mayara; Chacon, Diana; Luchiari, Ana

    2015-04-01

    Natural environment imposes many challenges to animals, which have to use cognitive abilities to cope with and exploit it to enhance their fitness. Since zebrafish is a well-established model for cognitive studies and high-throughput screening for drugs and diseases that affect cognition, we tested their ability for ambient color preference and 3D objects discrimination to establish a protocol for memory evaluation. For the color preference test, zebrafish were observed in a multiple-chamber tank with different environmental color options. Zebrafish showed preference for blue and green, and avoided yellow and red. For the 3D objects discrimination, zebrafish were allowed to explore two equal objects and then observed in a one-trial test in which a new color, size, or shape of the object was presented. Zebrafish showed discrimination for color, shape, and color+shape combined, but not size. These results imply that zebrafish seem to use some categorical system to discriminate items, and distracters affect their ability for discrimination. The type of variables available (color and shape) may favor zebrafish objects perception and facilitate discrimination processing. We suggest that this easy and simple memory test could serve as a useful screening tool for cognitive dysfunction and neurotoxicological studies.

  6. Zebrafish xenograft models of cancer and metastasis for drug discovery.

    PubMed

    Brown, Hannah K; Schiavone, Kristina; Tazzyman, Simon; Heymann, Dominique; Chico, Timothy Ja

    2017-04-01

    Patients with metastatic cancer suffer the highest rate of cancer-related death, but existing animal models of metastasis have disadvantages that limit our ability to understand this process. The zebrafish is increasingly used for cancer modelling, particularly xenografting of human cancer cell lines, and drug discovery, and may provide novel scientific and therapeutic insights. However, this model system remains underexploited. Areas covered: The authors discuss the advantages and disadvantages of the zebrafish xenograft model for the study of cancer, metastasis and drug discovery. They summarise previous work investigating the metastatic cascade, such as tumour-induced angiogenesis, intravasation, extravasation, dissemination and homing, invasion at secondary sites, assessing metastatic potential and evaluation of cancer stem cells in zebrafish. Expert opinion: The practical advantages of zebrafish for basic biological study and drug discovery are indisputable. However, their ability to sufficiently reproduce and predict the behaviour of human cancer and metastasis remains unproven. For this to be resolved, novel mechanisms must to be discovered in zebrafish that are subsequently validated in humans, and for therapeutic interventions that modulate cancer favourably in zebrafish to successfully translate to human clinical studies. In the meantime, more work is required to establish the most informative methods in zebrafish.

  7. Electroretinogram analysis of the visual response in zebrafish larvae.

    PubMed

    Chrispell, Jared D; Rebrik, Tatiana I; Weiss, Ellen R

    2015-03-16

    The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

  8. Study on acute toxicity of amoxicillin wastewater to Zebrafish

    NASA Astrophysics Data System (ADS)

    Xie, Weifang; Shen, Hongyan

    2017-12-01

    The main research in this paper is to obtain the effect of pharmaceutical wastewater on the acute toxicity of Zebrafish. The experimental method of exposure is used in this research. Experiments were carried out with different groups of pharmaceutical wastewater. Zebrafish was cultivated in a five liter fish tank. In the experiment, according to mortality, initially a 96h preliminary test was carried out at exposure concentrations to determine if the amoxicillin wastewater was toxic and to define the concentration range (24h LC100, 96h LC0) to be employed in the definitive tests. Based on the half lethal concentration of Zebrafish, the acute toxicity of amoxicillin wastewater to Zebrafish was calculated and the toxicity grade of wastewater was determined. In the experiment, the Zebrafish was exposed with amoxicillin wastewater during 96h. The 24h, 48h, 72h and 96h LC50 of amoxicillin wastewater on the Zebrafish were 63.10%, 53.70%, 41.69% and 40.74%, respectively. At 96h, the test time is the longest, and the value of LC50 is the smallest. In the observation period of 96 hours, the LC50 of amoxicillin wastewater were in the range of 40% ~ 60% and the value of Tua is 1 ~ 2. It indicates amoxicillin wastewater is low toxic wastewater when the experimental time is shorter than 48h, amoxicillin wastewater is moderate toxicity wastewater when the experimental time is higher than 48h. According to the experimental data, with the exposure time and the volume percentage of amoxicillin wastewater increases, the mortality rate of Zebrafish is gradually increased and the toxicity of amoxicillin wastewater increases. It indicates that the toxicity of amoxicillin wastewater is the biggest and the effect of wastewater on Zebrafish is greatest. In some ways, the toxicity of amoxicillin wastewater can be affected by the test time.

  9. Modeling Leukemogenesis in the Zebrafish Using Genetic and Xenograft Models.

    PubMed

    Rajan, Vinothkumar; Dellaire, Graham; Berman, Jason N

    2016-01-01

    The zebrafish is a widely accepted model to study leukemia. The major advantage of studying leukemogenesis in zebrafish is attributed to its short life cycle and superior imaging capacity. This chapter highlights using transgenic- and xenograft-based models in zebrafish to study a specific leukemogenic mutation and analyze therapeutic responses in vivo.

  10. Influenza A Virus Infection Damages Zebrafish Skeletal Muscle and Exacerbates Disease in Zebrafish Modeling Duchenne Muscular Dystrophy

    PubMed Central

    Goody, Michelle; Jurczyszak, Denise; Kim, Carol; Henry, Clarissa

    2017-01-01

    INTRODUCTION: Both genetic and infectious diseases can result in skeletal muscle degeneration, inflammation, pain, and/or weakness. Duchenne muscular dystrophy (DMD) is the most common congenital muscle disease. DMD causes progressive muscle wasting due to mutations in Dystrophin. Influenza A and B viruses are frequently associated with muscle complications, especially in children. Infections activate an immune response and immunosuppressant drugs reduce DMD symptoms. These data suggest that the immune system may contribute to muscle pathology. However, roles of the immune response in DMD and Influenza muscle complications are not well understood. Zebrafish with dmd mutations are a well-characterized model in which to study the molecular and cellular mechanisms of DMD pathology. We recently showed that zebrafish can be infected by human Influenza A virus (IAV). Thus, the zebrafish is a powerful system with which to ask questions about the etiology and mechanisms of muscle damage due to genetic and/or infectious diseases. METHODS: We infected zebrafish with IAV and assayed muscle tissue structure, sarcolemma integrity, cell-extracellular matrix (ECM) attachment, and molecular and cellular markers of inflammation in response to IAV infection alone or in the context of DMD. RESULTS: We find that IAV-infected zebrafish display mild muscle degeneration with sarcolemma damage and compromised ECM adhesion. An innate immune response is elicited in muscle in IAV-infected zebrafish: NFkB signaling is activated, pro-inflammatory cytokine expression is upregulated, and neutrophils localize to sites of muscle damage. IAV-infected dmd mutants display more severe muscle damage than would be expected from an additive effect of dmd mutation and IAV infection, suggesting that muscle damage caused by Dystrophin-deficiency and IAV infection is synergistic. DISCUSSION: These data demonstrate the importance of preventing IAV infections in individuals with genetic muscle diseases

  11. Influenza A Virus Infection Damages Zebrafish Skeletal Muscle and Exacerbates Disease in Zebrafish Modeling Duchenne Muscular Dystrophy.

    PubMed

    Goody, Michelle; Jurczyszak, Denise; Kim, Carol; Henry, Clarissa

    2017-10-25

    Both genetic and infectious diseases can result in skeletal muscle degeneration, inflammation, pain, and/or weakness. Duchenne muscular dystrophy (DMD) is the most common congenital muscle disease. DMD causes progressive muscle wasting due to mutations in Dystrophin. Influenza A and B viruses are frequently associated with muscle complications, especially in children. Infections activate an immune response and immunosuppressant drugs reduce DMD symptoms. These data suggest that the immune system may contribute to muscle pathology. However, roles of the immune response in DMD and Influenza muscle complications are not well understood. Zebrafish with dmd mutations are a well-characterized model in which to study the molecular and cellular mechanisms of DMD pathology. We recently showed that zebrafish can be infected by human Influenza A virus (IAV). Thus, the zebrafish is a powerful system with which to ask questions about the etiology and mechanisms of muscle damage due to genetic and/or infectious diseases. We infected zebrafish with IAV and assayed muscle tissue structure, sarcolemma integrity, cell-extracellular matrix (ECM) attachment, and molecular and cellular markers of inflammation in response to IAV infection alone or in the context of DMD. We find that IAV-infected zebrafish display mild muscle degeneration with sarcolemma damage and compromised ECM adhesion. An innate immune response is elicited in muscle in IAV-infected zebrafish: NFkB signaling is activated, pro-inflammatory cytokine expression is upregulated, and neutrophils localize to sites of muscle damage. IAV-infected dmd mutants display more severe muscle damage than would be expected from an additive effect of dmd mutation and IAV infection, suggesting that muscle damage caused by Dystrophin-deficiency and IAV infection is synergistic. These data demonstrate the importance of preventing IAV infections in individuals with genetic muscle diseases. Elucidating the mechanisms of immune

  12. Zebrafish Craniofacial Development: A Window into Early Patterning

    PubMed Central

    Mork, Lindsey; Crump, Gage

    2016-01-01

    The formation of the face and skull involves a complex series of developmental events mediated by cells derived from the neural crest, endoderm, mesoderm, and ectoderm. Although vertebrates boast an enormous diversity of adult facial morphologies, the fundamental signaling pathways and cellular events that sculpt the nascent craniofacial skeleton in the embryo have proven to be highly conserved from fish to man. The zebrafish Danio rerio, a small freshwater cyprinid fish from eastern India, has served as a popular model of craniofacial development since the 1990s. Unique strengths of the zebrafish model include a simplified skeleton during larval stages, access to rapidly developing embryos for live imaging, and amenability to transgenesis and complex genetics. In this chapter, we describe the anatomy of the zebrafish craniofacial skeleton; its applications as models for the mammalian jaw, middle ear, palate, and cranial sutures; the superior imaging technology available in fish that has provided unprecedented insights into the dynamics of facial morphogenesis; the use of the zebrafish to decipher the genetic underpinnings of craniofacial biology; and finally a glimpse into the most promising future applications of zebrafish craniofacial research. PMID:26589928

  13. Zebrafish Axenic Larvae Colonization with Human Intestinal Microbiota.

    PubMed

    Arias-Jayo, Nerea; Alonso-Saez, Laura; Ramirez-Garcia, Andoni; Pardo, Miguel A

    2018-04-01

    The human intestine hosts a vast and complex microbial community that is vital for maintaining several functions related with host health. The processes that determine the gut microbiome composition are poorly understood, being the interaction between species, the external environment, and the relationship with the host the most feasible. Animal models offer the opportunity to understand the interactions between the host and the microbiota. There are different gnotobiotic mice or rat models colonized with the human microbiota, however, to our knowledge, there are no reports on the colonization of germ-free zebrafish with a complex human intestinal microbiota. In the present study, we have successfully colonized 5 days postfertilization germ-free zebrafish larvae with the human intestinal microbiota previously extracted from a donor and analyzed by high-throughput sequencing the composition of the transferred microbial communities that established inside the zebrafish gut. Thus, we describe for first time which human bacteria phylotypes are able to colonize the zebrafish digestive tract. Species with relevant interest because of their linkage to dysbiosis in different human diseases, such as Akkermansia muciniphila, Eubacterium rectale, Faecalibacterium prausnitzii, Prevotella spp., or Roseburia spp. have been successfully transferred inside the zebrafish digestive tract.

  14. Method for somatic cell nuclear transfer in zebrafish.

    PubMed

    Siripattarapravat, Kannika; Cibelli, Jose B

    2011-01-01

    Somatic cell nuclear transfer (SCNT) has been a well-known technique for decades and widely applied to generate identical animals, including ones with genetic alterations. The system has been demonstrated successfully in zebrafish. The elaborated requirements of SCNT, however, limit reproducibility of the established model to a few groups in zebrafish research community. In this chapter, we meticulously outline each step of the published protocol as well as preparations of equipments and reagents used in zebrafish SCNT. All describable detailed-tips are elaborated in texts and figures. Copyright © 2011 Elsevier Inc. All rights reserved.

  15. Anxiogenic-like effects of chronic nicotine exposure in zebrafish.

    PubMed

    Stewart, Adam Michael; Grossman, Leah; Collier, Adam D; Echevarria, David J; Kalueff, Allan V

    2015-12-01

    Nicotine is one of the most widely used and abused legal drugs. Although its pharmacological profile has been extensively investigated in humans and rodents, nicotine CNS action remains poorly understood. The importance of finding evolutionarily conserved signaling pathways, and the need to apply high-throughput in vivo screens for CNS drug discovery, necessitate novel efficient experimental models for nicotine research. Zebrafish (Danio rerio) are rapidly emerging as an excellent organism for studying drug abuse, neuropharmacology and toxicology and have recently been applied to testing nicotine. Anxiolytic, rewarding and memory-modulating effects of acute nicotine treatment in zebrafish are consistently reported in the literature. However, while nicotine abuse is more relevant to long-term exposure models, little is known about chronic effects of nicotine on zebrafish behavior. In the present study, chronic 4-day exposure to 1-2mg/L nicotine mildly increased adult zebrafish shoaling but did not alter baseline cortisol levels. We also found that chronic exposure to nicotine evokes robust anxiogenic behavioral responses in zebrafish tested in the novel tank test paradigm. Generally paralleling clinical and rodent data on anxiogenic effects of chronic nicotine, our study supports the developing utility of zebrafish for nicotine research. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Zebrafish Models of Human Leukemia: Technological Advances and Mechanistic Insights.

    PubMed

    Harrison, Nicholas R; Laroche, Fabrice J F; Gutierrez, Alejandro; Feng, Hui

    2016-01-01

    Insights concerning leukemic pathophysiology have been acquired in various animal models and further efforts to understand the mechanisms underlying leukemic treatment resistance and disease relapse promise to improve therapeutic strategies. The zebrafish (Danio rerio) is a vertebrate organism with a conserved hematopoietic program and unique experimental strengths suiting it for the investigation of human leukemia. Recent technological advances in zebrafish research including efficient transgenesis, precise genome editing, and straightforward transplantation techniques have led to the generation of a number of leukemia models. The transparency of the zebrafish when coupled with improved lineage-tracing and imaging techniques has revealed exquisite details of leukemic initiation, progression, and regression. With these advantages, the zebrafish represents a unique experimental system for leukemic research and additionally, advances in zebrafish-based high-throughput drug screening promise to hasten the discovery of novel leukemia therapeutics. To date, investigators have accumulated knowledge of the genetic underpinnings critical to leukemic transformation and treatment resistance and without doubt, zebrafish are rapidly expanding our understanding of disease mechanisms and helping to shape therapeutic strategies for improved outcomes in leukemic patients.

  17. Zebrafish Models of Human Leukemia: Technological Advances and Mechanistic Insights

    PubMed Central

    Harrison, Nicholas R.; Laroche, Fabrice J.F.; Gutierrez, Alejandro

    2016-01-01

    Insights concerning leukemic pathophysiology have been acquired in various animal models and further efforts to understand the mechanisms underlying leukemic treatment resistance and disease relapse promise to improve therapeutic strategies. The zebrafish (Danio rerio) is a vertebrate organism with a conserved hematopoietic program and unique experimental strengths suiting it for the investigation of human leukemia. Recent technological advances in zebrafish research including efficient transgenesis, precise genome editing, and straightforward transplantation techniques have led to the generation of a number of leukemia models. The transparency of the zebrafish when coupled with improved lineage-tracing and imaging techniques has revealed exquisite details of leukemic initiation, progression, and regression. With these advantages, the zebrafish represents a unique experimental system for leukemic research and additionally, advances in zebrafish-based high-throughput drug screening promise to hasten the discovery of novel leukemia therapeutics. To date, investigators have accumulated knowledge of the genetic underpinnings critical to leukemic transformation and treatment resistance and without doubt, zebrafish are rapidly expanding our understanding of disease mechanisms and helping to shape therapeutic strategies for improved outcomes in leukemic patients. PMID:27165361

  18. Production of zebrafish cardiospheres and cardiac progenitor cells in vitro and three-dimensional culture of adult zebrafish cardiac tissue in scaffolds.

    PubMed

    Zeng, Wendy R; Beh, Siew-Joo; Bryson-Richardson, Robert J; Doran, Pauline M

    2017-09-01

    The hearts of adult zebrafish (Danio rerio) are capable of complete regeneration in vivo even after major injury, making this species of particular interest for understanding the growth and differentiation processes required for cardiac tissue engineering. To date, little research has been carried out on in vitro culture of adult zebrafish cardiac cells. In this work, progenitor-rich cardiospheres suitable for cardiomyocyte differentiation and myocardial regeneration were produced from adult zebrafish hearts. The cardiospheres contained a mixed population of c-kit + and Mef2c + cells; proliferative peripheral cells of possible mesenchymal lineage were also observed. Cellular outgrowth from cardiac explants and cardiospheres was enhanced significantly using conditioned medium harvested from cultures of a rainbow trout cell line, suggesting that fish-specific trophic factors are required for zebrafish cardiac cell expansion. Three-dimensional culture of zebrafish heart cells in fibrous polyglycolic acid (PGA) scaffolds was carried out under dynamic fluid flow conditions. High levels of cell viability and cardiomyocyte differentiation were maintained within the scaffolds. Expression of cardiac troponin T, a marker of differentiated cardiomyocytes, increased during the first 7 days of scaffold culture; after 15 days, premature disintegration of the biodegradable scaffolds led to cell detachment and a decline in differentiation status. This work expands our technical capabilities for three-dimensional zebrafish cardiac cell culture with potential applications in tissue engineering, drug and toxicology screening, and ontogeny research. Biotechnol. Bioeng. 2017;114: 2142-2148. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  19. [10]-Gingerdiols as the major metabolites of [10]-gingerol in zebrafish embryos and in humans and their hematopoietic effects in zebrafish embryos

    PubMed Central

    Chen, Huadong; Soroka, Dominique N.; Haider, Jamil; Ferri-Lagneau, Karine F.; Leung, TinChung; Sang, Shengmin

    2013-01-01

    Gingerols are a series of major constituents in fresh ginger with the most abundant being [6]-, [8]-, and [10]-gingerols (6G, 8G, and 10G). We previously found that ginger extract and its purified components, especially 10G, potentially stimulate both the primitive and definitive waves of hematopoiesis (blood cell formation) in zebrafish embryos. However, it is still unclear if the metabolites of 10G retain the efficacy of the parent compound towards pathological anemia treatment. In the present study, we first investigated the metabolism of 10G in zebrafish embryos, and then explored the biotransformation of 10G in humans. Our results show that 10G was extensively metabolized in both zebrafish embryos and in humans, in which two major metabolites, (3S,5S)-[10]-gingerdiol and (3R,5S)-[10]-gingerdiol, were identified by analysis of the MSn spectra and comparison to authentic standards that we synthesized. After 24 hours of treatment of zebrafish embryos, 10G was mostly converted to its metabolites. Our results clearly indicate the reductive pathway is a major metabolic route for 10G in both zebrafish embryos and in humans. Furthermore, we investigated the hematopoietic effect of 10G and its two metabolites, which show similar hematopoietic effects as 10G in zebrafish embryos. PMID:23701129

  20. Zebrafish for the Study of the Biological Effects of Nicotine

    PubMed Central

    Klee, Eric W.; Schneider, Henning; Hurt, Richard D.; Ekker, Stephen C.

    2011-01-01

    Introduction: Zebrafish are emerging as a powerful animal model for studying the molecular and physiological effects of nicotine exposure. The zebrafish have many advantageous physical characteristics, including small size, high fecundity rates, and externally developing transparent embryos. When combined with a battery of molecular–genetic tools and behavioral assays, these attributes enable studies to be conducted that are not practical using traditional animal models. Methods: We reviewed the literature on the application of the zebrafish model as a preclinical model to study the biological effects of nicotine exposure. Results: The identified studies used zebrafish to examine the effects of nicotine exposure on early development, addiction, anxiety, and learning. The methods used included green fluorescent protein–labeled proteins to track in vivo nicotine-altered neuron development, nicotine-conditioned place preference, and locomotive sensitization linked with high-throughput molecular and genetic screens and behavioral models of learning and stress response to nicotine. Data are presented on the complete homology of all known human neural nicotinic acetylcholine receptors in zebrafish and on the biological similarity of human and zebrafish dopaminergic signaling. Conclusions: Tobacco dependence remains a major health problem worldwide. Further understanding of the molecular effects of nicotine exposure and genetic contributions to dependence may lead to improvement in patient treatment strategies. While there are limitations to the use of zebrafish as a preclinical model, it should provide a valuable tool to complement existing model systems. The reviewed studies demonstrate the enormous opportunity zebrafish have to advance the science of nicotine and tobacco research. PMID:21385906

  1. In vivo physiological recording from the lateral line of juvenile zebrafish

    PubMed Central

    Olt, Jennifer; Allen, Claire E.

    2016-01-01

    Key points Zebrafish provide a unique opportunity to investigate in vivo sensory transduction in mature hair cells.We have developed a method for studying the biophysical properties of mature hair cells from the lateral line of juvenile zebrafish.The method involves application of the anaesthetic benzocaine and intubation to maintain ventilation and oxygenation through the gills.The same approach could be used for in vivo functional studies in other sensory and non‐sensory systems from juvenile and adult zebrafish. Abstract Hair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post‐fertilization (dpf) at 28.5°C). In larval zebrafish, the functional properties of most of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l−1 was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l−1 did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS‐222, which reduces the size of basolateral membrane K+ currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live

  2. A review of monoaminergic neuropsychopharmacology in zebrafish.

    PubMed

    Maximino, Caio; Herculano, Anderson Manoel

    2010-12-01

    Monoamine neurotransmitters are the major regulatory mechanisms in the vertebrate brain, involved in the adjustment of motivation, emotion, and cognition. The chemical anatomy of these systems is thought to be highly conserved in the brain of all vertebrates, including zebrafish. Recently, the development of behavioral assays in zebrafish allowed the neuropsychopharmacological investigation of these circuits and its functions. Here we review neuroanatomical, genetic, neurochemical, and psychopharmacological evidence regarding the roles of histaminergic, dopaminergic, noradrenergic, serotonergic, and melatonergic systems in this species. We conclude that, in spite of species differences, zebrafish are suitable for the investigation of neuropsychopharmacology of drugs that affect theses systems; nonetheless, more thorough validation of behavioral methods is still needed.

  3. Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish.

    PubMed

    Egan, Rupert J; Bergner, Carisa L; Hart, Peter C; Cachat, Jonathan M; Canavello, Peter R; Elegante, Marco F; Elkhayat, Salem I; Bartels, Brett K; Tien, Anna K; Tien, David H; Mohnot, Sopan; Beeson, Esther; Glasgow, Eric; Amri, Hakima; Zukowska, Zofia; Kalueff, Allan V

    2009-12-14

    The zebrafish (Danio rerio) is emerging as a promising model organism for experimental studies of stress and anxiety. Here we further validate zebrafish models of stress by analyzing how environmental and pharmacological manipulations affect their behavioral and physiological phenotypes. Experimental manipulations included exposure to alarm pheromone, chronic exposure to fluoxetine, acute exposure to caffeine, as well as acute and chronic exposure to ethanol. Acute (but not chronic) alarm pheromone and acute caffeine produced robust anxiogenic effects, including reduced exploration, increased erratic movements and freezing behavior in zebrafish tested in the novel tank diving test. In contrast, ethanol and fluoxetine had robust anxiolytic effects, including increased exploration and reduced erratic movements. The behavior of several zebrafish strains was also quantified to ascertain differences in their behavioral profiles, revealing high-anxiety (leopard, albino) and low-anxiety (wild type) strains. We also used LocoScan (CleverSys Inc.) video-tracking tool to quantify anxiety-related behaviors in zebrafish, and dissect anxiety-related phenotypes from locomotor activity. Finally, we developed a simple and effective method of measuring zebrafish physiological stress responses (based on a human salivary cortisol assay), and showed that alterations in whole-body cortisol levels in zebrafish parallel behavioral indices of anxiety. Collectively, our results confirm zebrafish as a valid, reliable, and high-throughput model of stress and affective disorders.

  4. Using zebrafish in systems toxicology for developmental toxicity testing.

    PubMed

    Nishimura, Yuhei; Inoue, Atsuto; Sasagawa, Shota; Koiwa, Junko; Kawaguchi, Koki; Kawase, Reiko; Maruyama, Toru; Kim, Soonih; Tanaka, Toshio

    2016-01-01

    With the high cost and the long-term assessment of developmental toxicity testing in mammals, the vertebrate zebrafish has become a useful alternative model organism for high-throughput developmental toxicity testing. Zebrafish is also very favorable for the 3R perspective in toxicology; however, the methodologies used by research groups vary greatly, posing considerable challenges to integrative analysis. In this review, we discuss zebrafish developmental toxicity testing, focusing on the methods of chemical exposure, the assessment of morphological abnormalities, housing conditions and their effects on the production of healthy embryos, and future directions. Zebrafish as a systems toxicology model has the potential to elucidate developmental toxicity pathways, and to provide a sound basis for human health risk assessments. © 2015 Japanese Teratology Society.

  5. Social learning of an associative foraging task in zebrafish

    NASA Astrophysics Data System (ADS)

    Zala, Sarah M.; Määttänen, Ilmari

    2013-05-01

    The zebrafish ( Danio rerio) is increasingly becoming an important model species for studies on the genetic and neural mechanisms controlling behaviour and cognition. Here, we utilized a conditioned place preference (CPP) paradigm to study social learning in zebrafish. We tested whether social interactions with conditioned demonstrators enhance the ability of focal naïve individuals to learn an associative foraging task. We found that the presence of conditioned demonstrators improved focal fish foraging behaviour through the process of social transmission, whereas the presence of inexperienced demonstrators interfered with the learning of the control focal fish. Our results indicate that zebrafish use social learning for finding food and that this CPP paradigm is an efficient assay to study social learning and memory in zebrafish.

  6. Tracking zebrafish larvae in group – Status and perspectives☆

    PubMed Central

    Martineau, Pierre R.; Mourrain, Philippe

    2013-01-01

    Video processing is increasingly becoming a standard procedure in zebrafish behavior investigations as it enables higher research throughput and new or better measures. This trend, fostered by the ever increasing performance-to-price ratio of the required recording and processing equipment, should be expected to continue in the foreseeable future, with video-processing based methods permeating more and more experiments and, as a result, expanding the very role of behavioral studies in zebrafish research. To assess whether the routine video tracking of zebrafish larvae directly in the Petri dish is a capability that can be expected in the near future, the key processing concepts are discussed and illustrated on published zebrafish studies when available or other animals when not. PMID:23707495

  7. Kidney organogenesis in the zebrafish: insights into vertebrate nephrogenesis and regeneration

    PubMed Central

    Gerlach, Gary F.; Wingert, Rebecca A.

    2012-01-01

    Vertebrates form a progressive series of up to three kidney organs during development—the pronephros, mesonephros, and metanephros. Each kidney derives from the intermediate mesoderm and is comprised of conserved excretory units called nephrons. The zebrafish is a powerful model for vertebrate developmental genetics, and recent studies have illustrated that zebrafish and mammals share numerous similarities in nephron composition and physiology. The zebrafish embryo forms an architecturally simple pronephros that has two nephrons, and these eventually become a scaffold onto which a mesonephros of several hundred nephrons is constructed during larval stages. In adult zebrafish, the mesonephros exhibits ongoing nephrogenesis, generating new nephrons from a local pool of renal progenitors during periods of growth or following kidney injury. The characteristics of the zebrafish pronephros and mesonephros make them genetically tractable kidney systems in which to study the functions of renal genes and address outstanding questions about the mechanisms of nephrogenesis. Here, we provide an overview of the formation and composition of these zebrafish kidney organs, and discuss how various zebrafish mutants, gene knockdowns, and transgenic models have created frameworks in which to further delineate nephrogenesis pathways. PMID:24014448

  8. Cardiac Ca2+ signalling in zebrafish: Translation of findings to man.

    PubMed

    van Opbergen, Chantal J M; van der Voorn, Stephanie M; Vos, Marc A; de Boer, Teun P; van Veen, Toon A B

    2018-05-07

    Sudden cardiac death is a leading cause of death worldwide, mainly caused by highly disturbed electrical activation patterns in the heart. Currently, murine models are the most popular model to study underlying molecular mechanisms of inherited or acquired cardiac electrical abnormalities, although the numerous electrophysiological discrepancies between mouse and human raise the question whether mice are the optimal model to study cardiac rhythm disorders. Recently it has been uncovered that the zebrafish cardiac electrophysiology seems surprisingly similar to the human heart, mainly because the zebrafish AP contains a clear plateau phase and ECG characteristics show alignment with the human ECG. Although, before using zebrafish as a model to study cardiac arrhythmogenesis, however, it is very important to gain a better insight into the electrophysiological characteristics of the zebrafish heart. In this review we outline the electrophysiological machinery of the zebrafish cardiomyocytes, with a special focus on the intracellular Ca 2+ dynamics and excitation-contraction coupling. We debate the potential of zebrafish as a model to study human cardiovascular diseases and postulate steps to employ zebrafish into a more 'humanized' model. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. The ontogeny of sleep-wake cycles in zebrafish: a comparison to humans

    PubMed Central

    Sorribes, Amanda; Þorsteinsson, Haraldur; Arnardóttir, Hrönn; Jóhannesdóttir, Ingibjörg Þ.; Sigurgeirsson, Benjamín; de Polavieja, Gonzalo G.; Karlsson, Karl Æ.

    2013-01-01

    Zebrafish (Danio rerio) are used extensively in sleep research; both to further understanding of sleep in general and also as a model of human sleep. To date, sleep studies have been performed in larval and adult zebrafish but no efforts have been made to document the ontogeny of zebrafish sleep–wake cycles. Because sleep differs across phylogeny and ontogeny it is important to validate the use of zebrafish in elucidating the neural substrates of sleep. Here we describe the development of sleep and wake across the zebrafish lifespan and how it compares to humans. We find power-law distributions to best fit wake bout data but demonstrate that exponential distributions, previously used to describe sleep bout distributions, fail to adequately account for the data in either species. Regardless, the data reveal remarkable similarities in the ontogeny of sleep cycles in zebrafish and humans. Moreover, as seen in other organisms, zebrafish sleep levels are highest early in ontogeny and sleep and wake bouts gradually consolidate to form the adult sleep pattern. Finally, sleep percentage, bout duration, bout number, and sleep fragmentation are shown to allow for meaningful comparisons between zebrafish and human sleep. PMID:24312015

  10. Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

    PubMed

    Kuriya, Kenji; Higashiyama, Eriko; Avşar-Ban, Eriko; Tamaru, Yutaka; Ogata, Shin; Takebayashi, Shin-ichiro; Ogata, Masato; Okumura, Katsuzumi

    2015-12-01

    Spatiotemporal regulation of DNA replication in the S-phase nucleus has been extensively studied in mammalian cells because it is tightly coupled with the regulation of other nuclear processes such as transcription. However, little is known about the replication dynamics in nonmammalian cells. Here, we analyzed the DNA replication processes of zebrafish (Danio rerio) cells through the direct visualization of replicating DNA in the nucleus and on DNA fiber molecules isolated from the nucleus. We found that zebrafish chromosomal DNA at the nuclear interior was replicated first, followed by replication of DNA at the nuclear periphery, which is reminiscent of the spatiotemporal regulation of mammalian DNA replication. However, the relative duration of interior DNA replication in zebrafish cells was longer compared to mammalian cells, possibly reflecting zebrafish-specific genomic organization. The rate of replication fork progression and ori-to-ori distance measured by the DNA combing technique were ∼ 1.4 kb/min and 100 kb, respectively, which are comparable to those in mammalian cells. To our knowledge, this is a first report that measures replication dynamics in zebrafish cells.

  11. Myomaker mediates fusion of fast myocytes in zebrafish embryos

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Landemaine, Aurélie; Rescan, Pierre-Yves; Gabillard, Jean-Charles, E-mail: Jean-charles.gabillard@rennes.inra.fr

    2014-09-05

    Highlights: • Myomaker is transiently expressed in fast myocytes during embryonic myogenesis. • Myomaker is essential for fast myocyte fusion in zebrafish. • The function of myomaker is conserved among Teleostomi. - Abstract: Myomaker (also called Tmem8c), a new membrane activator of myocyte fusion was recently discovered in mice. Using whole mount in situ hybridization on zebrafish embryos at different stages of embryonic development, we show that myomaker is transiently expressed in fast myocytes forming the bulk of zebrafish myotome. Zebrafish embryos injected with morpholino targeted against myomaker were alive after yolk resorption and appeared morphologically normal, but they weremore » unable to swim, even under effect of a tactile stimulation. Confocal observations showed a marked phenotype characterized by the persistence of mononucleated muscle cells in the fast myotome at developmental stages where these cells normally fuse to form multinucleated myotubes. This indicates that myomaker is essential for myocyte fusion in zebrafish. Thus, there is an evolutionary conservation of myomaker expression and function among Teleostomi.« less

  12. Development of the zebrafish mesonephros.

    PubMed

    Diep, Cuong Q; Peng, Zhenzhen; Ukah, Tobechukwu K; Kelly, Paul M; Daigle, Renee V; Davidson, Alan J

    2015-01-01

    The vertebrate kidney plays an essential role in removing metabolic waste and balancing water and salt. This is carried out by nephrons, which comprise a blood filter attached to an epithelial tubule with proximal and distal segments. In zebrafish, two nephrons are first formed as part of the embryonic kidney (pronephros) and hundreds are formed later to make up the adult kidney (mesonephros). Previous studies have focused on the development of the pronephros while considerably less is known about how the mesonephros is formed. Here, we characterize mesonephros development in zebrafish and examine the nephrons that form during larval metamorphosis. These nephrons, arising from proliferating progenitor cells that express the renal transcription factor genes wt1b, pax2a, and lhx1a, form on top of the pronephric tubules and develop a segmentation pattern similar to pronephric nephrons. We find that the pronephros acts as a scaffold for the mesonephros, where new nephrons fuse with the distal segments of the pronephric tubules to form the final branching network that characterizes the adult zebrafish kidney. © 2015 Wiley Periodicals, Inc.

  13. Development of the zebrafish mesonephros

    PubMed Central

    Diep, Cuong Q.; Peng, Zhenzhen; Ukah, Tobechukwu K.; Kelly, Paul M.; Daigle, Renee V.; Davidson, Alan J.

    2015-01-01

    The vertebrate kidney plays an essential role in removing metabolic waste and balancing water and salt. This is carried out by nephrons, which comprise a blood filter attached to an epithelial tubule with proximal and distal segments. In zebrafish, two nephrons are first formed as part of the embryonic kidney (pronephros) and hundreds are formed later to make up the adult kidney (mesonephros). Previous studies have focused on the development of the pronephros while considerably less is known about how the mesonephros is formed. Here, we characterize mesonephros development in zebrafish and examine the nephrons that form during larval metamorphosis. These nephrons, arising from proliferating progenitor cells that express the renal transcription factor genes wt1b, pax2a, and lhx1a, form on top of the pronephric tubules and develop a segmentation pattern similar to pronephric nephrons. We find that the pronephros acts as a scaffold for the mesonephros, where new nephrons fuse with the distal segments of the pronephric tubules to form the final branching network that characterizes the adult zebrafish kidney. PMID:25677367

  14. Differential expression of neuroligin genes in the nervous system of zebrafish.

    PubMed

    Davey, Crystal; Tallafuss, Alexandra; Washbourne, Philip

    2010-02-01

    The establishment and maturation of appropriate synaptic connections is crucial in the development of neuronal circuits. Cellular adhesion is believed to play a central role in this process. Neuroligins are neuronal cell adhesion molecules that are hypothesized to act in the initial formation and maturation of synaptic connections. In order to establish the zebrafish as a model to investigate the in vivo role of Neuroligin proteins in nervous system development, we identified the zebrafish orthologs of neuroligin family members and characterized their expression. Zebrafish possess seven neuroligin genes. Synteny analysis and sequence comparisons show that NLGN2, NLGN3, and NLGN4X are duplicated in zebrafish, but NLGN1 has a single zebrafish ortholog. All seven zebrafish neuroligins are expressed in complex patterns in the developing nervous system and in the adult brain. The spatial and temporal expression patterns of these genes suggest that they occupy a role in nervous system development and maintenance.

  15. Zebrafish: an important tool for liver disease research.

    PubMed

    Goessling, Wolfram; Sadler, Kirsten C

    2015-11-01

    As the incidence of hepatobiliary diseases increases, we must improve our understanding of the molecular, cellular, and physiological factors that contribute to the pathogenesis of liver disease. Animal models help us identify disease mechanisms that might be targeted therapeutically. Zebrafish (Danio rerio) have traditionally been used to study embryonic development but are also important to the study of liver disease. Zebrafish embryos develop rapidly; all of their digestive organs are mature in larvae by 5 days of age. At this stage, they can develop hepatobiliary diseases caused by developmental defects or toxin- or ethanol-induced injury and manifest premalignant changes within weeks. Zebrafish are similar to humans in hepatic cellular composition, function, signaling, and response to injury as well as the cellular processes that mediate liver diseases. Genes are highly conserved between humans and zebrafish, making them a useful system to study the basic mechanisms of liver disease. We can perform genetic screens to identify novel genes involved in specific disease processes and chemical screens to identify pathways and compounds that act on specific processes. We review how studies of zebrafish have advanced our understanding of inherited and acquired liver diseases as well as liver cancer and regeneration. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

  16. Zebrafish: An Important Tool for Liver Disease Research

    PubMed Central

    Goessling, Wolfram; Sadler, Kirsten C.

    2016-01-01

    As the incidence of hepatobiliary diseases increases, we must improve our understanding of the molecular, cellular, and physiological factors that contribute to the pathogenesis of liver disease. Animal models help us identify disease mechanisms that might be targeted therapeutically. Zebrafish (Danio rerio) have traditionally been used to study embryonic development but are also important to the study of liver disease. Zebrafish embryos develop rapidly; all of their digestive organs are mature in larvae by 5 days of age. At this stage, they can develop hepatobiliary diseases caused by developmental defects or toxin- or ethanol-induced injury and manifest premalignant changes within weeks. Zebrafish are similar to humans in hepatic cellular composition, function, signaling, and response to injury as well as the cellular processes that mediate liver diseases. Genes are highly conserved between humans and zebrafish, making them a useful system to study the basic mechanisms of liver disease. We can perform genetic screens to identify novel genes involved in specific disease processes and chemical screens to identify pathways and compounds that act on specific processes. We review how studies of zebrafish have advanced our understanding of inherited and acquired liver diseases as well as liver cancer and regeneration. PMID:26319012

  17. Heart-specific expression of laminopathic mutations in transgenic zebrafish.

    PubMed

    Verma, Ajay D; Parnaik, Veena K

    2017-07-01

    Lamins are key determinants of nuclear organization and function in the metazoan nucleus. Mutations in human lamin A cause a spectrum of genetic diseases that affect cardiac muscle and skeletal muscle as well as other tissues. A few laminopathies have been modeled using the mouse. As zebrafish is a well established model for the study of cardiac development and disease, we have investigated the effects of heart-specific lamin A mutations in transgenic zebrafish. We have developed transgenic lines of zebrafish expressing conserved lamin A mutations that cause cardiac dysfunction in humans. Expression of zlamin A mutations Q291P and M368K in the heart was driven by the zebrafish cardiac troponin T2 promoter. Homozygous mutant embryos displayed nuclear abnormalities in cardiomyocyte nuclei. Expression analysis showed the upregulation of genes involved in heart regeneration in transgenic mutant embryos and a cell proliferation marker was increased in adult heart tissue. At the physiological level, there was deviation of up to 20% from normal heart rate in transgenic embryos expressing mutant lamins. Adult homozygous zebrafish were fertile and did not show signs of early mortality. Our results suggest that transgenic zebrafish models of heart-specific laminopathies show cardiac regeneration and moderate deviations in heart rate during embryonic development. © 2017 International Federation for Cell Biology.

  18. Whole-body and multispectral photoacoustic imaging of adult zebrafish

    NASA Astrophysics Data System (ADS)

    Huang, Na; Xi, Lei

    2016-10-01

    Zebrafish is a top vertebrate model to study developmental biology and genetics, and it is becoming increasingly popular for studying human diseases due to its high genome similarity to that of humans and the optical transparency in embryonic stages. However, it becomes difficult for pure optical imaging techniques to volumetric visualize the internal organs and structures of wild-type zebrafish in juvenile and adult stages with excellent resolution and penetration depth. Even with the establishment of mutant lines which remain transparent over the life cycle, it is still a challenge for pure optical imaging modalities to image the whole body of adult zebrafish with micro-scale resolution. However, the method called photoacoustic imaging that combines all the advantages of the optical imaging and ultrasonic imaging provides a new way to image the whole body of the zebrafish. In this work, we developed a non-invasive photoacoustic imaging system with optimized near-infrared illumination and cylindrical scanning to image the zebrafish. The lateral and axial resolution yield to 80 μm and 600 μm, respectively. Multispectral strategy with wavelengths from 690 nm to 930 nm was employed to image various organs inside the zebrafish. From the reconstructed images, most major organs and structures inside the body can be precisely imaged. Quantitative and statistical analysis of absorption for organs under illumination with different wavelengths were carried out.

  19. Sleep–wake regulation and hypocretin–melatonin interaction in zebrafish

    PubMed Central

    Appelbaum, Lior; Wang, Gordon X.; Maro, Geraldine S.; Mori, Rotem; Tovin, Adi; Marin, Wilfredo; Yokogawa, Tohei; Kawakami, Koichi; Smith, Stephen J.; Gothilf, Yoav; Mignot, Emmanuel; Mourrain, Philippe

    2009-01-01

    In mammals, hypocretin/orexin (HCRT) neuropeptides are important sleep–wake regulators and HCRT deficiency causes narcolepsy. In addition to fragmented wakefulness, narcoleptic mammals also display sleep fragmentation, a less understood phenotype recapitulated in the zebrafish HCRT receptor mutant (hcrtr−/−). We therefore used zebrafish to study the potential mediators of HCRT-mediated sleep consolidation. Similar to mammals, zebrafish HCRT neurons express vesicular glutamate transporters indicating conservation of the excitatory phenotype. Visualization of the entire HCRT circuit in zebrafish stably expressing hcrt:EGFP revealed parallels with established mammalian HCRT neuroanatomy, including projections to the pineal gland, where hcrtr mRNA is expressed. As pineal-produced melatonin is a major sleep-inducing hormone in zebrafish, we further studied how the HCRT and melatonin systems interact functionally. mRNA level of arylalkylamine-N-acetyltransferase (AANAT2), a key enzyme of melatonin synthesis, is reduced in hcrtr−/− pineal gland during the night. Moreover, HCRT perfusion of cultured zebrafish pineal glands induces melatonin release. Together these data indicate that HCRT can modulate melatonin production at night. Furthermore, hcrtr−/− fish are hypersensitive to melatonin, but not other hypnotic compounds. Subthreshold doses of melatonin increased the amount of sleep and consolidated sleep in hcrtr−/− fish, but not in the wild-type siblings. These results demonstrate the existence of a functional HCRT neurons-pineal gland circuit able to modulate melatonin production and sleep consolidation. PMID:19966231

  20. Ionic channels underlying the ventricular action potential in zebrafish embryo.

    PubMed

    Alday, Aintzane; Alonso, Hiart; Gallego, Monica; Urrutia, Janire; Letamendia, Ainhoa; Callol, Carles; Casis, Oscar

    2014-06-01

    Over the last years zebrafish has become a popular model in the study of cardiac physiology, pathology and pharmacology. Recently, the application of the 3Rs regulation and the characteristics of the embryo have reduced the use of adult zebrafish use in many studies. However, the zebrafish embryo cardiac physiology is poorly characterized since most works have used indirect techniques and direct recordings of cardiac action potential and ionic currents are scarce. In order to optimize the zebrafish embryo model, we used electrophysiological, pharmacological and immunofluorescence tools to identify the characteristics and the ionic channels involved in the ventricular action potentials of zebrafish embryos. The application of Na(+) or T-type Ca(+2) channel blockers eliminated the cardiac electrical activity, indicating that the action potential upstroke depends on Na(+) and T-type Ca(+2) currents. The plateau phase depends on L-type Ca(+2) channels since it is abolished by specific blockade. The direct channel blockade indicates that the action potential repolarization and diastolic potential depends on ERG K(+) channels. The presence in the embryonic heart of the Nav1.5, Cav1.2, Cav3.2 and ERG channels was also confirmed by immunofluorescence, while the absence of effect of specific blockers and immunostaining indicate that two K(+) repolarizing currents present in human heart, Ito and IKs, are absent in the embryonic zebrafish heart. Our results describe the ionic channels present and its role in the zebrafish embryo heart and support the use of zebrafish embryos to study human diseases and their use for drug testing. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Dihydroartemisinin promotes angiogenesis during the early embryonic development of zebrafish

    PubMed Central

    Ba, Qian; Duan, Juan; Tian, Jia-qiang; Wang, Zi-liang; Chen, Tao; Li, Xiao-guang; Chen, Pei-zhan; Wu, Song-jie; Xiang, Li; Li, Jing-quan; Chu, Rui-ai; Wang, Hui

    2013-01-01

    Aim: To investigate the embryotoxicity of dihydroartemisinin (DHA), the main active metabolite of artemisinin, in zebrafish, and explore the corresponding mechanisms. Methods: The embryos of wild type and TG (flk1:GFP) transgenic zebrafish were exposed to DHA. Developmental phenotypes of the embryos were observed. Development of blood vessels was directly observed in living embryos of TG (flk1:GFP) transgenic zebrafish under fluorescence microscope. The expression of angiogenesis marker genes vegfa, flk1, and flt1 in the embryos was detected using real-time PCR and RNA in situ hybridization assays. Results: Exposure to DHA (1–10 mg/L) dose-dependently caused abnormal zebrafish embryonic phenotypes in the early developmental stage. Furthermore, exposure to DHA (10 mg/L) resulted in more pronounced embryonic angiogenesis in TG (flk1:GFP) zebrafish line. Exposure to DHA (10 mg/L) significantly increased the mRNA expression of vegfa, flk1, and flt1 in the embryos. Knockdown of the flk1 protein partially blocked the effects of DHA on embryogenesis. Conclusion: DHA causes abnormal embryonic phenotypes and promotes angiogenesis in zebrafish early embryonic development, demonstrating the potential embryotoxicity of DHA. PMID:23708556

  2. Biologically inspired robots elicit a robust fear response in zebrafish

    NASA Astrophysics Data System (ADS)

    Ladu, Fabrizio; Bartolini, Tiziana; Panitz, Sarah G.; Butail, Sachit; Macrı, Simone; Porfiri, Maurizio

    2015-03-01

    We investigate the behavioral response of zebrafish to three fear-evoking stimuli. In a binary choice test, zebrafish are exposed to a live allopatric predator, a biologically-inspired robot, and a computer-animated image of the live predator. A target tracking algorithm is developed to score zebrafish behavior. Unlike computer-animated images, the robotic and live predator elicit a robust avoidance response. Importantly, the robotic stimulus elicits more consistent inter-individual responses than the live predator. Results from this effort are expected to aid in hypothesis-driven studies on zebrafish fear response, by offering a valuable approach to maximize data-throughput and minimize animal subjects.

  3. Combinatorial Wnt control of zebrafish midbrain-hindbrain boundary formation.

    PubMed

    Buckles, Gerri R; Thorpe, Christopher J; Ramel, Marie-Christine; Lekven, Arne C

    2004-05-01

    Wnt signaling is known to be required for the normal development of the vertebrate midbrain and hindbrain, but genetic loss of function analyses in the mouse and zebrafish yield differing results regarding the relative importance of specific Wnt loci. In the zebrafish, Wnt1 and Wnt10b functionally overlap in their control of gene expression in the ventral midbrain-hindbrain boundary (MHB), but they are not required for the formation of the MHB constriction. Whether other wnt loci are involved in zebrafish MHB development is unclear, although the expression of at least two wnts, wnt3a and wnt8b, is maintained in wnt1/wnt10b mutants. In order to address the role of wnt3a in zebrafish, we have isolated a full length cDNA and examined its expression and function via knockdown by morpholino antisense oligonucleotide (MO)-mediated knockdown. The expression pattern of wnt3a appears to be evolutionarily conserved between zebrafish and mouse, and MO knockdown shows that Wnt3a, while not uniquely required for MHB development, is required in the absence of Wnt1 and Wnt10b for the formation of the MHB constriction. In zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b, the expression of engrailed orthologs, pax2a and fgf8 is not maintained after mid-somitogenesis. In contrast to acerebellar and no isthmus mutants, in which midbrain and hindbrain cells acquire new fates but cell number is not significantly affected until late in embryogenesis, zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b undergo extensive apoptosis in the midbrain and cerebellum anlagen beginning in mid-somitogenesis, which results in the absence of a significant portion of the midbrain and cerebellum. Thus, the requirement for Wnt signaling in forming the MHB constriction is evolutionarily conserved in vertebrates and it is possible in zebrafish to dissect the relative impact of multiple Wnt loci in midbrain and hindbrain development.

  4. The neurogenetic frontier--lessons from misbehaving zebrafish.

    PubMed

    Burgess, Harold A; Granato, Michael

    2008-11-01

    One of the central questions in neuroscience is how refined patterns of connectivity in the brain generate and monitor behavior. Genetic mutations can influence neural circuits by disrupting differentiation or maintenance of component neuronal cells or by altering functional patterns of nervous system connectivity. Mutagenesis screens therefore have the potential to reveal not only the molecular underpinnings of brain development and function, but to illuminate the cellular basis of behavior. Practical considerations make the zebrafish an organism of choice for undertaking forward genetic analysis of behavior. The powerful array of experimental tools at the disposal of the zebrafish researcher makes it possible to link molecular function to neuronal properties that underlie behavior. This review focuses on specific challenges to isolating and analyzing behavioral mutants in zebrafish.

  5. Automated image-based phenotypic analysis in zebrafish embryos

    PubMed Central

    Vogt, Andreas; Cholewinski, Andrzej; Shen, Xiaoqiang; Nelson, Scott; Lazo, John S.; Tsang, Michael; Hukriede, Neil A.

    2009-01-01

    Presently, the zebrafish is the only vertebrate model compatible with contemporary paradigms of drug discovery. Zebrafish embryos are amenable to automation necessary for high-throughput chemical screens, and optical transparency makes them potentially suited for image-based screening. However, the lack of tools for automated analysis of complex images presents an obstacle to utilizing the zebrafish as a high-throughput screening model. We have developed an automated system for imaging and analyzing zebrafish embryos in multi-well plates regardless of embryo orientation and without user intervention. Images of fluorescent embryos were acquired on a high-content reader and analyzed using an artificial intelligence-based image analysis method termed Cognition Network Technology (CNT). CNT reliably detected transgenic fluorescent embryos (Tg(fli1:EGFP)y1) arrayed in 96-well plates and quantified intersegmental blood vessel development in embryos treated with small molecule inhibitors of anigiogenesis. The results demonstrate it is feasible to adapt image-based high-content screening methodology to measure complex whole organism phenotypes. PMID:19235725

  6. Functional inhibition of UQCRB suppresses angiogenesis in zebrafish

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cho, Yoon Sun; Jung, Hye Jin; Seok, Seung Hyeok

    2013-04-19

    Highlights: ► This is the first functional characterization of UQCRB in vivo model. ► Angiogenesis is inhibited with UQCRB loss of function in zebrafish. ► UQCRB is introduced as a prognostic marker for mitochondria- and angiogenesis-related diseases. -- Abstract: As a subunit of mitochondrial complex III, UQCRB plays an important role in complex III stability, electron transport, and cellular oxygen sensing. Herein, we report UQCRB function regarding angiogenesis in vivo with the zebrafish (Danio rerio). UQCRB knockdown inhibited angiogenesis in zebrafish leading to the suppression of VEGF expression. Moreover, the UQCRB-targeting small molecule terpestacin also inhibited angiogenesis and VEGF levelsmore » in zebrafish, supporting the role of UQCRB in angiogenesis. Collectively, UQCRB loss of function by either genetic and pharmacological means inhibited angiogenesis, indicating that UQCRB plays a key role in this process and can be a prognostic marker of angiogenesis- and mitochondria-related diseases.« less

  7. Evolution of complexity in the zebrafish synapse proteome

    PubMed Central

    Bayés, Àlex; Collins, Mark O.; Reig-Viader, Rita; Gou, Gemma; Goulding, David; Izquierdo, Abril; Choudhary, Jyoti S.; Emes, Richard D.; Grant, Seth G. N.

    2017-01-01

    The proteome of human brain synapses is highly complex and is mutated in over 130 diseases. This complexity arose from two whole-genome duplications early in the vertebrate lineage. Zebrafish are used in modelling human diseases; however, its synapse proteome is uncharacterized, and whether the teleost-specific genome duplication (TSGD) influenced complexity is unknown. We report the characterization of the proteomes and ultrastructure of central synapses in zebrafish and analyse the importance of the TSGD. While the TSGD increases overall synapse proteome complexity, the postsynaptic density (PSD) proteome of zebrafish has lower complexity than mammals. A highly conserved set of ∼1,000 proteins is shared across vertebrates. PSD ultrastructural features are also conserved. Lineage-specific proteome differences indicate that vertebrate species evolved distinct synapse types and functions. The data sets are a resource for a wide range of studies and have important implications for the use of zebrafish in modelling human synaptic diseases. PMID:28252024

  8. Zebrafish (Danio rerio): A Potential Model for Toxinological Studies.

    PubMed

    Vargas, Rafael Antonio; Sarmiento, Karen; Vásquez, Isabel Cristina

    2015-10-01

    Zebrafish are an emerging basic biomedical research model that has multiple advantages compared with other research models. Given that biotoxins, such as toxins, poisons, and venoms, represent health hazards to animals and humans, a low-cost biological model that is highly sensitive to biotoxins is useful to understand the damage caused by such agents and to develop biological tests to prevent and reduce the risk of poisoning in potential cases of bioterrorism or food contamination. In this article, a narrative review of the general aspects of zebrafish as a model in basic biomedical research and various studies in the field of toxinology that have used zebrafish as a biological model are presented. This information will provide useful material to beginner students and researchers who are interested in developing toxinological studies with the zebrafish model.

  9. The zebrafish as a model for complex tissue regeneration

    PubMed Central

    Gemberling, Matthew; Bailey, Travis J.; Hyde, David R.; Poss, Kenneth D.

    2013-01-01

    For centuries, philosophers and scientists have been fascinated by the principles and implications of regeneration in lower vertebrate species. Two features have made zebrafish an informative model system for determining mechanisms of regenerative events. First, they are highly regenerative, able to regrow amputated fins, as well as a lesioned brain, retina, spinal cord, heart, and other tissues. Second, they are amenable to both forward and reverse genetic approaches, with a research toolset regularly updated by an expanding community of zebrafish researchers. Zebrafish studies have helped identify new mechanistic underpinnings of regeneration in multiple tissues, and in some cases have served as a guide for contemplating regenerative strategies in mammals. Here, we review the recent history of zebrafish as a genetic model system for understanding how and why tissue regeneration occurs. PMID:23927865

  10. Husbandry stress exacerbates mycobacterial infections in adult zebrafish, Danio rerio (Hamilton)

    USGS Publications Warehouse

    Ramsay, J.M.; Watral, Virginia G.; Schreck, C.B.; Kent, M.L.

    2009-01-01

    Mycobacteria are significant pathogens of laboratory zebrafish, Danio rerio (Hamilton). Stress is often implicated in clinical disease and morbidity associated with mycobacterial infections but has yet to be examined with zebrafish. The aim of this study was to examine the effects of husbandry stressors on zebrafish infected with mycobacteria. Adult zebrafish were exposed to Mycobacterium marinum or Mycobacterium chelonae, two species that have been associated with disease in zebrafish. Infected fish and controls were then subjected to chronic crowding and handling stressors and examined over an 8-week period. Whole-body cortisol was significantly elevated in stressed fish compared to non-stressed fish. Fish infected with M. marinum ATCC 927 and subjected to husbandry stressors had 14% cumulative mortality while no mortality occurred among infected fish not subjected to husbandry stressors. Stressed fish, infected with M. chelonae H1E2 from zebrafish, were 15-fold more likely to be infected than non-stressed fish at week 8 post-injection. Sub-acute, diffuse infections were more common among stressed fish infected with M. marinum or M. chelonae than non-stressed fish. This is the first study to demonstrate an effect of stress and elevated cortisol on the morbidity, prevalence, clinical disease and histological presentation associated with mycobacterial infections in zebrafish. Minimizing husbandry stress may be effective at reducing the severity of outbreaks of clinical mycobacteriosis in zebrafish facilities. ?? 2009 Blackwell Publishing Ltd.

  11. In vivo physiological recording from the lateral line of juvenile zebrafish.

    PubMed

    Olt, Jennifer; Allen, Claire E; Marcotti, Walter

    2016-10-01

    Zebrafish provide a unique opportunity to investigate in vivo sensory transduction in mature hair cells. We have developed a method for studying the biophysical properties of mature hair cells from the lateral line of juvenile zebrafish. The method involves application of the anaesthetic benzocaine and intubation to maintain ventilation and oxygenation through the gills. The same approach could be used for in vivo functional studies in other sensory and non-sensory systems from juvenile and adult zebrafish. Hair cells are sensory receptors responsible for transducing auditory and vestibular information into electrical signals, which are then transmitted with remarkable precision to afferent neurons. The zebrafish lateral line is emerging as an excellent in vivo model for genetic and physiological analysis of hair cells and neurons. However, research has been limited to larval stages because zebrafish become protected from the time of independent feeding under European law (from 5.2 days post-fertilization (dpf) at 28.5°C). In larval zebrafish, the functional properties of most of hair cells, as well as those of other excitable cells, are still immature. We have developed an experimental protocol to record electrophysiological properties from hair cells of the lateral line in juvenile zebrafish. We found that the anaesthetic benzocaine at 50 mg l(-1) was an effective and safe anaesthetic to use on juvenile zebrafish. Concentrations up to 300 mg l(-1) did not affect the electrical properties or synaptic vesicle release of juvenile hair cells, unlike the commonly used anaesthetic MS-222, which reduces the size of basolateral membrane K(+) currents. Additionally, we implemented a method to maintain gill movement, and as such respiration and blood oxygenation, via the intubation of > 21 dpf zebrafish. The combination of benzocaine and intubation provides an experimental platform to investigate the physiology of mature hair cells from live zebrafish. More

  12. Zebrafish in Toxicology and Environmental Health.

    PubMed

    Bambino, Kathryn; Chu, Jaime

    2017-01-01

    As manufacturing processes and development of new synthetic compounds increase to keep pace with the expanding global demand, environmental health, and the effects of toxicant exposure are emerging as critical public health concerns. Additionally, chemicals that naturally occur in the environment, such as metals, have profound effects on human and animal health. Many of these compounds are in the news: lead, arsenic, and endocrine disruptors such as bisphenol A have all been widely publicized as causing disease or damage to humans and wildlife in recent years. Despite the widespread appreciation that environmental toxins can be harmful, there is limited understanding of how many toxins cause disease. Zebrafish are at the forefront of toxicology research; this system has been widely used as a tool to detect toxins in water samples and to investigate the mechanisms of action of environmental toxins and their related diseases. The benefits of zebrafish for studying vertebrate development are equally useful for studying teratogens. Here, we review how zebrafish are being used both to detect the presence of some toxins as well as to identify how environmental exposures affect human health and disease. We focus on areas where zebrafish have been most effectively used in ecotoxicology and in environmental health, including investigation of exposures to endocrine disruptors, industrial waste byproducts, and arsenic. © 2017 Elsevier Inc. All rights reserved.

  13. Axonal regeneration in zebrafish spinal cord

    PubMed Central

    Hui, Subhra Prakash

    2018-01-01

    Abstract In the present review we discuss two interrelated events—axonal damage and repair—known to occur after spinal cord injury (SCI) in the zebrafish. Adult zebrafish are capable of regenerating axonal tracts and can restore full functionality after SCI. Unlike fish, axon regeneration in the adult mammalian central nervous system is extremely limited. As a consequence of an injury there is very little repair of disengaged axons and therefore functional deficit persists after SCI in adult mammals. In contrast, peripheral nervous system axons readily regenerate following injury and hence allow functional recovery both in mammals and fish. A better mechanistic understanding of these three scenarios could provide a more comprehensive insight into the success or failure of axonal regeneration after SCI. This review summarizes the present understanding of the cellular and molecular basis of axonal regeneration, in both the peripheral nervous system and the central nervous system, and large scale gene expression analysis is used to focus on different events during regeneration. The discovery and identification of genes involved in zebrafish spinal cord regeneration and subsequent functional experimentation will provide more insight into the endogenous mechanism of myelination and remyelination. Furthermore, precise knowledge of the mechanism underlying the extraordinary axonal regeneration process in zebrafish will also allow us to unravel the potential therapeutic strategies to be implemented for enhancing regrowth and remyelination of axons in mammals. PMID:29721326

  14. Zebrafish in Toxicology and Environmental Health

    PubMed Central

    Bambino, Kathryn; Chu, Jaime

    2018-01-01

    As manufacturing processes and development of new synthetic compounds increase to keep pace with the expanding global demand, environmental health, and the effects of toxicant exposure are emerging as critical public health concerns. Additionally, chemicals that naturally occur in the environment, such as metals, have profound effects on human and animal health. Many of these compounds are in the news: lead, arsenic, and endocrine disruptors such as bisphenol A have all been widely publicized as causing disease or damage to humans and wildlife in recent years. Despite the widespread appreciation that environmental toxins can be harmful, there is limited understanding of how many toxins cause disease. Zebrafish are at the forefront of toxicology research; this system has been widely used as a tool to detect toxins in water samples and to investigate the mechanisms of action of environmental toxins and their related diseases. The benefits of zebrafish for studying vertebrate development are equally useful for studying teratogens. Here, we review how zebrafish are being used both to detect the presence of some toxins as well as to identify how environmental exposures affect human health and disease. We focus on areas where zebrafish have been most effectively used in ecotoxicology and in environmental health, including investigation of exposures to endocrine disruptors, industrial waste byproducts, and arsenic. PMID:28335863

  15. Axonal regeneration in zebrafish spinal cord.

    PubMed

    Ghosh, Sukla; Hui, Subhra Prakash

    2018-03-01

    In the present review we discuss two interrelated events-axonal damage and repair-known to occur after spinal cord injury (SCI) in the zebrafish. Adult zebrafish are capable of regenerating axonal tracts and can restore full functionality after SCI. Unlike fish, axon regeneration in the adult mammalian central nervous system is extremely limited. As a consequence of an injury there is very little repair of disengaged axons and therefore functional deficit persists after SCI in adult mammals. In contrast, peripheral nervous system axons readily regenerate following injury and hence allow functional recovery both in mammals and fish. A better mechanistic understanding of these three scenarios could provide a more comprehensive insight into the success or failure of axonal regeneration after SCI. This review summarizes the present understanding of the cellular and molecular basis of axonal regeneration, in both the peripheral nervous system and the central nervous system, and large scale gene expression analysis is used to focus on different events during regeneration. The discovery and identification of genes involved in zebrafish spinal cord regeneration and subsequent functional experimentation will provide more insight into the endogenous mechanism of myelination and remyelination. Furthermore, precise knowledge of the mechanism underlying the extraordinary axonal regeneration process in zebrafish will also allow us to unravel the potential therapeutic strategies to be implemented for enhancing regrowth and remyelination of axons in mammals.

  16. Quantification of birefringence readily measures the level of muscle damage in zebrafish

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Berger, Joachim, E-mail: Joachim.Berger@Monash.edu; Sztal, Tamar; Currie, Peter D.

    2012-07-13

    Highlights: Black-Right-Pointing-Pointer Report of an unbiased quantification of the birefringence of muscle of fish larvae. Black-Right-Pointing-Pointer Quantification method readily identifies level of overall muscle damage. Black-Right-Pointing-Pointer Compare zebrafish muscle mutants for level of phenotype severity. Black-Right-Pointing-Pointer Proposed tool to survey treatments that aim to ameliorate muscular dystrophy. -- Abstract: Muscular dystrophies are a group of genetic disorders that progressively weaken and degenerate muscle. Many zebrafish models for human muscular dystrophies have been generated and analysed, including dystrophin-deficient zebrafish mutants dmd that model Duchenne Muscular Dystrophy. Under polarised light the zebrafish muscle can be detected as a bright area in anmore » otherwise dark background. This light effect, called birefringence, results from the diffraction of polarised light through the pseudo-crystalline array of the muscle sarcomeres. Muscle damage, as seen in zebrafish models for muscular dystrophies, can readily be detected by a reduction in the birefringence. Therefore, birefringence is a very sensitive indicator of overall muscle integrity within larval zebrafish. Unbiased documentation of the birefringence followed by densitometric measurement enables the quantification of the birefringence of zebrafish larvae. Thereby, the overall level of muscle integrity can be detected, allowing the identification and categorisation of zebrafish muscle mutants. In addition, we propose that the establish protocol can be used to analyse treatments aimed at ameliorating dystrophic zebrafish models.« less

  17. [Effect of rhynchophylline on behaviors of methamphetamine-dependent zebrafish and the mechanism].

    PubMed

    Chen, Yi-Fei; Peng, Ju; Fang, Miao; Liu, Yi; Nie, Ling-Hui; Mo, Zhi-Xian; Zhu, Ling-Ling

    2016-11-20

    To observe the effect of rhynchophylline on methamphetamine-dependent zebrafish and explore the possible mechanism. Zebrafish were divided into control group, amphetamine group, low- (50 mg/kg) and high (100 mg/kg)-dose rhynchophylline groups, and ketamine (150 mg/kg) group. Conditioned place preference (CPP) was induced in zebrafish with methamphetamine, and the staying time in the drug box and the tracking map of the zebrafish were observed with Noldus Ethovision XT system. The protein expressions of TH, NR2B and GLUR2 in the brain of zebrafish with CPP were detected with Western blotting. Compared with the control group, zebrafish in methamphetamine group showed significant variations in the staying time and swimming distance in the drug box after conditioning (P<0.05) with obvious alterations of NR2B, TH and GLUR2 expressions in the brain (P<0.05). Treatment of methamphetamine-dependent zebrafish with high-dose rhynchophylline significantly reduced the variations in the staying time and swimming distance in the drug box (P<0.05) and in the expressions of NR2B, TH and GLUR2 in the brain (P<0.05). Rhynchophylline can inhibit methamphetamine dependence in zebrafish, the mechanism of which may involve the expressions of TH, NR2B and GLUR2 proteins in the brain.

  18. Oceans of Opportunity: Exploring Vertebrate Hematopoiesis in Zebrafish

    PubMed Central

    Carroll, Kelli J.; North, Trista E.

    2015-01-01

    Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell (HSPC) biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor and effector cell emergence, expansion and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell-types can be identified and characterized. Further, a myriad of transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of HSPC biology during development, in response to infection or injury, or in the setting of hematological malignancy, continues to deepen, zebrafish will remain essential for exploring the spatio-temporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease. PMID:24816275

  19. BMP signaling modulates hepcidin expression in zebrafish embryos independent of hemojuvelin.

    PubMed

    Gibert, Yann; Lattanzi, Victoria J; Zhen, Aileen W; Vedder, Lea; Brunet, Frédéric; Faasse, Sarah A; Babitt, Jodie L; Lin, Herbert Y; Hammerschmidt, Matthias; Fraenkel, Paula G

    2011-01-21

    Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.

  20. The HDAC Inhibitor TSA Ameliorates a Zebrafish Model of Duchenne Muscular Dystrophy.

    PubMed

    Johnson, Nathan M; Farr, Gist H; Maves, Lisa

    2013-09-17

    Zebrafish are an excellent model for Duchenne muscular dystrophy. In particular, zebrafish provide a system for rapid, easy, and low-cost screening of small molecules that can ameliorate muscle damage in dystrophic larvae. Here we identify an optimal anti-sense morpholino cocktail that robustly knocks down zebrafish Dystrophin (dmd-MO). We use two approaches, muscle birefringence and muscle actin expression, to quantify muscle damage and show that the dmd-MO dystrophic phenotype closely resembles the zebrafish dmd mutant phenotype. We then show that the histone deacetylase (HDAC) inhibitor TSA, which has been shown to ameliorate the mdx mouse Duchenne model, can rescue muscle fiber damage in both dmd-MO and dmd mutant larvae. Our study identifies optimal morpholino and phenotypic scoring approaches for dystrophic zebrafish, further enhancing the zebrafish dmd model for rapid and cost-effective small molecule screening.

  1. Strategies to Mitigate a Mycobacterium marinum Outbreak in a Zebrafish Research Facility

    PubMed Central

    Snell, Kathy; Mittge, Erika; Melancon, Ellie; Montgomery, Rebecca; McFadden, Marcie; Camoriano, Javier; Kent, Michael L.; Whipps, Christopher M.; Peirce, Judy

    2016-01-01

    Abstract In 2011, the zebrafish research facility at the University of Oregon experienced an outbreak of Mycobacterium marinum that affected both research fish and facility staff. A thorough review of risks to personnel, the zebrafish veterinary care program, and zebrafish husbandry procedures at the research facility followed. In the years since 2011, changes have been implemented throughout the research facility to protect the personnel, the fish colony, and ultimately the continued success of the zebrafish model research program. In this study, we present the history of the outbreak, the changes we implemented, and recommendations to mitigate pathogen outbreaks in zebrafish research facilities. PMID:27351618

  2. Quaternary and tertiary aldoxime antidotes for organophosphate exposure in a zebrafish model system

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schmidt, Hayden R.; Radić, Zoran; Taylor, Palmer

    The zebrafish is rapidly becoming an important model system for screening of new therapeutics. Here we evaluated the zebrafish as a potential pharmacological model for screening novel oxime antidotes to organophosphate (OP)-inhibited acetylcholinesterase (AChE). The k{sub i} values determined for chlorpyrifos oxon (CPO) and dichlorvos (DDVP) showed that CPO was a more potent inhibitor of both human and zebrafish AChE, but overall zebrafish AChE was less sensitive to OP inhibition. In contrast, aldoxime antidotes, the quaternary ammonium 2-PAM and tertiary amine RS-194B, showed generally similar overall reactivation kinetics, k{sub r}, in both zebrafish and human AChE. However, differences between themore » K{sub ox} and k{sub 2} constants suggest that zebrafish AChE associates more tightly with oximes, but has a slower maximal reactivation rate than human AChE. Homology modeling suggests that these kinetic differences result from divergences in the amino acids lining the entrance to the active site gorge. Although 2-PAM had the more favorable in vitro reactivation kinetics, RS-194B was more effective antidote in vivo. In intact zebrafish embryos, antidotal treatment with RS-194B rescued embryos from OP toxicity, whereas 2-PAM had no effect. Dechorionation of the embryos prior to antidotal treatment allowed both 2-PAM and RS-194B to rescue zebrafish embryos from OP toxicity. Interestingly, RS-194B and 2-PAM alone increased cholinergic motor activity in dechorionated embryos possibly due to the reversible inhibition kinetics, K{sub i} and αK{sub i}, of the oximes. Together these results demonstrate that the zebrafish at various developmental stages provides an excellent model for investigating membrane penetrant antidotes to OP exposure. - Highlights: • Zebrafish AChE shares significant structural similarities with human AChE. • OP-inhibited zebrafish and human AChE exhibit similar reactivation kinetics. • The zebrafish chorion is permeable to BBB penetrant

  3. The zebrafish eye—a paradigm for investigating human ocular genetics

    PubMed Central

    Richardson, R; Tracey-White, D; Webster, A; Moosajee, M

    2017-01-01

    Although human epidemiological and genetic studies are essential to elucidate the aetiology of normal and aberrant ocular development, animal models have provided us with an understanding of the pathogenesis of multiple developmental ocular malformations. Zebrafish eye development displays in depth molecular complexity and stringent spatiotemporal regulation that incorporates developmental contributions of the surface ectoderm, neuroectoderm and head mesenchyme, similar to that seen in humans. For this reason, and due to its genetic tractability, external fertilisation, and early optical clarity, the zebrafish has become an invaluable vertebrate system to investigate human ocular development and disease. Recently, zebrafish have been at the leading edge of preclinical therapy development, with their amenability to genetic manipulation facilitating the generation of robust ocular disease models required for large-scale genetic and drug screening programmes. This review presents an overview of human and zebrafish ocular development, genetic methodologies employed for zebrafish mutagenesis, relevant models of ocular disease, and finally therapeutic approaches, which may have translational leads in the future. PMID:27612182

  4. Characterization of zebrafish dysferlin by morpholino knockdown

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kawahara, Genri; Serafini, Peter R.; Myers, Jennifer A.

    2011-09-23

    Highlights: {yields} cDNAs of zebrafish dysferlin were cloned (6.3 kb). {yields} The dysferlin expression was detected in skeletal muscle, heart and eye. {yields} Injection of antisense morpholinos to dysferlin caused marked muscle disorganization. {yields} Zebrafish dysferlin expression may be involved in stabilizing muscle structures. -- Abstract: Mutations in the gene encoding dysferlin cause two distinct muscular dystrophy phenotypes: limb-girdle muscular dystrophy type 2B (LGMD-2B) and Miyoshi myopathy (MM). Dysferlin is a large transmembrane protein involved in myoblast fusion and membrane resealing. Zebrafish represent an ideal animal model to use for studying muscle disease including abnormalities of dysferlin. cDNAs of zebrafishmore » dysferlin were cloned (6.3 kb) and the predicted amino acid sequences, showed 68% similarity to predicted amino acid sequences of mammalian dysferlin. The expression of dysferlin was mainly in skeletal muscle, heart and eye, and the expression could be detected as early as 11 h post fertilization (hpf). Three different antisense oligonucleotide morpholinos were targeted to inhibit translation of this dysferlin mRNA and the morpholino-injected fish showed marked muscle disorganization which could be detected by birefringence assay. Western blot analysis using dysferlin antibodies showed that the expression of dysferlin was reduced in each of the three morphants. Dysferlin expression was shown to be reduced at the myosepta of zebrafish muscle using immunohistochemistry, although the expression of other muscle membrane components, dystrophin, laminin, {beta}-dystroglycan were detected normally. Our data suggest that zebrafish dysferlin expression is involved in stabilizing muscle structures and its downregulation causes muscle disorganization.« less

  5. Can Zebrafish be used to Identify Developmentally Neurotoxic Chemicals

    EPA Science Inventory

    Can Zebrafish be Used to Identify Developmentally Neurotoxic Chemicals? The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals for developmental neurotoxicity. We are exploring behavioral methods using zebrafish by desig...

  6. Use of Gnotobiotic Zebrafish to Study Vibrio anguillarum Pathogenicity

    PubMed Central

    Oyarbide, Usua; Iturria, Iñaki; Rainieri, Sandra

    2015-01-01

    Abstract We evaluated the use of the gnotobiotic zebrafish system to study the effects of bacterial infection, and analyzed expression of genes involved in zebrafish innate immunity. Using a GFP-labeled strain of Vibrio anguillarum, we fluorescently monitored colonization of the zebrafish intestinal tract and used gene expression analysis to compare changes in genes involved in innate immunity between nongnotobiotic and gnotobiotic larvae. The experiments performed with the gnotobiotic zebrafish reveal new insights into V. anguillarum pathogenesis. Specifically, an alteration of the host immune system was detected through the suppression of a number of innate immune genes (NFKB, IL1B, TLR4, MPX, and TRF) during the first 3 h post infection. This immunomodulation can be indicative of a “stealth mechanism” of mucus invasion in which the pathogen found a sheltered niche, a typical trait of intracellular pathogens. PMID:25548877

  7. Teratogenic potential of antiepileptic drugs in the zebrafish model.

    PubMed

    Lee, Sung Hak; Kang, Jung Won; Lin, Tao; Lee, Jae Eun; Jin, Dong Il

    2013-01-01

    The zebrafish model is an attractive candidate for screening of developmental toxicity during early drug development. Antiepileptic drugs (AEDs) arouse concern for the risk of teratogenicity, but the data are limited. In this study, we evaluated the teratogenic potential of seven AEDs (carbamazepine (CBZ), ethosuximide (ETX), valproic acid (VPN), lamotrigine (LMT), lacosamide (LCM), levetiracetam (LVT), and topiramate (TPM)) in the zebrafish model. Zebrafish embryos were exposed to AEDs from initiation of gastrula (5.25 hours post-fertilization (hpf)) to termination of hatching (72 hpf) which mimic the mammalian teratogenic experimental design. The lethality and teratogenic index (TI) of AEDs were determined and the TI values of each drug were compared with the US FDA human pregnancy categories. Zebrafish model was useful screening model for teratogenic potential of antiepilepsy drugs and was in concordance with in vivo mammalian data and human clinical data.

  8. The Vital Relationship Between Nutrition and Health in Zebrafish.

    PubMed

    Watts, Stephen A; Lawrence, Christian; Powell, Mickie; D'Abramo, Louis R

    2016-07-01

    In the relatively short span of four decades, the zebrafish (Danio rerio) has emerged as an increasingly important model organism for biomedicine and other scientific disciplines. As the scale and sophistication of zebrafish research expands, so too does the need to develop standards that promote the production and maintenance of healthy animals for experiments. A major, but long overlooked, contributor to fish health is nutrition. Historically, feeding practices for laboratory zebrafish have been designed to promote growth and reproductive function. However, as the field matures, it is becoming increasingly clear that the nutritional goals for these animals should evolve beyond basic production to the maintenance of clinically healthy research subjects. This review outlines weaknesses and limitations of current approaches and provides a justification for the development of defined standardized diets that will strengthen and facilitate the continued growth of the zebrafish model system.

  9. Expression of CALR mutants causes mpl-dependent thrombocytosis in zebrafish.

    PubMed

    Lim, K-H; Chang, Y-C; Chiang, Y-H; Lin, H-C; Chang, C-Y; Lin, C-S; Huang, L; Wang, W-T; Gon-Shen Chen, C; Chou, W-C; Kuo, Y-Y

    2016-10-07

    CALR mutations are identified in about 30% of JAK2/MPL-unmutated myeloproliferative neoplasms (MPNs) including essential thrombocythemia (ET) and primary myelofibrosis. Although the molecular pathogenesis of CALR mutations leading to MPNs has been studied using in vitro cell lines models, how mutant CALR may affect developmental hematopoiesis remains unknown. Here we took advantage of the zebrafish model to examine the effects of mutant CALR on early hematopoiesis and model human CALR-mutated MPNs. We identified three zebrafish genes orthologous to human CALR, referred to as calr, calr3a and calr3b. The expression of CALR-del52 and CALR-ins5 mutants caused an increase in the hematopoietic stem/progenitor cells followed by thrombocytosis without affecting normal angiogenesis. The expression of CALR mutants also perturbed early developmental hematopoiesis in zebrafish. Importantly, morpholino knockdown of mpl but not epor or csf3r could significantly attenuate the effects of mutant CALR. Furthermore, the expression of mutant CALR caused jak-stat signaling activation in zebrafish that could be blocked by JAK inhibitors (ruxolitinib and fedratinib). These findings showed that mutant CALR activates jak-stat signaling through an mpl-dependent mechanism to mediate pathogenic thrombopoiesis in zebrafish, and illustrated that the signaling machinery related to mutant CALR tumorigenesis are conserved between human and zebrafish.

  10. Zebrafish Models of Prader-Willi Syndrome: Fast Track to Pharmacotherapeutics

    PubMed Central

    Spikol, Emma D.; Laverriere, Caroline E.; Robnett, Maya; Carter, Gabriela; Wolfe, Erin; Glasgow, Eric

    2016-01-01

    Prader-Willi syndrome (PWS) is a rare genetic neurodevelopmental disorder characterized by an insatiable appetite, leading to chronic overeating and obesity. Additional features include short stature, intellectual disability, behavioral problems and incomplete sexual development. Although significant progress has been made in understanding the genetic basis of PWS, the mechanisms underlying the pathogenesis of the disorder remain poorly understood. Treatment for PWS consists mainly of palliative therapies; curative therapies are sorely needed. Zebrafish, Danio rerio, represent a promising way forward for elucidating physiological problems such as obesity and identifying new pharmacotherapeutic options for PWS. Over the last decade, an increased appreciation for the highly conserved biology among vertebrates and the ability to perform high-throughput drug screening has seen an explosion in the use of zebrafish for disease modeling and drug discovery. Here, we review recent advances in developing zebrafish models of human disease. Aspects of zebrafish genetics and physiology that are relevant to PWS will be discussed, and the advantages and disadvantages of zebrafish models will be contrasted with current animal models for this syndrome. Finally, we will present a paradigm for drug screening in zebrafish that is potentially the fastest route for identifying and delivering curative pharmacotherapies to PWS patients. PMID:27857842

  11. Zebrafish heart failure models: opportunities and challenges.

    PubMed

    Shi, Xingjuan; Chen, Ru; Zhang, Yu; Yun, Junghwa; Brand-Arzamendi, Koroboshka; Liu, Xiangdong; Wen, Xiao-Yan

    2018-05-03

    Heart failure is a complex pathophysiological syndrome of pumping functional failure that results from injury, infection or toxin-induced damage on the myocardium, as well as genetic influence. Gene mutations associated with cardiomyopathies can lead to various pathologies of heart failure. In recent years, zebrafish, Danio rerio, has emerged as an excellent model to study human cardiovascular diseases such as congenital heart defects, cardiomyopathy, and preclinical development of drugs targeting these diseases. In this review, we will first summarize zebrafish genetic models of heart failure arose from cardiomyopathy, which is caused by mutations in sarcomere, calcium or mitochondrial-associated genes. Moreover, we outline zebrafish heart failure models triggered by chemical compounds. Elucidation of these models will improve the understanding of the mechanism of pathogenesis and provide potential targets for novel therapies.

  12. The neurogenetic frontier—lessons from misbehaving zebrafish

    PubMed Central

    Granato, Michael

    2008-01-01

    One of the central questions in neuroscience is how refined patterns of connectivity in the brain generate and monitor behavior. Genetic mutations can influence neural circuits by disrupting differentiation or maintenance of component neuronal cells or by altering functional patterns of nervous system connectivity. Mutagenesis screens therefore have the potential to reveal not only the molecular underpinnings of brain development and function, but to illuminate the cellular basis of behavior. Practical considerations make the zebrafish an organism of choice for undertaking forward genetic analysis of behavior. The powerful array of experimental tools at the disposal of the zebrafish researcher makes it possible to link molecular function to neuronal properties that underlie behavior. This review focuses on specific challenges to isolating and analyzing behavioral mutants in zebrafish. PMID:18836206

  13. Methods to study maternal regulation of germ cell specification in zebrafish

    PubMed Central

    Kaufman, O.H.; Marlow, F.L.

    2016-01-01

    The process by which the germ line is specified in the zebrafish embryo is under the control of maternal gene products that were produced during oogenesis. Zebrafish are highly amenable to microscopic observation of the processes governing maternal germ cell specification because early embryos are transparent, and the germ line is specified rapidly (within 4–5 h post fertilization). Advantages of zebrafish over other models used to study vertebrate germ cell formation include their genetic tractability, the large numbers of progeny, and the easily manipulable genome, all of which make zebrafish an ideal system for studying the genetic regulators and cellular basis of germ cell formation and maintenance. Classical molecular biology techniques, including expression analysis through in situ hybridization and forward genetic screens, have laid the foundation for our understanding of germ cell development in zebrafish. In this chapter, we discuss some of these classic techniques, as well as recent cutting-edge methodologies that have improved our ability to visualize the process of germ cell specification and differentiation, and the tracking of specific molecules involved in these processes. Additionally, we discuss traditional and novel technologies for manipulating the zebrafish genome to identify new components through loss-of-function studies of putative germ cell regulators. Together with the numerous aforementioned advantages of zebrafish as a genetic model for studying development, we believe these new techniques will continue to advance zebrafish to the forefront for investigation of the molecular regulators of germ cell specification and germ line biology. PMID:27312489

  14. An individual-based model of zebrafish population dynamics accounting for energy dynamics.

    PubMed

    Beaudouin, Rémy; Goussen, Benoit; Piccini, Benjamin; Augustine, Starrlight; Devillers, James; Brion, François; Péry, Alexandre R R

    2015-01-01

    Developing population dynamics models for zebrafish is crucial in order to extrapolate from toxicity data measured at the organism level to biological levels relevant to support and enhance ecological risk assessment. To achieve this, a dynamic energy budget for individual zebrafish (DEB model) was coupled to an individual based model of zebrafish population dynamics (IBM model). Next, we fitted the DEB model to new experimental data on zebrafish growth and reproduction thus improving existing models. We further analysed the DEB-model and DEB-IBM using a sensitivity analysis. Finally, the predictions of the DEB-IBM were compared to existing observations on natural zebrafish populations and the predicted population dynamics are realistic. While our zebrafish DEB-IBM model can still be improved by acquiring new experimental data on the most uncertain processes (e.g. survival or feeding), it can already serve to predict the impact of compounds at the population level.

  15. Mutagenesis and phenotyping resources in zebrafish for studying development and human disease

    PubMed Central

    Varshney, Gaurav Kumar

    2014-01-01

    The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts. PMID:24162064

  16. Dopamine receptor antagonism disrupts social preference in zebrafish, a strain comparison study

    PubMed Central

    Scerbina, Tanya; Chatterjee, Diptendu; Gerlai, Robert

    2012-01-01

    Zebrafish form shoals in nature and in the laboratory. The sight of conspecifics has been found reinforcing in zebrafish learning tasks. However, the mechanisms of shoaling, and those of its reinforcing properties, are not known. The dopaminergic system has been implicated in reward among other functions and it is also engaged by drugs of abuse as shown in a variety of vertebrates including zebrafish. The ontogenetic changes in dopamine levels and, to a lesser degree, in serotonin levels, have been found to accompany the maturation of shoaling in zebrafish. Thus, we hypothesized that the dopaminergic system may contribute to shoaling in zebrafish. To test this we employed a D1-receptor antagonist and quantified behavioral responses of our subjects using a social preference (shoaling) paradigm. We found significant reduction of social preference induced by the D1-R antagonist, SCH23390, in the AB strain of zebrafish, an alteration that was not accompanied by changes in motor function or vision. We also detected D1-R antagonist induced changes in the level of dopamine, DOPAC, serotonin and 5HIAA, respectively, in the brain of AB zebrafish as quantified by HPLC with electrochemical detection. We found the antagonist induced behavioral changes to be absent and the levels of these neurochemicals to be lower in another zebrafish population, SF, demonstrating naturally occurring genetic variability in these traits. We conclude that this variability may be utilized to unravel the mechanisms of social behavior in zebrafish, a line of research that may be extended to other vertebrates including our own species. PMID:22491827

  17. Evaluation of color preference in zebrafish for learning and memory.

    PubMed

    Avdesh, Avdesh; Martin-Iverson, Mathew T; Mondal, Alinda; Chen, Mengqi; Askraba, Sreten; Morgan, Newman; Lardelli, Michael; Groth, David M; Verdile, Giuseppe; Martins, Ralph N

    2012-01-01

    There is growing interest in using zebrafish (Danio rerio) as a model of neurodegenerative disorders such as Alzheimer's disease. A zebrafish model of tauopathies has recently been developed and characterized in terms of presence of the pathological hallmarks (i.e., neurofibrillary tangles and cell death). However, it is also necessary to validate these models for function by assessing learning and memory. The majority of tools to assess memory and learning in animal models involve visual stimuli, including color preference. The color preference of zebrafish has received little attention. To validate zebrafish as a model for color-associated-learning and memory, it is necessary to evaluate its natural preferences or any pre-existing biases towards specific colors. In the present study, we have used four different colors (red, yellow, green, and blue) to test natural color preferences of the zebrafish using two procedures: Place preference and T-maze. Results from both experiments indicate a strong aversion toward blue color relative to all other colors (red, yellow, and green) when tested in combinations. No preferences or biases were found among reds, yellows, and greens in the place preference procedure. However, red and green were equally preferred and both were preferred over yellow by zebrafish in the T-maze procedure. The results from the present study show a strong aversion towards blue color compared to red, green, and yellow, with yellow being less preferred relative to red and green. The findings from this study may underpin any further designing of color-based learning and memory paradigms or experiments involving aversion, anxiety, or fear in the zebrafish.

  18. Rapid quantification of neutral lipids and triglycerides during zebrafish embryogenesis.

    PubMed

    Yoganantharjah, Prusothman; Byreddy, Avinesh R; Fraher, Daniel; Puri, Munish; Gibert, Yann

    2017-01-01

    The zebrafish is a useful vertebrate model to study lipid metabolism. Oil Red-O (ORO) staining of zebrafish embryos, though sufficient for visualizing the localization of triglycerides, was previously inadequate to quantify neutral lipid abundance. For metabolic studies, it is crucial to be able to quantify lipids during embryogenesis. Currently no cost effective, rapid and reliable method exists to quantify the deposition of neutral lipids and triglycerides. Thin layer chromatography (TLC), gas chromatography and mass spectrometry can be used to accurately measure lipid levels, but are time consuming and costly in their use. Hence, we developed a rapid and reliable method to quantify neutral lipids and triglycerides. Zebrafish embryos were exposed to Rimonabant (Rimo) or WIN 55,212-2 mesylate (WIN), compounds previously shown to modify lipid content during zebrafish embryogenesis. Following this, ORO stain was extracted out of both the zebrafish body and yolk sac and optical density was measured to give an indication of neutral lipid and triglyceride accumulation. Embryos treated with 0.3 microM WIN resulted in increased lipid accumulation, whereas 3 microM Rimo caused a decrease in lipid accumulation during embryogenesis. TLC was performed on zebrafish bodies to validate the developed method. In addition, BODIPY free fatty acids were injected into zebrafish embryos to confirm quantification of changes in lipid content in the embryo. Previously, ORO was limited to qualitative assessment; now ORO can be used as a quantitative tool to directly determine changes in the levels of neutral lipids and triglycerides.

  19. A shifted repertoire of endocannabinoid genes in the zebrafish (Danio rerio).

    PubMed

    McPartland, J M; Glass, Michelle; Matias, Isabel; Norris, Ryan W; Kilpatrick, C William

    2007-05-01

    The zebrafish has served as a model organism for developmental biology. Sequencing its genome has expanded zebrafish research into physiology and drug-development testing. Several cannabinoid pharmaceuticals are in development, but expression of endocannabinoid receptors and enzymes remains unknown in this species. We conducted a bioinformatics analysis of the zebrafish genome using 17 human endocannabinoid genes as a reference set. Putative zebrafish orthologs were identified in filtered BLAST searches as reciprocal best hits. Orthology was confirmed by three in silico methods: phylogenetic testing, synteny analysis, and functional mapping. Zebrafish expressed orthologs of cannabinoid receptor 1, transient receptor potential channel vanilloid receptor 4, GPR55 receptor, fatty acid amide hydrolase 1, monoacylglycerol lipase, NAPE-selective phospholipase D, abhydrolase domain-containing protein 4, and diacylglycerol lipase alpha and beta; and paired paralogs of cannabinoid receptor 2, fatty acid amide hydrolase 2, peroxisome proliferator-activated receptor alpha, prostaglandin-endoperoxide synthase 2, and transient receptor potential cation channel subtype A1. Functional mapping suggested the orthologs of transient receptor potential vanilloid receptor 1 and peroxisome proliferator-activated receptor gamma lack specific amino acids critical for cannabinoid ligand binding. No orthologs of N-acylethanolamine acid amidase or protein tyrosine phosphatase, non-receptor type 22 were identified. In conclusion, the zebrafish genome expresses a shifted repertoire of endocannabinoid genes. In vitro analyses are warranted before using zebrafish for cannabinoid development testing.

  20. Oceans of opportunity: exploring vertebrate hematopoiesis in zebrafish.

    PubMed

    Carroll, Kelli J; North, Trista E

    2014-08-01

    Exploitation of the zebrafish model in hematology research has surged in recent years, becoming one of the most useful and tractable systems for understanding regulation of hematopoietic development, homeostasis, and malignancy. Despite the evolutionary distance between zebrafish and humans, remarkable genetic and phenotypic conservation in the hematopoietic system has enabled significant advancements in our understanding of blood stem and progenitor cell biology. The strengths of zebrafish in hematology research lie in the ability to perform real-time in vivo observations of hematopoietic stem, progenitor, and effector cell emergence, expansion, and function, as well as the ease with which novel genetic and chemical modifiers of specific hematopoietic processes or cell types can be identified and characterized. Further, myriad transgenic lines have been developed including fluorescent reporter systems to aid in the visualization and quantification of specified cell types of interest and cell-lineage relationships, as well as effector lines that can be used to implement a wide range of experimental manipulations. As our understanding of the complex nature of blood stem and progenitor cell biology during development, in response to infection or injury, or in the setting of hematologic malignancy continues to deepen, zebrafish will remain essential for exploring the spatiotemporal organization and integration of these fundamental processes, as well as the identification of efficacious small molecule modifiers of hematopoietic activity. In this review, we discuss the biology of the zebrafish hematopoietic system, including similarities and differences from mammals, and highlight important tools currently utilized in zebrafish embryos and adults to enhance our understanding of vertebrate hematology, with emphasis on findings that have impacted our understanding of the onset or treatment of human hematologic disorders and disease. Copyright © 2014 ISEH - International

  1. Zebrafish AID is capable of deaminating methylated deoxycytidines

    PubMed Central

    Abdouni, Hala; King, Justin J.; Suliman, Mussa; Quinlan, Matthew; Fifield, Heather; Larijani, Mani

    2013-01-01

    Activation-induced cytidine deaminase (AID) deaminates deoxycytidine (dC) to deoxyuracil (dU) at immunoglobulin loci in B lymphocytes to mediate secondary antibody diversification. Recently, AID has been proposed to also mediate epigenetic reprogramming by demethylating methylated cytidines (mC) possibly through deamination. AID overexpression in zebrafish embryos was shown to promote genome demethylation through G:T lesions, implicating a deamination-dependent mechanism. We and others have previously shown that mC is a poor substrate for human AID. Here, we examined the ability of bony fish AID to deaminate mC. We report that zebrafish AID was unique among all orthologs in that it efficiently deaminates mC. Analysis of domain-swapped and mutant AID revealed that mC specificity is independent of the overall high-catalytic efficiency of zebrafish AID. Structural modeling with or without bound DNA suggests that efficient deamination of mC by zebrafish AID is likely not due to a larger catalytic pocket allowing for better fit of mC, but rather because of subtle differences in the flexibility of its structure. PMID:23585279

  2. Embryonic senescence and laminopathies in a progeroid zebrafish model.

    PubMed

    Koshimizu, Eriko; Imamura, Shintaro; Qi, Jie; Toure, Jamal; Valdez, Delgado M; Carr, Christopher E; Hanai, Jun-ichi; Kishi, Shuji

    2011-03-30

    Mutations that disrupt the conversion of prelamin A to mature lamin A cause the rare genetic disorder Hutchinson-Gilford progeria syndrome and a group of laminopathies. Our understanding of how A-type lamins function in vivo during early vertebrate development through aging remains limited, and would benefit from a suitable experimental model. The zebrafish has proven to be a tractable model organism for studying both development and aging at the molecular genetic level. Zebrafish show an array of senescence symptoms resembling those in humans, which can be targeted to specific aging pathways conserved in vertebrates. However, no zebrafish models bearing human premature senescence currently exist. We describe the induction of embryonic senescence and laminopathies in zebrafish harboring disturbed expressions of the lamin A gene (LMNA). Impairments in these fish arise in the skin, muscle and adipose tissue, and sometimes in the cartilage. Reduced function of lamin A/C by translational blocking of the LMNA gene induced apoptosis, cell-cycle arrest, and craniofacial abnormalities/cartilage defects. By contrast, induced cryptic splicing of LMNA, which generates the deletion of 8 amino acid residues lamin A (zlamin A-Δ8), showed embryonic senescence and S-phase accumulation/arrest. Interestingly, the abnormal muscle and lipodystrophic phenotypes were common in both cases. Hence, both decrease-of-function of lamin A/C and gain-of-function of aberrant lamin A protein induced laminopathies that are associated with mesenchymal cell lineages during zebrafish early development. Visualization of individual cells expressing zebrafish progerin (zProgerin/zlamin A-Δ37) fused to green fluorescent protein further revealed misshapen nuclear membrane. A farnesyltransferase inhibitor reduced these nuclear abnormalities and significantly prevented embryonic senescence and muscle fiber damage induced by zProgerin. Importantly, the adult Progerin fish survived and remained fertile with

  3. Reproductive toxicity of azoxystrobin to adult zebrafish (Danio rerio).

    PubMed

    Cao, Fangjie; Zhu, Lizhen; Li, Hui; Yu, Song; Wang, Chengju; Qiu, Lihong

    2016-12-01

    In the past few decades, extensive application of azoxystrobin has led to great concern regarding its adverse effects on aquatic organisms. The objective of the present study was to evaluate the reproductive toxicity of azoxystrobin to zebrafish. After adult zebrafish of both sexes were exposed to 2, 20 and 200 μg/L azoxystrobin for 21 days, egg production, the fertilization rate, the gonadosomatic index (GSI) and hepatosomatic index (HSI), 17β-estradiol (E2), testosterone (T) and vitellogenin (Vtg) concentrations, and histological alterations in the gonads and livers were measured. Meanwhile, expression alterations of genes encoding gonadotropins and gonadotropin receptors (fshb, lhb, fshr and lhr), steroid hormone receptors (era, er2b and ar), steroidogenic enzymes (cyp11a, cyp11b, cyp17, cyp19a, cyp19b, hsd3b and hsd17b) in the hypothalamic-pituitary-gonad (HPG) axis and vitellogenin (vtg1 and vtg2) in the livers were also investigated. The results showed that reduced egg production and fertilization rates were observed at 200 μg/L azoxystrobin. In female zebrafish, reduced E2 and Vtg concentrations, decreased GSI, increased T concentrations, and histological alterations in the ovaries and livers were observed at 200 μg/L azoxystrobin, along with significant down-regulation of lhb, cyp19b, lhr, cyp19a, vtg1 and vtg2, and up-regulation of cyp17, hsd3b and hsd17b. In male zebrafish, increased E2 and Vtg concentrations, reduced T concentration and GSI, and histological alterations in the testes and livers were observed after exposure to 20 and 200 μg/L azoxystrobin, along with significant up-regulations of cyp19b, cyp11a, cyp17, cyp19a, hsd3b and hsd17b, vtg1 and vtg2. Moreover, cyp11a, hsd3b, cyp19a, vtg1 and vtg2 in male zebrafish were significantly up-regulated after treatment with 2 μg/L azoxystrobin. The results of the present study indicate that azoxystrobin led to reproductive toxicity in zebrafish and male zebrafish were more sensitive to

  4. Methods for studying the zebrafish brain: past, present and future.

    PubMed

    Wyatt, Cameron; Bartoszek, Ewelina M; Yaksi, Emre

    2015-07-01

    The zebrafish (Danio rerio) is one of the most promising new model organisms. The increasing popularity of this amazing small vertebrate is evident from the exponentially growing numbers of research articles, funded projects and new discoveries associated with the use of zebrafish for studying development, brain function, human diseases and screening for new drugs. Thanks to the development of novel technologies, the range of zebrafish research is constantly expanding with new tools synergistically enhancing traditional techniques. In this review we will highlight the past and present techniques which have made, and continue to make, zebrafish an attractive model organism for various fields of biology, with a specific focus on neuroscience. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  5. Optimizing multi-dimensional high throughput screening using zebrafish

    PubMed Central

    Truong, Lisa; Bugel, Sean M.; Chlebowski, Anna; Usenko, Crystal Y.; Simonich, Michael T.; Massey Simonich, Staci L.; Tanguay, Robert L.

    2016-01-01

    The use of zebrafish for high throughput screening (HTS) for chemical bioactivity assessments is becoming routine in the fields of drug discovery and toxicology. Here we report current recommendations from our experiences in zebrafish HTS. We compared the effects of different high throughput chemical delivery methods on nominal water concentration, chemical sorption to multi-well polystyrene plates, transcription responses, and resulting whole animal responses. We demonstrate that digital dispensing consistently yields higher data quality and reproducibility compared to standard plastic tip-based liquid handling. Additionally, we illustrate the challenges in using this sensitive model for chemical assessment when test chemicals have trace impurities. Adaptation of these better practices for zebrafish HTS should increase reproducibility across laboratories. PMID:27453428

  6. Behavioral effects of MDMA ('ecstasy') on adult zebrafish.

    PubMed

    Stewart, Adam; Riehl, Russell; Wong, Keith; Green, Jeremy; Cosgrove, Jessica; Vollmer, Karoly; Kyzar, Evan; Hart, Peter; Allain, Alexander; Cachat, Jonathan; Gaikwad, Siddharth; Hook, Molly; Rhymes, Kate; Newman, Alan; Utterback, Eli; Chang, Katie; Kalueff, Allan V

    2011-06-01

    3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') is a potent psychedelic drug inducing euphoria and hypersociability in humans, as well as hyperactivity and anxiety in rodents. Adult zebrafish (Danio rerio) have become a widely used species in neurobehavioral research. Here, we explore the effects of a wide range (0.25-120 mg/l) of acute MDMA doses on zebrafish behavior in the novel tank test. Although MDMA was inactive at lower doses (0.25-10 mg/l), higher doses reduced bottom swimming and immobility (40-120 mg/l) and impaired intrasession habituation (10-120 mg/l). MDMA also elevated brain c-fos expression, collectively confirming the usage of zebrafish models for screening of hallucinogenic compounds.

  7. An Individual-Based Model of Zebrafish Population Dynamics Accounting for Energy Dynamics

    PubMed Central

    Beaudouin, Rémy; Goussen, Benoit; Piccini, Benjamin; Augustine, Starrlight; Devillers, James; Brion, François; Péry, Alexandre R. R.

    2015-01-01

    Developing population dynamics models for zebrafish is crucial in order to extrapolate from toxicity data measured at the organism level to biological levels relevant to support and enhance ecological risk assessment. To achieve this, a dynamic energy budget for individual zebrafish (DEB model) was coupled to an individual based model of zebrafish population dynamics (IBM model). Next, we fitted the DEB model to new experimental data on zebrafish growth and reproduction thus improving existing models. We further analysed the DEB-model and DEB-IBM using a sensitivity analysis. Finally, the predictions of the DEB-IBM were compared to existing observations on natural zebrafish populations and the predicted population dynamics are realistic. While our zebrafish DEB-IBM model can still be improved by acquiring new experimental data on the most uncertain processes (e.g. survival or feeding), it can already serve to predict the impact of compounds at the population level. PMID:25938409

  8. Teratogenic Potential of Antiepileptic Drugs in the Zebrafish Model

    PubMed Central

    Lee, Sung Hak; Kang, Jung Won; Lin, Tao; Lee, Jae Eun; Jin, Dong Il

    2013-01-01

    The zebrafish model is an attractive candidate for screening of developmental toxicity during early drug development. Antiepileptic drugs (AEDs) arouse concern for the risk of teratogenicity, but the data are limited. In this study, we evaluated the teratogenic potential of seven AEDs (carbamazepine (CBZ), ethosuximide (ETX), valproic acid (VPN), lamotrigine (LMT), lacosamide (LCM), levetiracetam (LVT), and topiramate (TPM)) in the zebrafish model. Zebrafish embryos were exposed to AEDs from initiation of gastrula (5.25 hours post-fertilization (hpf)) to termination of hatching (72 hpf) which mimic the mammalian teratogenic experimental design. The lethality and teratogenic index (TI) of AEDs were determined and the TI values of each drug were compared with the US FDA human pregnancy categories. Zebrafish model was useful screening model for teratogenic potential of antiepilepsy drugs and was in concordance with in vivo mammalian data and human clinical data. PMID:24324971

  9. Functional and Structural Characterization of Zebrafish ASC.

    PubMed

    Li, Yajuan; Huang, Yi; Cao, Xiaocong; Yin, Xueying; Jin, Xiangyu; Liu, Sheng; Jiang, Jiansheng; Jiang, Wei; Xiao, Tsan Sam; Zhou, Rongbin; Cai, Gang; Hu, Bing; Jin, Tengchuan

    2018-05-23

    The zebrafish genome encodes homologs for most of the proteins involved in inflammatory pathways; however, the molecular components and activation mechanisms of fish inflammasomes are largely unknown. ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)) is the only adaptor involved in the formation of multiple types of inflammasomes. Here, we demonstrate that zASC is also involved in inflammasome activation in zebrafish. When overexpressed in vitro and in vivo in zebrafish, both the zASC and zASC pyrin domain (PYD) proteins form speck and filament structures. Importantly, the crystal structures of the N-terminal PYD and C-terminal CARD of zebrafish ASC were determined independently as two separate entities fused to maltose-binding protein (MBP). Structure-guided mutagenesis revealed the functional relevance of the PYD hydrophilic surface found in the crystal lattice. Finally, the fish caspase-1 homolog Caspy, but not the caspase-4/11 homolog Caspy2, interacts with zASC through homotypic PYD-PYD interactions, which differ from those in mammals. These observations establish the conserved and unique structural/functional features of the zASC-dependent inflammasome pathway. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  10. Zebrafish embryology and cartilage staining protocols for high school students.

    PubMed

    Emran, Farida; Brooks, Jacqueline M; Zimmerman, Steven R; Johnson, Susan L; Lue, Robert A

    2009-06-01

    The Life Sciences-Howard Hughes Medical Institute Outreach Program at Harvard University supports high school science education by offering an on-campus program for students and their teachers to participate in investigative, hands-on laboratory sessions. The outreach program has recently designed and launched a successful zebrafish embryology protocol that we present here. The main objectives of this protocol are to introduce students to zebrafish as a model research organism and to provide students with direct experience with current techniques used in embryological research. The content of the lab is designed to generate discussions on embryology, genetics, fertilization, natural selection, and animal adaptation. The protocol produces reliable results in a time-efficient manner using a minimum of reagents. The protocol presented here consists of three sections: observations of live zebrafish larvae at different developmental stages, cartilage staining of zebrafish larvae, and a mutant hunt involving identification of two zebrafish mutants (nacre and chokh). Here, we describe the protocol, show the results obtained for each section, and suggest possible alternatives for different lab settings.

  11. Pharmacological analyses of learning and memory in zebrafish (Danio rerio).

    PubMed

    Bailey, Jordan M; Oliveri, Anthony N; Levin, Edward D

    2015-12-01

    Over the last decade, zebrafish (Danio rerio) have become valuable as a complementary model in behavioral pharmacology, opening a new avenue for understanding the relationships between drug action and behavior. This species offers a useful intermediate approach bridging the gap between in vitro studies and traditional mammalian models. Zebrafish offer great advantages of economy compared to their rodent counterparts, their complex brains and behavioral repertoire offer great translational potential relative to in vitro models. The development and validation of a variety of tests to measure behavior, including cognition, in zebrafish have set the stage for the use of this animal for behavioral pharmacology studies. This has led to research into the basic mechanisms of cognitive function as well as screening for potential cognition-improving drug therapies, among other lines of research. As with all models, zebrafish have limitations, which span pharmacokinetic challenges to difficulties quantifying behavior. The use, efficacy and limitations associated with a zebrafish model of cognitive function are discussed in this review, within the context of behavioral pharmacology. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Automated phenotype pattern recognition of zebrafish for high-throughput screening.

    PubMed

    Schutera, Mark; Dickmeis, Thomas; Mione, Marina; Peravali, Ravindra; Marcato, Daniel; Reischl, Markus; Mikut, Ralf; Pylatiuk, Christian

    2016-07-03

    Over the last years, the zebrafish (Danio rerio) has become a key model organism in genetic and chemical screenings. A growing number of experiments and an expanding interest in zebrafish research makes it increasingly essential to automatize the distribution of embryos and larvae into standard microtiter plates or other sample holders for screening, often according to phenotypical features. Until now, such sorting processes have been carried out by manually handling the larvae and manual feature detection. Here, a prototype platform for image acquisition together with a classification software is presented. Zebrafish embryos and larvae and their features such as pigmentation are detected automatically from the image. Zebrafish of 4 different phenotypes can be classified through pattern recognition at 72 h post fertilization (hpf), allowing the software to classify an embryo into 2 distinct phenotypic classes: wild-type versus variant. The zebrafish phenotypes are classified with an accuracy of 79-99% without any user interaction. A description of the prototype platform and of the algorithms for image processing and pattern recognition is presented.

  13. Midline signals regulate retinal neurogenesis in zebrafish.

    PubMed

    Masai, I; Stemple, D L; Okamoto, H; Wilson, S W

    2000-08-01

    In zebrafish, neuronal differentiation progresses across the retina in a pattern that is reminiscent of the neurogenic wave that sweeps across the developing eye in Drosophila. We show that expression of a zebrafish homolog of Drosophila atonal, ath5, sweeps across the eye predicting the wave of neuronal differentiation. By analyzing the regulation of ath5 expression, we have elucidated the mechanisms that regulate initiation and spread of neurogenesis in the retina. ath5 expression is lost in Nodal pathway mutant embryos lacking axial tissues that include the prechordal plate. A likely role for axial tissue is to induce optic stalk cells that subsequently regulate ath5 expression. Our results suggest that a series of inductive events, initiated from the prechordal plate and progressing from the optic stalks, regulates the spread of neuronal differentiation across the zebrafish retina.

  14. Using Zebrafish to Study Podocyte Genesis During Kidney Development and Regeneration

    PubMed Central

    Kroeger, Paul T.; Wingert, Rebecca A.

    2014-01-01

    SUMMARY During development, vertebrates form a progression of up to three different kidneys that are comprised of functional units termed nephrons. Nephron composition is highly conserved across species, and an increasing appreciation of the similarities between zebrafish and mammalian nephron cell types has positioned the zebrafish as a relevant genetic system for nephrogenesis studies. A key component of the nephron blood filter is a specialized epithelial cell known as the podocyte. Podocyte research is of the utmost importance as a vast majority of renal diseases initiate with the dysfunction or loss of podocytes, resulting in a condition known as proteinuria that causes nephron degeneration and eventually leads to kidney failure. Understanding how podocytes develop during organogenesis may elucidate new ways to promote nephron health by stimulating podocyte replacement in kidney disease patients. In this review, we discuss how the zebrafish model can be used to study kidney development, and how zebrafish research has provided new insights into podocyte lineage specification and differentiation. Further, we discuss the recent discovery of podocyte regeneration in adult zebrafish, and explore how continued basic research using zebrafish can provide important knowledge about podocyte genesis in embryonic and adult environments. PMID:24920186

  15. Functional Development of the Circadian Clock in the Zebrafish Pineal Gland

    PubMed Central

    Ben-Moshe, Zohar; Foulkes, Nicholas S.

    2014-01-01

    The zebrafish constitutes a powerful model organism with unique advantages for investigating the vertebrate circadian timing system and its regulation by light. In particular, the remarkably early and rapid development of the zebrafish circadian system has facilitated exploring the factors that control the onset of circadian clock function during embryogenesis. Here, we review our understanding of the molecular basis underlying functional development of the central clock in the zebrafish pineal gland. Furthermore, we examine how the directly light-entrainable clocks in zebrafish cell lines have facilitated unravelling the general mechanisms underlying light-induced clock gene expression. Finally, we summarize how analysis of the light-induced transcriptome and miRNome of the zebrafish pineal gland has provided insight into the regulation of the circadian system by light, including the involvement of microRNAs in shaping the kinetics of light- and clock-regulated mRNA expression. The relative contributions of the pineal gland central clock and the distributed peripheral oscillators to the synchronization of circadian rhythms at the whole animal level are a crucial question that still remains to be elucidated in the zebrafish model. PMID:24839600

  16. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human.

    PubMed

    Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

    2016-04-13

    Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish.

  17. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human

    PubMed Central

    Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

    2016-01-01

    Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish. PMID:27071722

  18. Glutathione S-Transferase Protein Expression in Different Life Stages of Zebrafish (Danio rerio)

    PubMed Central

    Tierbach, Alena; Groh, Ksenia J; Schönenberger, René; Schirmer, Kristin

    2018-01-01

    Abstract Zebrafish is a widely used animal model in biomedical sciences and toxicology. Although evidence for the presence of phases I and II xenobiotic defense mechanisms in zebrafish exists on the transcriptional and enzyme activity level, little is known about the protein expression of xenobiotic metabolizing enzymes. Given the important role of glutathione S-transferases (GSTs) in phase II biotransformation, we analyzed cytosolic GST proteins in zebrafish early life stages and different organs of adult male and female fish, using a targeted proteomics approach. The established multiple reaction monitoring-based assays enable the measurement of the relative abundance of specific GST isoenzymes and GST classes in zebrafish through a combination of proteotypic peptides and peptides shared within the same class. GSTs of the classes alpha, mu, pi and rho are expressed in zebrafish embryo as early as 4 h postfertilization (hpf). The majority of GST enzymes are present at 72 hpf followed by a continuous increase in expression thereafter. In adult zebrafish, GST expression is organ dependent, with most of the GST classes showing the highest expression in the liver. The expression of a wide range of cytosolic GST isoenzymes and classes in zebrafish early life stages and adulthood supports the use of zebrafish as a model organism in chemical-related investigations. PMID:29361160

  19. Short-term developmental effects and potential mechanisms of azoxystrobin in larval and adult zebrafish (Danio rerio).

    PubMed

    Cao, Fangjie; Wu, Peizhuo; Huang, Lan; Li, Hui; Qian, Le; Pang, Sen; Qiu, Lihong

    2018-05-01

    Previous study indicated that azoxystrobin had high acute toxicity to zebrafish, and larval zebrafish were more sensitive to azoxystrobin than adult zebrafish. The objective of the present study was to investigate short-term developmental effects and potential mechanisms of azoxystrobin in larval and adult zebrafish. After zebrafish embryos and adults were exposed to 0.01, 0.05 and 0.20 mg/L azoxystrobin (equal to 25, 124 and 496 nM azoxystrobin, respectively) for 8 days, the lethal effect, physiological responses, liver histology, mitochondrial ultrastructure, and expression alteration of genes related to mitochondrial respiration, oxidative stress, cell apoptosis and innate immune response were determined. The results showed that there was no significant effect on larval and adult zebrafish after exposure to 0.01 mg/L azoxystrobin. However, increased ROS, MDA concentration and il1b in larval zebrafish, as well as increased il1b, il8 and cxcl-c1c in adult zebrafish were induced after exposure to 0.05 mg/L azoxystrobin. Reduced mitochondrial complex III activity and ATP concentration, increased SOD activity, ROS and MDA concentration, decreased cytb, as well as increased sod1, sod2, cat, il1b, il8 and cxcl-c1c were observed both in larval and adult zebrafish after exposure to 0.20 mg/L azoxystrobin; meanwhile, increased p53, bax, apaf1 and casp9, alteration of liver histology and mitochondrial ultrastructure in larval zebrafish, and alteration of mitochondrial ultrastructure in adult zebrafish were also induced. The results demonstrated that azoxytrobin induced short-term developmental effects on larval zebrafish and adult zebrafish, including mitochondrial dysfunction, oxidative stress, cell apoptosis and innate immune response. Statistical analysis indicated that azoxystrobin induced more negative effects on larval zebrafish, which might be the reason for the differences of developmental toxicity between larval and adult zebrafish caused by

  20. Preparing for a Semiannual IACUC Inspection of a Satellite Zebrafish (Danio rerio) Facility

    PubMed Central

    Koerber, Amy S; Kalishman, Jennifer

    2009-01-01

    Institutions worldwide have experienced a rapid growth in the use of zebrafish as a research model for a variety of molecular and genetic studies of vertebrate development. This expansion in zebrafish research essentially has outpaced the establishment of specific recommendations for the care and use of fish in research. In some cases, this situation has created a dilemma where an Institutional Animal Care and Use Committee, which is responsible for oversight of vertebrate animal research, is not fully prepared to undertake this role for a decentralized zebrafish facility. IACUC inspectors will be more equipped to ask pertinent questions by understanding the basic principles of zebrafish health and facility management. Concurrently, zebrafish facility managers can contribute to the progress of a semiannual facility inspection by maintaining fully accessible operating records. In the context of presenting a well-established and useful model of zebrafish management and recordkeeping to the zebrafish facility operator, the information we present here also prepares a potential IACUC inspector to conduct a constructive and positive inspection. PMID:19245754

  1. DRUG EFFECTS ON THE LOCOMOTOR ACTIVITY OF LARVAL ZEBRAFISH.

    EPA Science Inventory

    As part of an effort to develop a rapid in vivo screen for EPA’s prioritization of toxic chemicals, we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae and the effects of prototype drugs. Zebrafish larvae (6-7 days post-fertilization) were indiv...

  2. Regulatory effect of hydroquinone-tetraethylene glycol conjugates on zebrafish pigmentation.

    PubMed

    Le, Hoa Thi; Hong, Bin Na; Lee, Yeong Ro; Cheon, Ji Hyun; Kang, Tong Ho; Kim, Tae Woo

    2016-01-15

    We synthesized two hydroquinone-tetraethylene glycol conjugates (HQ-TGs) and investigated their logP, photophysical stability, and redox chemical stability. HQ-TGs are a little more hydrophilic than hydroquinone (HQ) and show an enhanced photophysical and redox chemical stability compared with HQ. In addition we studied the effect of HQ-TGs on cell viability and on zebrafish pigmentation. MTT assay in HF-16 cells showed HQ-TGs are less cytotoxic than HQ. The phenotype-based image analysis of zebrafish larvae suggests that HQ-TGs suppress the pigmentation of zebrafish in a dose-dependent manner. The comparative experiments on stability, cytotoxicity, and zebrafish pigmentation between HQ and HQ-TGs suggest that mono tetraethylene glycol-functionalization of HQ is an alternative solution to overcome the adverse effect of HQ. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Can zebrafish be used as animal model to study Alzheimer's disease?

    PubMed Central

    Santana, Soraya; Rico, Eduardo P; Burgos, Javier S

    2012-01-01

    Zebrafish is rapidly emerging as a promising model organism to study various central nervous system (CNS) disorders, including Alzheimer’s disease (AD). AD is the main cause of dementia in the human population and there is an urgency to understand the causes of this neurodegenerative disease. In this respect, the development of new animal models to study the underlying neurodegenerative mechanisms of AD is an urgent need. In this review we analyze the current situation in the use of zebrafish as a model for AD, discussing the reasons to use this experimental paradigm in CNS investigation and analyzing the several strategies adopted to induce an AD-like pathology in zebrafish. We discuss the strategies of performing interventions to cause damage in the zebrafish brain by altering the major neurotransmitter systems (such as cholinergic, glutamatergic or GABAergic circuits). We also analyze the several transgenic zebrafish constructed for the AD study, discussing both the familial-AD models based on APP processing pathway (APP and presenilins) and in the TAU hyperphosphorylation, together with the genes involved in sporadic-AD, as apolipoprotein E. We conclude that zebrafish is in a preliminary stage of development in the AD field, and that the transgenic animals must be improved to use this fish as an optimal model for AD research. Furthermore, a deeper knowledge of the zebrafish brain and a better characterization of the injury caused by alterations in the major neurotransmitter systems are needed. PMID:23383380

  4. Lxr regulates lipid metabolic and visual perception pathways during zebrafish development

    PubMed Central

    Pinto, Caroline Lucia; Kalasekar, Sharanya Maanasi; McCollum, Catherine W.; Riu, Anne; Jonsson, Philip; Lopez, Justin; Swindell, Eric; Bouhlatouf, Abdel; Balaguer, Patrick; Bondesson, Maria; Gustafsson, Jan-Åke

    2015-01-01

    The Liver X Receptors (LXRs) play important roles in multiple metabolic pathways, including fatty acid, cholesterol, carbohydrate and energy metabolism. To expand the knowledge of the functions of LXR signaling during embryonic development, we performed a whole-genome microarray analysis of Lxr target genes in zebrafish larvae treated with either one of the synthetic LXR ligands T0901317 or GW3965. Assessment of the biological processes enriched by differentially expressed genes revealed a prime role for Lxr in regulating lipid metabolic processes, similarly to the function of LXR in mammals. In addition, exposure to the Lxr ligands induced changes in expression of genes in the neural retina and lens of the zebrafish eye, including the photoreceptor guanylate cyclase activators and lens gamma crystallins, suggesting a potential novel role for Lxr in modulating the transcription of genes associated with visual function in zebrafish. The regulation of expression of metabolic genes was phenotypically reflected in an increased absorption of yolk in the zebrafish larvae, and changes in the expression of genes involved in visual perception were associated with morphological alterations in the retina and lens of the developing zebrafish eye. The regulation of expression of both lipid metabolic and eye specific genes was sustained in 1 month old fish. The transcriptional networks demonstrated several conserved effects of LXR activation between zebrafish and mammals, and also identified potential novel functions of Lxr, supporting zebrafish as a promising model for investigating the role of Lxr during development. PMID:26427652

  5. Correlation between photoreceptor injury-regeneration and behavior in a zebrafish model.

    PubMed

    Wang, Ya-Jie; Cai, Shi-Jiao; Cui, Jian-Lin; Chen, Yang; Tang, Xin; Li, Yu-Hao

    2017-05-01

    Direct exposure to intensive visible light can lead to solar retinopathy, including macular injury. The signs and symptoms include central scotoma, metamorphopsia, and decreased vision. However, there have been few studies examining retinal injury due to intensive light stimulation at the cellular level. Neural network arrangements and gene expression patterns in zebrafish photoreceptors are similar to those observed in humans, and photoreceptor injury in zebrafish can induce stem cell-based cellular regeneration. Therefore, the zebrafish retina is considered a useful model for studying photoreceptor injury in humans. In the current study, the central retinal photoreceptors of zebrafish were selectively ablated by stimulation with high-intensity light. Retinal injury, cell proliferation and regeneration of cones and rods were assessed at 1, 3 and 7 days post lesion with immunohistochemistry and in situ hybridization. Additionally, a light/dark box test was used to assess zebrafish behavior. The results revealed that photoreceptors were regenerated by 7 days after the light-induced injury. However, the regenerated cells showed a disrupted arrangement at the lesion site. During the injury-regeneration process, the zebrafish exhibited reduced locomotor capacity, weakened phototaxis and increased movement angular velocity. These behaviors matched the morphological changes of retinal injury and regeneration in a number of ways. This study demonstrates that the zebrafish retina has a robust capacity for regeneration. Visual impairment and stress responses following high-intensity light stimulation appear to contribute to the alteration of behaviors.

  6. Antibiotic toxicity and absorption in zebrafish using liquid chromatography-tandem mass spectrometry.

    PubMed

    Zhang, Fan; Qin, Wei; Zhang, Jing-Pu; Hu, Chang-Qin

    2015-01-01

    Evaluation of drug toxicity is necessary for drug safety, but in vivo drug absorption is varied; therefore, a rapid, sensitive and reliable method for measuring drugs is needed. Zebrafish are acceptable drug toxicity screening models; we used these animals with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method in a multiple reaction monitoring mode to quantify drug uptake in zebrafish to better estimate drug toxicity. Analytes were recovered from zebrafish homogenate by collecting supernatant. Measurements were confirmed for drugs in the range of 10-1,000 ng/mL. Four antibiotics with different polarities were tested to explore any correlation of drug polarity, absorption, and toxicity. Zebrafish at 3 days post-fertilization (dpf) absorbed more drug than those at 6 h post-fertilization (hpf), and different developmental periods appeared to be differentially sensitive to the same compound. By observing abnormal embryos and LD50 values, zebrafish embryos at 6 hpf were considered to be suitable for evaluating embryotoxicity. Also, larvae at 3 dpf were adapted to measure acute drug toxicity in adult mammals. Thus, we can exploit zebrafish to study drug toxicity and can reliably quantify drug uptake with LC-MS/MS. This approach will be helpful for future studies of toxicology in zebrafish.

  7. Evidence for a core gut microbiota in the zebrafish

    PubMed Central

    Roeselers, Guus; Mittge, Erika K; Stephens, W Zac; Parichy, David M; Cavanaugh, Colleen M; Guillemin, Karen; Rawls, John F

    2011-01-01

    Experimental analysis of gut microbial communities and their interactions with vertebrate hosts is conducted predominantly in domesticated animals that have been maintained in laboratory facilities for many generations. These animal models are useful for studying coevolved relationships between host and microbiota only if the microbial communities that occur in animals in lab facilities are representative of those that occur in nature. We performed 16S rRNA gene sequence-based comparisons of gut bacterial communities in zebrafish collected recently from their natural habitat and those reared for generations in lab facilities in different geographic locations. Patterns of gut microbiota structure in domesticated zebrafish varied across different lab facilities in correlation with historical connections between those facilities. However, gut microbiota membership in domesticated and recently caught zebrafish was strikingly similar, with a shared core gut microbiota. The zebrafish intestinal habitat therefore selects for specific bacterial taxa despite radical differences in host provenance and domestication status. PMID:21472014

  8. Zebrafish models of human eye and inner ear diseases.

    PubMed

    Blanco-Sánchez, B; Clément, A; Phillips, J B; Westerfield, M

    2017-01-01

    Eye and inner ear diseases are the most common sensory impairments that greatly impact quality of life. Zebrafish have been intensively employed to understand the fundamental mechanisms underlying eye and inner ear development. The zebrafish visual and vestibulo-acoustic systems are very similar to these in humans, and although not yet mature, they are functional by 5days post-fertilization (dpf). In this chapter, we show how the zebrafish has significantly contributed to the field of biomedical research and how researchers, by establishing disease models and meticulously characterizing their phenotypes, have taken the first steps toward therapies. We review here models for (1) eye diseases, (2) ear diseases, and (3) syndromes affecting eye and/or ear. The use of new genome editing technologies and high-throughput screening systems should increase considerably the speed at which knowledge from zebrafish disease models is acquired, opening avenues for better diagnostics, treatments, and therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. A bioenergetic model for zebrafish Danio rerio (Hamilton)

    USGS Publications Warehouse

    Chizinski, C.J.; Sharma, Bibek; Pope, K.L.; Patino, R.

    2008-01-01

    A bioenergetics model was developed from observed consumption, respiration and growth rates for zebrafish Danio rerio across a range (18-32?? C) of water temperatures, and evaluated with a 50 day laboratory trial at 28?? C. No significant bias in variable estimates was found during the validation trial; namely, predicted zebrafish mass generally agreed with observed mass. ?? 2008 The Authors.

  10. Determining Zebrafish Epitope Reactivity to Commercially Available Antibodies.

    PubMed

    Villarreal, Michael A; Biediger, Nicole M; Bonner, Natalie A; Miller, Jennifer N; Zepeda, Samantha K; Ricard, Benjamin J; García, Dana M; Lewis, Karen A

    2017-08-01

    Antibodies raised against mammalian proteins may exhibit cross-reactivity with zebrafish proteins, making these antibodies useful for fish studies. However, zebrafish may express multiple paralogues of similar sequence and size, making them difficult to distinguish by traditional Western blot analysis. To identify the zebrafish proteins that are recognized by an antimammalian antibody, we developed a system to screen putative epitopes by cloning the sequences between the yeast SUMO protein and a C-terminal 6xHis tag. The recombinant fusion protein was expressed in Escherichia coli and analyzed by Western blot to conclusively identify epitopes that exhibit cross-reactivity with the antibodies of interest. This approach can be used to determine the species cross-reactivity and epitope specificity of a wide variety of peptide antigen-derived antibodies.

  11. Optimizing multi-dimensional high throughput screening using zebrafish.

    PubMed

    Truong, Lisa; Bugel, Sean M; Chlebowski, Anna; Usenko, Crystal Y; Simonich, Michael T; Simonich, Staci L Massey; Tanguay, Robert L

    2016-10-01

    The use of zebrafish for high throughput screening (HTS) for chemical bioactivity assessments is becoming routine in the fields of drug discovery and toxicology. Here we report current recommendations from our experiences in zebrafish HTS. We compared the effects of different high throughput chemical delivery methods on nominal water concentration, chemical sorption to multi-well polystyrene plates, transcription responses, and resulting whole animal responses. We demonstrate that digital dispensing consistently yields higher data quality and reproducibility compared to standard plastic tip-based liquid handling. Additionally, we illustrate the challenges in using this sensitive model for chemical assessment when test chemicals have trace impurities. Adaptation of these better practices for zebrafish HTS should increase reproducibility across laboratories. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Zebrafish models of cardiovascular diseases and their applications in herbal medicine research.

    PubMed

    Seto, Sai-Wang; Kiat, Hosen; Lee, Simon M Y; Bensoussan, Alan; Sun, Yu-Ting; Hoi, Maggie P M; Chang, Dennis

    2015-12-05

    The zebrafish (Danio rerio) has recently become a powerful animal model for cardiovascular research and drug discovery due to its ease of maintenance, genetic manipulability and ability for high-throughput screening. Recent advances in imaging techniques and generation of transgenic zebrafish have greatly facilitated in vivo analysis of cellular events of cardiovascular development and pathogenesis. More importantly, recent studies have demonstrated the functional similarity of drug metabolism systems between zebrafish and humans, highlighting the clinical relevance of employing zebrafish in identifying lead compounds in Chinese herbal medicine with potential beneficial cardiovascular effects. This paper seeks to summarise the scope of zebrafish models employed in cardiovascular studies and the application of these research models in Chinese herbal medicine to date. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

  13. In-silico experiments of zebrafish behaviour: modeling swimming in three dimensions

    NASA Astrophysics Data System (ADS)

    Mwaffo, Violet; Butail, Sachit; Porfiri, Maurizio

    2017-01-01

    Zebrafish is fast becoming a species of choice in biomedical research for the investigation of functional and dysfunctional processes coupled with their genetic and pharmacological modulation. As with mammals, experimentation with zebrafish constitutes a complicated ethical issue that calls for the exploration of alternative testing methods to reduce the number of subjects, refine experimental designs, and replace live animals. Inspired by the demonstrated advantages of computational studies in other life science domains, we establish an authentic data-driven modelling framework to simulate zebrafish swimming in three dimensions. The model encapsulates burst-and-coast swimming style, speed modulation, and wall interaction, laying the foundations for in-silico experiments of zebrafish behaviour. Through computational studies, we demonstrate the ability of the model to replicate common ethological observables such as speed and spatial preference, and anticipate experimental observations on the correlation between tank dimensions on zebrafish behaviour. Reaching to other experimental paradigms, our framework is expected to contribute to a reduction in animal use and suffering.

  14. The Study of Glioma by Xenotransplantation in Zebrafish Early Life Stages

    PubMed Central

    Motaln, Helena; Turnšek, Tamara Lah

    2015-01-01

    Zebrafish (Danio rerio) and their transparent embryos are becoming an increasingly popular tool for studying processes involved in tumor progression and in the search for novel tumor treatment approaches. The xenotransplantation of fluorescently labeled mammalian cancer cells into zebrafish embryos is an approach enabling relatively high-throughput in vivo analyses. The small size of the embryos as well as the relative simplicity of their manipulation and maintenance allow for large numbers of embryos to be processed efficiently in a short time and at low cost. Furthermore, the possibility of fluorescence microscopic imaging of tumor progression within zebrafish embryos and larvae holds unprecedented potential for the real-time visualization of these processes in vivo. This review presents the methodologies of xenotransplantation studies on zebrafish involving research on tumor invasion, proliferation, tumor-induced angiogenesis and screening for antitumor therapeutics. We further focus on the application of these zebrafish to the study of glioma; in particular, its most common and malignant form, glioblastoma. PMID:26109632

  15. In-silico experiments of zebrafish behaviour: modeling swimming in three dimensions

    PubMed Central

    Mwaffo, Violet; Butail, Sachit; Porfiri, Maurizio

    2017-01-01

    Zebrafish is fast becoming a species of choice in biomedical research for the investigation of functional and dysfunctional processes coupled with their genetic and pharmacological modulation. As with mammals, experimentation with zebrafish constitutes a complicated ethical issue that calls for the exploration of alternative testing methods to reduce the number of subjects, refine experimental designs, and replace live animals. Inspired by the demonstrated advantages of computational studies in other life science domains, we establish an authentic data-driven modelling framework to simulate zebrafish swimming in three dimensions. The model encapsulates burst-and-coast swimming style, speed modulation, and wall interaction, laying the foundations for in-silico experiments of zebrafish behaviour. Through computational studies, we demonstrate the ability of the model to replicate common ethological observables such as speed and spatial preference, and anticipate experimental observations on the correlation between tank dimensions on zebrafish behaviour. Reaching to other experimental paradigms, our framework is expected to contribute to a reduction in animal use and suffering. PMID:28071731

  16. Behavioral and Molecular Analysis of Nicotine-Conditioned Place Preference in Zebrafish

    PubMed Central

    Kedikian, Ximena; Faillace, Maria Paula; Bernabeu, Ramón

    2013-01-01

    Studies using mice and rats have demonstrated that nicotine induces a conditioned place preference (CPP), with more effective results obtained by using biased procedures. Zebrafish have also been used as a model system to identify factors influencing nicotine-associated reward by using an unbiased design. Here, we report that zebrafish exhibited putative nicotine biased CPP to an initially aversive compartment (nicotine-paired group). A counterbalanced nicotine-exposed control group did not show a significant preference shift, providing evidence that the preference shift in the nicotine-paired group was not due to a reduction of aversion for this compartment. Zebrafish preference was corroborated by behavioral analysis of several indicators of drug preference, such as time spent in the drug-paired side, number of entries to the drug-paired side, and distance traveled. These results provided strong evidence that zebrafish may actually develop a preference for nicotine, although the drug was administrated in an aversive place for the fish, which was further supported by molecular studies. Reverse transcription-quantitative real-time PCR analysis depicted a significant increase in the expression of α7 and α6 but not α4 and β2 subunits of the nicotinic receptor in nicotine-paired zebrafish brains. In contrast, zebrafish brains from the counterbalanced nicotine group showed no significant changes. Moreover, CREB phosphorylation, an indicator of neural activity, accompanied the acquisition of nicotine-CPP. Our studies offered an incremental value to the drug addiction field, because they further describe behavioral features of CPP to nicotine in zebrafish. The results suggested that zebrafish exposed to nicotine in an unfriendly environment can develop a preference for that initially aversive place, which is likely due to the rewarding effect of nicotine. Therefore, this model can be used to screen exogenous and endogenous molecules involved in nicotine

  17. Anxiolytic-like effects of noribogaine in zebrafish.

    PubMed

    Kalueff, Allan V; Kaluyeva, Aleksandra; Maillet, Emeline L

    2017-07-14

    Noribogaine is the main psychoactive metabolite of the hallucinogenic drug ibogaine, and is a particularly interesting compound potentially useful to treat dependence and various psychiatric disorders. Here, we report the effects of noribogaine on anxiety and locomotion in zebrafish (Danio rerio), a new promising model organism in neurobehavioral and psychopharmacological research. Adult zebrafish were subjected to the 5min novel tank test (NTT) following an acute, 20-min drug immersion in 1, 5 and 10mg/L noribogaine. Overall, noribogaine produced robust anxiolytic-like behavior in zebrafish (increasing the time spent and transitions to the top half compartment and reducing freezing bouts) without overt effects on fish locomotion. Taken together, these results indicate that noribogaine modulates the components of the acute stress response related to emotionality and anxiety behaviors, implicating this drug as a potentially useful non-sedative anxiolytic agent. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. The photoreceptive cells of the pineal gland in adult zebrafish (Danio rerio).

    PubMed

    Laurà, Rosaria; Magnoli, Domenico; Zichichi, Rosalia; Guerrera, Maria Cristina; De Carlos, Felix; Suárez, Alberto Álvarez; Abbate, Francesco; Ciriaco, Emilia; Vega, Jose Antonio; Germanà, Antonino

    2012-03-01

    The zebrafish pineal gland plays a fundamental role in the regulation of the circadian rhythm through the melatonin secretion. The pinealocytes, also called photoreceptive cells, are considered the morphofunctional unit of pineal gland. In literature, the anatomical features, the cellular characteristics, and the pinealocytes morphology of zebrafish pineal gland have not been previously described in detail. Therefore, this study was undertaken to analyze the structure and ultrastructure, as well as the immunohistochemical profile of the zebrafish pineal gland with particular reference to the pinealocytes. Here, we demonstrated, using RT-PCR, immunohistochemistry and transmission electron microscopy, the expression of the mRNA for rhodopsin in the pineal gland of zebrafish, as well as its cellular localization exclusively in the pinealocytes of adult zebrafish. Moreover, the ultrastructural observations demonstrated that the pinealocytes were constituted by an outer segment with numerous lamellar membranes, an inner segment with many mitochondria, and a basal pole with the synapses. Our results taken together demonstrated a central role of zebrafish pinealocytes in the control of pineal gland functions. Copyright © 2011 Wiley Periodicals, Inc.

  19. Graph theoretical model of a sensorimotor connectome in zebrafish.

    PubMed

    Stobb, Michael; Peterson, Joshua M; Mazzag, Borbala; Gahtan, Ethan

    2012-01-01

    Mapping the detailed connectivity patterns (connectomes) of neural circuits is a central goal of neuroscience. The best quantitative approach to analyzing connectome data is still unclear but graph theory has been used with success. We present a graph theoretical model of the posterior lateral line sensorimotor pathway in zebrafish. The model includes 2,616 neurons and 167,114 synaptic connections. Model neurons represent known cell types in zebrafish larvae, and connections were set stochastically following rules based on biological literature. Thus, our model is a uniquely detailed computational representation of a vertebrate connectome. The connectome has low overall connection density, with 2.45% of all possible connections, a value within the physiological range. We used graph theoretical tools to compare the zebrafish connectome graph to small-world, random and structured random graphs of the same size. For each type of graph, 100 randomly generated instantiations were considered. Degree distribution (the number of connections per neuron) varied more in the zebrafish graph than in same size graphs with less biological detail. There was high local clustering and a short average path length between nodes, implying a small-world structure similar to other neural connectomes and complex networks. The graph was found not to be scale-free, in agreement with some other neural connectomes. An experimental lesion was performed that targeted three model brain neurons, including the Mauthner neuron, known to control fast escape turns. The lesion decreased the number of short paths between sensory and motor neurons analogous to the behavioral effects of the same lesion in zebrafish. This model is expandable and can be used to organize and interpret a growing database of information on the zebrafish connectome.

  20. Expression of sall4 in taste buds of zebrafish.

    PubMed

    Jackson, Robyn; Braubach, Oliver R; Bilkey, Jessica; Zhang, Jing; Akimenko, Marie-Andrée; Fine, Alan; Croll, Roger P; Jonz, Michael G

    2013-07-01

    We characterized the expression of sall4, a gene encoding a zinc finger transcription factor involved in the maintenance of embryonic stem cells, in taste buds of zebrafish (Danio rerio). Using an enhancer trap line (ET5), we detected enhanced green fluorescent protein (EGFP) in developing and adult transgenic zebrafish in regions containing taste buds: the lips, branchial arches, and the nasal and maxillary barbels. Localization of EGFP to taste cells of the branchial arches and lips was confirmed by co-immunolabeling with antibodies against calretinin and serotonin, and a zebrafish-derived neuronal marker (zn-12). Transgenic insertion of the ET construct into the zebrafish genome was evaluated and mapped to chromosome 23 in proximity (i.e. 23 kb) to the sall4 gene. In situ hybridization and expression analysis between 24 and 96 h post-fertilization (hpf) demonstrated that transgenic egfp expression in ET5 zebrafish was correlated with the spatial and temporal pattern of expression of sall4 in the wild-type. Expression was first observed in the central nervous system and branchial arches at 24 hpf. At 48 hpf, sall4 and egfp expression was observed in taste bud primordia surrounding the mouth and branchial arches. At 72 and 96 hpf, expression was detected in the upper and lower lips and branchial arches. Double fluorescence in situ hybridization at 3 and 10 dpf confirmed colocalization of sall4 and egfp in the lips and branchial arches. These studies reveal sall4 expression in chemosensory cells and implicate this transcription factor in the development and renewal of taste epithelia in zebrafish. Copyright © 2013 Wiley Periodicals, Inc.

  1. Zebrafish Discoveries in Cancer Epigenetics.

    PubMed

    Chernyavskaya, Yelena; Kent, Brandon; Sadler, Kirsten C

    2016-01-01

    The cancer epigenome is fundamentally different than that of normal cells. How these differences arise in and contribute to carcinogenesis is not known, and studies using model organisms such as zebrafish provide an opportunity to address these important questions. Modifications of histones and DNA comprise the complex epigenome, and these influence chromatin structure, genome stability and gene expression, all of which are fundamental to the cellular changes that cause cancer. The cancer genome atlas covers the wide spectrum of genetic changes associated with nearly every cancer type, however, this catalog is currently uni-dimensional. As the pattern of epigenetic marks and chromatin structure in cancer cells is described and overlaid on the mutational landscape, the map of the cancer genome becomes multi-dimensional and highly complex. Two major questions remain in the field: (1) how the epigenome becomes repatterned in cancer and (2) which of these changes are cancer-causing. Zebrafish provide a tractable in vivo system to monitor the epigenome during transformation and to identify epigenetic drivers of cancer. In this chapter, we review principles of cancer epigenetics and discuss recent work using zebrafish whereby epigenetic modifiers were established as cancer driver genes, thus providing novel insights into the mechanisms of epigenetic reprogramming in cancer.

  2. Zebrafish antipredatory responses: A future for translational research?

    PubMed Central

    Gerlai, Robert

    2011-01-01

    Human neuropsychiatric conditions associated with abnormally exaggerated or misdirected fear (anxiety disorders and phobias) still represent a large unmet medical need because the biological mechanisms underlying these diseases are not well understood. Animal models have been proposed to facilitate this research. Here I review the literature with a focus on zebrafish, an upcoming laboratory organism in behavioral brain research. I argue that abnormal human fear responses are likely the result of the malfunction of neurobiological mechanisms (brain areas, circuits and/or molecular mechanisms) that originally evolved to support avoidance of predators or other harm in nature. I also argue that the understanding of the normal as well as pathological functioning of such mechanisms may be best achieved if one utilizes naturalistic experimental approaches. In case of laboratory model organisms, this may entail presenting stimuli associated with predators and measuring species-specific antipredatory responses. Although zebrafish is a relatively new subject of such inquiry, I review the recently rapidly increasing number of zebrafish studies in this area, and conclude that zebrafish is a promising research tool for the analysis of the neurobiology and genetics of vertebrate fear responses. PMID:19836422

  3. Egfl6 is involved in zebrafish notochord development.

    PubMed

    Wang, Xueqian; Wang, Xin; Yuan, Wei; Chai, Renjie; Liu, Dong

    2015-08-01

    The epidermal growth factor (EGF) repeat motif defines a superfamily of diverse protein involved in regulating a variety of cellular and physiological processes, such as cell cycle, cell adhesion, proliferation, migration, and neural development. Egfl6, an EGF protein, also named MAGE was first cloned in human tissue. Up to date, the study of zebrafish Egfl6 expression pattern and functional analysis of Egfl6 involved in embryonic development of vertebrate in vivo is thus far lacking. Here we reported that Egfl6 was involved in zebrafish notochord development. It was shown that Egfl6 mRNA was expressed in zebrafish, developing somites, fin epidermis, pharyngeal arches, and hindbrain region. Particularly the secreted Egfl6 protein was significantly accumulated in notochord. Loss of Egfl6 function in zebrafish embryos resulted in curved body with distorted notochord in the posterior trunk. It was observed that expression of all Notch ligand and receptors in notochord of 28 hpf Egfl6 morphants was not affected, except notch2, which was up-regulated. We found that inhibition of Notch signaling by DAPT efficiently rescued notochord developmental defect of Egfl6 deficiency embryos.

  4. Halogenated Bisphenol-A Analogs Act as Obesogens in Zebrafish Larvae (Danio rerio)

    PubMed Central

    Pinto, Caroline L.; Grimaldi, Marina; Hillenweck, Anne; Perdu, Elisabeth; Zalko, Daniel; Bernard, Laure; Laudet, Vincent; Balaguer, Patrick; Bondesson, Maria; Gustafsson, Jan-Ake

    2014-01-01

    Obesity has increased dramatically over the past decades, reaching epidemic proportions. The reasons are likely multifactorial. One of the suggested causes is the accelerated exposure to obesity-inducing chemicals (obesogens). However, out of the tens of thousands of industrial chemicals humans are exposed to, very few have been tested for their obesogenic potential, mostly due to the limited availability of appropriate in vivo screening models. In this study, we investigated whether two commonly used flame retardants, the halogenated bisphenol-A (BPA) analogs tetrabromobisphenol-A (TBBPA) and tetrachlorobisphenol-A (TCBPA), could act as obesogens using zebrafish larvae as an in vivo animal model. The effect of embryonic exposure to these chemicals on lipid accumulation was analyzed by Oil Red-O staining, and correlated to their capacity to activate human and zebrafish peroxisome proliferator-activated receptor gamma (PPARγ) in zebrafish and in reporter cell lines. Then, the metabolic fate of TBBPA and TCBPA in zebrafish larvae was analyzed by high-performance liquid chromatography (HPLC) . TBBPA and TCBPA were readily taken up by the fish embryo and both compounds were biotransformed to sulfate-conjugated metabolites. Both halogenated-BPAs, as well as TBBPA-sulfate induced lipid accumulation in zebrafish larvae. TBBPA and TCBPA also induced late-onset weight gain in juvenile zebrafish. These effects correlated to their capacity to act as zebrafish PPARγ agonists. Screening of chemicals for inherent obesogenic capacities through the zebrafish lipid accumulation model could facilitate prioritizing chemicals for further investigations in rodents, and ultimately, help protect humans from exposure to environmental obesogens. PMID:24591153

  5. Genetic Disruption of 21-Hydroxylase in Zebrafish Causes Interrenal Hyperplasia.

    PubMed

    Eachus, Helen; Zaucker, Andreas; Oakes, James A; Griffin, Aliesha; Weger, Meltem; Güran, Tülay; Taylor, Angela; Harris, Abigail; Greenfield, Andy; Quanson, Jonathan L; Storbeck, Karl-Heinz; Cunliffe, Vincent T; Müller, Ferenc; Krone, Nils

    2017-12-01

    Congenital adrenal hyperplasia is a group of common inherited disorders leading to glucocorticoid deficiency. Most cases are caused by 21-hydroxylase deficiency (21OHD). The systemic consequences of imbalanced steroid hormone biosynthesis due to severe 21OHD remains poorly understood. Therefore, we developed a zebrafish model for 21OHD, which focuses on the impairment of glucocorticoid biosynthesis. A single 21-hydroxylase gene (cyp21a2) is annotated in the zebrafish genome based on sequence homology. Our in silico analysis of the 21-hydroxylase (Cyp21a2) protein sequence suggests a sufficient degree of similarity for the usage of zebrafish cyp21a2 to model aspects of human 21OHD in vivo. We determined the spatiotemporal expression patterns of cyp21a2 by whole-mount in situ hybridization and reverse transcription polymerase chain reaction throughout early development. Early cyp21a2 expression is restricted to the interrenal gland (zebrafish adrenal counterpart) and the brain. To further explore the in vivo consequences of 21OHD we created several cyp21a2 null-allele zebrafish lines by using a transcription activator-like effector nuclease genomic engineering strategy. Homozygous mutant zebrafish larvae showed an upregulation of the hypothalamic-pituitary-interrenal (HPI) axis and interrenal hyperplasia. Furthermore, Cyp21a2-deficient larvae had a typical steroid profile, with reduced concentrations of cortisol and increased concentrations of 17-hydroxyprogesterone and 21-deoxycortisol. Affected larvae showed an upregulation of the HPI axis and interrenal hyperplasia. Downregulation of the glucocorticoid-responsive genes pck1 and fkbp5 indicated systemic glucocorticoid deficiency. Our work demonstrates the crucial role of Cyp21a2 in glucocorticoid biosynthesis in zebrafish larvae and establishes an in vivo model allowing studies of systemic consequences of altered steroid hormone synthesis.

  6. Identification of oocyte progenitor cells in the zebrafish ovary.

    PubMed

    Draper, Bruce W

    2012-01-01

    Zebrafish breed year round and females are capable of producing thousands of eggs during their lifetime. This amazing fecundity is due to the fact that the adult ovary, contains premeiotic oocyte progenitor cells, called oogonia, which produce a continuous supply of new oocytes throughout adult life. Oocyte progenitor cells can be easily identified based on their expression of Vasa, and their characteristic nuclear morphology. Thus, the zebrafish ovary provides a unique and powerful system to study the genetic regulation of oocyte production in a vertebrate animal. A method is presented here for identifying oocyte progenitor cells in the zebrafish ovary using whole-mount confocal immunofluorescence that is simple and accurate.

  7. The Visual System of Zebrafish and its Use to Model Human Ocular Diseases

    PubMed Central

    Gestri, Gaia; Link, Brian A; Neuhauss, Stephan CF

    2011-01-01

    Free swimming zebrafish larvae depend mainly on their sense of vision to evade predation and to catch prey. Hence there is strong selective pressure on the fast maturation of visual function and indeed the visual system already supports a number of visually-driven behaviors in the newly hatched larvae. The ability to exploit the genetic and embryonic accessibility of the zebrafish in combination with a behavioral assessment of visual system function has made the zebrafish a popular model to study vision and its diseases. Here, we review the anatomy, physiology and development of the zebrafish eye as the basis to relate the contributions of the zebrafish to our understanding of human ocular diseases. PMID:21595048

  8. Zebrafish Development: High-throughput Test Systems to Assess Developmental Toxicity

    EPA Science Inventory

    Abstract Because of its developmental concordance, ease of handling and rapid development, the small teleost, zebrafish (Danio rerio), is frequently promoted as a vertebrate model for medium-throughput developmental screens. This present chapter discusses zebrafish as an altern...

  9. Retinoid regulation of the zebrafish cyp26a1 promoter.

    PubMed

    Hu, Ping; Tian, Miao; Bao, Jie; Xing, Guangdong; Gu, Xingxing; Gao, Xiang; Linney, Elwood; Zhao, Qingshun

    2008-12-01

    Cyp26A1 is a major enzyme that controls retinoic acid (RA) homeostasis by metabolizing RA into bio-inactive metabolites. Previous research revealed that the mouse Cyp26A1 promoter has two canonical RA response elements (RAREs) that underlie the regulation of the gene by RA. Analyzing the 2,533-base pairs (2.5 k) genomic sequence upstream of zebrafish cyp26a1 start codon, we report that the two RAREs are conserved in zebrafish cyp26a1 promoter. Mutagenesis demonstrated that the two RAREs work synergistically in RA inducibility of cyp26a1. Fusing the 2.5 k (kilobase pairs) fragment to the enhanced yellow fluorescent protein (eYFP) reporter gene, we have generated two transgenic lines of zebrafish [Tg(cyp26a1:eYFP)]. The transgenic zebrafish display expression patterns similar to that of cyp26a1 gene in vivo. Consistent with the in vitro results, the reporter activity is RA inducible in embryos. Taken together, our results demonstrate that the 2.5 k fragment underlies the regulation of the zebrafish cyp26a1 gene by RA. (c) 2008 Wiley-Liss, Inc.

  10. Zebrafish Locomotor Responses Predict Irritant Potential of ...

    EPA Pesticide Factsheets

    Over the past few decades, the drying and warming trends of global climate change have increased wildland fire (WF) season length, as well as geographic area impacted. Consequently, exposures to WF fine particulate matter (PM2.5; aerodynamic diameter <2.5 µm) are likely to increase in frequency and duration, contributing to a growing public health burden. Given the influence of fuel type and combustion conditions on WFPM2.5 composition, there is pressing need to identify the biomass fuel sources and emission constituents that drive toxicity. Previously, we reported the utility of 6-day post-fertilization (dpf) zebrafish larvae in evaluating diesel exhaust PM-induced irritation, demonstrating responses analogous to those in mammals. In the present study, combustions, separated by smoldering or flaming conditions, of pine needles, red oak, pine, eucalyptus, and peat were achieved using an automated tube furnace paired with a cryo-trapping apparatus to collect condensates of emissions. The condensates were extracted and prepared for use in zebrafish assays. We hypothesized that 1) the extractable organic fractions of biomass smoke PM will elicit dose-dependent irritant responses in 6-dpf zebrafish larvae, and 2) the relative potencies will vary across biomass emissions, potentially driven by varying chemical composition of fuel sources. Six-dpf zebrafish (n= 28-32/group) were exposed acutely to PM extracts (5 concentrations; 0.3-30 µg/ml; half-log intervals) and

  11. A two-scale model for correlation between B cell VDJ usage in zebrafish

    NASA Astrophysics Data System (ADS)

    Pan, Keyao; Deem, Michael

    2011-03-01

    The zebrafish (Danio rerio) is one of the model animals for study of immunology. The dynamics of the adaptive immune system in zebrafish is similar to that in higher animals. In this work, we built a two-scale model to simulate the dynamics of B cells in primary and secondary immune reactions in zebrafish and to explain the reported correlation between VDJ usage of B cell repertoires in distinct zebrafish. The first scale of the model consists of a generalized NK model to simulate the B cell maturation process in the 10-day primary immune response. The second scale uses a delay ordinary differential equation system to model the immune responses in the 6-month lifespan of zebrafish. The generalized NK model shows that mature B cells specific to one antigen mostly possess a single VDJ recombination. The probability that mature B cells in two zebrafish have the same VDJ recombination increases with the B cell population size or the B cell selection intensity and decreases with the B cell hypermutation rate. The ODE model shows a distribution of correlation in the VDJ usage of the B cell repertoires in two six-month-old zebrafish that is highly similar to that from experiment. This work presents a simple theory to explain the experimentally observed correlation in VDJ usage of distinct zebrafish B cell repertoires after an immune response.

  12. Textile dyes induce toxicity on zebrafish early life stages.

    PubMed

    de Oliveira, Gisele Augusto Rodrigues; de Lapuente, Joaquín; Teixidó, Elisabet; Porredón, Constança; Borràs, Miquel; de Oliveira, Danielle Palma

    2016-02-01

    Textile manufacturing is one of the most polluting industrial sectors because of the release of potentially toxic compounds, such as synthetic dyes, into the environment. Depending on the class of the dyes, their loss in wastewaters can range from 2% to 50% of the original dye concentration. Consequently, uncontrolled use of such dyes can negatively affect human health and the ecological balance. The present study assessed the toxicity of the textile dyes Direct Black 38 (DB38), Reactive Blue 15 (RB15), Reactive Orange 16 (RO16), and Vat Green 3 (VG3) using zebrafish (Danio rerio) embryos for 144 h postfertilization (hpf). At the tested conditions, none of the dyes caused significant mortality. The highest RO16 dose significantly delayed or inhibited the ability of zebrafish embryos to hatch from the chorion after 96 hpf. From 120 hpf to 144 hpf, all the dyes impaired the gas bladder inflation of zebrafish larvae, DB38 also induced curved tail, and VG3 led to yolk sac edema in zebrafish larvae. Based on these data, DB38, RB15, RO16, and VG3 can induce malformations during embryonic and larval development of zebrafish. Therefore, it is essential to remove these compounds from wastewater or reduce their concentrations to safe levels before discharging textile industry effluents into the aquatic environment. © 2015 SETAC.

  13. Understanding spatio-temporal strategies of adult zebrafish exploration in the open field test.

    PubMed

    Stewart, Adam Michael; Gaikwad, Siddharth; Kyzar, Evan; Kalueff, Allan V

    2012-04-27

    Zebrafish (Danio rerio) are emerging as a useful model organism for neuroscience research. Mounting evidence suggests that various traditional rodent paradigms may be adapted for testing zebrafish behavior. The open field test is a popular rodent test of novelty exploration, recently applied to zebrafish research. To better understand fish novelty behavior, we exposed adult zebrafish to two different open field arenas for 30 min, assessing the amount and temporal patterning of their exploration. While (similar to rodents) zebrafish scale their locomotory activity depending on the size of the tank, the temporal patterning of their activity was independent of arena size. These observations strikingly parallel similar rodent behaviors, suggesting that spatio-temporal strategies of animal exploration may be evolutionarily conserved across vertebrate species. In addition, we found interesting oscillations in zebrafish exploration, with the per-minute distribution of their horizontal activity demonstrating sinusoidal-like patterns. While such patterning is not reported for rodents and other higher vertebrates, a nonlinear regression analysis confirmed the oscillation patterning of all assessed zebrafish behavioral endpoints in both open field arenas, revealing a potentially important aspect of novelty exploration in lower vertebrates. Copyright © 2012 Elsevier B.V. All rights reserved.

  14. Lxr regulates lipid metabolic and visual perception pathways during zebrafish development.

    PubMed

    Pinto, Caroline Lucia; Kalasekar, Sharanya Maanasi; McCollum, Catherine W; Riu, Anne; Jonsson, Philip; Lopez, Justin; Swindell, Eric C; Bouhlatouf, Abdel; Balaguer, Patrick; Bondesson, Maria; Gustafsson, Jan-Åke

    2016-01-05

    The Liver X Receptors (LXRs) play important roles in multiple metabolic pathways, including fatty acid, cholesterol, carbohydrate and energy metabolism. To expand the knowledge of the functions of LXR signaling during embryonic development, we performed a whole-genome microarray analysis of Lxr target genes in zebrafish larvae treated with either one of the synthetic LXR ligands T0901317 or GW3965. Assessment of the biological processes enriched by differentially expressed genes revealed a prime role for Lxr in regulating lipid metabolic processes, similarly to the function of LXR in mammals. In addition, exposure to the Lxr ligands induced changes in expression of genes in the neural retina and lens of the zebrafish eye, including the photoreceptor guanylate cyclase activators and lens gamma crystallins, suggesting a potential novel role for Lxr in modulating the transcription of genes associated with visual function in zebrafish. The regulation of expression of metabolic genes was phenotypically reflected in an increased absorption of yolk in the zebrafish larvae, and changes in the expression of genes involved in visual perception were associated with morphological alterations in the retina and lens of the developing zebrafish eye. The regulation of expression of both lipid metabolic and eye specific genes was sustained in 1 month old fish. The transcriptional networks demonstrated several conserved effects of LXR activation between zebrafish and mammals, and also identified potential novel functions of Lxr, supporting zebrafish as a promising model for investigating the role of Lxr during development. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  15. TSH Receptor Function Is Required for Normal Thyroid Differentiation in Zebrafish

    PubMed Central

    Opitz, Robert; Maquet, Emilie; Zoenen, Maxime; Dadhich, Rajesh

    2011-01-01

    TSH is the primary physiological regulator of thyroid gland function. The effects of TSH on thyroid cells are mediated via activation of its membrane receptor [TSH receptor (TSHR)]. In this study, we examined functional thyroid differentiation in zebrafish and characterized the role of TSHR signaling during thyroid organogenesis. Cloning of a cDNA encoding zebrafish Tshr showed conservation of primary structure and functional properties between zebrafish and mammalian TSHR. In situ hybridization confirmed that the thyroid is the major site of tshr expression during zebrafish development. In addition, we identified tpo, iyd, duox, and duoxa as novel thyroid differentiation markers in zebrafish. Temporal analyses of differentiation marker expression demonstrated the induction of an early thyroid differentiation program along with thyroid budding, followed by a delayed onset of duox and duoxa expression coincident with thyroid hormone synthesis. Furthermore, comparative analyses in mouse and zebrafish revealed for the first time a thyroid-enriched expression of cell death regulators of the B-cell lymphoma 2 family during early thyroid morphogenesis. Knockdown of tshr function by morpholino microinjection into embryos did not affect early thyroid morphogenesis but caused defects in later functional differentiation. The thyroid phenotype observed in tshr morphants at later stages comprised a reduction in number and size of functional follicles, down-regulation of differentiation markers, as well as reduced thyroid transcription factor expression. A comparison of our results with phenotypes observed in mouse models of defective TSHR and cAMP signaling highlights the value of zebrafish as a model to enhance the understanding of functional differentiation in the vertebrate thyroid. PMID:21737742

  16. Using local chromatin structure to improve CRISPR/Cas9 efficiency in zebrafish.

    PubMed

    Chen, Yunru; Zeng, Shiyang; Hu, Ruikun; Wang, Xiangxiu; Huang, Weilai; Liu, Jiangfang; Wang, Luying; Liu, Guifen; Cao, Ying; Zhang, Yong

    2017-01-01

    Although the CRISPR/Cas9 has been successfully applied in zebrafish, considerable variations in efficiency have been observed for different gRNAs. The workload and cost of zebrafish mutant screening is largely dependent on the mutation rate of injected embryos; therefore, selecting more effective gRNAs is especially important for zebrafish mutant construction. Besides the sequence features, local chromatin structures may have effects on CRISPR/Cas9 efficiency, which remain largely unexplored. In the only related study in zebrafish, nucleosome organization was not found to have an effect on CRISPR/Cas9 efficiency, which is inconsistent with recent studies in vitro and in mammalian cell lines. To understand the effects of local chromatin structure on CRISPR/Cas9 efficiency in zebrafish, we first determined that CRISPR/Cas9 introduced genome editing mainly before the dome stage. Based on this observation, we reanalyzed our published nucleosome organization profiles and generated chromatin accessibility profiles in the 256-cell and dome stages using ATAC-seq technology. Our study demonstrated that chromatin accessibility showed positive correlation with CRISPR/Cas9 efficiency, but we did not observe a clear correlation between nucleosome organization and CRISPR/Cas9 efficiency. We constructed an online database for zebrafish gRNA selection based on local chromatin structure features that could prove beneficial to zebrafish homozygous mutant construction via CRISPR/Cas9.

  17. Effect of Cefotaxime on the CAT Activities and GSH Contents of Zebrafish

    NASA Astrophysics Data System (ADS)

    Wang, Yaxue; Shen, Hong-Yan

    2018-05-01

    In order to define eco-toxicity effect of cefotaxime on zebrafish, the indoor exposure method was used to study the impact of cefotaxime on zebrafish. In this study, zebrafish was exposed to cefotaxime of 1mg/L, 5mg/L, 25mg/L and 125mg/L for 15 days to study the effect of Catalase (CAT) activities and Glutathione (GSH) contents. According to the experimental data, the CAT activities and GSH contents in zebrafish muscle tissue had changed significantly during the period of exposure. The experimental results show that the activities of CAT in four concentration groups were significantly inhibited (P<0.01). The CAT activities in the 1 mg/L and 5 mg/L groups showed the "Λ" type change, inhibited first and induced later. But in the 25 mg/L and 125 mg/L groups, the CAT activities were inhibited all the time. Cefotaxime had a significant effect on GSH content in the muscle tissue of the zebrafish at the early stage of exposure, rapidly increase to the maximum at the early stage and rapidly decrease to the minimum on the 6th day. During 6th day to 15th day, the contents of GSH in the zebrafish were basically stable at the level of control. The experimental results show that the CAT activities and GSH contents in zebrafish muscle tissue had changed significantly.

  18. Identification and characterization of circular RNAs in zebrafish.

    PubMed

    Shen, Yudong; Guo, Xianwu; Wang, Weimin

    2017-01-01

    Circular RNA (circRNA), a class of RNAs with circular structure, has received little attention until recently, when some new features and functions were discovered. In the present study, we sequenced circRNAs in zebrafish (Danio rerio) and identified 3868 circRNAs using three algorithms (find_circ, CIRI, segemehl). The analysis of microRNA target sites on circRNAs shows that some circRNAs may function as miRNA sponges. Furthermore, we identified the existence of reverse complementary sequences in the flanking regions of only 25 (2.64%) exonic circRNAs, indicating that the mechanism of zebrafish exonic circRNA biogenesis might be different from that in mammals. Moreover, 1122 (29%) zebrafish circRNA sequences showed homology with human, mouse and coelacanth circRNAs. © 2016 Federation of European Biochemical Societies.

  19. Tributyltin and Zebrafish: Swimming in Dangerous Water

    PubMed Central

    Berto-Júnior, Clemilson; de Carvalho, Denise Pires; Soares, Paula; Miranda-Alves, Leandro

    2018-01-01

    Zebrafish has been established as a reliable biological model with important insertion in academy (morphologic, biochemical, and pathophysiological studies) and pharmaceutical industry (toxicology and drug development) due to its molecular complexity and similar systems biology that recapitulate those from other organisms. Considering the toxicological aspects, many efforts using zebrafish models are being done in order to elucidate the effects of endocrine disruptors, and some of them are focused on tributyltin (TBT) and its mechanism of action. TBT is an antifouling agent applied in ship’s hull that is constantly released into the water and absorbed by marine organisms, leading to bioaccumulation and biomagnification effects. Thus, several findings of malformations and changes in the normal biochemical and physiologic aspects of these marine animals have been related to TBT contamination. In the present review, we have compiled the most significant studies related to TBT effects in zebrafish, also taking into consideration the effects found in other study models. PMID:29692757

  20. Use of zebrafish to study Shigella infection

    PubMed Central

    Duggan, Gina M.

    2018-01-01

    ABSTRACT Shigella is a leading cause of dysentery worldwide, responsible for up to 165 million cases of shigellosis each year. Shigella is also recognised as an exceptional model pathogen to study key issues in cell biology and innate immunity. Several infection models have been useful to explore Shigella biology; however, we still lack information regarding the events taking place during the Shigella infection process in vivo. Here, we discuss a selection of mechanistic insights recently gained from studying Shigella infection of zebrafish (Danio rerio), with a focus on cytoskeleton rearrangements and cellular immunity. We also discuss how infection of zebrafish can be used to investigate new concepts underlying infection control, including emergency granulopoiesis and the use of predatory bacteria to combat antimicrobial resistance. Collectively, these insights illustrate how Shigella infection of zebrafish can provide fundamental advances in our understanding of bacterial pathogenesis and vertebrate host defence. This information should also provide vital clues for the discovery of new therapeutic strategies against infectious disease in humans. PMID:29590642

  1. Disease modeling in genetic kidney diseases: zebrafish.

    PubMed

    Schenk, Heiko; Müller-Deile, Janina; Kinast, Mark; Schiffer, Mario

    2017-07-01

    Growing numbers of translational genomics studies are based on the highly efficient and versatile zebrafish (Danio rerio) vertebrate model. The increasing types of zebrafish models have improved our understanding of inherited kidney diseases, since they not only display pathophysiological changes but also give us the opportunity to develop and test novel treatment options in a high-throughput manner. New paradigms in inherited kidney diseases have been developed on the basis of the distinct genome conservation of approximately 70 % between zebrafish and humans in terms of existing gene orthologs. Several options are available to determine the functional role of a specific gene or gene sets. Permanent genome editing can be induced via complete gene knockout by using the CRISPR/Cas-system, among others, or via transient modification by using various morpholino techniques. Cross-species rescues succeeding knockdown techniques are employed to determine the functional significance of a target gene or a specific mutation. This article summarizes the current techniques and discusses their perspectives.

  2. Glyphosate and Roundup® alter morphology and behavior in zebrafish.

    PubMed

    Bridi, Daiane; Altenhofen, Stefani; Gonzalez, Jonas Brum; Reolon, Gustavo Kellermann; Bonan, Carla Denise

    2017-12-01

    Glyphosate has become the most widely used herbicide in the world, due to the wide scale adoption of transgenic glyphosate resistant crops after its introduction in 1996. Glyphosate may be used alone, but it is commonly applied as an active ingredient of the herbicide Roundup ® . This pesticide contains several adjuvants, which may promote an unknown toxicity. The indiscriminate application poses numerous problems, both for the health of the applicators and consumers, and for the environment, contaminating the soil, water and leading to the death of plants and animals. Zebrafish (Danio rerio) is quickly gaining popularity in behavioral research, because of physiological similarity to mammals, sensitivity to pharmacological factors, robust performance, low cost, short spawning intervals, external fertilization, transparency of embryos through larval stages, and rapid development. The aim of this study was evaluate the effects of glyphosate and Roundup ® on behavioral and morphological parameters in zebrafish larvae and adults. Zebrafish larvae at 3days post-fertilization and adults were exposed to glyphosate (0.01, 0.065, and 0.5mg/L) or Roundup ® (0.01, 0.065, and 0.5mg/L) for 96h. Immediately after the exposure, we performed the analysis of locomotor activity, aversive behavior, and morphology for the larvae and exploratory behavior, aggression and inhibitory avoidance memory for adult zebrafish. In zebrafish larvae, there were significant differences in the locomotor activity and aversive behavior after glyphosate or Roundup ® exposure when compared to the control group. Our findings demonstrated that exposure to glyphosate at the concentration of 0.5mg/L, Roundup ® at 0.065 or 0.5mg/L reduced the distance traveled, the mean speed and the line crossings in adult zebrafish. A decreased ocular distance was observed for larvae exposed at 0.5mg/L of glyphosate. We verified that at 0.5mg/L of Roundup ® -treated adult zebrafish demonstrated a significant

  3. Identifying Structural Alerts Based on Zebrafish Developmental Morphological Toxicity (TDS)

    EPA Science Inventory

    Zebrafish constitute a powerful alternative animal model for chemical hazard evaluation. To provide an in vivo complement to high-throughput screening data from the ToxCast program, zebrafish developmental toxicity screens were conducted on the ToxCast Phase I (Padilla et al., 20...

  4. High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope

    PubMed Central

    Blackburn, Jessica S; Liu, Sali; Raimondi, Aubrey R; Ignatius, Myron S; Salthouse, Christopher D; Langenau, David M

    2011-01-01

    Zebrafish are a useful vertebrate model for the study of development, behavior, disease and cancer. A major advantage of zebrafish is that large numbers of animals can be economically used for experimentation; however, high-throughput methods for imaging live adult zebrafish had not been developed. Here, we describe protocols for building a light-emitting diode (LED) fluorescence macroscope and for using it to simultaneously image up to 30 adult animals that transgenically express a fluorescent protein, are transplanted with fluorescently labeled tumor cells or are tagged with fluorescent elastomers. These protocols show that the LED fluorescence macroscope is capable of distinguishing five fluorescent proteins and can image unanesthetized swimming adult zebrafish in multiple fluorescent channels simultaneously. The macroscope can be built and used for imaging within 1 day, whereas creating fluorescently labeled adult zebrafish requires 1 hour to several months, depending on the method chosen. The LED fluorescence macroscope provides a low-cost, high-throughput method to rapidly screen adult fluorescent zebrafish and it will be useful for imaging transgenic animals, screening for tumor engraftment, and tagging individual fish for long-term analysis. PMID:21293462

  5. Discrimination reversal and attentional sets in zebrafish (Danio rerio)

    PubMed Central

    Parker, Matthew O.; Gaviria, Jessica; Haigh, Alastair; Millington, Mollie E.; Brown, Verity J.; Combe, Fraser J.; Brennan, Caroline H.

    2014-01-01

    The potential of zebrafish as a comparative model in behavioural neuroscience is currently hampered only by the lack of reliable and validated behavioural assays available to researchers. In the present experiment, we describe the performance of zebrafish in a test of attentional set formation. The fish were initially trained on a two-choice colour discrimination. Upon reaching acquisition criterion, the reinforced alternative was switched to the previously unreinforced alternative. Again, upon reaching criterion, the cues were replaced with a novel pair of colours (intra-dimensional shift) and reversed again on reaching criteria. We found that zebrafish show a steady decrease in trials-to-criteria over the four phases of the experiment, suggesting that they are forming and maintaining an attentional set, as has previously been demonstrated with mammals. Reversal learning deficits have been implicated in a variety of human psychological disorders (e.g., disorders of impulse control) and as such, we propose that performance of zebrafish in this procedure may represent a useful comparative model to complement existing rodent models. PMID:22561034

  6. Chemokine guided angiogenesis directs coronary vasculature formation in zebrafish

    PubMed Central

    Harrison, Michael R.M.; Bussmann, Jeroen; Huang, Ying; Zhao, Long; Osorio, Arthela; Burns, C. Geoffrey; Burns, Caroline E.; Sucov, Henry M.; Siekmann, Arndt F.; Lien, Ching-Ling

    2015-01-01

    SUMMARY Interruption of coronary blood supply severely impairs heart function with often-fatal consequences for heart disease patients. However the formation and maturation of these coronary vessels is not fully understood. Here we provide a detailed analysis of coronary vessel development in zebrafish. We observe that coronary vessels form in zebrafish by angiogenic sprouting of arterial cells derived from the endocardium at the atrioventricular canal. Endothelial cells express the CXC-motif chemokine receptor Cxcr4a and migrate to vascularize the ventricle under the guidance of the myocardium-expressed ligand Cxcl12b. cxcr4a mutant zebrafish fail to form a vascular network, whereas ectopic expression of Cxcl12b ligand induces coronary vessel formation. Importantly, cxcr4a mutant zebrafish fail to undergo heart regeneration following injury. Our results suggest that chemokine-signaling has an essential role in coronary vessel formation by directing migration of endocardium-derived endothelial cells. Poorly developed vasculature in cxcr4a mutants likely underlies decreased regenerative potential in adults. PMID:26017769

  7. It's time to swim! Zebrafish and the circadian clock.

    PubMed

    Vatine, Gad; Vallone, Daniela; Gothilf, Yoav; Foulkes, Nicholas S

    2011-05-20

    The zebrafish represents a fascinating model for studying key aspects of the vertebrate circadian timing system. Easy access to early embryonic development has made this species ideal for investigating how the clock is first established during embryogenesis. In particular, the molecular basis for the functional development of the zebrafish pineal gland has received much attention. In addition to this dedicated clock and photoreceptor organ, and unlike the situation in mammals, the clocks in zebrafish peripheral tissues and even cell lines are entrainable by direct exposure to light thus providing unique insight into the function and evolution of the light input pathway. Finally, the small size, low maintenance costs and high fecundity of this fish together with the availability of genetic tools make this an attractive model for forward genetic analysis of the circadian clock. Here, we review the work that has established the zebrafish as a valuable clock model organism and highlight the key questions that will shape the future direction of research. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  8. Advances in the Study of Heart Development and Disease Using Zebrafish

    PubMed Central

    Brown, Daniel R.; Samsa, Leigh Ann; Qian, Li; Liu, Jiandong

    2016-01-01

    Animal models of cardiovascular disease are key players in the translational medicine pipeline used to define the conserved genetic and molecular basis of disease. Congenital heart diseases (CHDs) are the most common type of human birth defect and feature structural abnormalities that arise during cardiac development and maturation. The zebrafish, Danio rerio, is a valuable vertebrate model organism, offering advantages over traditional mammalian models. These advantages include the rapid, stereotyped and external development of transparent embryos produced in large numbers from inexpensively housed adults, vast capacity for genetic manipulation, and amenability to high-throughput screening. With the help of modern genetics and a sequenced genome, zebrafish have led to insights in cardiovascular diseases ranging from CHDs to arrhythmia and cardiomyopathy. Here, we discuss the utility of zebrafish as a model system and summarize zebrafish cardiac morphogenesis with emphasis on parallels to human heart diseases. Additionally, we discuss the specific tools and experimental platforms utilized in the zebrafish model including forward screens, functional characterization of candidate genes, and high throughput applications. PMID:27335817

  9. A zebrafish (Danio rerio) model of infectious spleen and kidney necrosis virus (ISKNV) infection

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xu Xiaopeng; Zhang Lichun; Weng Shaoping

    2008-06-20

    Zebrafish is a model animal for studies of genetics, development, toxicology, oncology, and immunology. In this study, infectious spleen and kidney necrosis virus (ISKNV) was used to establish an infection in zebrafish, and the experimental conditions were established and characterized. Mortality of adult zebrafish infected with ISKNV by intraperitoneal (i.p.) injection exceeded 60%. ISKNV can be passed stably in zebrafish for over ten passages. The ailing zebrafish displayed petechial hemorrhaging and scale protrusion. Histological analysis of moribund fish revealed necrosis of tissue and enlarged cells in kidney and spleen. The real-time RT-PCR analysis of mRNA level confirmed that ISKNV wasmore » replicated in zebrafish. Immunohistochemistry and immunofluorescence analyses further confirmed the presence of ISKNV-infected cells in almost all organs of the infected fish. Electron microscope analyses showed that the ISKNV particle was present in the infected tissues. The establishment of zebrafish infection model of ISKNV can offer a valuable tool for studying the interactions between ISKNV and its host.« less

  10. A Dynamic Anesthesia System for Long-Term Imaging in Adult Zebrafish

    PubMed Central

    Wynd, Brenen M.; Watson, Claire J.; Patil, Karuna; Sanders, George E.

    2017-01-01

    Abstract Long-term in vivo imaging in adult zebrafish (i.e., 1–24 h) has been limited by the fact that regimens for long-term anesthesia in embryos and larvae are ineffective in adults. Here, we examined the potential for dynamic administration of benzocaine to enable long-term anesthesia in adult zebrafish. We developed a computer-controlled perfusion system comprised of programmable peristaltic pumps that enabled automatic exchange between anesthetic and system water. Continuous administration of benzocaine in adult zebrafish resulted in a mean time to respiratory arrest of 5.0 h and 8-h survival of 14.3%. We measured characteristic sedation and recovery times in response to benzocaine, and used them to devise an intermittent dosing regimen consisting of 14.5 min of benzocaine followed by 5.5 min of system water. Intermittent benzocaine administration in adult zebrafish resulted in a mean time to respiratory arrest of 7.6 h and 8-h survival of 71.4%. Finally, we performed a single 24-h trial and found that intermittent dosing maintained anesthesia in an adult zebrafish over the entire 24-h period. In summary, our studies demonstrate the potential for dynamic administration of benzocaine to enable prolonged anesthesia in adult zebrafish, expanding the potential for imaging in adult physiologies that unfold over 1–24 h. PMID:27409411

  11. A Dynamic Anesthesia System for Long-Term Imaging in Adult Zebrafish.

    PubMed

    Wynd, Brenen M; Watson, Claire J; Patil, Karuna; Sanders, George E; Kwon, Ronald Y

    2017-02-01

    Long-term in vivo imaging in adult zebrafish (i.e., 1-24 h) has been limited by the fact that regimens for long-term anesthesia in embryos and larvae are ineffective in adults. Here, we examined the potential for dynamic administration of benzocaine to enable long-term anesthesia in adult zebrafish. We developed a computer-controlled perfusion system comprised of programmable peristaltic pumps that enabled automatic exchange between anesthetic and system water. Continuous administration of benzocaine in adult zebrafish resulted in a mean time to respiratory arrest of 5.0 h and 8-h survival of 14.3%. We measured characteristic sedation and recovery times in response to benzocaine, and used them to devise an intermittent dosing regimen consisting of 14.5 min of benzocaine followed by 5.5 min of system water. Intermittent benzocaine administration in adult zebrafish resulted in a mean time to respiratory arrest of 7.6 h and 8-h survival of 71.4%. Finally, we performed a single 24-h trial and found that intermittent dosing maintained anesthesia in an adult zebrafish over the entire 24-h period. In summary, our studies demonstrate the potential for dynamic administration of benzocaine to enable prolonged anesthesia in adult zebrafish, expanding the potential for imaging in adult physiologies that unfold over 1-24 h.

  12. Angiopoietin-like 3 regulates hepatocyte proliferation and lipid metabolism in zebrafish

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, So-Hyun; Department of Biology, Chungnam National University, Daejeon; So, Ju-Hoon

    2014-04-18

    Highlights: • angptl3 is specifically expressed in the liver of developing zebrafish. • Knockdown of Angptl3 decreases liver size in developing zebrafish. • Knockdown of zebrafish Angptl3 elicits a hypocholesterolemia phenotype. - Abstract: Loss-of-function mutations in angiopoietin-like 3 (ANGPTL3) cause familial hypobetalipoproteinemia type 2 (FHBL2) in humans. ANGPTL3 belongs to the angiopoietin-like family, the vascular endothelial growth factor family that is structurally similar to angiopoietins and is known for a regulator of lipid and glucose metabolism, although it is unclear how mutations in ANGPTL3 lead to defect in liver development in the vertebrates. We report here that angptl3 is primarilymore » expressed in the zebrafish developing liver and that morpholino (MO) knockdown of Angptl3 reduces the size of the developing liver, which is caused by suppression of cell proliferation, but not by enhancement of apoptosis. However, MO knockdown of Angptl3 did not alter angiogenesis in the developing liver. Additionally, disruption of zebrafish Angptl3 elicits the hypocholesterolemia phenotype that is characteristic of FHBL2 in humans. Together, our findings propose a novel role for Angptl3 in liver cell proliferation and maintenance during zebrafish embryogenesis. Finally, angptl3 morphants will serve as a good model for understanding the pathophysiology of FHBL2.« less

  13. Acute ethanol administration affects zebrafish preference for a biologically inspired robot.

    PubMed

    Spinello, Chiara; Macrì, Simone; Porfiri, Maurizio

    2013-08-01

    Preclinical animal models constitute a cornerstone against which the reward processes involved in drug addiction are often studied and dissected. While rodents have traditionally represented the species of choice, a growing body of literature indicates that zebrafish are emerging as a valuable model organism. Specifically, several studies demonstrate that the effects of ethanol at the level of emotional- and cognitive-related domains can be reliably investigated using zebrafish. The rapidly evolving nature of these efforts allows substantial room for the development of novel experimental paradigms suited to this freshwater species. The field of ethorobotics may prove particularly beneficial, due to its ability to convey fully controllable and easily reproducible experimental tools. In this study, we addressed the possibility of using a biologically inspired robot to investigate the emotionally related properties of ethanol in a preference task in zebrafish. To this aim, we evaluated wild-type zebrafish preference toward a robotic stimulus and addressed whether ethanol administration (0.25% and 1.00% ethanol/water concentration) may alter such preferences. In accordance with our previous studies, we observed that zebrafish exhibit a natural attraction toward the robot. Additionally, in agreement with our predictions, we showed that ethanol administration abolishes such preferences. This work is the first to demonstrate that robotic stimuli can be used in zebrafish to investigate the reward-related properties of alcohol. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Chamber Specific Gene Expression Landscape of the Zebrafish Heart

    PubMed Central

    Singh, Angom Ramcharan; Sivadas, Ambily; Sabharwal, Ankit; Vellarikal, Shamsudheen Karuthedath; Jayarajan, Rijith; Verma, Ankit; Kapoor, Shruti; Joshi, Adita; Scaria, Vinod; Sivasubbu, Sridhar

    2016-01-01

    The organization of structure and function of cardiac chambers in vertebrates is defined by chamber-specific distinct gene expression. This peculiarity and uniqueness of the genetic signatures demonstrates functional resolution attributed to the different chambers of the heart. Altered expression of the cardiac chamber genes can lead to individual chamber related dysfunctions and disease patho-physiologies. Information on transcriptional repertoire of cardiac compartments is important to understand the spectrum of chamber specific anomalies. We have carried out a genome wide transcriptome profiling study of the three cardiac chambers in the zebrafish heart using RNA sequencing. We have captured the gene expression patterns of 13,396 protein coding genes in the three cardiac chambers—atrium, ventricle and bulbus arteriosus. Of these, 7,260 known protein coding genes are highly expressed (≥10 FPKM) in the zebrafish heart. Thus, this study represents nearly an all-inclusive information on the zebrafish cardiac transcriptome. In this study, a total of 96 differentially expressed genes across the three cardiac chambers in zebrafish were identified. The atrium, ventricle and bulbus arteriosus displayed 20, 32 and 44 uniquely expressing genes respectively. We validated the expression of predicted chamber-restricted genes using independent semi-quantitative and qualitative experimental techniques. In addition, we identified 23 putative novel protein coding genes that are specifically restricted to the ventricle and not in the atrium or bulbus arteriosus. In our knowledge, these 23 novel genes have either not been investigated in detail or are sparsely studied. The transcriptome identified in this study includes 68 differentially expressing zebrafish cardiac chamber genes that have a human ortholog. We also carried out spatiotemporal gene expression profiling of the 96 differentially expressed genes throughout the three cardiac chambers in 11 developmental stages and 6

  15. Phylogeny of zebrafish, a "model species," within Danio, a "model genus".

    PubMed

    McCluskey, Braedan M; Postlethwait, John H

    2015-03-01

    Zebrafish (Danio rerio) is an important model for vertebrate development, genomics, physiology, behavior, toxicology, and disease. Additionally, work on numerous Danio species is elucidating evolutionary mechanisms for morphological development. Yet, the relationships of zebrafish and its closest relatives remain unclear possibly due to incomplete lineage sorting, speciation with gene flow, and interspecies hybridization. To clarify these relationships, we first constructed phylogenomic data sets from 30,801 restriction-associated DNA (RAD)-tag loci (483,026 variable positions) with clear orthology to a single location in the sequenced zebrafish genome. We then inferred a well-supported species tree for Danio and tested for gene flow during the diversification of the genus. An approach independent of the sequenced zebrafish genome verified all inferred relationships. Although identification of the sister taxon to zebrafish has been contentious, multiple RAD-tag data sets and several analytical methods provided strong evidence for Danio aesculapii as the most closely related extant zebrafish relative studied to date. Data also displayed patterns consistent with gene flow during speciation and postspeciation introgression in the lineage leading to zebrafish. The incorporation of biogeographic data with phylogenomic analyses put these relationships in a phylogeographic context and supplied additional support for D. aesculapii as the sister species to D. rerio. The clear resolution of this study establishes a framework for investigating the evolutionary biology of Danio and the heterogeneity of genome evolution in the recent history of a model organism within an emerging model genus for genetics, development, and evolution. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Simultaneous assessment of glomerular filtration and barrier function in live zebrafish

    PubMed Central

    Kotb, Ahmed M.; Müller, Tobias; Xie, Jing; Anand-Apte, Bela; Endlich, Nicole

    2014-01-01

    The zebrafish pronephros is a well-established model to study glomerular development, structure, and function. A few methods have been described to evaluate glomerular barrier function in zebrafish larvae so far. However, there is a need to assess glomerular filtration as well. In the present study, we extended the available methods by simultaneously measuring the intravascular clearances of Alexa fluor 647-conjugated 10-kDa dextran and FITC-conjugated 500-kDa dextran as indicators of glomerular filtration and barrier function, respectively. After intravascular injection of the dextrans, mean fluorescence intensities of both dextrans were measured in the cardinal vein of living zebrafish (4 days postfertilization) by confocal microscopy over time. We demonstrated that injected 10-kDa dextran was rapidly cleared from the circulation, became visible in the lumen of the pronephric tubule, quickly accumulated in tubular cells, and was detectably excreted at the cloaca. In contrast, 500-kDa dextran could not be visualized in the tubule at any time point. To check whether alterations in glomerular function can be quantified by our method, we injected morpholino oligonucleotides (MOs) against zebrafish nonmuscle myosin heavy chain IIA (zMyh9) or apolipoprotein L1 (zApol1). While glomerular filtration was reduced in zebrafish nonmuscle myosin heavy chain IIA MO-injected larvae, glomerular barrier function remained intact. In contrast, in zebrafish apolipoprotein L1 MO-injected larvae, glomerular barrier function was compromised as 500-kDa dextran disappeared from the circulation and became visible in tubular cells. In summary, we present a novel method that allows to simultaneously assess glomerular filtration and barrier function in live zebrafish. PMID:25298528

  17. Strain differences in the collective behaviour of zebrafish (Danio rerio) in heterogeneous environment

    PubMed Central

    Collignon, Bertrand

    2016-01-01

    Recent studies show differences in individual motion and shoaling tendency between strains of the same species. Here, we analyse collective motion and response to visual stimuli in two morphologically different strains (TL and AB) of zebrafish. For both strains, we observed 10 groups of 5 and 10 zebrafish swimming freely in a large experimental tank with two identical landmarks (cylinders or discs) for 1 h. We tracked the positions of the fish by an automated tracking method and compute several metrics at the group level. First, the probability of the presence shows that both strains avoid free space and are more likely to swim in the vicinity of the walls of the tank and the landmarks. Second, the analysis of landmarks occupancy shows that AB zebrafish are more present in their vicinity than TL ones and that both strains regularly transit from one to the other one with no preference on the long duration. Finally, TL zebrafish show a higher cohesion than AB zebrafish. Thus, environmental heterogeneity and duration of the trials allow to reveal individual and collective behavioural variabilities among different strains of zebrafish. These results provide a new insight into the need to take into account individual variability of zebrafish strains for studying collective behaviour. PMID:27853558

  18. Retinoic Acid Metabolic Genes, Meiosis, and Gonadal Sex Differentiation in Zebrafish

    PubMed Central

    Rodríguez-Marí, Adriana; Cañestro, Cristian; BreMiller, Ruth A.; Catchen, Julian M.; Yan, Yi-Lin; Postlethwait, John H.

    2013-01-01

    To help understand the elusive mechanisms of zebrafish sex determination, we studied the genetic machinery regulating production and breakdown of retinoic acid (RA) during the onset of meiosis in gonadogenesis. Results uncovered unexpected mechanistic differences between zebrafish and mammals. Conserved synteny and expression analyses revealed that cyp26a1 in zebrafish and its paralog Cyp26b1 in tetrapods independently became the primary genes encoding enzymes available for gonadal RA-degradation, showing lineage-specific subfunctionalization of vertebrate genome duplication (VGD) paralogs. Experiments showed that zebrafish express aldh1a2, which encodes an RA-synthesizing enzyme, in the gonad rather than in the mesonephros as in mouse. Germ cells in bipotential gonads of all zebrafish analyzed were labeled by the early meiotic marker sycp3, suggesting that in zebrafish, the onset of meiosis is not sexually dimorphic as it is in mouse and is independent of Stra8, which is required in mouse but was lost in teleosts. Analysis of dead-end knockdown zebrafish depleted of germ cells revealed the germ cell-independent onset and maintenance of gonadal aldh1a2 and cyp26a1 expression. After meiosis initiated, somatic cell expression of cyp26a1 became sexually dimorphic: up-regulated in testes but not ovaries. Meiotic germ cells expressing the synaptonemal complex gene sycp3 occupied islands of somatic cells that lacked cyp26a1 expression, as predicted by the hypothesis that Cyp26a1 acts as a meiosis-inhibiting factor. Consistent with this hypothesis, females up-regulated cyp26a1 in oocytes that entered prophase-I meiotic arrest, and down-regulated cyp26a1 in oocytes resuming meiosis. Co-expression of cyp26a1 and the pluripotent germ cell stem cell marker pou5f1(oct4) in meiotically arrested oocytes was consistent with roles in mouse to promote germ cell survival and to prevent apoptosis, mechanisms that are central for tipping the sexual fate of gonads towards the female

  19. Glutathione and zebrafish: Old assays to address a current issue.

    PubMed

    Massarsky, Andrey; Kozal, Jordan S; Di Giulio, Richard T

    2017-02-01

    Several xenobiotic agents (e.g. metals, polycyclic aromatic hydrocarbons, nanoparticles, etc.) commonly involve the generation of reactive oxygen species (ROS) and oxidative stress as part of their toxic mode of action. Among piscine models, the zebrafish is a popular vertebrate model to study toxicity of various xenobiotic agents. Similarly to other vertebrates, zebrafish possess an extensive antioxidant system, including the reduced form of glutathione (GSH), which is an important antioxidant that acts alone or in conjunction with enzymes, such as glutathione peroxidase (GPx). Upon interaction with ROS, GSH is oxidized, resulting in the formation of glutathione disulfide (GSSG). GSSG is recycled by an auxiliary antioxidant enzyme glutathione reductase (GR). This article outlines detailed methods to measure the concentrations of GSH and GSSG, as well as the activities of GPx and GR in zebrafish larvae as robust and economical means to assess oxidative stress. The studies that have assessed these endpoints in zebrafish and alternative methods are also discussed. We conclude that the availability of these robust and economical methods support the use of zebrafish as a model organism in studies evaluating redox biology, as well as the induction of oxidative stress following exposure to toxic agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. An automated device for appetitive conditioning in zebrafish (Danio rerio).

    PubMed

    Manabe, Kazuchika; Dooling, R J; Takaku, Shinichi

    2013-12-01

    An automated device and a procedure for the operant conditioning individual zebrafish were developed. The key feature of this procedure was the construction of a simple, inexpensive feeder that can deliver extremely small amounts of food, thus preventing rapid satiation. This allows the experimenter to run multiple trails in a single test session and multiple sessions in one day. In addition, small response keys made from acryl rods and fiber sensors were developed that were sufficiently sensitive to detect fish contact. To illustrate the efficiency and utility of the device for traditional learning paradigms, we trained zebrafish in a fixed ratio schedule where subjects were reinforced with food after 10 responses. Zebrafish reliably responded on the response key for sessions that lasted as long 80-reinforcements. They also showed the traditional "break and run" response pattern that has been found in many species. These results show that this system will be valuable for behavioral studies with zebrafish, especially for experiments that need many repeated trials using food reinforcer in a session. The present system can be used for sensory and learning investigations, as well applications in behavioral pharmacology, behavioral genetics, and toxicology where the zebrafish is becoming the vertebrate model of choice.

  1. Comparison of Antemortem and Environmental Samples for Zebrafish Health Monitoring and Quarantine.

    PubMed

    Crim, Marcus J; Lawrence, Christian; Livingston, Robert S; Rakitin, Andrei; Hurley, Shane J; Riley, Lela K

    2017-07-01

    Molecular diagnostic assays offer both exquisite sensitivity and the ability to test a wide variety of sample types. Various types of environmental sample, such as detritus and concentrated water, might provide a useful adjunct to sentinels in routine zebrafish health monitoring. Similarly, antemortem sampling would be advantageous for expediting zebrafish quarantine, without euthanasia of valuable fish. We evaluated the detection of Mycobacterium chelonae, M. fortuitum, M. peregrinum, Pseudocapillaria tomentosa, and Pseudoloma neurophilia in zebrafish, detritus, pooled feces, and filter membranes after filtration of 1000-, 500-, and 150-mL water samples by real-time PCR analysis. Sensitivity varied according to sample type and pathogen, and environmental sampling was significantly more sensitive than zebrafish sampling for detecting Mycobacterium spp. but not for Pseudocapillaria neurophilia or Pseudoloma tomentosa. The results of these experiments provide strong evidence of the utility of multiple sample types for detecting pathogens according to each pathogen's life cycle and ecological niche within zebrafish systems. In a separate experiment, zebrafish subclinically infected with M. chelonae, M. marinum, Pleistophora hyphessobryconis, Pseudocapillaria tomentosa, or Pseudoloma neurophilia were pair-spawned and individually tested with subsets of embryos from each clutch that received no rinse, a fluidizing rinse, or were surface-disinfected with sodium hypochlorite. Frequently, one or both parents were subclinically infected with pathogen(s) that were not detected in any embryo subset. Therefore, negative results from embryo samples may not reflect the health status of the parent zebrafish.

  2. Comparison of Antemortem and Environmental Samples for Zebrafish Health Monitoring and Quarantine

    PubMed Central

    Crim, Marcus J; Lawrence, Christian; Livingston, Robert S; Rakitin, Andrei; Hurley, Shane J; Riley, Lela K

    2017-01-01

    Molecular diagnostic assays offer both exquisite sensitivity and the ability to test a wide variety of sample types. Various types of environmental sample, such as detritus and concentrated water, might provide a useful adjunct to sentinels in routine zebrafish health monitoring. Similarly, antemortem sampling would be advantageous for expediting zebrafish quarantine, without euthanasia of valuable fish. We evaluated the detection of Mycobacterium chelonae, M. fortuitum, M. peregrinum, Pseudocapillaria tomentosa, and Pseudoloma neurophilia in zebrafish, detritus, pooled feces, and filter membranes after filtration of 1000-, 500-, and 150-mL water samples by real-time PCR analysis. Sensitivity varied according to sample type and pathogen, and environmental sampling was significantly more sensitive than zebrafish sampling for detecting Mycobacterium spp. but not for Pseudocapillaria neurophilia or Pseudoloma tomentosa. The results of these experiments provide strong evidence of the utility of multiple sample types for detecting pathogens according to each pathogen's life cycle and ecological niche within zebrafish systems. In a separate experiment, zebrafish subclinically infected with M. chelonae, M. marinum, Pleistophora hyphessobryconis, Pseudocapillaria tomentosa, or Pseudoloma neurophilia were pair-spawned and individually tested with subsets of embryos from each clutch that received no rinse, a fluidizing rinse, or were surface-disinfected with sodium hypochlorite. Frequently, one or both parents were subclinically infected with pathogen(s) that were not detected in any embryo subset. Therefore, negative results from embryo samples may not reflect the health status of the parent zebrafish. PMID:28724491

  3. Altered Glycolysis and Mitochondrial Respiration in a Zebrafish Model of Dravet Syndrome.

    PubMed

    Kumar, Maneesh G; Rowley, Shane; Fulton, Ruth; Dinday, Matthew T; Baraban, Scott C; Patel, Manisha

    2016-01-01

    Altered metabolism is an important feature of many epileptic syndromes but has not been reported in Dravet syndrome (DS), a catastrophic childhood epilepsy associated with mutations in a voltage-activated sodium channel, Nav1.1 (SCN1A). To address this, we developed novel methodology to assess real-time changes in bioenergetics in zebrafish larvae between 4 and 6 d postfertilization (dpf). Baseline and 4-aminopyridine (4-AP) stimulated glycolytic flux and mitochondrial respiration were simultaneously assessed using a Seahorse Biosciences extracellular flux analyzer. Scn1Lab mutant zebrafish showed a decrease in baseline glycolytic rate and oxygen consumption rate (OCR) compared to controls. A ketogenic diet formulation rescued mutant zebrafish metabolism to control levels. Increasing neuronal excitability with 4-AP resulted in an immediate increase in glycolytic rates in wild-type zebrafish, whereas mitochondrial OCR increased slightly and quickly recovered to baseline values. In contrast, scn1Lab mutant zebrafish showed a significantly slower and exaggerated increase of both glycolytic rates and OCR after 4-AP. The underlying mechanism of decreased baseline OCR in scn1Lab mutants was not because of altered mitochondrial DNA content or dysfunction of enzymes in the electron transport chain or tricarboxylic acid cycle. Examination of glucose metabolism using a PCR array identified five glycolytic genes that were downregulated in scn1Lab mutant zebrafish. Our findings in scn1Lab mutant zebrafish suggest that glucose and mitochondrial hypometabolism contribute to the pathophysiology of DS.

  4. Shoaling develops with age in Zebrafish (Danio rerio)

    PubMed Central

    Buske, Christine; Gerlai, Robert

    2010-01-01

    The biological mechanisms of human social behavior are complex. Animal models may facilitate the understanding of these mechanisms and may help one to develop treatment strategies for abnormal human social behavior, a core symptom in numerous clinical conditions. The zebrafish is perhaps the most social vertebrate among commonly used laboratory species. Given its practical features and the numerous genetic tools developed for it, it should be a promising tool. Zebrafish shoal, i.e. form tight multimember groups, but the ontogenesis of this behavior has not been described. Analyzing the development of shoaling is a step towards discovering the mechanisms of this behavior. Here we study age-dependent changes of shoaling in zebrafish from day 7 post fertilization to over 5 months of age by measuring the distance between all pairs of fish in freely swimming groups of ten subjects. Our longitudinal (repeated measure within subject) and cross sectional (non-repeated measure between subject) analyses both demonstrated a significant increase of shoaling with age (decreased distance between shoal members). Given the sophisticated genetic and developmental biology methods already available for zebrafish, we argue that our behavioral results open a new avenue towards the understanding of the development of vertebrate social behavior and of its mechanisms and abnormalities. PMID:20837077

  5. A Sketch of the Taiwan Zebrafish Core Facility.

    PubMed

    You, May-Su; Jiang, Yun-Jin; Yuh, Chiou-Hwa; Wang, Chien-Ming; Tang, Chih-Hao; Chuang, Yung-Jen; Lin, Bo-Hung; Wu, Jen-Leih; Hwang, Sheng-Ping L

    2016-07-01

    In the past three decades, the number of zebrafish laboratories has significantly increased in Taiwan. The Taiwan Zebrafish Core Facility (TZCF), a government-funded core facility, was launched to serve this growing community. The Core Facility was built on two sites, one located at the National Health Research Institutes (NHRI, called Taiwan Zebrafish Core Facility at NHRI or TZeNH) and the other is located at the Academia Sinica (Taiwan Zebrafish Core Facility at AS a.k.a. TZCAS). The total surface area of the TZCF is about 180 m(2) encompassing 2880 fish tanks. Each site has a separate quarantine room and centralized water recirculating systems, monitoring key water parameters. To prevent diseases, three main strategies have been implemented: (1) imported fish must be quarantined; (2) only bleached embryos are introduced into the main facilities; and (3) working practices were implemented to minimize pathogen transfer between stocks and facilities. Currently, there is no health program in place; however, a fourth measure for the health program, specific regular pathogen tests, is being planned. In March 2015, the TZCF at NHRI has been AAALAC accredited. It is our goal to ensure that we provide "disease-free" fish and embryos to the Taiwanese research community.

  6. Imaging Subcellular Structures in the Living Zebrafish Embryo.

    PubMed

    Engerer, Peter; Plucinska, Gabriela; Thong, Rachel; Trovò, Laura; Paquet, Dominik; Godinho, Leanne

    2016-04-02

    In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

  7. Curcumin affects development of zebrafish embryo.

    PubMed

    Wu, Jheng-Yu; Lin, Chin-Yi; Lin, Tien-Wei; Ken, Chuian-Fu; Wen, Yu-Der

    2007-07-01

    Embryotoxic and teratogenic effects of curcumin on the development of zebrafish embryo were investi-gated in this study. The LD(50) values of curcumin (24-h incubation) were estimated at 7.5 microM and 5 microM for embryos and larvae, respectively. The developmental defects caused by curcumin treatments include bent or hook-like tails, spinal column curving, edema in pericardial sac, retarded yolk sac resorption, and shorter body length. In curcumin-treated larvae, fluorescence signals of curcumin were found in edamae sac and some skin cells. Together, these results indicate that zebrafish are suitable model organisms to study the toxic effects of curcumin.

  8. Habenular kisspeptin modulates fear in the zebrafish

    PubMed Central

    Ogawa, Satoshi; Nathan, Fatima M.; Parhar, Ishwar S.

    2014-01-01

    Kisspeptin, a neuropeptide encoded by the KISS1/Kiss1, and its cognate G protein-coupled receptor, GPR54 (kisspeptin receptor, Kiss-R), are critical for the control of reproduction in vertebrates. We have previously identified two kisspeptin genes (kiss1 and kiss2) in the zebrafish, of which kiss1 neurons are located in the habenula, which project to the median raphe. kiss2 neurons are located in the hypothalamic nucleus and send axonal projections to gonadotropin-releasing hormone neurons and regulate reproductive functions. However, the physiological significance of the Kiss1 expressed in the habenula remains unknown. Here we demonstrate the role of habenular Kiss1 in alarm substance (AS)-induced fear response in the zebrafish. We found that AS-evoked fear experience significantly reduces kiss1 and serotonin-related genes (plasmacytoma expressed transcript 1 and solute carrier family 6, member 4) in the zebrafish. Furthermore, Kiss1 administration suppressed the AS-evoked fear response. To further evaluate the role of Kiss1 in fear response, zebrafish Kiss1 peptide was conjugated to saporin (SAP) to selectively inactivate Kiss-R1-expressing neurons. The Kiss1-SAP injection significantly reduced Kiss1 immunoreactivity and c-fos mRNA in the habenula and the raphe compared with control. Furthermore, 3 d after Kiss1-SAP injection, the fish had a significantly reduced AS-evoked fear response. These findings provide an insight into the role of the habenular kisspeptin system in inhibiting fear. PMID:24567386

  9. Grading and quantification of dental fluorosis in zebrafish larva.

    PubMed

    Zhang, Yutao; Zhang, Yanli; Zheng, Xueni; Xu, Rongchen; He, Huiming; Duan, Xiaohong

    2016-10-01

    The prevalence and severity of dental fluorosis in primary teeth are different from permanent teeth. Previous animal models of dental fluorosis mainly focus on juvenile rats, mice and zebrafish. Our experiment aims to set a dental fluorosis model using zebrafish larva and explore the characteristics of the first generation teeth by fluoride treatment. After the zebrafish eggs were laid, they were exposed to excess fluoride (19ppm, 38ppm and 76ppm) for five days. The morphological characteristics of first generation teeth were examined by H&E staining, whole-mount alizarin red and alcian blue staining, and scanning electron microscope (SEM) technique. With whole-mount alizarin red and alcian blue staining, the tooth cusps presented red in normal control. 19ppm and 38ppmm fluoride resulted in extensive red staining from tooth cusps to the lower 1/3 of teeth. 76ppm fluoride caused malformed teeth with uneven red staining. H&E staining showed that excess fluoride caused cystic-like changes in 38ppm and 76ppm groups. SEM revealed the dose dependent pathological changes in zebrafish enameloid with fluoride treatment. Based on SEM findings, we set 0-4 dental fluorosis index (DFI) score to label the severity of dental fluorosis. Excess fluoride presented a dose dependent fluorosis changes in the teeth of zebrafish larva. The DFI scores in our experiment reflect dose dependent fluorosis changes in a good way and will benefit the future research of dental fluorosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Using the zebrafish to understand tendon development and repair

    PubMed Central

    Chen, Jessica W.; Galloway, Jenna L.

    2017-01-01

    Tendons are important components of our musculoskeletal system. Injuries to these tissues are very common, resulting from occupational-related injuries, sports-related trauma, and age-related degeneration. Unfortunately, there are few treatment options, and current therapies rarely restore injured tendons to their original function. An improved understanding of the pathways regulating their development and repair would have significant impact in stimulating the formulation of regenerative-based approaches for tendon injury. The zebrafish provides an ideal system in which to perform genetic and chemical screens to identify new pathways involved in tendon biology. Until recently, there had been few descriptions of tendons and ligaments in the zebrafish and their similarity to mammalian tendon tissues. In this chapter, we describe the development of the zebrafish tendon and ligament tissues in the context of their gene expression, structure, and interactions with neighboring musculoskeletal tissues. We highlight the similarities with tendon development in higher vertebrates, showing that the craniofacial tendons and ligaments in zebrafish morphologically, molecularly, and structurally resemble mammalian tendons and ligaments from embryonic to adult stages. We detail methods for fluorescent in situ hybridization and immunohistochemistry as an assay to examine morphological changes in the zebrafish musculoskeleton. Staining assays such as these could provide the foundation for screen-based approaches to identify new regulators of tendon development, morphogenesis, and repair. These discoveries would provide new targets and pathways to study in the context of regenerative medicine-based approaches to improve tendon healing. PMID:28129848

  11. Melatonin mitigates neomycin-induced hair cell injury in zebrafish.

    PubMed

    Oh, Kyoung Ho; Rah, Yoon Chan; Hwang, Kyu Ho; Lee, Seung Hoon; Kwon, Soon Young; Cha, Jae Hyung; Choi, June

    2017-10-01

    Ototoxicity due to medications, such as aminoglycosides, is irreversible, and free radicals in the inner ear are assumed to play a major role. Because melatonin has an antioxidant property, we hypothesize that it might mitigate hair cell injury by aminoglycosides. The objective of this study was to evaluate whether melatonin has an alleviative effect on neomycin-induced hair cell injury in zebrafish (Danio rerio). Various concentrations of melatonin were administered to 5-day post-fertilization zebrafish treated with 125 μM neomycin for 1 h. Surviving hair cells within four neuromasts were compared with that of a control group. Apoptosis was assessed via terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The changes of ultrastructure were confirmed using a scanning electron microscope. Melatonin alleviated neomycin-induced hair cell injury in neuromasts (neomycin + melatonin 100 μM: 13.88 ± 0.91 cells, neomycin only: 7.85 ± 0.90 cells; n = 10, p < 0.05) and reduced neomycin-induced apoptosis in the TUNEL assay. In ultrastructural analysis, hair cells within the neuromasts in zebrafish were preserved exposed to 125 μM neomycin and 100 μM melatonin for 1 h in SEM findings. Melatonin is effective in alleviating aminoglycoside-induced hair cell injury in zebrafish. The results of this study demonstrated that melatonin has the potential to reduce apoptosis induced by aminoglycosides in zebrafish.

  12. Genome-wide Gene Expression Profiling of Acute Metal Exposures in Male Zebrafish

    DTIC Science & Technology

    2014-10-23

    Data in Brief Genome-wide gene expression profiling of acute metal exposures in male zebrafish Christine E. Baer a,⁎, Danielle L. Ippolito b, Naissan... Zebrafish Whole organism Nickel Chromium Cobalt Toxicogenomics To capture global responses to metal poisoning and mechanistic insights into metal...toxicity, gene expression changes were evaluated in whole adult male zebrafish following acute 24 h high dose exposure to three metals with known human

  13. Effects of abnormal light-rearing conditions on retinal physiology in larvae zebrafish.

    PubMed

    Saszik, S; Bilotta, J

    1999-11-01

    Anatomic studies have found that zebrafish retinal neurons develop in a sequential fashion. In addition, exposure to abnormal light-rearing conditions produces deficits in visual behavior of larvae zebrafish, even though there appears to be little effect of the light-rearing conditions on the gross morphology of the retina. The purpose of this study was to assess the effects of abnormal light-rearing conditions on larvae zebrafish retinal physiology. Larvae zebrafish (Danio rerio) were exposed to constant light (LL), constant dark (DD), or normal cyclic light (LD) from fertilization to 6 days postfertilization (dpf). After 6 days, the animals were placed into normal cyclic light and tested at 6 to 8, 13 to 15, and 21 to 24 dpf. Electroretinogram (ERG) responses to visual stimuli, consisting of various wavelengths and irradiances, were recorded. Comparisons were made across the three age groups and the three light-rearing conditions. Deficits from the light-rearing conditions were seen immediately after exposure (6 8 dpf). The LL-condition subjects showed the greatest deficit in the UV and short-wavelength areas and the DD-condition subjects showed a slight deficit across the entire spectrum. At 13 to 15 dpf, the LL and DD groups showed an increase in sensitivity and by 21 to 24 dpf, the groups no longer differed from controls. Abnormal lighting environments can adversely influence the physiological development of the larvae zebrafish retina. The pattern of damage that was seen in zebrafish is similar to that found in other vertebrates, including higher vertebrates. However, unlike higher vertebrates, the zebrafish appears to be capable of regeneration. This suggests that the zebrafish would be a viable model for light environment effects and neural regeneration.

  14. Effect of acute ethanol administration on zebrafish tail-beat motion.

    PubMed

    Bartolini, Tiziana; Mwaffo, Violet; Butail, Sachit; Porfiri, Maurizio

    2015-11-01

    Zebrafish is becoming a species of choice in neurobiological and behavioral studies of alcohol-related disorders. In these efforts, the activity of adult zebrafish is typically quantified using indirect activity measures that are either scored manually or identified automatically from the fish trajectory. The analysis of such activity measures has produced important insight into the effect of acute ethanol exposure on individual and social behavior of this vertebrate species. Here, we leverage a recently developed tracking algorithm that reconstructs fish body shape to investigate the effect of acute ethanol administration on zebrafish tail-beat motion in terms of amplitude and frequency. Our results demonstrate a significant effect of ethanol on the tail-beat amplitude as well as the tail-beat frequency, both of which were found to robustly decrease for high ethanol concentrations. Such a direct measurement of zebrafish motor functions is in agreement with evidence based on indirect activity measures, offering a complementary perspective in behavioral screening. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Elucidating the mechanism of action of tributyltin (TBT) in zebrafish.

    PubMed

    McGinnis, Courtney L; Crivello, Joseph F

    2011-05-01

    Tributyltin (TBT), an antifouling agent, has been implicated in the masculinization of fish species worldwide, but the masculinizing mechanism is not fully understood. We have examined the actions of TBT as an endocrine disruptor in zebrafish (Danio rerio). In HeLa cells transiently co-transfected with plasmid constructs containing the zebrafish estrogen receptors (zfERα, zfERβ(1) and zfERβ(2)) and the zebrafish estrogen response element (zfERE-tk-luc), ethinyl estradiol (EE2) induced luciferase activity 4 to 6-fold and was inhibited by TBT. In HeLa cells transiently co-transfected with the zebrafish androgen receptor (zfAR) and the murine androgen receptor response element (ARE-slp-luc), testosterone induced luciferase activity was not inhibited by TBT. In HeLa cells co-transfected with zfERα, zfERβ(1) and zfERβ(2) and a plasmid containing zebrafish aromatase (zfCyp19b-luc), TBT inhibited luciferase activity. In zebrafish exposed to 1mg/kg and 5mg/kg TBT in vivo, there was a increase in liver sulfotransferase and a decrease acyl-CoA testosterone acyltransferase activity. Real-time PCR analysis of sexual differentiation markers in fish exposed to TBT in vivo revealed a tissue-specific response. In brain there was increased production of Sox9, Dax1, and SF1 mRNA, an androgenizing effect, while in the liver there was increased production of Dax1, Cyp19a and zfERβ(1) mRNA but decreased production of Sox9 mRNA, a feminizing effect. In the gonads there was increased production of zfERα and zfCyp19a mRNA, again a feminizing effect. TBT has an overall masculinizing effect but the masculinizing effect is tempered by a feminizing effect on gene transcription in certain tissues. These results are discussed in the context of TBT as an endocrine disruptor in zebrafish. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. Multiple zebrafish atoh1 genes specify a diversity of neuronal types in the zebrafish cerebellum.

    PubMed

    Kidwell, Chelsea U; Su, Chen-Ying; Hibi, Masahiko; Moens, Cecilia B

    2018-06-01

    A single Atoh1 basic-helix-loop-helix transcription factor specifies multiple neuron types in the mammalian cerebellum and anterior hindbrain. The zebrafish genome encodes three paralagous atoh1 genes whose functions in cerebellum and anterior hindbrain development we explore here. With use of a transgenic reporter, we report that zebrafish atoh1c-expressing cells are organized in two distinct domains that are separated both by space and developmental time. An early isthmic expression domain gives rise to an extracerebellar population in rhombomere 1 and an upper rhombic lip domain gives rise to granule cell progenitors that migrate to populate all four granule cell territories of the fish cerebellum. Using genetic mutants we find that of the three zebrafish atoh1 paralogs, atoh1c and atoh1a are required for the full complement of granule neurons. Surprisingly, the two genes are expressed in non-overlapping granule cell progenitor populations, indicating that fish use duplicate atoh1 genes to generate granule cell diversity that is not detected in mammals. Finally, live imaging of granule cell migration in wildtype and atoh1c mutant embryos reveals that while atoh1c is not required for granule cell specification per se, it is required for granule cells to delaminate and migrate away from the rhombic lip. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. The zebrafish reference genome sequence and its relationship to the human genome.

    PubMed

    Howe, Kerstin; Clark, Matthew D; Torroja, Carlos F; Torrance, James; Berthelot, Camille; Muffato, Matthieu; Collins, John E; Humphray, Sean; McLaren, Karen; Matthews, Lucy; McLaren, Stuart; Sealy, Ian; Caccamo, Mario; Churcher, Carol; Scott, Carol; Barrett, Jeffrey C; Koch, Romke; Rauch, Gerd-Jörg; White, Simon; Chow, William; Kilian, Britt; Quintais, Leonor T; Guerra-Assunção, José A; Zhou, Yi; Gu, Yong; Yen, Jennifer; Vogel, Jan-Hinnerk; Eyre, Tina; Redmond, Seth; Banerjee, Ruby; Chi, Jianxiang; Fu, Beiyuan; Langley, Elizabeth; Maguire, Sean F; Laird, Gavin K; Lloyd, David; Kenyon, Emma; Donaldson, Sarah; Sehra, Harminder; Almeida-King, Jeff; Loveland, Jane; Trevanion, Stephen; Jones, Matt; Quail, Mike; Willey, Dave; Hunt, Adrienne; Burton, John; Sims, Sarah; McLay, Kirsten; Plumb, Bob; Davis, Joy; Clee, Chris; Oliver, Karen; Clark, Richard; Riddle, Clare; Elliot, David; Eliott, David; Threadgold, Glen; Harden, Glenn; Ware, Darren; Begum, Sharmin; Mortimore, Beverley; Mortimer, Beverly; Kerry, Giselle; Heath, Paul; Phillimore, Benjamin; Tracey, Alan; Corby, Nicole; Dunn, Matthew; Johnson, Christopher; Wood, Jonathan; Clark, Susan; Pelan, Sarah; Griffiths, Guy; Smith, Michelle; Glithero, Rebecca; Howden, Philip; Barker, Nicholas; Lloyd, Christine; Stevens, Christopher; Harley, Joanna; Holt, Karen; Panagiotidis, Georgios; Lovell, Jamieson; Beasley, Helen; Henderson, Carl; Gordon, Daria; Auger, Katherine; Wright, Deborah; Collins, Joanna; Raisen, Claire; Dyer, Lauren; Leung, Kenric; Robertson, Lauren; Ambridge, Kirsty; Leongamornlert, Daniel; McGuire, Sarah; Gilderthorp, Ruth; Griffiths, Coline; Manthravadi, Deepa; Nichol, Sarah; Barker, Gary; Whitehead, Siobhan; Kay, Michael; Brown, Jacqueline; Murnane, Clare; Gray, Emma; Humphries, Matthew; Sycamore, Neil; Barker, Darren; Saunders, David; Wallis, Justene; Babbage, Anne; Hammond, Sian; Mashreghi-Mohammadi, Maryam; Barr, Lucy; Martin, Sancha; Wray, Paul; Ellington, Andrew; Matthews, Nicholas; Ellwood, Matthew; Woodmansey, Rebecca; Clark, Graham; Cooper, James D; Cooper, James; Tromans, Anthony; Grafham, Darren; Skuce, Carl; Pandian, Richard; Andrews, Robert; Harrison, Elliot; Kimberley, Andrew; Garnett, Jane; Fosker, Nigel; Hall, Rebekah; Garner, Patrick; Kelly, Daniel; Bird, Christine; Palmer, Sophie; Gehring, Ines; Berger, Andrea; Dooley, Christopher M; Ersan-Ürün, Zübeyde; Eser, Cigdem; Geiger, Horst; Geisler, Maria; Karotki, Lena; Kirn, Anette; Konantz, Judith; Konantz, Martina; Oberländer, Martina; Rudolph-Geiger, Silke; Teucke, Mathias; Lanz, Christa; Raddatz, Günter; Osoegawa, Kazutoyo; Zhu, Baoli; Rapp, Amanda; Widaa, Sara; Langford, Cordelia; Yang, Fengtang; Schuster, Stephan C; Carter, Nigel P; Harrow, Jennifer; Ning, Zemin; Herrero, Javier; Searle, Steve M J; Enright, Anton; Geisler, Robert; Plasterk, Ronald H A; Lee, Charles; Westerfield, Monte; de Jong, Pieter J; Zon, Leonard I; Postlethwait, John H; Nüsslein-Volhard, Christiane; Hubbard, Tim J P; Roest Crollius, Hugues; Rogers, Jane; Stemple, Derek L

    2013-04-25

    Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.

  18. The essential role of endogenous ghrelin in growth hormone expression during zebrafish adenohypophysis development.

    PubMed

    Li, Xi; He, Jiangyan; Hu, Wei; Yin, Zhan

    2009-06-01

    Ghrelin, a multifunctional hormone, including potent GH stimulation activity, has been suggested to be important during embryonic development. Expression of ghrelin has been confirmed in the zebrafish pancreas during embryonic stages. Interfering with ghrelin function using two specific antisense morpholino oligonucleotides causes defects during zebrafish embryonic development. In ghrelin morphants the expression of GH was abolished in zebrafish somatotropes, whereas the expression patterns of the other key molecules involved in hypothalamic-pituitary development and distinct pituitary hormones genes remain largely intact at the appropriate time during zebrafish adenohypophysis development. Effective rescue of the ghrelin morphants with exogenous ghrelin mRNA showed that the correct gene had been targeted. Moreover, by analyzing the efficiencies of the ghrelin morphants rescue experiments with various forms of exogenous mutant ghrelin mRNAs, we also demonstrated the essentiality of the form acyl-ghrelin on GH stimulation during zebrafish adenohypophysis development. Our in vivo experiments, for the first time, also provided evidence of the existence of functional obestatin in the C-terminal part of zebrafish proghrelin peptides. Our research here has demonstrated that zebrafish is a unique model for functional studies of endogenous ghrelin, especially during embryonic development.

  19. Cortisol elevation post-hatch affects behavioural performance in zebrafish larvae.

    PubMed

    Best, Carol; Vijayan, Mathilakath M

    2018-02-01

    Maternal cortisol is essential for cortisol stress axis development and de novo production of this steroid commences only after hatch in zebrafish (Danio rerio). However, very little is known about the effect of elevated cortisol levels, during the critical period of stress axis activation, on larval performance. We tested the hypothesis that elevated cortisol levels post-hatch affect behavioural performance and this is mediated by glucocorticoid receptor (GR) activation in zebrafish larvae. The behavioural response included measuring larval activity in response to alternating light and dark cycles, as well as thigmotaxis. Zebrafish larvae at 3days post-fertilization were exposed to waterborne cortisol for 24h to mimic a steroid response to an early-life stressor exposure. Also, larvae were exposed to waterborne RU-486 (a GR antagonist) either in the presence or absence of cortisol to confirm GR activation. Co-treatment with RU-486 completely abolished the upregulation of cortisol-induced 11β-hydroxysteroid dehydrogenase type 2 transcript abundance, confirming GR signalling. Cortisol-exposed larvae displayed increased locomotor activity irrespective of light condition, but showed no changes in thigmotaxis. This cortisol-mediated behavioural response was not affected by co-treatment with RU-486. Cortisol exposure also did not modify the transcript abundances of GR and mineralocorticoid receptor (MR) in zebrafish larvae. Altogether, cortisol stress axis activation post-hatch increases locomotor activity in zebrafish larvae. Our results suggest that GR signalling may not be involved in this behavioural response, leading to the proposal that cortisol action via MR signalling may influence locomotor activity in zebrafish larvae. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Developmental Neurotoxicity of Methamidophos in the Embryo-Larval Stages of Zebrafish.

    PubMed

    He, Xiaowei; Gao, Jiawei; Dong, Tianyu; Chen, Minjian; Zhou, Kun; Chang, Chunxin; Luo, Jia; Wang, Chao; Wang, Shoulin; Chen, Daozhen; Zhou, Zuomin; Tian, Ying; Xia, Yankai; Wang, Xinru

    2016-12-28

    Methamidophos is a representative organophosphate insecticide. The knowledge of its developmental neurotoxicity is limited, especially for zebrafish in the early stages of their life. Four hour post-fertilization (hpf) zebrafish embryos were exposed to several environmentally relevant concentrations of methamidophos (0, 25, and 500 μg/L) for up to 72 hpf. Locomotor behavior was then studied in the zebrafish larvae at this timepoint. Acridine orange (AO) staining was carried out in the zebrafish larvae, and the mRNA levels of genes associated with neural development ( mbp and syn2a ) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The number of escape responders for mechanical stimulation was significantly decreased in exposed groups. AO staining showed noticeable signs of apoptosis mainly in the brain. In addition, the mRNA levels of mbp and syn2a were both significantly down-regulated in exposed groups. Our study provides the first evidence that methamidophos exposure can cause developmental neurotoxicity in the early stages of zebrafish life, which may be caused by the effect of methamidophos on neurodevelopmental genes and the activation of cell apoptosis in the brain.

  1. Guided inquiry lab exercises in development and oxygen consumption using zebrafish.

    PubMed

    Bagatto, Brian

    2009-06-01

    Zebrafish have become a model organism in many areas of research and are now being used with more frequency in the classroom to teach important biological concepts. The two guided inquiry exercises in this article are each aimed at a different level of instruction, but each can be modified to fit the needs of many high school or college-level courses. The "Zebrafish Development and Environment" exercise teaches high school students about zebrafish development by presenting a series of embryos at different ages. Without access to visual references, students are asked to rank developing zebrafish by age and explain their choices. The students also learn about the heart and circulatory system and the effects of temperature on physiological processes. The second exercise, "Oxygen Consumption," is a 2-week laboratory designed for introductory college biology majors and involves the concept of oxygen consumption as a predictor of metabolic rate. During the first week of lab, students are introduced to the concept and learn how to measure oxygen consumption in zebrafish. In the second week, they perform an instructor-approved experiment of their own design, analyze the results using statistics, and write a report.

  2. Developmental social isolation affects adult behavior, social interaction, and dopamine metabolite levels in zebrafish.

    PubMed

    Shams, Soaleha; Amlani, Shahid; Buske, Christine; Chatterjee, Diptendu; Gerlai, Robert

    2018-01-01

    The zebrafish is a social vertebrate and an excellent translational model for a variety of human disorders. Abnormal social behavior is a hallmark of several human brain disorders. Social behavioral problems can arise as a result of adverse early social environment. Little is known about the effects of early social isolation in adult zebrafish. We compared zebrafish that were isolated for either short (7 days) or long duration (180 days) to socially housed zebrafish, testing their behavior across ontogenesis (ages 10, 30, 60, 90, 120, 180 days), and shoal cohesion and whole-brain monoamines and their metabolites in adulthood. Long social isolation increased locomotion and decreased shoal cohesion and anxiety in the open-field in adult. Additionally, both short and long social isolation reduced dopamine metabolite levels in response to social stimuli. Thus, early social isolation has lasting effects in zebrafish, and may be employed to generate zebrafish models of human neuropsychiatric conditions. © 2017 Wiley Periodicals, Inc.

  3. A multi-scale model for correlation in B cell VDJ usage of zebrafish

    NASA Astrophysics Data System (ADS)

    Pan, Keyao; Deem, Michael W.

    2011-10-01

    The zebrafish (Danio rerio) is one of the model animals used for the study of immunology because the dynamics in the adaptive immune system of zebrafish are similar to that in higher animals. In this work, we built a multi-scale model to simulate the dynamics of B cells in the primary and secondary immune responses of zebrafish. We use this model to explain the reported correlation between VDJ usage of B cell repertoires in individual zebrafish. We use a delay ordinary differential equation (ODE) system to model the immune responses in the 6-month lifespan of a zebrafish. This mean field theory gives the number of high-affinity B cells as a function of time during an infection. The sequences of those B cells are then taken from a distribution calculated by a 'microscopic' random energy model. This generalized NK model shows that mature B cells specific to one antigen largely possess a single VDJ recombination. The model allows first-principle calculation of the probability, p, that two zebrafish responding to the same antigen will select the same VDJ recombination. This probability p increases with the B cell population size and the B cell selection intensity. The probability p decreases with the B cell hypermutation rate. The multi-scale model predicts correlations in the immune system of the zebrafish that are highly similar to that from experiment.

  4. Repairing quite swimmingly: advances in regenerative medicine using zebrafish.

    PubMed

    Goessling, Wolfram; North, Trista E

    2014-07-01

    Regenerative medicine has the promise to alleviate morbidity and mortality caused by organ dysfunction, longstanding injury and trauma. Although regenerative approaches for a few diseases have been highly successful, some organs either do not regenerate well or have no current treatment approach to harness their intrinsic regenerative potential. In this Review, we describe the modeling of human disease and tissue repair in zebrafish, through the discovery of disease-causing genes using classical forward-genetic screens and by modulating clinically relevant phenotypes through chemical genetic screening approaches. Furthermore, we present an overview of those organ systems that regenerate well in zebrafish in contrast to mammalian tissue, as well as those organs in which the regenerative potential is conserved from fish to mammals, enabling drug discovery in preclinical disease-relevant models. We provide two examples from our own work in which the clinical translation of zebrafish findings is either imminent or has already proven successful. The promising results in multiple organs suggest that further insight into regenerative mechanisms and novel clinically relevant therapeutic approaches will emerge from zebrafish research in the future. © 2014. Published by The Company of Biologists Ltd.

  5. Hypothalamic Projections to the Optic Tectum in Larval Zebrafish.

    PubMed

    Heap, Lucy A; Vanwalleghem, Gilles C; Thompson, Andrew W; Favre-Bulle, Itia; Rubinsztein-Dunlop, Halina; Scott, Ethan K

    2017-01-01

    The optic tectum of larval zebrafish is an important model for understanding visual processing in vertebrates. The tectum has been traditionally viewed as dominantly visual, with a majority of studies focusing on the processes by which tectal circuits receive and process retinally-derived visual information. Recently, a handful of studies have shown a much more complex role for the optic tectum in larval zebrafish, and anatomical and functional data from these studies suggest that this role extends beyond the visual system, and beyond the processing of exclusively retinal inputs. Consistent with this evolving view of the tectum, we have used a Gal4 enhancer trap line to identify direct projections from rostral hypothalamus (RH) to the tectal neuropil of larval zebrafish. These projections ramify within the deepest laminae of the tectal neuropil, the stratum album centrale (SAC)/stratum griseum periventriculare (SPV), and also innervate strata distinct from those innervated by retinal projections. Using optogenetic stimulation of the hypothalamic projection neurons paired with calcium imaging in the tectum, we find rebound firing in tectal neurons consistent with hypothalamic inhibitory input. Our results suggest that tectal processing in larval zebrafish is modulated by hypothalamic inhibitory inputs to the deep tectal neuropil.

  6. Strategies for Analyzing Cardiac Phenotypes in the Zebrafish Embryo

    PubMed Central

    Houk, Andrew R.; Yelon, Deborah

    2017-01-01

    The molecular mechanisms underlying cardiogenesis are of critical biomedical importance due to the high prevalence of cardiac birth defects. Over the past two decades, the zebrafish has served as a powerful model organism for investigating heart development, facilitated by its powerful combination of optical access to the embryonic heart and plentiful opportunities for genetic analysis. Work in zebrafish has identified numerous factors that are required for various aspects of heart formation, including the specification and differentiation of cardiac progenitor cells, the morphogenesis of the heart tube, cardiac chambers, and atrioventricular canal, and the establishment of proper cardiac function. However, our current roster of regulators of cardiogenesis is by no means complete. It is therefore valuable for ongoing studies to continue pursuit of additional genes and pathways that control the size, shape, and function of the zebrafish heart. An extensive arsenal of techniques is available to distinguish whether particular mutations, morpholinos, or small molecules disrupt specific processes during heart development. In this chapter, we provide a guide to the experimental strategies that are especially effective for the characterization of cardiac phenotypes in the zebrafish embryo. PMID:27312497

  7. Zebrafish heart regeneration: 15 years of discoveries

    PubMed Central

    González‐Rosa, Juan Manuel; Burns, Caroline E.

    2017-01-01

    Abstract Cardiovascular disease is the leading cause of death worldwide. Compared to other organs such as the liver, the adult human heart lacks the capacity to regenerate on a macroscopic scale after injury. As a result, myocardial infarctions are responsible for approximately half of all cardiovascular related deaths. In contrast, the zebrafish heart regenerates efficiently upon injury through robust myocardial proliferation. Therefore, deciphering the mechanisms that underlie the zebrafish heart's endogenous regenerative capacity represents an exciting avenue to identify novel therapeutic strategies for inducing regeneration of the human heart. This review provides a historical overview of adult zebrafish heart regeneration. We summarize 15 years of research, with a special focus on recent developments from this fascinating field. We discuss experimental findings that address fundamental questions of regeneration research. What is the origin of regenerated muscle? How is regeneration controlled from a genetic and molecular perspective? How do different cell types interact to achieve organ regeneration? Understanding natural models of heart regeneration will bring us closer to answering the ultimate question: how can we stimulate myocardial regeneration in humans? PMID:28979788

  8. Biotransformation of ginsenosides F4 and Rg6 in zebrafish.

    PubMed

    Shen, Wen-Wen; Zhang, Hai-Xia; Qiu, Shou-Bei; Wei, Ying-Jie; Zhu, Fen-Xia; Wang, Jing; Wang, Dan-Dan; Jia, Xiao-Bin; Tang, Dao-Quan; Chen, Bin

    2017-03-28

    Ginsenosides F 4 and Rg 6 (GF 4 and GRg 6 ), two main active components of steamed notoginseng or red ginseng, are dehydrated disaccharide saponins. In this work, biotransformation of ginsenosides F 4 and Rg 6 in zebrafish was investigated by qualitatively identifying their metabolites and then proposing their possible metabolic pathways. The prediction of possible metabolism of ginsenosides F 4 and Rg 6 using zebrafish model which can effectively simulate existing mammals model was early and quickly performed. Metabolites of ginsenosides F 4 and Rg 6 after exposing to zebrafish for 24 h were identified by Ultraperformance Liquid Chromatography/Quadrupole-Time-of-Flight Mass Spectrometry. A total of 8 and 6 metabolites of ginsenosides F 4 and Rg 6 were identified in zebrafish, respectively. Of these, 7 and 5, including M1, M3-M5, M7-M9 and N1 (N5), N2, N4 (N9), N7-N8 were reported for the first time as far as we know. The mechanisms of their biotransformation involved were further deduced to be desugarization, glucuronidation, sulfation, dehydroxylation, loss of C-17 and/or C-23 residue pathways. It was concluded that loss of rhamnose at position C-6 and glucuronidation at position C-3 in zebrafish were considered as the main physiologic and metabolic processes of ginsenosides F 4 and ginsenosides Rg 6 , respectively.

  9. A Retrospective Study of the Prevalence and Classification of Intestinal Neoplasia in Zebrafish (Danio Rerio)

    PubMed Central

    Paquette, Colleen E.; Buchner, Cari; Tanguay, Robert L.; Guillemin, Karen; Mason, Timothy J.; Peterson, Tracy S.

    2013-01-01

    Abstract For over a decade, spontaneous intestinal neoplasia has been observed in zebrafish (Danio rerio) submitted to the ZIRC (Zebrafish International Resource Center) diagnostic service. In addition, zebrafish displayed preneoplastic intestinal changes including hyperplasia, dysplasia, and enteritis. A total of 195 zebrafish, representing 2% of the total fish submitted to the service, were diagnosed with these lesions. Neoplastic changes were classified either as adenocarcinoma or small cell carcinoma, with a few exceptions (carcinoma not otherwise specified, tubular adenoma, and tubulovillous adenoma). Tumor prevalence appeared similarly distributed between sexes and generally occurred in zebrafish greater than 1 year of age, although neoplastic changes were observed in fish 6 months of age. Eleven lines displayed these preneoplastic and neoplastic changes, including wild-types and mutants. Affected zebrafish originated from 18 facilities, but the majority of fish were from a single zebrafish research facility (hereafter referred to as the primary facility) that has submitted numerous samples to the ZIRC diagnostic service. Zebrafish from the primary facility submitted as normal sentinel fish demonstrate that these lesions are most often subclinical. Fish fed the diet from the primary facility and held at another location did not develop intestinal lesions, indicating that diet is not the etiologic agent. PMID:23544991

  10. Mitragynine Attenuates Withdrawal Syndrome in Morphine-Withdrawn Zebrafish

    PubMed Central

    Khor, Beng-Siang; Amar Jamil, Mohd Fadzly; Adenan, Mohamad Ilham; Chong Shu-Chien, Alexander

    2011-01-01

    A major obstacle in treating drug addiction is the severity of opiate withdrawal syndrome, which can lead to unwanted relapse. Mitragynine is the major alkaloid compound found in leaves of Mitragyna speciosa, a plant widely used by opiate addicts to mitigate the harshness of drug withdrawal. A series of experiments was conducted to investigate the effect of mitragynine on anxiety behavior, cortisol level and expression of stress pathway related genes in zebrafish undergoing morphine withdrawal phase. Adult zebrafish were subjected to two weeks chronic morphine exposure at 1.5 mg/L, followed by withdrawal for 24 hours prior to tests. Using the novel tank diving tests, we first showed that morphine-withdrawn zebrafish display anxiety-related swimming behaviors such as decreased exploratory behavior and increased erratic movement. Morphine withdrawal also elevated whole-body cortisol levels, which confirms the phenotypic stress-like behaviors. Exposing morphine-withdrawn fish to mitragynine however attenuates majority of the stress-related swimming behaviors and concomitantly lower whole-body cortisol level. Using real-time PCR gene expression analysis, we also showed that mitragynine reduces the mRNA expression of corticotropin releasing factor receptors and prodynorphin in zebrafish brain during morphine withdrawal phase, revealing for the first time a possible link between mitragynine's ability to attenuate anxiety during opiate withdrawal with the stress-related corticotropin pathway. PMID:22205946

  11. Automated processing of zebrafish imaging data: a survey.

    PubMed

    Mikut, Ralf; Dickmeis, Thomas; Driever, Wolfgang; Geurts, Pierre; Hamprecht, Fred A; Kausler, Bernhard X; Ledesma-Carbayo, María J; Marée, Raphaël; Mikula, Karol; Pantazis, Periklis; Ronneberger, Olaf; Santos, Andres; Stotzka, Rainer; Strähle, Uwe; Peyriéras, Nadine

    2013-09-01

    Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines.

  12. Automated Processing of Zebrafish Imaging Data: A Survey

    PubMed Central

    Dickmeis, Thomas; Driever, Wolfgang; Geurts, Pierre; Hamprecht, Fred A.; Kausler, Bernhard X.; Ledesma-Carbayo, María J.; Marée, Raphaël; Mikula, Karol; Pantazis, Periklis; Ronneberger, Olaf; Santos, Andres; Stotzka, Rainer; Strähle, Uwe; Peyriéras, Nadine

    2013-01-01

    Abstract Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines. PMID:23758125

  13. Short-term memory in zebrafish (Danio rerio).

    PubMed

    Jia, Jason; Fernandes, Yohaan; Gerlai, Robert

    2014-08-15

    Learning and memory represent perhaps the most complex behavioral phenomena. Although their underlying mechanisms have been extensively analyzed, only a fraction of the potential molecular components have been identified. The zebrafish has been proposed as a screening tool with which mechanisms of complex brain functions may be systematically uncovered. However, as a relative newcomer in behavioral neuroscience, the zebrafish has not been well characterized for its cognitive and mnemonic features, thus learning and/or memory screens with adults have not been feasible. Here we study short-term memory of adult zebrafish. We show animated images of conspecifics (the stimulus) to the experimental subject during 1 min intervals on ten occasions separated by different (2, 4, 8 or 16 min long) inter-stimulus intervals (ISI), a between subject experimental design. We quantify the distance of the subject from the image presentation screen during each stimulus presentation interval, during each of the 1-min post-stimulus intervals immediately following the stimulus presentations and during each of the 1-min intervals furthest away from the last stimulus presentation interval and just before the next interval (pre-stimulus interval), respectively. Our results demonstrate significant retention of short-term memory even in the longest ISI group but suggest no acquisition of reference memory. Because in the employed paradigm both stimulus presentation and behavioral response quantification is computer automated, we argue that high-throughput screening for drugs or mutations that alter short-term memory performance of adult zebrafish is now becoming feasible. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Expression of Glycosaminoglycan Epitopes During Zebrafish Skeletogenesis

    PubMed Central

    Hayes, Anthony J; Mitchell, Ruth E; Bashford, Andrew; Reynolds, Scott; Caterson, Bruce; Hammond, Chrissy L

    2013-01-01

    Background: The zebrafish is an important developmental model. Surprisingly, there are few studies that describe the glycosaminoglycan composition of its extracellular matrix during skeletogenesis. Glycosaminoglycans on proteoglycans contribute to the material properties of musculo skeletal connective tissues, and are important in regulating signalling events during morphogenesis. Sulfation motifs within the chain structure of glycosaminoglycans on cell-associated and extracellular matrix proteoglycans allow them to bind and regulate the sequestration/presentation of bioactive signalling molecules important in musculo-skeletal development. Results: We describe the spatio-temporal expression of different glycosaminoglycan moieties during zebrafish skeletogenesis with antibodies recognising (1) native sulfation motifs within chondroitin and keratan sulfate chains, and (2) enzyme-generated neoepitope sequences within the chain structure of chondroitin sulfate (i.e., 0-, 4-, and 6-sulfated isoforms) and heparan sulfate glycosaminoglycans. We show that all the glycosaminoglycan moieties investigated are expressed within the developing skeletal tissues of larval zebrafish. However, subtle changes in their patterns of spatio-temporal expression over the period examined suggest that their expression is tightly and dynamically controlled during development. Conclusions: The subtle differences observed in the domains of expression between different glycosaminoglycan moieties suggest differences in their functional roles during establishment of the primitive analogues of the skeleton. Developmental Dynamics 242:778–789, 2013. © 2013 Wiley Periodicals, Inc. Key Findings The developing zebrafish skeleton expresses many different glycosaminoglycan modifications. Multiple different glycosaminoglycan epitopes are dynamically expressed in the craniofacial skeleton. Expression of chondroitin sulfate moieties are dynamically expressed in the vertebral column and precede

  15. Fish from Head to Tail: The 9th European Zebrafish Meeting in Oslo.

    PubMed

    Griffiths, Gareth; Müller, Ferenc; Ledin, Johan; Patton, E Elizabeth; Gjøen, Tor; Lobert, Viola Hélène; Winther-Larsen, Hanne Cecilie; Mullins, Mary; Joly, Jean-Stephane; Weltzien, Finn-Arne; Press, Charles McLean; Aleström, Peter

    2016-04-01

    The 9th European Zebrafish Meeting took place recently in Oslo (June 28-July 2, 2015). A total of 650 participants came to hear the latest research news focused on the zebrafish, Danio rerio, and to its distant evolutionary relative medaka, Oryzias latipes. The packed program included keynote and plenary talks, short oral presentations and poster sessions, workshops, and strategic discussions. The meeting was a great success and revealed dramatically how important the zebrafish in particular has become as a model system for topics, such as developmental biology, functional genomics, biomedicine, toxicology, and drug development. A new emphasis was given to its potential as a model for aquaculture, a topic of great economic interest to the host country Norway and for the future global food supply in general. Zebrafish husbandry as well as its use in teaching were also covered in separate workshops. As has become a tradition in these meetings, there was a well-attended Wellcome Trust Sanger Institute and ZFIN workshop focused on Zebrafish Genome Resources on the first day. The full EZM 2015 program with abstracts can be read and downloaded from the EZM 2015 Web site zebrafish2015.org .

  16. Social dominance modulates eavesdropping in zebrafish

    PubMed Central

    Abril-de-Abreu, Rodrigo; Cruz, Ana S.; Oliveira, Rui F.

    2015-01-01

    Group living animals may eavesdrop on signalling interactions between conspecifics and integrate it with their own past social experience in order to optimize the use of relevant information from others. However, little is known about this interplay between public (eavesdropped) and private social information. To investigate it, we first manipulated the dominance status of bystander zebrafish. Next, we either allowed or prevented bystanders from observing a fight. Finally, we assessed their behaviour towards the winners and losers of the interaction, using a custom-made video-tracking system and directional analysis. We found that only dominant bystanders who had seen the fight revealed a significant increase in directional focus (a measure of attention) towards the losers of the fights. Furthermore, our results indicate that information about the fighters' acquired status was collected from the signalling interaction itself and not from post-interaction status cues, which implies the existence of individual recognition in zebrafish. Thus, we show for the first time that zebrafish, a highly social model organism, eavesdrop on conspecific agonistic interactions and that this process is modulated by the eavesdroppers' dominance status. We suggest that this type of integration of public and private information may be ubiquitous in social learning processes. PMID:26361550

  17. Non-invasive electrocardiogram detection of in vivo zebrafish embryos using electric potential sensors

    NASA Astrophysics Data System (ADS)

    Rendon-Morales, E.; Prance, R. J.; Prance, H.; Aviles-Espinosa, R.

    2015-11-01

    In this letter, we report the continuous detection of the cardiac electrical activity in embryonic zebrafish using a non-invasive approach. We present a portable and cost-effective platform based on the electric potential sensing technology, to monitor in vivo electrocardiogram activity from the zebrafish heart. This proof of principle demonstration shows how electrocardiogram measurements from the embryonic zebrafish may become accessible by using electric field detection. We present preliminary results using the prototype, which enables the acquisition of electrophysiological signals from in vivo 3 and 5 days-post-fertilization zebrafish embryos. The recorded waveforms show electrocardiogram traces including detailed features such as QRS complex, P and T waves.

  18. Comparative Analyses of Zebrafish Anxiety-Like Behavior Using Conflict-Based Novelty Tests.

    PubMed

    Kysil, Elana V; Meshalkina, Darya A; Frick, Erin E; Echevarria, David J; Rosemberg, Denis B; Maximino, Caio; Lima, Monica Gomes; Abreu, Murilo S; Giacomini, Ana C; Barcellos, Leonardo J G; Song, Cai; Kalueff, Allan V

    2017-06-01

    Modeling of stress and anxiety in adult zebrafish (Danio rerio) is increasingly utilized in neuroscience research and central nervous system (CNS) drug discovery. Representing the most commonly used zebrafish anxiety models, the novel tank test (NTT) focuses on zebrafish diving in response to potentially threatening stimuli, whereas the light-dark test (LDT) is based on fish scototaxis (innate preference for dark vs. bright areas). Here, we systematically evaluate the utility of these two tests, combining meta-analyses of published literature with comparative in vivo behavioral and whole-body endocrine (cortisol) testing. Overall, the NTT and LDT behaviors demonstrate a generally good cross-test correlation in vivo, whereas meta-analyses of published literature show that both tests have similar sensitivity to zebrafish anxiety-like states. Finally, NTT evokes higher levels of cortisol, likely representing a more stressful procedure than LDT. Collectively, our study reappraises NTT and LDT for studying anxiety-like states in zebrafish, and emphasizes their developing utility for neurobehavioral research. These findings can help optimize drug screening procedures by choosing more appropriate models for testing anxiolytic or anxiogenic drugs.

  19. An integrative analysis of ethanol tolerance and withdrawal in zebrafish (Danio rerio)

    PubMed Central

    Tran, Steven; Chatterjee, Diptendu; Gerlai, Robert

    2014-01-01

    The zebrafish is emerging as a popular animal model for alcohol (ethanol or EtOH) addiction due to its simplicity and practical advantages. Two phenomena associated with ethanol addiction are the development of tolerance and withdrawal. Using a multi-level approach in the current study, we characterize ethanol tolerance and withdrawal in zebrafish. We first investigate the temporal trajectory of ethanol concentration in the zebrafish brain in response to an acute exposure and during withdrawal. We report that ethanol concentrations approach a steady state within 60 minutes of exposure to 0.50% and 1.00% v/v ethanol and rapidly decline and return to zero within 60 minutes following withdrawal from chronic ethanol exposure (0.50% v/v). We characterize the changes associated with ethanol tolerance and withdrawal in zebrafish by focusing on 3 domains relevant to ethanol addiction: motor patterns, physiological responses (i.e. cortisol levels), and neurochemical alterations. The use of multiple domains of investigation allowed an in-depth analysis of ethanol induced changes in zebrafish. PMID:24598276

  20. Limb regeneration is impaired in an adult zebrafish model of diabetes mellitus.

    PubMed

    Olsen, Ansgar S; Sarras, Michael P; Intine, Robert V

    2010-01-01

    The zebrafish (Danio rerio) is an established model organism for the study of developmental processes, human disease, and tissue regeneration. We report that limb regeneration is severely impaired in our newly developed adult zebrafish model of type I diabetes mellitus. Intraperitoneal streptozocin injection of adult, wild-type zebrafish results in a sustained hyperglycemic state as determined by elevated fasting blood glucose values and increased glycation of serum protein. Serum insulin levels are also decreased and pancreas immunohistochemisty revealed a decreased amount of insulin signal in hyperglycemic fish. Additionally, the diabetic complications of retinal thinning and glomerular basement membrane thickening (early signs of retinopathy and nephropathy) resulting from the hyperglycemic state were evident in streptozocin-injected fish at 3 weeks. Most significantly, limb regeneration, following caudal fin amputation, is severely impaired in diabetic zebrafish and nonspecific toxic effects outside the pancreas were not found to contribute to impaired limb regeneration. This experimental system using adult zebrafish facilitates a broad spectrum of genetic and molecular approaches to study regeneration in the diabetic background. © 2010 by the Wound Healing Society.

  1. Protective Role of Comfrey Leave Extracts on UV-induced Zebrafish Fin Damage

    PubMed Central

    Cheng, Chien-Chung; Chou, Chi-Yuan; Chang, Yao-Chin; Wang, Hsuan-Wen; Wen, Chi-Chung; Chen, Yau-Hung

    2014-01-01

    In zebrafish, UV exposure leads to fin malformation phenotypes including fin reduction or absence. The present study evaluated UV-protective activities of comfrey leaves extracts in a zebrafish model by recording fin morphological changes. Chemopreventive effects of comfrey leave extracts were evaluated using Kaplan-Meier analysis and Cox proportional hazards regression. The results showed that (1) the mean times of return to normal fin in the UV+comfrey (50 and 100 ppm) groups were 3.43 and 2.86 days and were quicker compared with that in the UV only group (4.21 days); (2) zebrafish fins in the UV+comfrey (50 and 100 ppm) groups were 2.05 and 3.25 times more likely to return to normal than those in the UV only group; and (3) comfrey leave extracts had UV-absorbance abilities and significantly reduced ROS production in UV-exposed zebrafish embryos, which may attenuate UV-mediated apoptosis. In conclusion, comfrey leaves extracts may have the potential to be developed as UV-protective agents to protect zebrafish embryos from UV-induced damage. PMID:25352712

  2. Time-lapse imaging of neural development: zebrafish lead the way into the fourth dimension.

    PubMed

    Rieger, Sandra; Wang, Fang; Sagasti, Alvaro

    2011-07-01

    Time-lapse imaging is often the only way to appreciate fully the many dynamic cell movements critical to neural development. Zebrafish possess many advantages that make them the best vertebrate model organism for live imaging of dynamic development events. This review will discuss technical considerations of time-lapse imaging experiments in zebrafish, describe selected examples of imaging studies in zebrafish that revealed new features or principles of neural development, and consider the promise and challenges of future time-lapse studies of neural development in zebrafish embryos and adults. Copyright © 2011 Wiley-Liss, Inc.

  3. Enumerating Hematopoietic Stem and Progenitor Cells in Zebrafish Embryos.

    PubMed

    Esain, Virginie; Cortes, Mauricio; North, Trista E

    2016-01-01

    Over the past 20 years, zebrafish have proven to be a valuable model to dissect the signaling pathways involved in hematopoiesis, including Hematopoietic Stem and Progenitor Cell (HSPC) formation and homeostasis. Despite tremendous efforts to generate the tools necessary to characterize HSPCs in vitro and in vivo the zebrafish community still lacks standardized methods to quantify HSPCs across laboratories. Here, we describe three methods used routinely in our lab, and in others, to reliably enumerate HSPCs in zebrafish embryos: large-scale live imaging of transgenic reporter lines, Fluorescence-Activated Cell Sorting (FACS), and in vitro cell culture. While live imaging and FACS analysis allows enumeration of total or site-specific HSPCs, the cell culture assay provides the unique opportunity to test the functional potential of isolated HSPCs, similar to those employed in mammals.

  4. Neuronal expression of fibroblast growth factor receptors in zebrafish.

    PubMed

    Rohs, Patricia; Ebert, Alicia M; Zuba, Ania; McFarlane, Sarah

    2013-12-01

    Fibroblast growth factor (FGF) signaling is important for a host of developmental processes such as proliferation, differentiation, tissue patterning, and morphogenesis. In vertebrates, FGFs signal through a family of four fibroblast growth factor receptors (FGFR 1-4), one of which is duplicated in zebrafish (FGFR1). Here we report the mRNA expression of the five known zebrafish fibroblast growth factor receptors at five developmental time points (24, 36, 48, 60, and 72h postfertilization), focusing on expression within the central nervous system. We show that the receptors have distinct and dynamic expression in the developing zebrafish brain, eye, inner ear, lateral line, and pharynx. In many cases, the expression patterns are similar to those of homologous FGFRs in mouse, chicken, amphibians, and other teleosts. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Developmental and Persistent Toxicities of Maternally Deposited Selenomethionine in Zebrafish (Danio rerio).

    PubMed

    Thomas, Jith K; Janz, David M

    2015-08-18

    The objectives of this study were (1) to establish egg selenium (Se) toxicity thresholds for mortality and deformities in early life stages of zebrafish (Danio rerio) after exposure to excess selenomethionine (SeMet, the dominant chemical species of Se in diets) via in ovo maternal transfer and (2) to investigate the persistent effects of developmental exposure to excess SeMet on swim performance and metabolic capacities in F1-generation adult zebrafish. Adult zebrafish were fed either control food (1.3 μg Se/g, dry mass or d.m.) or food spiked with increasing measured concentrations of Se (3.4, 9.8, or 27.5 μg Se/g, d.m.) in the form of SeMet for 90 d. In ovo exposure to SeMet increased mortality and deformities in larval zebrafish in a concentration-dependent fashion with threshold egg Se concentrations (EC10s) of 7.5 and 7.0 μg Se/g d.m., respectively. Impaired swim performance and greater respiration and metabolic rates were observed in F1-generation zebrafish exposed in ovo to 6.8 and 12.7 μg Se/g d.m and raised to adulthood in clean water. A species sensitivity distribution (SSD) based on egg Se developmental toxicity thresholds suggests that zebrafish are the most sensitive fish species studied to date.

  6. The zebrafish reference genome sequence and its relationship to the human genome

    PubMed Central

    Howe, Kerstin; Clark, Matthew D.; Torroja, Carlos F.; Torrance, James; Berthelot, Camille; Muffato, Matthieu; Collins, John E.; Humphray, Sean; McLaren, Karen; Matthews, Lucy; McLaren, Stuart; Sealy, Ian; Caccamo, Mario; Churcher, Carol; Scott, Carol; Barrett, Jeffrey C.; Koch, Romke; Rauch, Gerd-Jörg; White, Simon; Chow, William; Kilian, Britt; Quintais, Leonor T.; Guerra-Assunção, José A.; Zhou, Yi; Gu, Yong; Yen, Jennifer; Vogel, Jan-Hinnerk; Eyre, Tina; Redmond, Seth; Banerjee, Ruby; Chi, Jianxiang; Fu, Beiyuan; Langley, Elizabeth; Maguire, Sean F.; Laird, Gavin K.; Lloyd, David; Kenyon, Emma; Donaldson, Sarah; Sehra, Harminder; Almeida-King, Jeff; Loveland, Jane; Trevanion, Stephen; Jones, Matt; Quail, Mike; Willey, Dave; Hunt, Adrienne; Burton, John; Sims, Sarah; McLay, Kirsten; Plumb, Bob; Davis, Joy; Clee, Chris; Oliver, Karen; Clark, Richard; Riddle, Clare; Eliott, David; Threadgold, Glen; Harden, Glenn; Ware, Darren; Mortimer, Beverly; Kerry, Giselle; Heath, Paul; Phillimore, Benjamin; Tracey, Alan; Corby, Nicole; Dunn, Matthew; Johnson, Christopher; Wood, Jonathan; Clark, Susan; Pelan, Sarah; Griffiths, Guy; Smith, Michelle; Glithero, Rebecca; Howden, Philip; Barker, Nicholas; Stevens, Christopher; Harley, Joanna; Holt, Karen; Panagiotidis, Georgios; Lovell, Jamieson; Beasley, Helen; Henderson, Carl; Gordon, Daria; Auger, Katherine; Wright, Deborah; Collins, Joanna; Raisen, Claire; Dyer, Lauren; Leung, Kenric; Robertson, Lauren; Ambridge, Kirsty; Leongamornlert, Daniel; McGuire, Sarah; Gilderthorp, Ruth; Griffiths, Coline; Manthravadi, Deepa; Nichol, Sarah; Barker, Gary; Whitehead, Siobhan; Kay, Michael; Brown, Jacqueline; Murnane, Clare; Gray, Emma; Humphries, Matthew; Sycamore, Neil; Barker, Darren; Saunders, David; Wallis, Justene; Babbage, Anne; Hammond, Sian; Mashreghi-Mohammadi, Maryam; Barr, Lucy; Martin, Sancha; Wray, Paul; Ellington, Andrew; Matthews, Nicholas; Ellwood, Matthew; Woodmansey, Rebecca; Clark, Graham; Cooper, James; Tromans, Anthony; Grafham, Darren; Skuce, Carl; Pandian, Richard; Andrews, Robert; Harrison, Elliot; Kimberley, Andrew; Garnett, Jane; Fosker, Nigel; Hall, Rebekah; Garner, Patrick; Kelly, Daniel; Bird, Christine; Palmer, Sophie; Gehring, Ines; Berger, Andrea; Dooley, Christopher M.; Ersan-Ürün, Zübeyde; Eser, Cigdem; Geiger, Horst; Geisler, Maria; Karotki, Lena; Kirn, Anette; Konantz, Judith; Konantz, Martina; Oberländer, Martina; Rudolph-Geiger, Silke; Teucke, Mathias; Osoegawa, Kazutoyo; Zhu, Baoli; Rapp, Amanda; Widaa, Sara; Langford, Cordelia; Yang, Fengtang; Carter, Nigel P.; Harrow, Jennifer; Ning, Zemin; Herrero, Javier; Searle, Steve M. J.; Enright, Anton; Geisler, Robert; Plasterk, Ronald H. A.; Lee, Charles; Westerfield, Monte; de Jong, Pieter J.; Zon, Leonard I.; Postlethwait, John H.; Nüsslein-Volhard, Christiane; Hubbard, Tim J. P.; Crollius, Hugues Roest; Rogers, Jane; Stemple, Derek L.

    2013-01-01

    Zebrafish have become a popular organism for the study of vertebrate gene function1,2. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease3–5. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes6, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination. PMID:23594743

  7. Best practices for germ-free derivation and gnotobiotic zebrafish husbandry

    PubMed Central

    Melancon, E.; De La Torre Canny, S. Gomez; Sichel, S.; Kelly, M.; Wiles, T.J.; Rawls, J.F.; Eisen, J.S.; Guillemin, K.

    2017-01-01

    All animals are ecosystems with resident microbial communities, referred to as microbiota, which play profound roles in host development, physiology, and evolution. Enabled by new DNA sequencing technologies, there is a burgeoning interest in animal–microbiota interactions, but dissecting the specific impacts of microbes on their hosts is experimentally challenging. Gnotobiology, the study of biological systems in which all members are known, enables precise experimental analysis of the necessity and sufficiency of microbes in animal biology by deriving animals germ-free (GF) and inoculating them with defined microbial lineages. Mammalian host models have long dominated gnotobiology, but we have recently adapted gnotobiotic approaches to the zebrafish (Danio rerio), an important aquatic model. Zebrafish offer several experimental attributes that enable rapid, large-scale gnotobiotic experimentation with high replication rates and exquisite optical resolution. Here we describe detailed protocols for three procedures that form the foundation of zebrafish gnotobiology: derivation of GF embryos, microbial association of GF animals, and long-term, GF husbandry. Our aim is to provide sufficient guidance in zebrafish gnotobiotic methodology to expand and enrich this exciting field of research. PMID:28129860

  8. Comprehensive analysis of coding-lncRNA gene co-expression network uncovers conserved functional lncRNAs in zebrafish.

    PubMed

    Chen, Wen; Zhang, Xuan; Li, Jing; Huang, Shulan; Xiang, Shuanglin; Hu, Xiang; Liu, Changning

    2018-05-09

    Zebrafish is a full-developed model system for studying development processes and human disease. Recent studies of deep sequencing had discovered a large number of long non-coding RNAs (lncRNAs) in zebrafish. However, only few of them had been functionally characterized. Therefore, how to take advantage of the mature zebrafish system to deeply investigate the lncRNAs' function and conservation is really intriguing. We systematically collected and analyzed a series of zebrafish RNA-seq data, then combined them with resources from known database and literatures. As a result, we obtained by far the most complete dataset of zebrafish lncRNAs, containing 13,604 lncRNA genes (21,128 transcripts) in total. Based on that, a co-expression network upon zebrafish coding and lncRNA genes was constructed and analyzed, and used to predict the Gene Ontology (GO) and the KEGG annotation of lncRNA. Meanwhile, we made a conservation analysis on zebrafish lncRNA, identifying 1828 conserved zebrafish lncRNA genes (1890 transcripts) that have their putative mammalian orthologs. We also found that zebrafish lncRNAs play important roles in regulation of the development and function of nervous system; these conserved lncRNAs present a significant sequential and functional conservation, with their mammalian counterparts. By integrative data analysis and construction of coding-lncRNA gene co-expression network, we gained the most comprehensive dataset of zebrafish lncRNAs up to present, as well as their systematic annotations and comprehensive analyses on function and conservation. Our study provides a reliable zebrafish-based platform to deeply explore lncRNA function and mechanism, as well as the lncRNA commonality between zebrafish and human.

  9. Two-photon-based photoactivation in live zebrafish embryos.

    PubMed

    Russek-Blum, Niva; Nabel-Rosen, Helit; Levkowitz, Gil

    2010-12-24

    Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

  10. Using Transgenic Zebrafish to Study Muscle Stem/Progenitor Cells.

    PubMed

    Nguyen, Phong D; Currie, Peter D

    2017-01-01

    Understanding muscle stem cell behaviors can potentially provide insights into how these cells act and respond during normal growth and diseased contexts. The zebrafish is an ideal model organism to examine these behaviors in vivo where it would normally be technically challenging in other mammalian models. This chapter will describe the procedures required to successfully conduct live imaging of zebrafish transgenics that has specifically been adapted for skeletal muscle.

  11. Genotoxicity and oxidative stress induced by the fungicide azoxystrobin in zebrafish (Danio rerio) livers.

    PubMed

    Han, Yingnan; Liu, Tong; Wang, Jinhua; Wang, Jun; Zhang, Cheng; Zhu, Lusheng

    2016-10-01

    Azoxystrobin is a frequently used fungicide in agriculture. Its toxicological effects on non-target organisms have aroused attention. In the present work, the toxic effects of azoxystrobin on zebrafish (Danio rerio) were investigated. Male and female zebrafish were separately exposed to a control solution and three azoxystrobin treatments (1, 10, and 100μg/L) and were sampled on days 7, 14, 21, and 28. Reactive oxygen species (ROS) were accumulated in excess in the zebrafish livers. Superoxide dismutase (SOD) activity was significantly inhibited in the male zebrafish. Moreover, a notable decrease was also observed after day 21 in the female zebrafish. Catalase (CAT) activity was induced by the azoxystrobin treatments with the exception of the 1μg/L treatment. A significant increase in glutathione-S-transferase (GST) activity was observed after day 21. Lipid peroxidation (LPO) was generated, and DNA damage was enhanced in a concentration-dependent manner. In conclusion, azoxystrobin induced oxidative stress and genotoxicity in zebrafish livers. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Tensile properties of craniofacial tendons in the mature and aged zebrafish

    PubMed Central

    Shah, Rishita R.; Nerurkar, Nandan L.; Wang, Calvin; Galloway, Jenna L.

    2015-01-01

    The zebrafish Danio rerio is a powerful model for the study of development, regenerative biology, and human disease. However, the analysis of load-bearing tissues such as tendons and ligaments has been limited in this system. This is largely due to technical limitations that preclude accurate measurement of their mechanical properties. Here, we present a custom tensile testing system that applies nano-Newton scale forces to zebrafish tendons as small as 1 mm in length. Tendon properties were remarkably similar to mammalian tendons, including stress-strain nonlinearity and a linear modulus (515±152 MPa) that aligned closely with mammalian data. Additionally, a simple exponential constitutive law used to describe tendon mechanics was successfully fit to zebrafish tendons; the associated material constants agreed with literature values for mammalian tendons. Finally, mature and aged zebrafish comparisons revealed a significant decline in mechanical function with age. Based on the exponential constitutive model, age related changes were primarily caused by a reduction in nonlinearity (e.g. changes in collagen crimp or fiber recruitment). These findings demonstrate the utility of zebrafish as a model to study tendon biomechanics in health and disease. Moreover, these findings suggest that tendon mechanical behavior is highly conserved across vertebrates. PMID:25665155

  13. 4-dimensional functional profiling in the convulsant-treated larval zebrafish brain.

    PubMed

    Winter, Matthew J; Windell, Dylan; Metz, Jeremy; Matthews, Peter; Pinion, Joe; Brown, Jonathan T; Hetheridge, Malcolm J; Ball, Jonathan S; Owen, Stewart F; Redfern, Will S; Moger, Julian; Randall, Andrew D; Tyler, Charles R

    2017-07-26

    Functional neuroimaging, using genetically-encoded Ca 2+ sensors in larval zebrafish, offers a powerful combination of high spatiotemporal resolution and higher vertebrate relevance for quantitative neuropharmacological profiling. Here we use zebrafish larvae with pan-neuronal expression of GCaMP6s, combined with light sheet microscopy and a novel image processing pipeline, for the 4D profiling of chemoconvulsant action in multiple brain regions. In untreated larvae, regions associated with autonomic functionality, sensory processing and stress-responsiveness, consistently exhibited elevated spontaneous activity. The application of drugs targeting different convulsant mechanisms (4-Aminopyridine, Pentylenetetrazole, Pilocarpine and Strychnine) resulted in distinct spatiotemporal patterns of activity. These activity patterns showed some interesting parallels with what is known of the distribution of their respective molecular targets, but crucially also revealed system-wide neural circuit responses to stimulation or suppression. Drug concentration-response curves of neural activity were identified in a number of anatomically-defined zebrafish brain regions, and in vivo larval electrophysiology, also conducted in 4dpf larvae, provided additional measures of neural activity. Our quantification of network-wide chemoconvulsant drug activity in the whole zebrafish brain illustrates the power of this approach for neuropharmacological profiling in applications ranging from accelerating studies of drug safety and efficacy, to identifying pharmacologically-altered networks in zebrafish models of human neurological disorders.

  14. Characterization of Sleep in Zebrafish and Insomnia in Hypocretin Receptor Mutants

    PubMed Central

    Yokogawa, Tohei; Marin, Wilfredo; Faraco, Juliette; Pézeron, Guillaume; Appelbaum, Lior; Zhang, Jian; Rosa, Frédéric; Mourrain, Philippe; Mignot, Emmanuel

    2007-01-01

    Sleep is a fundamental biological process conserved across the animal kingdom. The study of how sleep regulatory networks are conserved is needed to better understand sleep across evolution. We present a detailed description of a sleep state in adult zebrafish characterized by reversible periods of immobility, increased arousal threshold, and place preference. Rest deprivation using gentle electrical stimulation is followed by a sleep rebound, indicating homeostatic regulation. In contrast to mammals and similarly to birds, light suppresses sleep in zebrafish, with no evidence for a sleep rebound. We also identify a null mutation in the sole receptor for the wake-promoting neuropeptide hypocretin (orexin) in zebrafish. Fish lacking this receptor demonstrate short and fragmented sleep in the dark, in striking contrast to the excessive sleepiness and cataplexy of narcolepsy in mammals. Consistent with this observation, we find that the hypocretin receptor does not colocalize with known major wake-promoting monoaminergic and cholinergic cell groups in the zebrafish. Instead, it colocalizes with large populations of GABAergic neurons, including a subpopulation of Adra2a-positive GABAergic cells in the anterior hypothalamic area, neurons that could assume a sleep modulatory role. Our study validates the use of zebrafish for the study of sleep and indicates molecular diversity in sleep regulatory networks across vertebrates. PMID:17941721

  15. Cadherin-17 is required to maintain pronephric duct integrity during zebrafish development.

    PubMed

    Horsfield, Julia; Ramachandran, Anassuya; Reuter, Katja; LaVallie, Edward; Collins-Racie, Lisa; Crosier, Kathryn; Crosier, Philip

    2002-07-01

    We have isolated a zebrafish cadherin that is orthologous to human LI-cadherin (CDH17). Zebrafish cdh17 is expressed exclusively in the pronephric ducts during embryogenesis, and in the mesonephros during larval development and adulthood. Like its mammalian ortholog, cdh17 is also expressed in liver and intestine in adult zebrafish. We show that cdh17-positive mesodermal cells do not contribute to the hematopoietic system. Consistent with a cell adhesion role for Cdh17, depletion of Cdh17 function using antisense morpholino oligonucleotides compromised cell cohesion during pronephric duct formation. Our results indicate that Cdh17 is necessary for maintaining the integrity of the pronephric ducts during zebrafish embryogenesis. This finding contrasts with the role of mammalian CDH17, which does not appear to be involved in nephric development.

  16. Differences of acute versus chronic ethanol exposure on anxiety-like behavioral responses in zebrafish.

    PubMed

    Mathur, Priya; Guo, Su

    2011-06-01

    Zebrafish, a vertebrate model organism amenable to high throughput screening, is an attractive system to model and study the mechanisms underlying human diseases. Alcoholism and alcoholic medical disorders are among the most debilitating diseases, yet the mechanisms by which ethanol inflicts the disease states are not well understood. In recent years zebrafish behavior assays have been used to study learning and memory, fear and anxiety, and social behavior. It is important to characterize the effects of ethanol on zebrafish behavioral repertoires in order to successfully harvest the strength of zebrafish for alcohol research. One prominent effect of alcohol in humans is its effect on anxiety, with acute intermediate doses relieving anxiety and withdrawal from chronic exposure increasing anxiety, both of which have significant contributions to alcohol dependence. In this study, we assess the effects of both acute and chronic ethanol exposure on anxiety-like behaviors in zebrafish, using two behavioral paradigms, the Novel Tank Diving Test and the Light/Dark Choice Assay. Acute ethanol exposure exerted significant dose-dependent anxiolytic effects. However, withdrawal from repeated intermittent ethanol exposure disabled recovery from heightened anxiety. These results demonstrate that zebrafish exhibit different anxiety-like behavioral responses to acute and chronic ethanol exposure, which are remarkably similar to these effects of alcohol in humans. Because of the accessibility of zebrafish to high throughput screening, our results suggest that genes and small molecules identified in zebrafish will be of relevance to understand how acute versus chronic alcohol exposure have opposing effects on the state of anxiety in humans. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Translating Discovery in Zebrafish Pancreatic Development to Human Pancreatic Cancer: Biomarkers, Targets, Pathogenesis, and Therapeutics

    PubMed Central

    Kazi, Abid A.; Yee, Rosemary K.

    2013-01-01

    Abstract Experimental studies in the zebrafish have greatly facilitated understanding of genetic regulation of the early developmental events in the pancreas. Various approaches using forward and reverse genetics, chemical genetics, and transgenesis in zebrafish have demonstrated generally conserved regulatory roles of mammalian genes and discovered novel genetic pathways in exocrine pancreatic development. Accumulating evidence has supported the use of zebrafish as a model of human malignant diseases, including pancreatic cancer. Studies have shown that the genetic regulators of exocrine pancreatic development in zebrafish can be translated into potential clinical biomarkers and therapeutic targets in human pancreatic adenocarcinoma. Transgenic zebrafish expressing oncogenic K-ras and zebrafish tumor xenograft model have emerged as valuable tools for dissecting the pathogenetic mechanisms of pancreatic cancer and for drug discovery and toxicology. Future analysis of the pancreas in zebrafish will continue to advance understanding of the genetic regulation and biological mechanisms during organogenesis. Results of those studies are expected to provide new insights into how aberrant developmental pathways contribute to formation and growth of pancreatic neoplasia, and hopefully generate valid biomarkers and targets as well as effective and safe therapeutics in pancreatic cancer. PMID:23682805

  18. Translating discovery in zebrafish pancreatic development to human pancreatic cancer: biomarkers, targets, pathogenesis, and therapeutics.

    PubMed

    Yee, Nelson S; Kazi, Abid A; Yee, Rosemary K

    2013-06-01

    Abstract Experimental studies in the zebrafish have greatly facilitated understanding of genetic regulation of the early developmental events in the pancreas. Various approaches using forward and reverse genetics, chemical genetics, and transgenesis in zebrafish have demonstrated generally conserved regulatory roles of mammalian genes and discovered novel genetic pathways in exocrine pancreatic development. Accumulating evidence has supported the use of zebrafish as a model of human malignant diseases, including pancreatic cancer. Studies have shown that the genetic regulators of exocrine pancreatic development in zebrafish can be translated into potential clinical biomarkers and therapeutic targets in human pancreatic adenocarcinoma. Transgenic zebrafish expressing oncogenic K-ras and zebrafish tumor xenograft model have emerged as valuable tools for dissecting the pathogenetic mechanisms of pancreatic cancer and for drug discovery and toxicology. Future analysis of the pancreas in zebrafish will continue to advance understanding of the genetic regulation and biological mechanisms during organogenesis. Results of those studies are expected to provide new insights into how aberrant developmental pathways contribute to formation and growth of pancreatic neoplasia, and hopefully generate valid biomarkers and targets as well as effective and safe therapeutics in pancreatic cancer.

  19. Zebrafish (Danio rerio) embryos as a model for testing proteratogens.

    PubMed

    Weigt, Stefan; Huebler, Nicole; Strecker, Ruben; Braunbeck, Thomas; Broschard, Thomas H

    2011-03-15

    Zebrafish embryos have been shown to be a useful model for the detection of direct acting teratogens. This communication presents a protocol for a 3-day in vitro zebrafish embryo teratogenicity assay and describes results obtained for 10 proteratogens: 2-acetylaminofluorene, benzo[a]pyrene, aflatoxin B(1), carbamazepine, phenytoin, trimethadione, cyclophosphamide, ifosfamide, tegafur and thio-TEPA. The selection of the test substances accounts for differences in structure, origin, metabolism and water solubility. Apart from 2-acetylaminofluorene, which mainly produces lethal effects, all proteratogens tested were teratogenic in zebrafish embryos exposed for 3 days. The test substances and/or the substance class produced characteristic patterns of fingerprint endpoints. Several substances produced effects that could be identified already at 1 dpf (days post fertilization), whereas the effects of others could only be identified unambiguously after hatching at ≥ 3 dpf. The LC₅₀ and EC₅₀ values were used to calculate the teratogenicity index (TI) for the different substances, and the EC₂₀ values were related to human plasma concentrations. Results lead to the conclusion that zebrafish embryos are able to activate proteratogenic substances without addition of an exogenous metabolic activation system. Moreover, the teratogenic effects were observed at concentrations relevant to human exposure data. Along with other findings, our results indicate that zebrafish embryos are a useful alternative method for traditional teratogenicity testing with mammalian species. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  20. Zebrafish Developmental Screening of the ToxCast™ Phase I Chemical Library

    EPA Science Inventory

    Zebrafish (Danio rerio) is an emerging toxicity screening model for both human health and ecology. As part of the Computational Toxicology Research Program of the U.S. EPA, the toxicity of the 309 ToxCast™ Phase I chemicals was assessed using a zebrafish screen for developmental ...

  1. Hypothalamic Projections to the Optic Tectum in Larval Zebrafish

    PubMed Central

    Heap, Lucy A.; Vanwalleghem, Gilles C.; Thompson, Andrew W.; Favre-Bulle, Itia; Rubinsztein-Dunlop, Halina; Scott, Ethan K.

    2018-01-01

    The optic tectum of larval zebrafish is an important model for understanding visual processing in vertebrates. The tectum has been traditionally viewed as dominantly visual, with a majority of studies focusing on the processes by which tectal circuits receive and process retinally-derived visual information. Recently, a handful of studies have shown a much more complex role for the optic tectum in larval zebrafish, and anatomical and functional data from these studies suggest that this role extends beyond the visual system, and beyond the processing of exclusively retinal inputs. Consistent with this evolving view of the tectum, we have used a Gal4 enhancer trap line to identify direct projections from rostral hypothalamus (RH) to the tectal neuropil of larval zebrafish. These projections ramify within the deepest laminae of the tectal neuropil, the stratum album centrale (SAC)/stratum griseum periventriculare (SPV), and also innervate strata distinct from those innervated by retinal projections. Using optogenetic stimulation of the hypothalamic projection neurons paired with calcium imaging in the tectum, we find rebound firing in tectal neurons consistent with hypothalamic inhibitory input. Our results suggest that tectal processing in larval zebrafish is modulated by hypothalamic inhibitory inputs to the deep tectal neuropil. PMID:29403362

  2. Zebrafish response to a robotic replica in three dimensions

    PubMed Central

    Ruberto, Tommaso; Mwaffo, Violet; Singh, Sukhgewanpreet; Neri, Daniele

    2016-01-01

    As zebrafish emerge as a species of choice for the investigation of biological processes, a number of experimental protocols are being developed to study their social behaviour. While live stimuli may elicit varying response in focal subjects owing to idiosyncrasies, tiredness and circadian rhythms, video stimuli suffer from the absence of physical input and rely only on two-dimensional projections. Robotics has been recently proposed as an alternative approach to generate physical, customizable, effective and consistent stimuli for behavioural phenotyping. Here, we contribute to this field of investigation through a novel four-degree-of-freedom robotics-based platform to manoeuvre a biologically inspired three-dimensionally printed replica. The platform enables three-dimensional motions as well as body oscillations to mimic zebrafish locomotion. In a series of experiments, we demonstrate the differential role of the visual stimuli associated with the biologically inspired replica and its three-dimensional motion. Three-dimensional tracking and information-theoretic tools are complemented to quantify the interaction between zebrafish and the robotic stimulus. Live subjects displayed a robust attraction towards the moving replica, and such attraction was lost when controlling for its visual appearance or motion. This effort is expected to aid zebrafish behavioural phenotyping, by offering a novel approach to generate physical stimuli moving in three dimensions. PMID:27853566

  3. A Fully Automated High-Throughput Zebrafish Behavioral Ototoxicity Assay.

    PubMed

    Todd, Douglas W; Philip, Rohit C; Niihori, Maki; Ringle, Ryan A; Coyle, Kelsey R; Zehri, Sobia F; Zabala, Leanne; Mudery, Jordan A; Francis, Ross H; Rodriguez, Jeffrey J; Jacob, Abraham

    2017-08-01

    Zebrafish animal models lend themselves to behavioral assays that can facilitate rapid screening of ototoxic, otoprotective, and otoregenerative drugs. Structurally similar to human inner ear hair cells, the mechanosensory hair cells on their lateral line allow the zebrafish to sense water flow and orient head-to-current in a behavior called rheotaxis. This rheotaxis behavior deteriorates in a dose-dependent manner with increased exposure to the ototoxin cisplatin, thereby establishing itself as an excellent biomarker for anatomic damage to lateral line hair cells. Building on work by our group and others, we have built a new, fully automated high-throughput behavioral assay system that uses automated image analysis techniques to quantify rheotaxis behavior. This novel system consists of a custom-designed swimming apparatus and imaging system consisting of network-controlled Raspberry Pi microcomputers capturing infrared video. Automated analysis techniques detect individual zebrafish, compute their orientation, and quantify the rheotaxis behavior of a zebrafish test population, producing a powerful, high-throughput behavioral assay. Using our fully automated biological assay to test a standardized ototoxic dose of cisplatin against varying doses of compounds that protect or regenerate hair cells may facilitate rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

  4. Data-driven modelling of social forces and collective behaviour in zebrafish.

    PubMed

    Zienkiewicz, Adam K; Ladu, Fabrizio; Barton, David A W; Porfiri, Maurizio; Bernardo, Mario Di

    2018-04-14

    Zebrafish are rapidly emerging as a powerful model organism in hypothesis-driven studies targeting a number of functional and dysfunctional processes. Mathematical models of zebrafish behaviour can inform the design of experiments, through the unprecedented ability to perform pilot trials on a computer. At the same time, in-silico experiments could help refining the analysis of real data, by enabling the systematic investigation of key neurobehavioural factors. Here, we establish a data-driven model of zebrafish social interaction. Specifically, we derive a set of interaction rules to capture the primary response mechanisms which have been observed experimentally. Contrary to previous studies, we include dynamic speed regulation in addition to turning responses, which together provide attractive, repulsive and alignment interactions between individuals. The resulting multi-agent model provides a novel, bottom-up framework to describe both the spontaneous motion and individual-level interaction dynamics of zebrafish, inferred directly from experimental observations. Copyright © 2018 Elsevier Ltd. All rights reserved.

  5. Dampened Hedgehog signaling but normal Wnt signaling in zebrafish without cilia

    PubMed Central

    Huang, Peng; Schier, Alexander F.

    2009-01-01

    Summary Cilia have been implicated in Hedgehog (Hh) and Wnt signaling in mouse but not in Drosophila. To determine whether the role of cilia is conserved in zebrafish, we generated maternal-zygotic (MZ) oval (ovl; ift88) mutants that lack all cilia. MZovl mutants display normal canonical and non-canonical Wnt signaling but show defects in Hh signaling. As in mouse, zebrafish cilia are required to mediate the activities of Hh, Ptc, Smo and PKA. However, in contrast to mouse Ift88 mutants, which show a dramatic reduction in Hh signaling, zebrafish MZovl mutants display dampened, but expanded, Hh pathway activity. This activity is largely due to gli1, the expression of which is fully dependent on Hh signaling in mouse but not in zebrafish. These results reveal a conserved requirement for cilia in transducing the activity of upstream regulators of Hh signaling but distinct phenotypic effects due to differential regulation and differing roles of transcriptional mediators. PMID:19700616

  6. LITTLE FISH, BIG DATA: ZEBRAFISH AS A MODEL FOR CARDIOVASCULAR AND METABOLIC DISEASE.

    PubMed

    Gut, Philipp; Reischauer, Sven; Stainier, Didier Y R; Arnaout, Rima

    2017-07-01

    The burden of cardiovascular and metabolic diseases worldwide is staggering. The emergence of systems approaches in biology promises new therapies, faster and cheaper diagnostics, and personalized medicine. However, a profound understanding of pathogenic mechanisms at the cellular and molecular levels remains a fundamental requirement for discovery and therapeutics. Animal models of human disease are cornerstones of drug discovery as they allow identification of novel pharmacological targets by linking gene function with pathogenesis. The zebrafish model has been used for decades to study development and pathophysiology. More than ever, the specific strengths of the zebrafish model make it a prime partner in an age of discovery transformed by big-data approaches to genomics and disease. Zebrafish share a largely conserved physiology and anatomy with mammals. They allow a wide range of genetic manipulations, including the latest genome engineering approaches. They can be bred and studied with remarkable speed, enabling a range of large-scale phenotypic screens. Finally, zebrafish demonstrate an impressive regenerative capacity scientists hope to unlock in humans. Here, we provide a comprehensive guide on applications of zebrafish to investigate cardiovascular and metabolic diseases. We delineate advantages and limitations of zebrafish models of human disease and summarize their most significant contributions to understanding disease progression to date. Copyright © 2017 the American Physiological Society.

  7. The role of Sox6 in zebrafish muscle fiber type specification.

    PubMed

    Jackson, Harriet E; Ono, Yosuke; Wang, Xingang; Elworthy, Stone; Cunliffe, Vincent T; Ingham, Philip W

    2015-01-01

    The transcription factor Sox6 has been implicated in regulating muscle fiber type-specific gene expression in mammals. In zebrafish, loss of function of the transcription factor Prdm1a results in a slow to fast-twitch fiber type transformation presaged by ectopic expression of sox6 in slow-twitch progenitors. Morpholino-mediated Sox6 knockdown can suppress this transformation but causes ectopic expression of only one of three slow-twitch specific genes assayed. Here, we use gain and loss of function analysis to analyse further the role of Sox6 in zebrafish muscle fiber type specification. The GAL4 binary misexpression system was used to express Sox6 ectopically in zebrafish embryos. Cis-regulatory elements were characterized using transgenic fish. Zinc finger nuclease mediated targeted mutagenesis was used to analyse the effects of loss of Sox6 function in embryonic, larval and adult zebrafish. Zebrafish transgenic for the GCaMP3 Calcium reporter were used to assay Ca2+ transients in wild-type and mutant muscle fibres. Ectopic Sox6 expression is sufficient to downregulate slow-twitch specific gene expression in zebrafish embryos. Cis-regulatory elements upstream of the slow myosin heavy chain 1 (smyhc1) and slow troponin c (tnnc1b) genes contain putative Sox6 binding sites required for repression of the former but not the latter. Embryos homozygous for sox6 null alleles expressed tnnc1b throughout the fast-twitch muscle whereas other slow-specific muscle genes, including smyhc1, were expressed ectopically in only a subset of fast-twitch fibers. Ca2+ transients in sox6 mutant fast-twitch fibers were intermediate in their speed and amplitude between those of wild-type slow- and fast-twitch fibers. sox6 homozygotes survived to adulthood and exhibited continued misexpression of tnnc1b as well as smaller slow-twitch fibers. They also exhibited a striking curvature of the spine. The Sox6 transcription factor is a key regulator of fast-twitch muscle fiber differentiation

  8. The Morphogenesis of Cranial Sutures in Zebrafish

    PubMed Central

    Topczewska, Jolanta M.; Shoela, Ramy A.; Tomaszewski, Joanna P.; Mirmira, Rupa B.; Gosain, Arun K.

    2016-01-01

    Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births. PMID:27829009

  9. Ethanol Exposure Causes Muscle Degeneration in Zebrafish

    PubMed Central

    Coffey, Elizabeth C.; Pasquarella, Maggie E.; Goody, Michelle F.

    2018-01-01

    Alcoholic myopathies are characterized by neuromusculoskeletal symptoms such as compromised movement and weakness. Although these symptoms have been attributed to neurological damage, EtOH may also target skeletal muscle. EtOH exposure during zebrafish primary muscle development or adulthood results in smaller muscle fibers. However, the effects of EtOH exposure on skeletal muscle during the growth period that follows primary muscle development are not well understood. We determined the effects of EtOH exposure on muscle during this phase of development. Strikingly, muscle fibers at this stage are acutely sensitive to EtOH treatment: EtOH induces muscle degeneration. The severity of EtOH-induced muscle damage varies but muscle becomes more refractory to EtOH as muscle develops. NF-kB induction in muscle indicates that EtOH triggers a pro-inflammatory response. EtOH-induced muscle damage is p53-independent. Uptake of Evans blue dye shows that EtOH treatment causes sarcolemmal instability before muscle fiber detachment. Dystrophin-null sapje mutant zebrafish also exhibit sarcolemmal instability. We tested whether Trichostatin A (TSA), which reduces muscle degeneration in sapje mutants, would affect EtOH-treated zebrafish. We found that TSA and EtOH are a lethal combination. EtOH does, however, exacerbate muscle degeneration in sapje mutants. EtOH also disrupts adhesion of muscle fibers to their extracellular matrix at the myotendinous junction: some detached muscle fibers retain beta-Dystroglycan indicating failure of muscle end attachments. Overexpression of Paxillin, which reduces muscle degeneration in zebrafish deficient for beta-Dystroglycan, is not sufficient to rescue degeneration. Taken together, our results suggest that EtOH exposure has pleiotropic deleterious effects on skeletal muscle. PMID:29615556

  10. Increased cell proliferation and neural activity by physostigmine in the telencephalon of adult zebrafish.

    PubMed

    Lee, Yunkyoung; Lee, Bongkyu; Jeong, Sumin; Park, Ji-Won; Han, Inn-Oc; Lee, Chang-Joong

    2016-08-26

    Physostigmine, an acetylcholinesterase inhibitor, is known to affect the brain function in various aspects. This study was conducted to test whether physostigmine affects cell proliferation in the telencephalon of zebrafish. BrdU-labeled cells was prominently observed in the ventral zone of the ventral telencephalon of zebrafish. The increased number of BrdU- and proliferating cell nuclear antigen-labeled cells were shown in zebrafish treated with 200μM physostigmine, which was inhibited by pretreatment with 200μM scopolamine. iNOS mRNA expression was increased in the brain of zebrafish treated with 200μM physostigmine. Consistently, aminoguanidine, an iNOS inhibitor, attenuated the increase in the number of BrdU-labeled cells by physostigmine treatment. Zebrafish also showed seizure-like locomotor activity characterized by a rapid and abrupt movement during a 30min treatment with 200μM physostigmine. Neural activity in response to an electrical stimulus was increased in the isolated telencephalon of zebrafish continuously perfused with 200μM physostigmine. None of the number of BrdU-labeled cells, neural activity, or locomotor activity was affected by treatment with 20μM physostigmine. These results suggest that 200μM physostigmine increased neural activity and induced cell proliferation via nitric oxide production in zebrafish. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. Modelling Delta-Notch perturbations during zebrafish somitogenesis.

    PubMed

    Murray, Philip J; Maini, Philip K; Baker, Ruth E

    2013-01-15

    The discovery over the last 15 years of molecular clocks and gradients in the pre-somitic mesoderm of numerous vertebrate species has added significant weight to Cooke and Zeeman's 'clock and wavefront' model of somitogenesis, in which a travelling wavefront determines the spatial position of somite formation and the somitogenesis clock controls periodicity (Cooke and Zeeman, 1976). However, recent high-throughput measurements of spatiotemporal patterns of gene expression in different zebrafish mutant backgrounds allow further quantitative evaluation of the clock and wavefront hypothesis. In this study we describe how our recently proposed model, in which oscillator coupling drives the propagation of an emergent wavefront, can be used to provide mechanistic and testable explanations for the following observed phenomena in zebrafish embryos: (a) the variation in somite measurements across a number of zebrafish mutants; (b) the delayed formation of somites and the formation of 'salt and pepper' patterns of gene expression upon disruption of oscillator coupling; and (c) spatial correlations in the 'salt and pepper' patterns in Delta-Notch mutants. In light of our results, we propose a number of plausible experiments that could be used to further test the model. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Aspartame-fed zebrafish exhibit acute deaths with swimming defects and saccharin-fed zebrafish have elevation of cholesteryl ester transfer protein activity in hypercholesterolemia.

    PubMed

    Kim, Jae-Yong; Seo, Juyi; Cho, Kyung-Hyun

    2011-11-01

    Although many artificial sweeteners (AS) have safety issues, the AS have been widely used in industry. To determine the physiologic effect of AS in the presence of hyperlipidemia, zebrafish were fed aspartame or saccharin with a high-cholesterol diet (HCD). After 12 days, 30% of zebrafish, which consumed aspartame and HCD, died with exhibiting swimming defects. The aspartame group had 65% survivability, while the control and saccharin groups had 100% survivability. Under HCD, the saccharin-fed groups had the highest increase in the serum cholesterol level (599 mg/dL). Aspartame-fed group showed a remarkable increase in serum glucose (up to 125 mg/dL), which was 58% greater than the increase in the HCD alone group. The saccharin and HCD groups had the highest cholesteryl ester transfer protein (CETP) activity (52% CE-transfer), while the HCD alone group had 42% CE-transfer. Histologic analysis revealed that the aspartame and HCD groups showed more infiltration of inflammatory cells in the brain and liver sections. Conclusively, under presence of hyperlipidemia, aspartame-fed zebrafish exhibited acute swimming defects with an increase in brain inflammation. Saccharin-fed zebrafish had an increased atherogenic serum lipid profile with elevation of CETP activity. Copyright © 2011 Elsevier Ltd. All rights reserved.

  13. Imaging a seizure model in zebrafish with structured illumination light sheet microscopy

    NASA Astrophysics Data System (ADS)

    Liu, Yang; Dale, Savannah; Ball, Rebecca; VanLeuven, Ariel J.; Baraban, Scott; Sornborger, Andrew; Lauderdale, James D.; Kner, Peter

    2018-02-01

    Zebrafish are a promising vertebrate model for elucidating how neural circuits generate behavior under normal and pathological conditions. The Baraban group first demonstrated that zebrafish larvae are valuable for investigating seizure events and can be used as a model for epilepsy in humans. Because of their small size and transparency, zebrafish embryos are ideal for imaging seizure activity using calcium indicators. Light-sheet microscopy is well suited to capturing neural activity in zebrafish because it is capable of optical sectioning, high frame rates, and low excitation intensities. We describe work in our lab to use light-sheet microscopy for high-speed long-time imaging of neural activity in wildtype and mutant zebrafish to better understand the connectivity and activity of inhibitory neural networks when GABAergic signaling is altered in vivo. We show that, with light-sheet microscopy, neural activity can be recorded at 23 frames per second in twocolors for over 10 minutes allowing us to capture rare seizure events in mutants. We have further implemented structured illumination to increase resolution and contrast in the vertical and axial directions during high-speed imaging at an effective frame rate of over 7 frames per second.

  14. A Rotifer-Based Technique to Rear Zebrafish Larvae in Small Academic Settings.

    PubMed

    Allen, Raymond L; Wallace, Robert L; Sisson, Barbara E

    2016-08-01

    Raising zebrafish from larvae to juveniles can be laborious, requiring frequent water exchanges and continuous culturing of live feed. This task becomes even more difficult for small institutions that do not have access to the necessary funding, equipment, or personnel to maintain large-scale systems usually employed in zebrafish husbandry. To open this opportunity to smaller institutions, a cost-efficient protocol was developed to culture Nannochloropsis to feed the halophilic, planktonic rotifer Brachionus plicatilis; the rotifers were then used to raise larval zebrafish to juveniles. By using these methods, small institutions can easily raise zebrafish embryos in a cost-efficient manner without the need to establish an extensive fish-raising facility. In addition, culturing rotifers provides a micrometazoan that serves as a model organism for teaching and undergraduate research studies for a variety of topics, including aging, toxicology, and predator-prey dynamics.

  15. Generation and detection of plasmonic nanobubbles in zebrafish.

    PubMed

    Lukianova-Hleb, E Y; Santiago, C; Wagner, D S; Hafner, J H; Lapotko, D O

    2010-06-04

    The zebrafish embryo has been evaluated as an in vivo model for plasmonic nanobubble (PNB) generation and detection at nanoscale. The embryo is easily observed and manipulated utilizing the same methodology as for application of PNBs in vitro. Injection and irradiation of gold nanoparticles with a short laser pulse resulted in generation of PNBs in zebrafish with similar parameters as for PNBs generated in water and cultured living cells. These PNBs do not result in systemic damage, thus we demonstrated an in vivo model for rapid and precise testing of plasmonic nanotechnologies.

  16. Zebrafish: A marvel of high-throughput biology for 21st century toxicology.

    PubMed

    Bugel, Sean M; Tanguay, Robert L; Planchart, Antonio

    2014-09-07

    The evolutionary conservation of genomic, biochemical and developmental features between zebrafish and humans is gradually coming into focus with the end result that the zebrafish embryo model has emerged as a powerful tool for uncovering the effects of environmental exposures on a multitude of biological processes with direct relevance to human health. In this review, we highlight advances in automation, high-throughput (HT) screening, and analysis that leverage the power of the zebrafish embryo model for unparalleled advances in our understanding of how chemicals in our environment affect our health and wellbeing.

  17. Zebrafish: A marvel of high-throughput biology for 21st century toxicology

    PubMed Central

    Bugel, Sean M.; Tanguay, Robert L.; Planchart, Antonio

    2015-01-01

    The evolutionary conservation of genomic, biochemical and developmental features between zebrafish and humans is gradually coming into focus with the end result that the zebrafish embryo model has emerged as a powerful tool for uncovering the effects of environmental exposures on a multitude of biological processes with direct relevance to human health. In this review, we highlight advances in automation, high-throughput (HT) screening, and analysis that leverage the power of the zebrafish embryo model for unparalleled advances in our understanding of how chemicals in our environment affect our health and wellbeing. PMID:25678986

  18. Effectiveness of Rapid Cooling as a Method of Euthanasia for Young Zebrafish (Danio rerio).

    PubMed

    Wallace, Chelsea K; Bright, Lauren A; Marx, James O; Andersen, Robert P; Mullins, Mary C; Carty, Anthony J

    2018-01-01

    Despite increased use of zebrafish (Danio rerio) in biomedical research, consistent information regarding appropriate euthanasia methods, particularly for embryos, is sparse. Current literature indicates that rapid cooling is an effective method of euthanasia for adult zebrafish, yet consistent guidelines regarding zebrafish younger than 6 mo are unavailable. This study was performed to distinguish the age at which rapid cooling is an effective method of euthanasia for zebrafish and the exposure times necessary to reliably euthanize zebrafish using this method. Zebrafish at 3, 4, 7, 14, 16, 19, 21, 28, 60, and 90 d postfertilization (dpf) were placed into an ice water bath for 5, 10, 30, 45, or 60 min (n = 12 to 40 per group). In addition, zebrafish were placed in ice water for 12 h (age ≤14 dpf) or 30 s (age ≥14 dpf). After rapid cooling, fish were transferred to a recovery tank and the number of fish alive at 1, 4, and 12-24 h after removal from ice water was documented. Euthanasia was defined as a failure when evidence of recovery was observed at any point after removal from ice water. Results showed that younger fish required prolonged exposure to rapid cooling for effective euthanasia, with the required exposure time decreasing as fish age. Although younger fish required long exposure times, animals became immobilized immediately upon exposure to the cold water, and behavioral indicators of pain or distress rarely occurred. We conclude that zebrafish 14 dpf and younger require as long as 12 h, those 16 to 28 dpf of age require 5 min, and those older than 28 dpf require 30 s minimal exposure to rapid cooling for reliable euthanasia.

  19. Alkbh4 and Atrn Act Maternally to Regulate Zebrafish Epiboly

    PubMed Central

    Sun, Qingrui; Liu, Xingfeng; Gong, Bo; Wu, Di; Meng, Anming; Jia, Shunji

    2017-01-01

    During embryonic gastrulation, coordinated cell movements occur to bring cells to their correct position. Among them, epiboly produces the first distinct morphological changes, which is essential for the early development of zebrafish. Despite its fundamental importance, little is known to understand the underlying molecular mechanisms. By generating maternal mutant lines with CRISPR/Cas9 technology and using morpholino knockdown strategy, we showed that maternal Alkbh4 depletion leads to severe epiboly defects in zebrafish. Immunofluorescence assays revealed that Alkbh4 promotes zebrafish embryonic epiboly through regulating actomyosin contractile ring formation, which is composed of Actin and non-muscular myosin II (NMII). To further investigate this process, yeast two hybridization assay was performed and Atrn was identified as a binding partner of Alkbh4. Combining with the functional results of Alkbh4, we found that maternal Atrn plays a similar role in zebrafish embryonic morphogenesis by regulating actomyosin formation. On the molecular level, our data revealed that Atrn prefers to interact with the active form of Alkbh4 and functions together with it to regulate the demethylation of Actin, the actomyosin formation, and subsequently the embryonic epiboly. PMID:28924386

  20. Netting Novel Regulators of Hematopoiesis and Hematologic Malignancies in Zebrafish.

    PubMed

    Kwan, Wanda; North, Trista E

    2017-01-01

    Zebrafish are one of the preeminent model systems for the study of blood development (hematopoiesis), hematopoietic stem and progenitor cell (HSPC) biology, and hematopathology. The zebrafish hematopoietic system shares strong similarities in functional populations, genetic regulators, and niche interactions with its mammalian counterparts. These evolutionarily conserved characteristics, together with emerging technologies in live imaging, compound screening, and genetic manipulation, have been employed to successfully identify and interrogate novel regulatory mechanisms and molecular pathways that guide hematopoiesis. Significantly, perturbations in many of the key developmental signals controlling hematopoiesis are associated with hematological disorders and disease, including anemia, bone marrow failure syndromes, and leukemia. Thus, understanding the regulatory pathways controlling HSPC production and function has important clinical implications. In this review, we describe how the blood system forms and is maintained in zebrafish, with particular focus on new insights into vertebrate hematological regulation gained using this model. The interplay of factors controlling development and disease in the hematopoietic system combined with the unique attributes of the zebrafish make this a powerful platform to discover novel targets for the treatment of hematological disease. © 2017 Elsevier Inc. All rights reserved.

  1. Vascular wall shear stress in zebrafish model of early atherosclerosis

    NASA Astrophysics Data System (ADS)

    Choi, Woorak; Seo, Eunseok; Yeom, Eunseop; Lee, Sang Joon

    2016-11-01

    Although atherosclerosis is a multifactorial disease, the role of hemodynamic force has strong influence on the outbreak of the disease. Low and oscillating wall shear stress (WSS) is associated with the incidence of atherosclerosis. Many researchers have investigated relationships between WSS and the occurrence of atherosclerosis using in vitro and in vivo models. However, these models possess technological limitations in mimicking real biophysiological conditions and monitoring the temporal progression of atherosclerosis. In this study, a hypercholesterolaemic zebrafish model was established as a novel model to resolve these technical limitations. WSS in blood vessels of 15 days post-fertilisation zebrafish was measured using a micro PIV technique, and the spatial distribution of lipids inside blood vessels was quantitatively visualized using a confocal microscopy. As a result, lipids are mainly deposited in the regions of low WSS. The oscillating WSS is not induced by blood flows in the zebrafish disease model. The present hypercholesterolaemic zebrafish model would be useful for understanding the effect of WSS on the early stage of atherosclerosis. This work was supported by the National Research Foundation of Korea (NRF) under a Grant funded by the Korean government (MSIP) (No. 2008-0061991).

  2. Recording the adult zebrafish cerebral field potential during pentylenetetrazole seizures

    PubMed Central

    Pineda, Ricardo; Beattie, Christine E.; Hall, Charles W.

    2017-01-01

    Although the zebrafish is increasingly used as a model organism to study epilepsy, no standard electrophysiological technique for recording electrographic seizures in adult fish exists. The purpose of this paper is to introduce a readily implementable technique for recording pentylenetetrazole seizures in the adult zebrafish. We find that we can consistently record a high quality field potential over the zebrafish cerebrum using an amplification of 5000 V/V and bandpass filtering at corner frequencies of 1.6 and 16 Hz. The cerebral field potential recordings show consistent features in the baseline, pre-seizure, seizure and post-seizure time periods that can be easily recognized by visual inspection as is the case with human and rodent electroencephalogram. Furthermore, numerical analysis of the field potential at the time of seizure onset reveals an increase in the total power, bandwidth and peak frequency in the power spectrum, as is also the case with human and rodent electroencephalogram. The techniques presented herein stand to advance the utility of the adult zebrafish in the study of epilepsy by affording an equivalent to the electroencephalogram used in mammalian models and human patients. PMID:21689682

  3. Digestive enzymatic activity during ontogenetic development in zebrafish (Danio rerio).

    PubMed

    Guerrera, Maria Cristina; De Pasquale, Francesca; Muglia, Ugo; Caruso, Gabriella

    2015-12-01

    Despite the growing importance of zebrafish (Danio rerio) as an experimental model in biomedical research, some aspect of physiological and related morphological age dependent changes in digestive system during larval development are still unknown. In this paper, a biochemical and morphological study of the digestive tract of zebrafish was undertaken to record the functional changes occurring in this species during its ontogenetic development, particularly from 24 hr to 47 days post fertilization (dpf). Endo- and exo-proteases, as well as α-amylase enzymes, were quantified in zebrafish larvae before first feeding (7 dpf). The most morphologically significant events during the ontogenesis of the gut occurred between 3 dpf (mouth opening) and 7 dpf (end of exocrine pancreas differentiation). The presence of a wide range of digestive enzymes, already active at earlier zebrafish larval stages, closely related with the omnivorous diet of this species. Increasing enzyme activities were found with increasing age, probably in relation with intestinal mucosa folding and consequent absorption surface increase. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 699-706, 2015. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

  4. Real-Time Monitoring and Analysis of Zebrafish Electrocardiogram with Anomaly Detection.

    PubMed

    Lenning, Michael; Fortunato, Joseph; Le, Tai; Clark, Isaac; Sherpa, Ang; Yi, Soyeon; Hofsteen, Peter; Thamilarasu, Geethapriya; Yang, Jingchun; Xu, Xiaolei; Han, Huy-Dung; Hsiai, Tzung K; Cao, Hung

    2017-12-28

    Heart disease is the leading cause of mortality in the U.S. with approximately 610,000 people dying every year. Effective therapies for many cardiac diseases are lacking, largely due to an incomplete understanding of their genetic basis and underlying molecular mechanisms. Zebrafish ( Danio rerio ) are an excellent model system for studying heart disease as they enable a forward genetic approach to tackle this unmet medical need. In recent years, our team has been employing electrocardiogram (ECG) as an efficient tool to study the zebrafish heart along with conventional approaches, such as immunohistochemistry, DNA and protein analyses. We have overcome various challenges in the small size and aquatic environment of zebrafish in order to obtain ECG signals with favorable signal-to-noise ratio (SNR), and high spatial and temporal resolution. In this paper, we highlight our recent efforts in zebrafish ECG acquisition with a cost-effective simplified microelectrode array (MEA) membrane providing multi-channel recording, a novel multi-chamber apparatus for simultaneous screening, and a LabVIEW program to facilitate recording and processing. We also demonstrate the use of machine learning-based programs to recognize specific ECG patterns, yielding promising results with our current limited amount of zebrafish data. Our solutions hold promise to carry out numerous studies of heart diseases, drug screening, stem cell-based therapy validation, and regenerative medicine.

  5. Real-Time Monitoring and Analysis of Zebrafish Electrocardiogram with Anomaly Detection

    PubMed Central

    Lenning, Michael; Fortunato, Joseph; Le, Tai; Clark, Isaac; Sherpa, Ang; Yi, Soyeon; Hofsteen, Peter; Thamilarasu, Geethapriya; Yang, Jingchun; Xu, Xiaolei; Hsiai, Tzung K.; Cao, Hung

    2017-01-01

    Heart disease is the leading cause of mortality in the U.S. with approximately 610,000 people dying every year. Effective therapies for many cardiac diseases are lacking, largely due to an incomplete understanding of their genetic basis and underlying molecular mechanisms. Zebrafish (Danio rerio) are an excellent model system for studying heart disease as they enable a forward genetic approach to tackle this unmet medical need. In recent years, our team has been employing electrocardiogram (ECG) as an efficient tool to study the zebrafish heart along with conventional approaches, such as immunohistochemistry, DNA and protein analyses. We have overcome various challenges in the small size and aquatic environment of zebrafish in order to obtain ECG signals with favorable signal-to-noise ratio (SNR), and high spatial and temporal resolution. In this paper, we highlight our recent efforts in zebrafish ECG acquisition with a cost-effective simplified microelectrode array (MEA) membrane providing multi-channel recording, a novel multi-chamber apparatus for simultaneous screening, and a LabVIEW program to facilitate recording and processing. We also demonstrate the use of machine learning-based programs to recognize specific ECG patterns, yielding promising results with our current limited amount of zebrafish data. Our solutions hold promise to carry out numerous studies of heart diseases, drug screening, stem cell-based therapy validation, and regenerative medicine. PMID:29283402

  6. Advancements in zebrafish applications for 21st century toxicology

    PubMed Central

    Garcia, Gloria R.; Noyes, Pamela D.; Tanguay, Robert L.

    2016-01-01

    The zebrafish model is the only available high-throughput vertebrate assessment system, and it is uniquely suited for studies of in vivo cell biology. A sequenced and annotated genome has revealed a large degree of evolutionary conservation in comparison to the human genome. Due to our shared evolutionary history, the anatomical and physiological features of fish are highly homologous to humans, which facilitates studies relevant to human health. In addition, zebrafish provide a very unique vertebrate data stream that allows researchers to anchor hypotheses at the biochemical, genetic, and cellular levels to observations at the structural, functional, and behavioral level in a high-throughput format. In this review, we will draw heavily from toxicological studies to highlight advances in zebrafish high-throughput systems. Breakthroughs in transgenic/reporter lines and methods for genetic manipulation, such as the CRISPR-Cas9 system, will be comprised of reports across diverse disciplines. PMID:27016469

  7. Circadian rhythmicity and light sensitivity of the zebrafish brain.

    PubMed

    Moore, Helen A; Whitmore, David

    2014-01-01

    Traditionally, circadian clocks have been thought of as a neurobiological phenomenon. This view changed somewhat over recent years with the discovery of peripheral tissue circadian oscillators. In mammals, however, the suprachiasmatic nucleus (SCN) in the hypothalamus still retains the critical role of a central synchronizer of biological timing. Zebrafish, in contrast, have always reflected a more highly decentralized level of clock organization, as individual cells and tissues contain directly light responsive circadian pacemakers. As a consequence, clock function in the zebrafish brain has remained largely unexplored, and the precise organization of rhythmic and light-sensitive neurons within the brain is unknown. To address this issue, we used the period3 (per3)-luciferase transgenic zebrafish to confirm that multiple brain regions contain endogenous circadian oscillators that are directly light responsive. In addition, in situ hybridization revealed localised neural expression of several rhythmic and light responsive clock genes, including per3, cryptochrome1a (cry1a) and per2. Adult brain nuclei showing significant clock gene expression include the teleost equivalent of the SCN, as well as numerous hypothalamic nuclei, the periventricular grey zone (PGZ) of the optic tectum, and granular cells of the rhombencephalon. To further investigate the light sensitive properties of neurons, expression of c-fos, a marker for neuronal activity, was examined. c-fos mRNA was upregulated in response to changing light conditions in different nuclei within the zebrafish brain. Furthermore, under constant dark (DD) conditions, c-fos shows a significant circadian oscillation. Taken together, these results show that there are numerous areas of the zebrafish central nervous system, which contain deep brain photoreceptors and directly light-entrainable circadian pacemakers. However, there are also multiple brain nuclei, which possess neither, demonstrating a degree of pacemaker

  8. Circadian Rhythmicity and Light Sensitivity of the Zebrafish Brain

    PubMed Central

    Moore, Helen A.; Whitmore, David

    2014-01-01

    Traditionally, circadian clocks have been thought of as a neurobiological phenomenon. This view changed somewhat over recent years with the discovery of peripheral tissue circadian oscillators. In mammals, however, the suprachiasmatic nucleus (SCN) in the hypothalamus still retains the critical role of a central synchronizer of biological timing. Zebrafish, in contrast, have always reflected a more highly decentralized level of clock organization, as individual cells and tissues contain directly light responsive circadian pacemakers. As a consequence, clock function in the zebrafish brain has remained largely unexplored, and the precise organization of rhythmic and light-sensitive neurons within the brain is unknown. To address this issue, we used the period3 (per3)-luciferase transgenic zebrafish to confirm that multiple brain regions contain endogenous circadian oscillators that are directly light responsive. In addition, in situ hybridization revealed localised neural expression of several rhythmic and light responsive clock genes, including per3, cryptochrome1a (cry1a) and per2. Adult brain nuclei showing significant clock gene expression include the teleost equivalent of the SCN, as well as numerous hypothalamic nuclei, the periventricular grey zone (PGZ) of the optic tectum, and granular cells of the rhombencephalon. To further investigate the light sensitive properties of neurons, expression of c-fos, a marker for neuronal activity, was examined. c-fos mRNA was upregulated in response to changing light conditions in different nuclei within the zebrafish brain. Furthermore, under constant dark (DD) conditions, c-fos shows a significant circadian oscillation. Taken together, these results show that there are numerous areas of the zebrafish central nervous system, which contain deep brain photoreceptors and directly light-entrainable circadian pacemakers. However, there are also multiple brain nuclei, which possess neither, demonstrating a degree of pacemaker

  9. Developmental nephrotoxicity of aristolochic acid in a zebrafish model

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ding, Yu-Ju; Chen, Yau-Hung, E-mail: yauhung@mail.tku.edu.tw

    2012-05-15

    Aristolochic acid (AA) is a component of Aristolochia plant extracts which is used as a treatment for different pathologies and their toxicological effects have not been sufficiently studied. The aim of this study was to evaluate AA-induced nephrotoxicity in zebrafish embryos. After soaking zebrafish embryos in AA, the embryos displayed malformed kidney phenotypes, such as curved, cystic pronephric tubes, pronephric ducts, and cases of atrophic glomeruli. The percentages of embryos with malformed kidney phenotypes increased as the exposure dosages of AA increased. Furthermore, AA-treated embryos exhibited significantly reduced glomerular filtration rates (GFRs) in comparison with mock-control littermates (mock-control: 100 ±more » 2.24% vs. 10 ppm AA treatment for 3–5 h: 71.48 ± 18.84% ∼ 39.41 ± 15.88%), indicating that AA treatment not only caused morphological kidney changes but also induced renal failure. In addition to kidney malformations, AA-treated zebrafish embryos also exhibited deformed hearts, swollen pericardiums, impaired blood circulation and the accumulation(s) of red blood cells. Whole-mount in situ hybridization studies using cmlc2 and wt1b as riboprobes indicated that the kidney is more sensitive than the heart to AA damage. Real-time PCR showed that AA can up-regulate the expression of proinflammatory genes like TNFα, cox2 and mpo. These results support the following conclusions: (1) AA-induced renal failure is mediated by inflammation, which causes circulation dysfunction followed by serious heart malformation; and (2) the kidney is more sensitive than the heart to AA injury. -- Highlights: ► Zebrafish were used to evaluate aristolochic acid (AA)-induced nephrotoxicity. ► AA-treated zebrafish embryos exhibited deformed heart as well as malformed kidney. ► Kidney is more sensitive to AA injury than the heart.« less

  10. Treatment with sodium benzoate leads to malformation of zebrafish larvae.

    PubMed

    Tsay, Huey-Jen; Wang, Yun-Hsin; Chen, Wei-Li; Huang, Mei-Yun; Chen, Yau-Hung

    2007-01-01

    Sodium benzoate (SB) is a commonly used food preservative and anti-microbial agent in many foods from soup to cereals. However, little is known about the SB-induced toxicity and teratogenicity during early embryonic development. Here, we used zebrafish as a model to test the toxicity and teratogenicity because of their transparent eggs; therefore, the organogenesis of zebrafish embryos is easy to observe. After low dosages of SB (1-1000 ppm) treatment, the zebrafish embryos exhibited a 100% survival rate. As the exposure dosages increased, the survival rates decreased. No embryos survived after treatment with 2000 ppm SB. The 50% lethal dose (LD(50)) of zebrafish is found to be in the range of 1400-1500 ppm. Gut abnormalities, malformation of pronephros, defective hatching gland and edema in pericardial sac were observed after treatment with SB. Compared to untreated littermates (vehicle-treated control), SB-treated embryos exhibited significantly reduced tactile sensitivity frequencies of touch-induced movement (vehicle-treated control: 27.60+/-1.98 v.s. 1000 ppm SB: 7.89+/-5.28; N=30). Subtle changes are easily observed by staining with specific monoclonal antibodies F59, Znp1 and alpha6F to detect morphology changes in muscle fibers, motor axons and pronephros, respectively. Our data showed that the treatment of SB led to misalignment of muscle fibers, motor neuron innervations, excess acetyl-choline receptor cluster and defective pronephric tubes. On the basis of these observations, we suggest that sodium benzoate is able to induce neurotoxicity and nephrotoxicity of zebrafish larvae.

  11. Production of Zebrafish Offspring from Cultured Female Germline Stem Cells

    PubMed Central

    Wong, Ten-Tsao; Tesfamichael, Abraham; Collodi, Paul

    2013-01-01

    Zebrafish female germline stem cell (FGSC) cultures were generated from a transgenic line of fish that expresses Neo and DsRed under the control of the germ cell specific promoter, ziwi [Tg(ziwi:neo);Tg(ziwi:DsRed)]. Homogeneous FGSC cultures were established by G418 selection and continued to express ziwi for more than 6 weeks along with the germ cell markers nanos3, dnd, dazl and vasa. A key component of the cell culture system was the use of a feeder cell line that was initiated from ovaries of a transgenic line of fish [Tg(gsdf:neo)] that expresses Neo controlled by the zebrafish gonadal soma derived factor (gsdf) promoter. The feeder cell line was selected in G418 and engineered to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and glial-cell-line derived neurotrophic factor (Gdnf). These factors were shown to significantly enhance FGSC growth, survival and germline competency in culture. Results from cell transplantation experiments revealed that the cultured FGSCs were able to successfully colonize the gonad of sterile recipient fish and generate functional gametes. Up to 20% of surviving recipient fish that were injected with the cultured FGSCs were fertile and generated multiple batches of normal offspring for at least 6 months. The FGSC cultures will provide an in vitro system for studies of zebrafish germ cell growth and differentiation and their high frequency of germline transmission following transplantation could form the basis of a stem cell-mediated strategy for gene transfer and manipulation of the zebrafish genome. PMID:23671620

  12. Sherrington's Model of Successive Induction for Comparative Analysis of Zebrafish Motor Response

    EPA Science Inventory

    The responses in motor activity of zebrafish to sudden changes in lighting conditions may be modeled by Sherrington’s model of successive induction. Fish left in the dark exhibit very little motion, when exposed to light zebrafish motion increases towards an apparent horizo...

  13. Comparison of the Exomes of Common Carp (Cyprinus carpio) and Zebrafish (Danio rerio)

    PubMed Central

    Henkel, Christiaan V.; Dirks, Ron P.; Jansen, Hans J.; Forlenza, Maria; Wiegertjes, Geert F.; Howe, Kerstin; van den Thillart, Guido E.E.J.M.

    2012-01-01

    Abstract Research on common carp, Cyprinus carpio, is beneficial for zebrafish research because of resources available owing to its large body size, such as the availability of sufficient organ material for transcriptomics, proteomics, and metabolomics. Here we describe the shot gun sequencing of a clonal double-haploid common carp line. The assembly consists of 511891 scaffolds with an N50 of 17 kb, predicting a total genome size of 1.4–1.5 Gb. A detailed analysis of the ten largest scaffolds indicates that the carp genome has a considerably lower repeat coverage than zebrafish, whilst the average intron size is significantly smaller, making it comparable to the fugu genome. The quality of the scaffolding was confirmed by comparisons with RNA deep sequencing data sets and a manual analysis for synteny with the zebrafish, especially the Hox gene clusters. In the ten largest scaffolds analyzed, the synteny of genes is almost complete. Comparisons of predicted exons of common carp with those of the zebrafish revealed only few genes specific for either zebrafish or carp, most of these being of unknown function. This supports the hypothesis of an additional genome duplication event in the carp evolutionary history, which—due to a higher degree of compactness—did not result in a genome larger than that of zebrafish. PMID:22715948

  14. Embryonic Alcohol Exposure Impairs the Dopaminergic System and Social Behavioral Responses in Adult Zebrafish

    PubMed Central

    Fernandes, Yohaan; Rampersad, Mindy

    2015-01-01

    Background: The zebrafish is a powerful neurobehavioral genetics tool with which complex human brain disorders including alcohol abuse and fetal alcohol spectrum disorders may be modeled and investigated. Zebrafish innately form social groups called shoals. Previously, it has been demonstrated that a single bath exposure (24 hours postfertilization) to low doses of alcohol (0, 0.25, 0.50, 0.75, and 1% vol/vol) for a short duration (2 hours) leads to impaired group forming, or shoaling, in adult zebrafish. Methods: In the current study, we immersed zebrafish eggs in a low concentration of alcohol (0.5% or 1% vol/vol) for 2 hours at 24 hours postfertilization and let the fish grow and reach adulthood. In addition to quantifying the behavioral response of the adult fish to an animated shoal, we also measured the amount of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid from whole brain extracts of these fish using high-pressure liquid chromatograph. Results: Here we confirm that embryonic alcohol exposure makes adult zebrafish increase their distance from the shoal stimulus in a dose-dependent manner. We also show that the shoal stimulus increases the amount of dopamine and 3,4-dihydroxyphenylacetic acid in the brain of control zebrafish but not in fish previously exposed to alcohol during their embryonic development. Conclusions: We speculate that one of the mechanisms that may explain the embryonic alcohol-induced impaired shoaling response in zebrafish is dysfunction of reward mechanisms subserved by the dopaminergic system. PMID:25568285

  15. Quantum Dot Nanotoxicity Assessment Using the Zebrafish Embryo

    PubMed Central

    King-Heiden, Tisha C.; Wiecinski, Paige N.; Mangham, Andrew N.; Metz, Kevin M.; Nesbit, Dorothy; Pedersen, Joel A.; Hamers, Robert J.; Heideman, Warren; Peterson, Richard E.

    2009-01-01

    Quantum dots (QDs) hold promise for several biomedical, life sciences and photovoltaic applications. Substantial production volumes and environmental release are anticipated. QD toxicity may be intrinsic to their physicochemical properties, or result from the release of toxic components during breakdown. We hypothesized that developing zebrafish could be used to identify and distinguish these different types of toxicity. Embryos were exposed to aqueous suspensions of CdSecore/ZnSshell QDs functionalized with either poly-L-lysine or poly(ethylene glycol) terminated with methoxy, carboxylate, or amine groups. Toxicity was influenced by the QD coating, which also contributed to the QD suspension stability. At sublethal concentrations, many QD preparations produced characteristic signs of Cd toxicity that weakly correlated with metallothionein expression, indicating that QDs are only slightly degraded in vivo. QDs also produced distinctly different toxicity that could not be explained by Cd release. Using the zebrafish model, we were able to distinguish toxicity intrinsic to QDs from that caused by released metal ions. We conclude that developing zebrafish provide a rapid, low- cost approach for assessing structure-toxicity relationships of nanoparticles. PMID:19350942

  16. Ion transport in the zebrafish kidney from a human disease angle: possibilities, considerations, and future perspectives.

    PubMed

    Kersten, Simone; Arjona, Francisco J

    2017-01-01

    Unique experimental advantages, such as its embryonic/larval transparency, high-throughput nature, and ease of genetic modification, underpin the rapid emergence of the zebrafish (Danio rerio) as a preeminent model in biomedical research. Particularly in the field of nephrology, the zebrafish provides a promising model for studying the physiological implications of human solute transport processes along consecutive nephron segments. However, although the zebrafish might be considered a valuable model for numerous renal ion transport diseases and functional studies of many channels and transporters, not all human renal electrolyte transport mechanisms and human diseases can be modeled in the zebrafish. With this review, we explore the ontogeny of zebrafish renal ion transport, its nephron structure and function, and thereby demonstrate the clinical translational value of this model. By critical assessment of genomic and amino acid conservation of human proteins involved in renal ion handling (channels, transporters, and claudins), kidney and nephron segment conservation, and renal electrolyte transport physiology in the zebrafish, we provide researchers and nephrologists with an indication of the possibilities and considerations of the zebrafish as a model for human renal ion transport. Combined with advanced techniques envisioned for the future, implementation of the zebrafish might expand beyond unraveling pathophysiological mechanisms that underlie distinct genetic or environmentally, i.e., pharmacological and lifestyle, induced renal transport deficits. Specifically, the ease of drug administration and the exploitation of improved genetic approaches might argue for the adoption of the zebrafish as a model for preclinical personalized medicine for distinct renal diseases and renal electrolyte transport proteins. Copyright © 2017 the American Physiological Society.

  17. Recommendations for Health Monitoring and Reporting for Zebrafish Research Facilities

    PubMed Central

    Crim, Marcus J.; Lieggi, Christine

    2016-01-01

    Abstract The presence of subclinical infection or clinical disease in laboratory zebrafish may have a significant impact on research results, animal health and welfare, and transfer of animals between institutions. As use of zebrafish as a model of disease increases, a harmonized method for monitoring and reporting the health status of animals will facilitate the transfer of animals, allow institutions to exclude diseases that may negatively impact their research programs, and improve animal health and welfare. All zebrafish facilities should implement a health monitoring program. In this study, we review important aspects of a health monitoring program, including choice of agents, samples for testing, available testing methodologies, housing and husbandry, cost, test subjects, and a harmonized method for reporting results. Facilities may use these recommendations to implement their own health monitoring program. PMID:26991393

  18. PFOS induces behavioral alterations, including spontaneous hyperactivity that is corrected by dexamfetamine in zebrafish larvae.

    PubMed

    Spulber, Stefan; Kilian, Pascal; Wan Ibrahim, Wan Norhamidah; Onishchenko, Natalia; Ulhaq, Mazhar; Norrgren, Leif; Negri, Sara; Di Tuccio, Marcello; Ceccatelli, Sandra

    2014-01-01

    Perfluorooctane sulfonate (PFOS) is a widely spread environmental contaminant. It accumulates in the brain and has potential neurotoxic effects. The exposure to PFOS has been associated with higher impulsivity and increased ADHD prevalence. We investigated the effects of developmental exposure to PFOS in zebrafish larvae, focusing on the modulation of activity by the dopaminergic system. We exposed zebrafish embryos to 0.1 or 1 mg/L PFOS (0.186 or 1.858 µM, respectively) and assessed swimming activity at 6 dpf. We analyzed the structure of spontaneous activity, the hyperactivity and the habituation during a brief dark period (visual motor response), and the vibrational startle response. The findings in zebrafish larvae were compared with historical data from 3 months old male mice exposed to 0.3 or 3 mg/kg/day PFOS throughout gestation. Finally, we investigated the effects of dexamfetamine on the alterations in spontaneous activity and startle response in zebrafish larvae. We found that zebrafish larvae exposed to 0.1 mg/L PFOS habituate faster than controls during a dark pulse, while the larvae exposed to 1 mg/L PFOS display a disorganized pattern of spontaneous activity and persistent hyperactivity. Similarly, mice exposed to 0.3 mg/kg/day PFOS habituated faster than controls to a new environment, while mice exposed to 3 mg/kg/day PFOS displayed more intense and disorganized spontaneous activity. Dexamfetamine partly corrected the hyperactive phenotype in zebrafish larvae. In conclusion, developmental exposure to PFOS in zebrafish induces spontaneous hyperactivity mediated by a dopaminergic deficit, which can be partially reversed by dexamfetamine in zebrafish larvae.

  19. Preliminary Evaluation on the Effects of Feeds on the Growth and Early Reproductive Performance of Zebrafish (Danio rerio)

    PubMed Central

    2012-01-01

    This study evaluated the effects of several commercially available feeds and different feeding regimes on the growth and early reproductive performance of zebrafish (Danio rerio). Juvenile zebrafish (n= 20; 5.06 ± 0.69 mg) were stocked into each of 24 tanks (volume, 2 L); 3 tanks were assigned to each of 8 feeding combinations for a period of 60 d. At the end of 60 d, 2 male and 2 female fish from each tank were pooled by dietary treatment (n = 6) and used to evaluate the effects of feeding combinations on early reproductive performance. Zebrafish fed dietary treatments 3 and 7 had significantly greater weight gain than zebrafish fed diet 5. Mean spawning success was significantly greater in zebrafish fed the control diet (Artemiaonly) than in those fed diet 1. Mean hatch rates were greater in zebrafish fed the control feed and diets 1, 2, 3, 5, and 6 than zebrafish fed diet 4. Additional results suggest that female zebrafish are sexually mature after 90 d post fertilization and that fertilization rates are the limiting factor in early reproduction. PMID:23043806

  20. Zebrafish models flex their muscles to shed light on muscular dystrophies.

    PubMed

    Berger, Joachim; Currie, Peter D

    2012-11-01

    Muscular dystrophies are a group of genetic disorders that specifically affect skeletal muscle and are characterized by progressive muscle degeneration and weakening. To develop therapies and treatments for these diseases, a better understanding of the molecular basis of muscular dystrophies is required. Thus, identification of causative genes mutated in specific disorders and the study of relevant animal models are imperative. Zebrafish genetic models of human muscle disorders often closely resemble disease pathogenesis, and the optical clarity of zebrafish embryos and larvae enables visualization of dynamic molecular processes in vivo. As an adjunct tool, morpholino studies provide insight into the molecular function of genes and allow rapid assessment of candidate genes for human muscular dystrophies. This unique set of attributes makes the zebrafish model system particularly valuable for the study of muscle diseases. This review discusses how recent research using zebrafish has shed light on the pathological basis of muscular dystrophies, with particular focus on the muscle cell membrane and the linkage between the myofibre cytoskeleton and the extracellular matrix.

  1. Atrogin-1 Deficiency Leads to Myopathy and Heart Failure in Zebrafish.

    PubMed

    Bühler, Anja; Kustermann, Monika; Bummer, Tiziana; Rottbauer, Wolfgang; Sandri, Marco; Just, Steffen

    2016-01-30

    Orchestrated protein synthesis and degradation is fundamental for proper cell function. In muscle, impairment of proteostasis often leads to severe cellular defects finally interfering with contractile function. Here, we analyze for the first time the role of Atrogin-1, a muscle-specific E3 ubiquitin ligase known to be involved in the regulation of protein degradation via the ubiquitin proteasome and the autophagy/lysosome systems, in the in vivo model system zebrafish (Danio rerio). We found that targeted inactivation of zebrafish Atrogin-1 leads to progressive impairment of heart and skeletal muscle function and disruption of muscle structure without affecting early cardiogenesis and skeletal muscle development. Autophagy is severely impaired in Atrogin-1-deficient zebrafish embryos resulting in the disturbance of the cytoarchitecture of cardiomyocytes and skeletal muscle cells. These observations are consistent with molecular and ultrastructural findings in an Atrogin-1 knockout mouse and demonstrate that the zebrafish is a suitable vertebrate model to study the molecular mechanisms of Atrogin-1-mediated autophagic muscle pathologies and to screen for novel therapeutically active substances in high-throughput in vivo small compound screens (SCS).

  2. Developmental neurotoxicity of pyrethroid insecticides in zebrafish embryos.

    PubMed

    DeMicco, Amy; Cooper, Keith R; Richardson, Jason R; White, Lori A

    2010-01-01

    Pyrethroid insecticides are one of the most commonly used residential and agricultural insecticides. Based on the increased use of pyrethroids and recent studies showing that pregnant women and children are exposed to pyrethroids, there are concerns over the potential for developmental neurotoxicity. However, there have been relatively few studies on the developmental neurotoxicity of pyrethroids. In this study, we sought to investigate the developmental toxicity of six common pyrethroids, three type I compounds (permethrin, resmethrin, and bifenthrin) and three type II compounds (deltamethrin, cypermethrin, and lambda-cyhalothrin), and to determine whether zebrafish embryos may be an appropriate model for studying the developmental neurotoxicity of pyrethroids. Exposure of zebrafish embryos to pyrethroids caused a dose-dependent increase in mortality and pericardial edema, with type II compounds being the most potent. At doses approaching the LC(50), permethrin and deltamethrin caused craniofacial abnormalities. These findings are consistent with mammalian studies demonstrating that pyrethroids are mildly teratogenic at very high doses. However, at lower doses, body axis curvature and spasms were observed, which were reminiscent of the classic syndromes observed with pyrethroid toxicity. Treatment with diazepam ameliorated the spasms, while treatment with the sodium channel antagonist MS-222 ameliorated both spasms and body curvature, suggesting that pyrethroid-induced neurotoxicity is similar in zebrafish and mammals. Taken in concert, these data suggest that zebrafish may be an appropriate alternative model to study the mechanism(s) responsible for the developmental neurotoxicity of pyrethroid insecticides and aid in identification of compounds that should be further tested in mammalian systems.

  3. Developmental Neurotoxicity of Pyrethroid Insecticides in Zebrafish Embryos

    PubMed Central

    DeMicco, Amy; Cooper, Keith R.; Richardson, Jason R.; White, Lori A.

    2010-01-01

    Pyrethroid insecticides are one of the most commonly used residential and agricultural insecticides. Based on the increased use of pyrethroids and recent studies showing that pregnant women and children are exposed to pyrethroids, there are concerns over the potential for developmental neurotoxicity. However, there have been relatively few studies on the developmental neurotoxicity of pyrethroids. In this study, we sought to investigate the developmental toxicity of six common pyrethroids, three type I compounds (permethrin, resmethrin, and bifenthrin) and three type II compounds (deltamethrin, cypermethrin, and λ-cyhalothrin), and to determine whether zebrafish embryos may be an appropriate model for studying the developmental neurotoxicity of pyrethroids. Exposure of zebrafish embryos to pyrethroids caused a dose-dependent increase in mortality and pericardial edema, with type II compounds being the most potent. At doses approaching the LC50, permethrin and deltamethrin caused craniofacial abnormalities. These findings are consistent with mammalian studies demonstrating that pyrethroids are mildly teratogenic at very high doses. However, at lower doses, body axis curvature and spasms were observed, which were reminiscent of the classic syndromes observed with pyrethroid toxicity. Treatment with diazepam ameliorated the spasms, while treatment with the sodium channel antagonist MS-222 ameliorated both spasms and body curvature, suggesting that pyrethroid-induced neurotoxicity is similar in zebrafish and mammals. Taken in concert, these data suggest that zebrafish may be an appropriate alternative model to study the mechanism(s) responsible for the developmental neurotoxicity of pyrethroid insecticides and aid in identification of compounds that should be further tested in mammalian systems. PMID:19861644

  4. Intrinsic Properties of Larval Zebrafish Neurons in Ethanol

    PubMed Central

    Ikeda, Hiromi; Delargy, Alison H.; Yokogawa, Tohei; Urban, Jason M.; Burgess, Harold A.; Ono, Fumihito

    2013-01-01

    The behavioral effects of ethanol have been studied in multiple animal models including zebrafish. Locomotion of zebrafish larvae is resistant to high concentrations of ethanol in bath solution. This resistance has been attributed to a lower systemic concentration of ethanol in zebrafish when compared with bath solution, although the mechanism to maintain such a steep gradient is unclear. Here we examined whether the intrinsic properties of neurons play roles in this resistance. In order to minimize the contribution of metabolism and diffusional barriers, larvae were hemisected and the anterior half immersed in a range of ethanol concentrations thereby ensuring the free access of bath ethanol to the brain. The response to vibrational stimuli of three types of reticulospinal neurons: Mauthner neurons, vestibulospinal neurons, and MiD3 neurons were examined using an intracellular calcium indicator. The intracellular [Ca2+] response in MiD3 neurons decreased in 100 mM ethanol, while Mauthner neurons and vestibulospinal neurons required >300 mM ethanol to elicit similar effects. The ethanol effect in Mauthner neurons was reversible following removal of ethanol. Interestingly, activities of MiD3 neurons displayed spontaneous recovery in 300 mM ethanol, suggestive of acute tolerance. Finally, we examined with mechanical vibration the startle response of free-swimming larvae in 300 mM ethanol. Ethanol treatment abolished long latency startle responses, suggesting a functional change in neural processing. These data support the hypothesis that individual neurons in larval zebrafish brains have distinct patterns of response to ethanol dictated by specific molecular targets. PMID:23658822

  5. The smell of "anxiety": Behavioral modulation by experimental anosmia in zebrafish.

    PubMed

    Abreu, Murilo S; Giacomini, Ana C V V; Kalueff, Allan V; Barcellos, Leonardo J G

    2016-04-01

    Olfaction is strongly involved in the regulation of fish behavior, including reproductive, defensive, social and migration behaviors. In fish, anosmia (the lack of olfaction) can be induced experimentally, impairing their ability to respond to various olfactory stimuli. Here, we examine the effects of experimental lidocaine-induced anosmia on anxiety-like behavior and whole-body cortisol levels in adult zebrafish (Danio rerio). We show that experimentally-induced anosmia reduces anxiolytic-like behavioral effects of fluoxetine and seems to interact with anxiogenic effect of stress also paralleling cortisol responses in zebrafish. These findings provide first experimental evidence that temporary anosmia modulates anxiety-like behaviors and physiology in adult zebrafish. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Rhodopsin expression in the zebrafish pineal gland from larval to adult stage.

    PubMed

    Magnoli, Domenico; Zichichi, Rosalia; Laurà, Rosaria; Guerrera, Maria Cristina; Campo, Salvatore; de Carlos, Felix; Suárez, Alberto Álvarez; Abbate, Francesco; Ciriaco, Emilia; Vega, Jose Antonio; Germanà, Antonino

    2012-03-09

    The zebrafish pineal gland plays an important role in different physiological functions including the regulation of the circadian clock. In the fish pineal gland the pinealocytes are made up of different segments: outer segment, inner segment and basal pole. Particularly, in the outer segment the rhodopsin participates in the external environment light reception that represents the first biochemical step in the melatonin production. It is well known that the rhodopsin in the adult zebrafish is well expressed in the pineal gland but both the expression and the cellular localization of this protein during development remain still unclear. In this study using qRT-PCR, sequencing and immunohistochemistry the expression as well as the protein localization of the rhodopsin in the zebrafish from larval (10 dpf) to adult stage (90 dpf) were demonstrated. The rhodopsin mRNA expression presents a peak of expression at 10 dpf, a further reduction to 50 dpf before increasing again in the adult stage. Moreover, the cellular localization of the rhodopsin-like protein was always localized in the pinealocyte at all ages examined. Our results demonstrated the involvement of the rhodopsin in the zebrafish pineal gland physiology particularly in the light capture during the zebrafish lifespan. Copyright © 2012 Elsevier B.V. All rights reserved.

  7. The French press: a repeatable and high-throughput approach to exercising zebrafish (Danio rerio).

    PubMed

    Usui, Takuji; Noble, Daniel W A; O'Dea, Rose E; Fangmeier, Melissa L; Lagisz, Malgorzata; Hesselson, Daniel; Nakagawa, Shinichi

    2018-01-01

    Zebrafish are increasingly used as a vertebrate model organism for various traits including swimming performance, obesity and metabolism, necessitating high-throughput protocols to generate standardized phenotypic information. Here, we propose a novel and cost-effective method for exercising zebrafish, using a coffee plunger and magnetic stirrer. To demonstrate the use of this method, we conducted a pilot experiment to show that this simple system provides repeatable estimates of maximal swim performance (intra-class correlation [ICC] = 0.34-0.41) and observe that exercise training of zebrafish on this system significantly increases their maximum swimming speed. We propose this high-throughput and reproducible system as an alternative to traditional linear chamber systems for exercising zebrafish and similarly sized fishes.

  8. Application of chrysophanol in zebrafish to reduce dietary introduced lipid and its possible mechanism

    PubMed Central

    Chen, Kan; Wang, Chang-Qian; Fan, Yu-Qi; Xie, Yu-Shui; Yin, Zhao-Fang; Xu, Zuo-Jun; Zhang, Hui-Li; Cao, Jia-Tian; Wang, Yue

    2015-01-01

    Purpose: To explore the therapeutic potential and mechanism of chrysophanol on lipid-lowering function. Methods: Zebrafish or larvae were employed to evaluate the effect of chrysophanol on lipid-lowering. Zebrafish of 5 day post fertilization (dpf, larva) and 13-week old were fed with high-cholesterol diet or high-fat to investigate the influence of chrysophanol comparing with atorvastain and co-administration of chrysophanol and atorvastain on subsistent blood lipid using the fluorescence microscope and lipid panel screen. Thereafter, we enrolled zebrafish of 7 dpf fed with high-fat diet to explore the lipid-lowering mechanism of chrysophanol basing on the frequency of peristalsis and the residual on the digestive wall. Results: Chrysophanol could significantly lower cholesterol both in zebrafish and larvae (P < 0.05), and the co-administration of chrysophanol and atorvastatin had the best performance in reducing cholesterol (P < 0.05). Chrysophanol and atorvastain could also significantly lower triglyceride. Moreover, we found that chrysophanol attached on the digestive wall for a long time and enhanced the frequency of peristalsis. Conclusions: Chrysophanol has lipid-lowering effect both in zebrafish and larvae which may be attributed to the effect on the frequency of peristalsis and fat absorption, and co-administration with atorvastain performs better lipid-lowering effect in zebrafish. PMID:26379845

  9. Cell Migration During Heart Regeneration in Zebrafish

    PubMed Central

    Tahara, Naoyuki; Brush, Michael; Kawakami, Yasuhiko

    2018-01-01

    Zebrafish possess the remarkable ability to regenerate injured hearts as adults, which contrasts the very limited ability in mammals. Although very limited, mammalian hearts do in fact have measurable levels of cardiomyocyte regeneration. Therefore, elucidating mechanisms of zebrafish heart regeneration would provide information of naturally occurring regeneration to potentially apply to mammalian studies, in addition to addressing this biologically interesting phenomenon in itself. Studies over the past 13 years have identified processes and mechanisms of heart regeneration in zebrafish. After heart injury, preexisting cardiomyocytes dedifferentiate, enter the cell cycle, and repair the injured myocardium. This process requires interaction with epicardial cells, endocardial cells, and vascular endothelial cells. Epicardial cells envelope the heart, while endocardial cells make up the inner lining of the heart. They provide paracrine signals to cardiomyocytes to regenerate the injured myocardium, which is vascularized during heart regeneration. In addition, accumulating results suggest that local migration of these major cardiac cell types have roles in heart regeneration. In this review, we summarize the characteristics of various heart injury methods used in the research community and regeneration of the major cardiac cell types. Then, we discuss local migration of these cardiac cell types and immune cells during heart regeneration. PMID:27085002

  10. Advancements in zebrafish applications for 21st century toxicology.

    PubMed

    Garcia, Gloria R; Noyes, Pamela D; Tanguay, Robert L

    2016-05-01

    The zebrafish model is the only available high-throughput vertebrate assessment system, and it is uniquely suited for studies of in vivo cell biology. A sequenced and annotated genome has revealed a large degree of evolutionary conservation in comparison to the human genome. Due to our shared evolutionary history, the anatomical and physiological features of fish are highly homologous to humans, which facilitates studies relevant to human health. In addition, zebrafish provide a very unique vertebrate data stream that allows researchers to anchor hypotheses at the biochemical, genetic, and cellular levels to observations at the structural, functional, and behavioral level in a high-throughput format. In this review, we will draw heavily from toxicological studies to highlight advances in zebrafish high-throughput systems. Breakthroughs in transgenic/reporter lines and methods for genetic manipulation, such as the CRISPR-Cas9 system, will be comprised of reports across diverse disciplines. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. A novel perivascular cell population in the zebrafish brain.

    PubMed

    Venero Galanternik, Marina; Castranova, Daniel; Gore, Aniket V; Blewett, Nathan H; Jung, Hyun Min; Stratman, Amber N; Kirby, Martha R; Iben, James; Miller, Mayumi F; Kawakami, Koichi; Maraia, Richard J; Weinstein, Brant M

    2017-04-11

    The blood-brain barrier is essential for the proper homeostasis and function of the CNS, but its mechanism of function is poorly understood. Perivascular cells surrounding brain blood vessels are thought to be important for blood-brain barrier establishment, but their roles are not well defined. Here, we describe a novel perivascular cell population closely associated with blood vessels on the zebrafish brain. Based on similarities in their morphology, location, and scavenger behavior, these cells appear to be the zebrafish equivalent of cells variably characterized as Fluorescent Granular Perithelial cells (FGPs), perivascular macrophages, or 'Mato Cells' in mammals. Despite their macrophage-like morphology and perivascular location, zebrafish FGPs appear molecularly most similar to lymphatic endothelium, and our imaging studies suggest that these cells emerge by differentiation from endothelium of the optic choroidal vascular plexus. Our findings provide the first report of a perivascular cell population in the brain derived from vascular endothelium.

  12. A novel perivascular cell population in the zebrafish brain

    PubMed Central

    Galanternik, Marina Venero; Castranova, Daniel; Gore, Aniket V; Blewett, Nathan H; Jung, Hyun Min; Stratman, Amber N; Kirby, Martha R; Iben, James; Miller, Mayumi F; Kawakami, Koichi; Maraia, Richard J; Weinstein, Brant M

    2017-01-01

    The blood-brain barrier is essential for the proper homeostasis and function of the CNS, but its mechanism of function is poorly understood. Perivascular cells surrounding brain blood vessels are thought to be important for blood-brain barrier establishment, but their roles are not well defined. Here, we describe a novel perivascular cell population closely associated with blood vessels on the zebrafish brain. Based on similarities in their morphology, location, and scavenger behavior, these cells appear to be the zebrafish equivalent of cells variably characterized as Fluorescent Granular Perithelial cells (FGPs), perivascular macrophages, or ‘Mato Cells’ in mammals. Despite their macrophage-like morphology and perivascular location, zebrafish FGPs appear molecularly most similar to lymphatic endothelium, and our imaging studies suggest that these cells emerge by differentiation from endothelium of the optic choroidal vascular plexus. Our findings provide the first report of a perivascular cell population in the brain derived from vascular endothelium. DOI: http://dx.doi.org/10.7554/eLife.24369.001 PMID:28395729

  13. Identification of Estrogen Target Genes during Zebrafish Embryonic Development through Transcriptomic Analysis

    EPA Science Inventory

    Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio) as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 μM 17β-estradiol (E2) or vehicle from 3 hours to 4 days post...

  14. Dissection and lateral mounting of zebrafish embryos: analysis of spinal cord development.

    PubMed

    Beck, Aaron P; Watt, Roland M; Bonner, Jennifer

    2014-02-28

    The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

  15. Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

    PubMed Central

    Beck, Aaron P.; Watt, Roland M.; Bonner, Jennifer

    2014-01-01

    The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue. PMID:24637734

  16. Relationships among msx gene structure and function in zebrafish and other vertebrates.

    PubMed

    Ekker, M; Akimenko, M A; Allende, M L; Smith, R; Drouin, G; Langille, R M; Weinberg, E S; Westerfield, M

    1997-10-01

    The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.

  17. Osteoblast Production by Reserved Progenitor Cells in Zebrafish Bone Regeneration and Maintenance.

    PubMed

    Ando, Kazunori; Shibata, Eri; Hans, Stefan; Brand, Michael; Kawakami, Atsushi

    2017-12-04

    Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Interordinal chimera formation between medaka and zebrafish for analyzing stem cell differentiation.

    PubMed

    Hong, Ni; Chen, Songlin; Ge, Ruowen; Song, Jianxing; Yi, Meisheng; Hong, Yunhan

    2012-08-10

    Chimera formation is a standard test for pluripotency of stem cells in vivo. Interspecific chimera formation between distantly related organisms offers also an attractive approach for propagating endangered species. Parameters influencing interspecies chimera formation have remained poorly elucidated. Here, we report interordinal chimera formation between medaka and zebrafish, which separated ∼320 million years ago and exhibit a more than 2-fold difference in developmental speed. We show that, on transplantation into zebrafish blastulae, both noncultivated blastomeres and long-term cultivated embryonic stem (ES) cells of medaka adopted the zebrafish developmental program and differentiated into physiologically functional cell types including pigment cells, blood cells, and cardiomyocytes. We also show that medaka ES cells express differentiation gene markers during chimeric embryogenesis. Therefore, the evolutionary distance and different embryogenesis speeds do not produce donor-host incompatibility to compromise chimera formation between medaka and zebrafish, and molecular markers are valuable for analyzing lineage commitment and cell differentiation in interspecific chimeric embryos.

  19. Interordinal Chimera Formation Between Medaka and Zebrafish for Analyzing Stem Cell Differentiation

    PubMed Central

    Hong, Ni; Chen, Songlin; Ge, Ruowen; Song, Jianxing

    2012-01-01

    Chimera formation is a standard test for pluripotency of stem cells in vivo. Interspecific chimera formation between distantly related organisms offers also an attractive approach for propagating endangered species. Parameters influencing interspecies chimera formation have remained poorly elucidated. Here, we report interordinal chimera formation between medaka and zebrafish, which separated ∼320 million years ago and exhibit a more than 2-fold difference in developmental speed. We show that, on transplantation into zebrafish blastulae, both noncultivated blastomeres and long-term cultivated embryonic stem (ES) cells of medaka adopted the zebrafish developmental program and differentiated into physiologically functional cell types including pigment cells, blood cells, and cardiomyocytes. We also show that medaka ES cells express differentiation gene markers during chimeric embryogenesis. Therefore, the evolutionary distance and different embryogenesis speeds do not produce donor-host incompatibility to compromise chimera formation between medaka and zebrafish, and molecular markers are valuable for analyzing lineage commitment and cell differentiation in interspecific chimeric embryos. PMID:22204449

  20. Zebrafish in the sea of mineral (iron, zinc, and copper) metabolism

    PubMed Central

    Zhao, Lu; Xia, Zhidan; Wang, Fudi

    2014-01-01

    Iron, copper, zinc, and eight other minerals are classified as essential trace elements because they present in minute in vivo quantities and are essential for life. Because either excess or insufficient levels of trace elements can be detrimental to life (causing human diseases such as iron-deficiency anemia, hemochromatosis, Menkes syndrome and Wilson's disease), the endogenous levels of trace minerals must be tightly regulated. Many studies have demonstrated the existence of systems that maintain trace element homeostasis, and these systems are highly conserved in multiple species ranging from yeast to mice. As a model for studying trace mineral metabolism, the zebrafish is indispensable to researchers. Several large-scale mutagenesis screens have been performed in zebrafish, and these screens led to the identification of a series of metal transporters and the generation of several mutagenesis lines, providing an in-depth functional analysis at the system level. Moreover, because of their developmental advantages, zebrafish have also been used in mineral metabolism-related chemical screens and toxicology studies. Here, we systematically review the major findings of trace element homeostasis studies using the zebrafish model, with a focus on iron, zinc, copper, selenium, manganese, and iodine. We also provide a homology analysis of trace mineral transporters in fish, mice and humans. Finally, we discuss the evidence that zebrafish is an ideal experimental tool for uncovering novel mechanisms of trace mineral metabolism and for improving approaches to treat mineral imbalance-related diseases. PMID:24639652

  1. Effect of Tbx1 knock-down on cardiac performance in zebrafish.

    PubMed

    Zhang, Li-feng; Gui, Yong-hao; Wang, Yue-xiang; Jiang, Qiu; Song, Hou-yan

    2010-05-05

    Tbx1 is the major candidate gene for DiGeorge syndrome (DGS). Similar to defects observed in DGS patients, the structures disrupted in Tbx1(-/-) animal models are derived from the neural crest cells during development. Although the morphological phenotypes of some Tbx1 knock-down animal models have been well described, analysis of the cardiac performance is limited. Therefore, myocardial performance was explored in Tbx1 morpholino injected zebrafish embryos. To elucidate these issues, Tbx1 specific morpholino was used to reduce the function of Tbx1 in zebrafish. The differentiation of the myocardial cells was observed using whole mount in situ hybridization. Heart rates were observed and recorded under the microscope from 24 to 72 hours post fertilization (hpf). The cardiac performance was analyzed by measuring ventricular shortening fraction and atrial shortening fraction. Tbx1 morpholino injected embryos were characterized by defects in the pharyngeal arches, otic vesicle, aortic arches and thymus. In addition, Tbx1 knock down reduced the amount of pharyngeal neural crest cells in zebrafish. Abnormal cardiac morphology was visible in nearly 20% of the Tbx1 morpholino injected embryos. The hearts in these embryos did not loop or loop incompletely. Importantly, cardiac performance and heart rate were reduced in Tbx1 morpholino injected embryos. Tbx1 might play an essential role in the development of pharyngeal neural crest cells in zebrafish. Cardiac performance is impaired by Tbx1 knock down in zebrafish.

  2. Zebrafish Pronephros Development.

    PubMed

    Naylor, Richard W; Qubisi, Sarah S; Davidson, Alan J

    The pronephros is the first kidney type to form in vertebrate embryos. The first step of pronephrogenesis in the zebrafish is the formation of the intermediate mesoderm during gastrulation, which occurs in response to secreted morphogens such as BMPs and Nodals. Patterning of the intermediate mesoderm into proximal and distal cell fates is induced by retinoic acid signaling with downstream transcription factors including wt1a, pax2a, pax8, hnf1b, sim1a, mecom, and irx3b. In the anterior intermediate mesoderm, progenitors of the glomerular blood filter migrate and fuse at the midline and recruit a blood supply. More posteriorly localized tubule progenitors undergo epithelialization and fuse with the cloaca. The Notch signaling pathway regulates the formation of multi-ciliated cells in the tubules and these cells help propel the filtrate to the cloaca. The lumenal sheer stress caused by flow down the tubule activates anterior collective migration of the proximal tubules and induces stretching and proliferation of the more distal segments. Ultimately these processes create a simple two-nephron kidney that is capable of reabsorbing and secreting solutes and expelling excess water-processes that are critical to the homeostasis of the body fluids. The zebrafish pronephric kidney provides a simple, yet powerful, model system to better understand the conserved molecular and cellular progresses that drive nephron formation, structure, and function.

  3. Heterozygous inactivation of tsc2 enhances tumorigenesis in p53 mutant zebrafish

    PubMed Central

    Kim, Seok-Hyung; Kowalski, Marie L.; Carson, Robert P.; Bridges, L. Richard; Ess, Kevin C.

    2013-01-01

    SUMMARY Tuberous sclerosis complex (TSC) is a multi-organ disorder caused by mutations of the TSC1 or TSC2 genes. A key function of these genes is to inhibit mTORC1 (mechanistic target of rapamycin complex 1) kinase signaling. Cells deficient for TSC1 or TSC2 have increased mTORC1 signaling and give rise to benign tumors, although, as a rule, true malignancies are rarely seen. In contrast, other disorders with increased mTOR signaling typically have overt malignancies. A better understanding of genetic mechanisms that govern the transformation of benign cells to malignant ones is crucial to understand cancer pathogenesis. We generated a zebrafish model of TSC and cancer progression by placing a heterozygous mutation of the tsc2 gene in a p53 mutant background. Unlike tsc2 heterozygous mutant zebrafish, which never exhibited cancers, compound tsc2;p53 mutants had malignant tumors in multiple organs. Tumorigenesis was enhanced compared with p53 mutant zebrafish. p53 mutants also had increased mTORC1 signaling that was further enhanced in tsc2;p53 compound mutants. We found increased expression of Hif1-α, Hif2-α and Vegf-c in tsc2;p53 compound mutant zebrafish compared with p53 mutant zebrafish. Expression of these proteins probably underlies the increased angiogenesis seen in compound mutant zebrafish compared with p53 mutants and might further drive cancer progression. Treatment of p53 and compound mutant zebrafish with the mTORC1 inhibitor rapamycin caused rapid shrinkage of tumor size and decreased caliber of tumor-associated blood vessels. This is the first report using an animal model to show interactions between tsc2, mTORC1 and p53 during tumorigenesis. These results might explain why individuals with TSC rarely have malignant tumors, but also suggest that cancer arising in individuals without TSC might be influenced by the status of TSC1 and/or TSC2 mutations and be potentially treatable with mTORC1 inhibitors. PMID:23580196

  4. Biological response of zebrafish after short-term exposure to azoxystrobin.

    PubMed

    Jiang, Jinhua; Shi, Yan; Yu, Ruixian; Chen, Liping; Zhao, Xueping

    2018-07-01

    Azoxystrobin (AZ) is a broad-spectrum systemic fungicide that widely used in the world. The present study investigated the toxicity effects on zebrafish after short-term exposure of AZ. Results demonstrated that the larval stage was most susceptible to AZ in the multiple life stages of zebrafish, with 96 h-LC 50 value of 0.777 mg/L. Zebrafish larvae were exposed to different AZ concentrations (0, 0.1, 1, 10, 100 μg/L) and examined on 24, 48 and 72 h. It was found that AZ induced ROS accumulation, increased GST, GPX and POD activity and the transcriptions of antioxidant and stress response related genes, while the opposite trend occurred for SOD and CAT activity in 24-h or 48-h exposure period. The increased E 2 and VTG levels in zebrafish larvae, and altered transcription levels of regulatory and steroidogenic genes in the hypothalamus-pituitary-gonad (HPG) axis indicated the endocrine disruption capacity of AZ. The transcripts of mdm2, p53, ogg1, bcl2, bbc3, cas8 and cas9 involved in cell apoptosis, and the mRNA levels of cytokines and chemokines such as cxcl-c1c, ccl, il-1β, il-8, ifn, and tnfα were in accordance with the trends of the examined genes involved in oxidative stress and endocrine system. The results suggested that short-term exposure to AZ might impose ecotoxicological effects on zebrafish larvae, and the information presented here also provide molecular strategies and increase mechanistic understanding of AZ-induced toxic response, and help elucidate the environmental risks of AZ. Copyright © 2018. Published by Elsevier Ltd.

  5. Assessing the impact of thermal acclimation on physiological condition in the zebrafish model.

    PubMed

    Vergauwen, Lucia; Knapen, Dries; Hagenaars, An; De Boeck, Gudrun; Blust, Ronny

    2013-01-01

    The zebrafish has become a valuable vertebrate model organism in a wide range of scientific disciplines, but current information concerning the physiological temperature response of adult zebrafish is rather scarce. In this study, zebrafish were experimentally acclimated for 28 days to 18, 26 or 34 °C and a suite of non-invasive and invasive methods was applied to determine the thermal dependence of zebrafish physiological condition. With decreasing temperature, the metabolic rate of zebrafish decreased, as shown by the decreasing oxygen uptake and ammonia excretion rates, limiting the critical swimming speed, probably due to a decreased muscle fibre power output. In response to exercise, fuel stores were mobilized to the liver as shown by the increased hepatosomatic index, liver total absolute energetic value and liver carbohydrate concentration but due to the low metabolic rate they could not be adequately addressed to power swimming activity at 18 °C. Conversely, the increased metabolic performance at high temperature came with an increased metabolic cost resulting in decreased energy status reflected particularly well by the non-invasive condition factor and invasive measures of carcass protein concentration, carcass total absolute energetic value and liver carbohydrate concentration. We showed that the combined measurement of the relative condition factor and critical swimming speed is a powerful non-invasive tool for long-term follow-up studies. Invasive methods were redundant for measuring general energy status but they provided detailed information concerning metabolic reorganization. With this study we proved that the usefulness of the zebrafish as a model organism can easily be expanded to include physiological studies and we provided a reference dataset for the selection of measures of physiological responses for future studies using the zebrafish.

  6. Beta-glucan enhances the response to SVCV infection in zebrafish.

    PubMed

    M Medina-Gali, Regla; Ortega-Villaizan, María Del Mar; Mercado, Luis; Novoa, Beatriz; Coll, Julio; Perez, Luis

    2018-07-01

    The antiviral effects of beta-glucan, an immunostimulatory agent were studied in zebrafish both in vitro and in vivo. Here we show that zebrafish ZF4 cells as well as whole fish primed with yeast β-glucan zymosan exhibited increased cytokine expression and elevated response to spring viremia of carp virus (SVCV) infection. In vitro, previous treatment of β-glucan enhanced ZF4 cell viability against SVCV infection which is associated to the activation of interferon signaling pathway and inflammatory cytokines gene expression. In vivo, the SVCV-infected fish primed with β-glucan had a higher survival rate (≈73%) than the control SVCV-infected group (≈33%). Additionally, up-regulation of the expression of a set of genes involved in innate immune response was detected in zebrafish intraperitoneally injected of β-glucan: il1b, il6, il8, il10 and tnfa transcripts showed increased expression that appear to be rapid (2 days) but not long-lived (less than 2 weeks). The present study is, to our knowledge, the first to combine cell culture and in vivo approaches to describe host response to β-glucan stimulation and viral infection in zebrafish. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. In vivo cell biology in zebrafish - providing insights into vertebrate development and disease.

    PubMed

    Vacaru, Ana M; Unlu, Gokhan; Spitzner, Marie; Mione, Marina; Knapik, Ela W; Sadler, Kirsten C

    2014-02-01

    Over the past decades, studies using zebrafish have significantly advanced our understanding of the cellular basis for development and human diseases. Zebrafish have rapidly developing transparent embryos that allow comprehensive imaging of embryogenesis combined with powerful genetic approaches. However, forward genetic screens in zebrafish have generated unanticipated findings that are mirrored by human genetic studies: disruption of genes implicated in basic cellular processes, such as protein secretion or cytoskeletal dynamics, causes discrete developmental or disease phenotypes. This is surprising because many processes that were assumed to be fundamental to the function and survival of all cell types appear instead to be regulated by cell-specific mechanisms. Such discoveries are facilitated by experiments in whole animals, where zebrafish provides an ideal model for visualization and manipulation of organelles and cellular processes in a live vertebrate. Here, we review well-characterized mutants and newly developed tools that underscore this notion. We focus on the secretory pathway and microtubule-based trafficking as illustrative examples of how studying cell biology in vivo using zebrafish has broadened our understanding of the role fundamental cellular processes play in embryogenesis and disease.

  8. [Application of zebrafish model organism in the research of Chinese materia medica].

    PubMed

    Chen, Lei; Liu, Yi; Liang, Sheng-Wang

    2012-04-01

    Zebrafish has become an important model organism in many fields of biomedical studies and been increasingly used in Chinese materia medica studies in recent years. This article summarized the achievements and prospect for zebrafish as a pharmacological and toxicological tool in the study and development of Chinese materia medica.

  9. Insights from zebrafish on human pigment cell disease and treatment.

    PubMed

    Cooper, Cynthia D

    2017-11-01

    Black pigment cells, melanocytes, arise early during development from multipotent neural crest cells. Melanocytes protect human skin from DNA damaging sunrays and provide color for hair, eyes, and skin. Several disorders and diseases originate from these cells, including the deadliest skin cell cancer, melanoma. Thus, melanocytes are critical for a healthy life and for protecting humans from disease. Due to the ease of visualizing pigment cells through transparent larvae skin and conserved roles for zebrafish melanophore genes to mammalian melanocyte genes, zebrafish larvae offer a biologically relevant model for understanding pigment cell development and disease in humans. This review discusses our current knowledge of melanophore biology and how zebrafish are contributing to improving how diseases of melanocytes are understood and treated in humans. Developmental Dynamics 246:889-896, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  10. Toxicity of multi-walled carbon nanotubes, graphene oxide, and reduced graphene oxide to zebrafish embryos.

    PubMed

    Liu, Xiao Tong; Mu, Xi Yan; Wu, Xiao Li; Meng, Li Xuan; Guan, Wen Bi; Ma, Yong Qiang; Sun, Hua; Wang, Cheng Ju; Li, Xue Feng

    2014-09-01

    This study was aimed to investigate the toxic effects of 3 nanomaterials, i.e. multi-walled carbon nanotubes (MWCNTs), graphene oxide (GO), and reduced graphene oxide (RGO), on zebrafish embryos. The 2-h post-fertilization (hpf) zebrafish embryos were exposed to MWCNTs, GO, and RGO at different concentrations (1, 5, 10, 50, 100 mg/L) for 96 h. Afterwards, the effects of the 3 nanomateria on spontaneous movement, heart rate, hatching rate, length of larvae, mortality, and malformations ls were evaluated. Statistical analysis indicated that RGO significantly inhibited the hatching of zebrafish embryos. Furthermore, RGO and MWCNTs decreased the length of the hatched larvae at 96 hpf. No obvious morphological malformation or mortality was observed in the zebrafish embryos after exposure to the three nanomaterials. MWCNTs, GO, and RGO were all toxic to zebrafish embryos to influence embryos hatching and larvae length. Although no obvious morphological malformation and mortality were observed in exposed zebrafish embryos, further studies on the toxicity of the three nanomaterials are still needed. Copyright © 2014 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  11. Identification of Chemical Inhibitors of β-Catenin-Driven Liver Tumorigenesis in Zebrafish

    PubMed Central

    Evason, Kimberley J.; Francisco, Macrina T.; Juric, Vladislava; Balakrishnan, Sanjeev; Lopez Pazmino, Maria del Pilar; Gordan, John D.; Kakar, Sanjay; Spitsbergen, Jan; Goga, Andrei; Stainier, Didier Y. R.

    2015-01-01

    Hepatocellular carcinoma (HCC) is one of the most lethal human cancers. The search for targeted treatments has been hampered by the lack of relevant animal models for the genetically diverse subsets of HCC, including the 20-40% of HCCs that are defined by activating mutations in the gene encoding β-catenin. To address this chemotherapeutic challenge, we created and characterized transgenic zebrafish expressing hepatocyte-specific activated β-catenin. By 2 months post fertilization (mpf), 33% of transgenic zebrafish developed HCC in their livers, and 78% and 80% of transgenic zebrafish showed HCC at 6 and 12 mpf, respectively. As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings. Using this novel transgenic model, we screened for druggable pathways that mediate β-catenin-induced liver growth and identified two c-Jun N-terminal kinase (JNK) inhibitors and two antidepressants (one tricyclic antidepressant, amitriptyline, and one selective serotonin reuptake inhibitor) that suppressed this phenotype. We further found that activated β-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC. In zebrafish larvae, JNK inhibition decreased liver size specifically in the presence of activated β-catenin. The β-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells. Our other class of hits, antidepressants, has been used in patient treatment for decades, raising the exciting possibility that these drugs could potentially be repurposed for cancer treatment. In support of this proposal, we found that amitriptyline decreased tumor burden in a mouse HCC model. Our studies implicate JNK inhibitors and antidepressants as potential therapeutics for β-catenin-induced liver tumors. PMID:26134322

  12. Miniaturized Embryo Array for Automated Trapping, Immobilization and Microperfusion of Zebrafish Embryos

    PubMed Central

    Akagi, Jin; Khoshmanesh, Khashayar; Evans, Barbara; Hall, Chris J.; Crosier, Kathryn E.; Cooper, Jonathan M.; Crosier, Philip S.; Wlodkowic, Donald

    2012-01-01

    Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale. PMID:22606275

  13. A plasmid library of full-length zebrafish rab proteins for in vivo cell biology.

    PubMed

    Hall, Thomas E; Martel, Nick; Lo, Harriet P; Xiong, Zherui; Parton, Robert G

    2017-01-01

    The zebrafish is an emerging model for highly sophisticated medium-throughput experiments such as genetic and chemical screens. However, studies of entire protein families within this context are often hampered by poor genetic resources such as clone libraries. Here we describe a complete collection of 76 full-length open reading frame clones for the zebrafish rab protein family. While the mouse genome contains 60 rab genes and the human genome 63, we find that 18 zebrafish rab genes have 2, and in the case of rab38, 3 paralogues. In contrast, we were unable to identify zebrafish orthologues of the mammalian Rab2b, Rab17 or Rab29. We make this resource available through the Addgene repository to facilitate cell biologic approaches using this model.

  14. The French press: a repeatable and high-throughput approach to exercising zebrafish (Danio rerio)

    PubMed Central

    Usui, Takuji; Noble, Daniel W.A.; O’Dea, Rose E.; Fangmeier, Melissa L.; Lagisz, Malgorzata; Hesselson, Daniel

    2018-01-01

    Zebrafish are increasingly used as a vertebrate model organism for various traits including swimming performance, obesity and metabolism, necessitating high-throughput protocols to generate standardized phenotypic information. Here, we propose a novel and cost-effective method for exercising zebrafish, using a coffee plunger and magnetic stirrer. To demonstrate the use of this method, we conducted a pilot experiment to show that this simple system provides repeatable estimates of maximal swim performance (intra-class correlation [ICC] = 0.34–0.41) and observe that exercise training of zebrafish on this system significantly increases their maximum swimming speed. We propose this high-throughput and reproducible system as an alternative to traditional linear chamber systems for exercising zebrafish and similarly sized fishes. PMID:29372124

  15. A critical period for functional vestibular development in zebrafish

    NASA Technical Reports Server (NTRS)

    Moorman, Stephen J.; Cordova, Rodolfo; Davies, Sarah A.

    2002-01-01

    We have determined a critical period for vestibular development in zebrafish by using a bioreactor designed by NASA to simulate microgravity for cells in culture. A critical period is defined as the briefest period of time during development when stimulus deprivation results in long lasting or permanent sensory deficits. Zebrafish eggs were collected within 3 hours of being laid and fertilized. In experiment 1, eggs were placed in the bioreactor at 3, 24, 30, 36, 48, or 72 hours postfertilization (hPF) and maintained in the bioreactor until 96 hPF. In experiment 2, eggs were placed in the bioreactor immediately after they were collected and maintained in the bioreactor until 24, 36, 48, 60, 66, 72, or 96 hPF. Beginning at 96 hPF, all larvae had their vestibulo-ocular reflexes (VOR) evaluated once each day for 5 days. Only larvae that hatched from eggs that were placed in the bioreactor before 30 hPF in experiment 1 or removed from the bioreactor later than 66 hPF in experiment 2 had VOR deficits that persisted for at least 5 days. These data suggest a critical period for vestibular development in the zebrafish that begins before 30 hPF and ends after 66 hPF. To confirm this, zebrafish eggs were placed in the bioreactor at 24 hPF and removed at 72 hPF. VORs were evaluated in these larvae once each day for 5 days beginning at 96 hPF. These larvae had VOR deficits that persisted for at least 5 days. In addition, larvae that had been maintained in the bioreactor from 24 to 66 hPF or from 30 to 72 hPF, had only temporary VOR deficits. In a final experiment, zebrafish eggs were placed in the bioreactor at 3 hPF and removed at 96 hPF but the bioreactor was turned off from 24 hPF to 72 hPF. These larvae had normal VORs when they were removed from the bioreactor at 96 hPF. Taken as a whole, these data support the idea that there is a critical period for functional maturation of the zebrafish vestibular system. The developmental period identified includes the timeframe

  16. Characterization of glutathione-S-transferases in zebrafish (Danio rerio).

    PubMed

    Glisic, Branka; Mihaljevic, Ivan; Popovic, Marta; Zaja, Roko; Loncar, Jovica; Fent, Karl; Kovacevic, Radmila; Smital, Tvrtko

    2015-01-01

    Glutathione-S-transferases (GSTs) are one of the key enzymes that mediate phase II of cellular detoxification. The aim of our study was a comprehensive characterization of GSTs in zebrafish (Danio rerio) as an important vertebrate model species frequently used in environmental research. A detailed phylogenetic analysis of GST superfamily revealed 27 zebrafish gst genes. Further insights into the orthology relationships between human and zebrafish GSTs/Gsts were obtained by the conserved synteny analysis. Expression of gst genes in six tissues (liver, kidney, gills, intestine, brain and gonads) of adult male and female zebrafish was determined using qRT-PCR. Functional characterization was performed on 9 cytosolic Gst enzymes after overexpression in E. coli and subsequent protein purification. Enzyme kinetics was measured for GSH and a series of model substrates. Our data revealed ubiquitously high expression of gstp, gstm (except in liver), gstr1, mgst3a and mgst3b, high expression of gsto2 in gills and ovaries, gsta in intestine and testes, gstt1a in liver, and gstz1 in liver, kidney and brain. All zebrafish Gsts catalyzed the conjugation of GSH to model GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and monochlorobimane (MCB), apart from Gsto2 and Gstz1 that catalyzed GSH conjugation to dehydroascorbate (DHA) and dichloroacetic acid (DCA), respectively. Affinity toward CDNB varied from 0.28 mM (Gstp2) to 3.69 mM (Gstm3), while affinity toward MCB was in the range of 5 μM (Gstt1a) to 250 μM (Gstp1). Affinity toward GSH varied from 0.27 mM (Gstz1) to 4.45 mM (Gstt1a). Turnover number for CDNB varied from 5.25s(-1) (Gstt1a) to 112s(-1) (Gstp2). Only Gst Pi enzymes utilized ethacrynic acid (ETA). We suggest that Gstp1, Gstp2, Gstt1a, Gstz1, Gstr1, Mgst3a and Mgst3b have important role in the biotransformation of xenobiotics, while Gst Alpha, Mu, Pi, Zeta and Rho classes are involved in the crucial physiological processes. In summary, this study provides the

  17. A MULTIFACETED, MEDIUM-THROUGHPUT APPROACH FOR DETECTING AND CHARACTERIZING DEVELOPMENTAL NEUROTOXICITY USING ZEBRAFISH.

    EPA Science Inventory

    To address the EPA's need to prioritize hundreds to thousands of chemicals for testing, we are developing a rapid, cost-effective in vivo screen for developmental neurotoxicity using zebrafish (Danio rerio), a small freshwater fish with external fertilization. Zebrafish embryos d...

  18. Stimulus-triggered enhancement of chilling tolerance in zebrafish embryos

    PubMed Central

    Szabó, Katalin; Budai, Csilla; Losonczi, Eszter; Bernáth, Gergely; Csenki-Bakos, Zsolt; Urbányi, Béla; Pribenszky, Csaba; Horváth, Ákos; Cserepes, Judit

    2017-01-01

    Background Cryopreservation of zebrafish embryos is still an unsolved problem despite market demand and massive efforts to preserve genetic variation among numerous existing lines. Chilled storage of embryos might be a step towards developing successful cryopreservation, but no methods to date have worked. Methods In the present study, we applied a novel strategy to improve the chilling tolerance of zebrafish embryos by introducing a preconditioning hydrostatic pressure treatment to the embryos. In our experiments, 26-somites and Prim-5 stage zebrafish embryos were chilled at 0°C for 24 hours after preconditioning. Embryo survival rate, ability to reach maturation and fertilizing capacity were tested. Results Our results indicate that applied preconditioning technology made it possible for the chilled embryos to develop normally until maturity, and to produce healthy offspring as normal, thus passing on their genetic material successfully. Treated embryos had a significantly higher survival and better developmental rate, moreover the treated group had a higher ratio of normal morphology during continued development. While all controls from chilled embryos died by 30 day-post-fertilization, the treated group reached maturity (~90–120 days) and were able to reproduce, resulting in offspring in expected quantity and quality. Conclusions Based on our results, we conclude that the preconditioning technology represents a significant improvement in zebrafish embryo chilling tolerance, thus enabling a long-time survival. Furthermore, as embryonic development is arrested during chilled storage this technology also provides a solution to synchronize or delay the development. PMID:28166301

  19. Behavioral Changes Over Time Following Ayahuasca Exposure in Zebrafish

    PubMed Central

    Savoldi, Robson; Polari, Daniel; Pinheiro-da-Silva, Jaquelinne; Silva, Priscila F.; Lobao-Soares, Bruno; Yonamine, Mauricio; Freire, Fulvio A. M.; Luchiari, Ana C.

    2017-01-01

    The combined infusion of Banisteriopsis caapi stem and Psychotria viridis leaves, known as ayahuasca, has been used for centuries by indigenous tribes. The infusion is rich in N, N-dimethyltryptamine (DMT) and monoamine oxidase inhibitors, with properties similar to those of serotonin. Despite substantial progress in the development of new drugs to treat anxiety and depression, current treatments have several limitations. Alternative drugs, such as ayahuasca, may shed light on these disorders. Here, we present time-course behavioral changes induced by ayahuasca in zebrafish, as first step toward establishing an ideal concentration for pre-clinical evaluations. We exposed adult zebrafish to five concentrations of the ayahuasca infusion: 0 (control), 0.1, 0.5, 1, and 3 ml/L (n = 14 each group), and behavior was recorded for 60 min. We evaluated swimming speed, distance traveled, freezing and bottom dwelling every min for 60 min. Swimming speed and distance traveled decreased with an increase in ayahuasca concentration while freezing increased with 1 and 3 ml/L. Bottom dwelling increased with 1 and 3 ml/L, but declined with 0.1 ml/L. Our data suggest that small amounts of ayahuasca do not affect locomotion and reduce anxiety-like behavior in zebrafish, while increased doses of the drug lead to crescent anxiogenic effects. We conclude that the temporal analysis of zebrafish behavior is a sensitive method for the study of ayahuasca-induced functional changes in the vertebrate brain. PMID:28804451

  20. Behavioral Changes Over Time Following Ayahuasca Exposure in Zebrafish.

    PubMed

    Savoldi, Robson; Polari, Daniel; Pinheiro-da-Silva, Jaquelinne; Silva, Priscila F; Lobao-Soares, Bruno; Yonamine, Mauricio; Freire, Fulvio A M; Luchiari, Ana C

    2017-01-01

    The combined infusion of Banisteriopsis caapi stem and Psychotria viridis leaves, known as ayahuasca, has been used for centuries by indigenous tribes. The infusion is rich in N , N -dimethyltryptamine (DMT) and monoamine oxidase inhibitors, with properties similar to those of serotonin. Despite substantial progress in the development of new drugs to treat anxiety and depression, current treatments have several limitations. Alternative drugs, such as ayahuasca, may shed light on these disorders. Here, we present time-course behavioral changes induced by ayahuasca in zebrafish, as first step toward establishing an ideal concentration for pre-clinical evaluations. We exposed adult zebrafish to five concentrations of the ayahuasca infusion: 0 (control), 0.1, 0.5, 1, and 3 ml/L ( n = 14 each group), and behavior was recorded for 60 min. We evaluated swimming speed, distance traveled, freezing and bottom dwelling every min for 60 min. Swimming speed and distance traveled decreased with an increase in ayahuasca concentration while freezing increased with 1 and 3 ml/L. Bottom dwelling increased with 1 and 3 ml/L, but declined with 0.1 ml/L. Our data suggest that small amounts of ayahuasca do not affect locomotion and reduce anxiety-like behavior in zebrafish, while increased doses of the drug lead to crescent anxiogenic effects. We conclude that the temporal analysis of zebrafish behavior is a sensitive method for the study of ayahuasca-induced functional changes in the vertebrate brain.

  1. Behavorial assessments of larval zebrafish neurotoxicology

    EPA Science Inventory

    Fishes have long been a popular organism in ecotoxicology research, and are increasingly used in human health research as an alternative animal model for chemical screening. Our laboratory incorporates a zebrafish (Danio rerio) embryo/larval assay to screen chemicals for developm...

  2. Myotonia congenita-associated mutations in chloride channel-1 affect zebrafish body wave swimming kinematics.

    PubMed

    Cheng, Wei; Tian, Jing; Burgunder, Jean-Marc; Hunziker, Walter; Eng, How-Lung

    2014-01-01

    Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1). Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita.

  3. Colonizing the embryonic zebrafish gut with anaerobic bacteria derived from the human gastrointestinal tract.

    PubMed

    Toh, Michael C; Goodyear, Mara; Daigneault, Michelle; Allen-Vercoe, Emma; Van Raay, Terence J

    2013-06-01

    The zebrafish has become increasingly popular for microbiological research. It has been used as an infection model for a variety of pathogens, and is also emerging as a tool for studying interactions between a host and its resident microbial communities. The mouse microbiota has been transplanted into the zebrafish gut, but to our knowledge, there has been no attempt to introduce a bacterial community derived from the human gut. We explored two methods for colonizing the developing gut of 5-day-old germ-free zebrafish larvae with a defined anaerobic microbial community derived from a single human fecal sample. Both environmental exposure (static immersion) and direct microinjection into the gut resulted in the establishment of two species-Lactobacillus paracasei and Eubacterium limosum-from a community of 30 strains consisting of 22 anaerobic species. Of particular interest is E. limosum, which, as a strict anaerobe, represents a group of bacteria which until now have not been shown to colonize the developing zebrafish gut. Our success here indicates that further investigation of zebrafish as a tool for studying human gut microbial communities is warranted.

  4. Acute toxicity and histopathological effects of naproxen in zebrafish (Danio rerio) early life stages.

    PubMed

    Li, Qian; Wang, Peipei; Chen, Ling; Gao, Hongwen; Wu, Lingling

    2016-09-01

    Zebrafish (Danio rerio) embryos and larvae were selected to investigate the potential risk and aquatic toxicity of a widely used pharmaceutical, naproxen. The acute toxicity of naproxen to embryos and larvae was measured, respectively. The histopathology was investigated in the liver of zebrafish larvae after 8-day embryo-larvae exposure to naproxen. The values of 96-h LC50 were 115.2 mg/L for embryos and 147.6 mg/L for larvae, indicating that zebrafish embryos were more sensitive than larvae to naproxen exposure. Large suites of symptoms were induced in zebrafish (D. rerio) early life stages by different dosages of naproxen, including hatching inhibition, lower heart rate, and morphological abnormalities. The most sensitive sub-lethal effect caused by naproxen was pericardial edema, the 72-h EC50 values of which for embryos and larvae were 98.3 and 149.0 mg/L, respectively. In addition, naproxen-treated zebrafish larvae exhibited histopathological liver damage, including swollen hepatocytes, vacuolar degeneration, and nuclei pycnosis. The results indicated that naproxen is a potential threat to aquatic organisms.

  5. Myotonia Congenita-Associated Mutations in Chloride Channel-1 Affect Zebrafish Body Wave Swimming Kinematics

    PubMed Central

    Cheng, Wei; Tian, Jing; Burgunder, Jean-Marc; Hunziker, Walter; Eng, How-Lung

    2014-01-01

    Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1). Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita. PMID:25083883

  6. The State of the Art of the Zebrafish Model for Toxicology and Toxicologic Pathology Research—Advantages and Current Limitations

    PubMed Central

    Spitsbergen, Jan M.; Kent, Michael L.

    2007-01-01

    The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. The zebrafish genome will be completely sequenced within the next 1–2 years. Together with the substantial historical database regarding basic developmental biology, toxicology, and gene transfer, the rich foundation of molecular genetic and genomic data makes zebrafish a powerful model system for clarifying mechanisms in toxicity. In contrast to the highly advanced knowledge base on molecular developmental genetics in zebrafish, our database regarding infectious and noninfectious diseases and pathologic lesions in zebrafish lags far behind the information available on most other domestic mammalian and avian species, particularly rodents. Currently, minimal data are available regarding spontaneous neoplasm rates or spontaneous aging lesions in any of the commonly used wild-type or mutant lines of zebrafish. Therefore, to fully utilize the potential of zebrafish as an animal model for understanding human development, disease, and toxicology we must greatly advance our knowledge on zebrafish diseases and pathology. PMID:12597434

  7. Zebrafish Embryo Toxicity Microscale Model for Ichthyotoxicity Evaluation of Marine Natural Products.

    PubMed

    Bai, Hong; Kong, Wen-Wen; Shao, Chang-Lun; Li, Yun; Liu, Yun-Zhang; Liu, Min; Guan, Fei-Fei; Wang, Chang-Yun

    2016-04-01

    Marine organisms often protect themselves against their predators by chemical defensive strategy. The second metabolites isolated from marine organisms and their symbiotic microbes have been proven to play a vital role in marine chemical ecology, such as ichthyotoxicity, allelopathy, and antifouling. It is well known that the microscale models for marine chemoecology assessment are urgently needed for trace quantity of marine natural products. Zebrafish model has been widely used as a microscale model in the fields of environment ecological evaluation and drug safety evaluation, but seldom reported for marine chemoecology assessment. In this work, zebrafish embryo toxicity microscale model was established for ichthyotoxicity evaluation of marine natural products by using 24-well microplate based on zebrafish embryo. Ichthyotoxicity was evaluated by observation of multiple toxicological endpoints, including coagulation egg, death, abnormal heartbeat, no spontaneous movement, delayed hatch, and malformation of the different organs during zebrafish embryogenesis periods at 24, 48, and 72 h post-fertilization (hpf). 3,4-Dichloroaniline was used as the positive control for method validation. Subsequently, the established model was applied to test the ichthyotoxic activity of the compounds isolated from corals and their symbiotic microbes and to isolate the bioactive secondary metabolites from the gorgonian Subergorgia mollis under bioassay guidance. It was suggested that zebrafish embryo toxicity microscale model is suitable for bioassay-guided isolation and preliminary bioactivity screening of marine natural products.

  8. First report of Fusarium oxysporum species complex infection in zebrafish culturing system.

    PubMed

    Kulatunga, D C M; Dananjaya, S H S; Park, B K; Kim, C-H; Lee, J; De Zoysa, M

    2017-04-01

    Fusarium oxysporum species complex (FOSC) is a highly diverse fungus. Recently, F. oxysporum infection was identified from zebrafish (Danio rerio) culturing system in Korea. Initially, a rapid whitish smudge was appeared in the water with the fungal blooming on walls of fish tanks. Microscopic studies were conducted on fungal hyphae, colony pigmentation and chlamydospore formation and the presence of macro- and microspores confirmed that the isolated fungus as F. oxysporum. Furthermore, isolated F. oxysporum was confirmed by internal transcribed spacer sequencing which matched (100%) to nine F. oxysporum sequences available in GenBank. Experimental hypodermic injection of F. oxysporum into adult zebrafish showed the development of fungal mycelium and pathogenicity similar to signs observed. Histopathologic results revealed a presence of F. oxysporum hyphae in zebrafish muscle. Fusarium oxysporum growth was increased with sea salt in a concentration-dependent manner. Antifungal susceptibility results revealed that F. oxysporum is resistant to copper sulphate (up to 200 μg mL -1 ) and sensitive to nystatin (up to 40 μg mL -1 ). This is the first report of FOSC from zebrafish culture system, suggesting it appears as an emerging pathogen, thus posing a significant risk on zebrafish facilities in the world. © 2016 John Wiley & Sons Ltd.

  9. Telomeres in aging and disease: lessons from zebrafish.

    PubMed

    Carneiro, Madalena C; de Castro, Inês Pimenta; Ferreira, Miguel Godinho

    2016-07-01

    Age is the highest risk factor for some of the most prevalent human diseases, including cancer. Telomere shortening is thought to play a central role in the aging process in humans. The link between telomeres and aging is highlighted by the fact that genetic diseases causing telomerase deficiency are associated with premature aging and increased risk of cancer. For the last two decades, this link has been mostly investigated using mice that have long telomeres. However, zebrafish has recently emerged as a powerful and complementary model system to study telomere biology. Zebrafish possess human-like short telomeres that progressively decline with age, reaching lengths in old age that are observed when telomerase is mutated. The extensive characterization of its well-conserved molecular and cellular physiology makes this vertebrate an excellent model to unravel the underlying relationship between telomere shortening, tissue regeneration, aging and disease. In this Review, we explore the advantages of using zebrafish in telomere research and discuss the primary discoveries made in this model that have contributed to expanding our knowledge of how telomere attrition contributes to cellular senescence, organ dysfunction and disease. © 2016. Published by The Company of Biologists Ltd.

  10. Analysis of a Gene Regulatory Cascade Mediating Circadian Rhythm in Zebrafish

    PubMed Central

    Wang, Haifang; Du, Jiulin; Yan, Jun

    2013-01-01

    In the study of circadian rhythms, it has been a puzzle how a limited number of circadian clock genes can control diverse aspects of physiology. Here we investigate circadian gene expression genome-wide using larval zebrafish as a model system. We made use of a spatial gene expression atlas to investigate the expression of circadian genes in various tissues and cell types. Comparison of genome-wide circadian gene expression data between zebrafish and mouse revealed a nearly anti-phase relationship and allowed us to detect novel evolutionarily conserved circadian genes in vertebrates. We identified three groups of zebrafish genes with distinct responses to light entrainment: fast light-induced genes, slow light-induced genes, and dark-induced genes. Our computational analysis of the circadian gene regulatory network revealed several transcription factors (TFs) involved in diverse aspects of circadian physiology through transcriptional cascade. Of these, microphthalmia-associated transcription factor a (mitfa), a dark-induced TF, mediates a circadian rhythm of melanin synthesis, which may be involved in zebrafish's adaptation to daily light cycling. Our study describes a systematic method to discover previously unidentified TFs involved in circadian physiology in complex organisms. PMID:23468616

  11. Humanizing the zebrafish liver shifts drug metabolic profiles and improves pharmacokinetics of CYP3A4 substrates.

    PubMed

    Poon, Kar Lai; Wang, Xingang; Ng, Ashley S; Goh, Wei Huang; McGinnis, Claudia; Fowler, Stephen; Carney, Tom J; Wang, Haishan; Ingham, Phillip W

    2017-03-01

    Understanding and predicting whether new drug candidates will be safe in the clinic is a critical hurdle in pharmaceutical development, that relies in part on absorption, distribution, metabolism, excretion and toxicology studies in vivo. Zebrafish is a relatively new model system for drug metabolism and toxicity studies, offering whole organism screening coupled with small size and potential for high-throughput screening. Through toxicity and absorption analyses of a number of drugs, we find that zebrafish is generally predictive of drug toxicity, although assay outcomes are influenced by drug lipophilicity which alters drug uptake. In addition, liver microsome assays reveal specific differences in metabolism of compounds between human and zebrafish livers, likely resulting from the divergence of the cytochrome P450 superfamily between species. To reflect human metabolism more accurately, we generated a transgenic "humanized" zebrafish line that expresses the major human phase I detoxifying enzyme, CYP3A4, in the liver. Here, we show that this humanized line shows an elevated metabolism of CYP3A4-specific substrates compared to wild-type zebrafish. The generation of this first described humanized zebrafish liver suggests such approaches can enhance the accuracy of the zebrafish model for toxicity prediction.

  12. Auditory sensitivity of larval zebrafish (Danio rerio) measured using a behavioral prepulse inhibition assay

    PubMed Central

    Bhandiwad, Ashwin A.; Zeddies, David G.; Raible, David W.; Rubel, Edwin W.; Sisneros, Joseph A.

    2013-01-01

    SUMMARY Zebrafish (Danio rerio) have become a valuable model for investigating the molecular genetics and development of the inner ear in vertebrates. In this study, we employed a prepulse inhibition (PPI) paradigm to assess hearing in larval wild-type (AB) zebrafish during early development at 5–6 days post-fertilization (d.p.f.). We measured the PPI of the acoustic startle response in zebrafish using a 1-dimensional shaker that simulated the particle motion component of sound along the fish's dorsoventral axis. The thresholds to startle-inducing stimuli were determined in 5–6 d.p.f. zebrafish, and their hearing sensitivity was then characterized using the thresholds of prepulse tone stimuli (90–1200 Hz) that inhibited the acoustic startle response to a reliable startle stimulus (820 Hz at 20 dB re. 1 m s−2). Hearing thresholds were defined as the minimum prepulse tone level required to significantly reduce the startle response probability compared with the baseline (no-prepulse) condition. Larval zebrafish showed greatest auditory sensitivity from 90 to 310 Hz with corresponding mean thresholds of −19 to −10 dB re. 1 m s−2, respectively. Hearing thresholds of prepulse tones were considerably lower than previously predicted by startle response assays. The PPI assay was also used to investigate the relative contribution of the lateral line to the detection of acoustic stimuli. After aminoglycoside-induced neuromast hair-cell ablation, we found no difference in PPI thresholds between treated and control fish. We propose that this PPI assay can be used to screen for novel zebrafish hearing mutants and to investigate the ontogeny of hearing in zebrafish and other fishes. PMID:23966590

  13. A novel TRIM family member, Trim69, regulates zebrafish development through p53-mediated apoptosis.

    PubMed

    Han, Ruiqin; Zhao, Qing; Zong, Shudong; Miao, Shiying; Song, Wei; Wang, Linfang

    2016-05-01

    Trim69 contains the hallmark domains of a tripartite motif (TRIM) protein, including a Ring-finger domain, B-box domain, and coiled-coil domain. Trim69 is structurally and evolutionarily conserved in zebrafish, mouse, rat, human, and chimpanzee. The role of this protein is unclear, however, so we investigated its function in zebrafish development. Trim69 is extensively expressed in zebrafish adults and developing embryos-particularly in the testis, brain, ovary, and heart-and its expression decreases in a time- and stage-dependent manner. Loss of trim69 in zebrafish induces apoptosis and activates apoptosis-related processes; indeed, the tp53 pathway was up-regulated in response to the knockdown. Expression of human trim69 rescued the apoptotic phenotype, while overexpression of trim69 does not increase cellular apoptosis. Taken together, our results suggest that trim69 participates in tp53-mediated apoptosis during zebrafish development. Mol. Reprod. Dev. 83: 442-454, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  14. Monitoring of injury induced brain regeneration of the adult zebrafish by using optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Yuan, Zhen; Zhang, Jian

    2018-02-01

    The adult zebrafish has pronounced regenerative capacity of the brain, which makes it an ideal model organism of vertebrate biology for the investigation of recovery of central nervous system injuries. The aim of this study was to employ spectral-domain optical coherence tomography (SD-OCT) system for long-term in vivo monitoring of tissue regeneration using an adult zebrafish model of brain injury. Based on a 1325 nm light source and two high-speed galvo mirrors, the SD-OCT system can offer a large field of view of the three-dimensional (3D) brain structures with high imaging resolution (12 μm axial and 13 μm lateral) at video rate. In vivo experiments based on this system were conducted to monitor the regeneration process of zebrafish brain after injury during a period of 43 days. To monitor and detect the process of tissue regeneration, we performed 3D in vivo imaging in a zebrafish model of adult brain injury during a period of 43 days. The coronal and sagittal views of the injured zebrafish brain at each time point (0 days, 10 days, 20 days and 43 days postlesion) were presented to show the changes of the brain lesion in detail. In addition, the 3D SD-OCT images for an injured zebrafish brain were also reconstructed at days 0 and days 43 post-lesion. We found that SD-OCT is able to effectively and noninvasively monitor the regeneration of the adult zebrafish brain after injury in real time with high 3D spatial resolution and good penetration depth. Our findings also suggested that the adult zebrafish has the extraordinary capability of brain regeneration and is able to repair itself after brain injury.

  15. Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato

    PubMed Central

    Palanca, Ana Marie S.; Sagasti, Alvaro

    2013-01-01

    Larval zebrafish are emerging as a model for describing the development and function of simple neural circuits. Due to their external fertilization, rapid development, and translucency, zebrafish are particularly well suited for optogenetic approaches to investigate neural circuit function. In this approach, light-sensitive ion channels are expressed in specific neurons, enabling the experimenter to activate or inhibit them at will and thus assess their contribution to specific behaviors. Applying these methods in larval zebrafish is conceptually simple but requires the optimization of technical details. Here we demonstrate a procedure for expressing a channelrhodopsin variant in larval zebrafish somatosensory neurons, photo-activating single cells, and recording the resulting behaviors. By introducing a few modifications to previously established methods, this approach could be used to elicit behavioral responses from single neurons activated up to at least 4 days post-fertilization (dpf). Specifically, we created a transgene using a somatosensory neuron enhancer, CREST3, to drive the expression of the tagged channelrhodopsin variant, ChEF-tdTomato. Injecting this transgene into 1-cell stage embryos results in mosaic expression in somatosensory neurons, which can be imaged with confocal microscopy. Illuminating identified cells in these animals with light from a 473 nm DPSS laser, guided through a fiber optic cable, elicits behaviors that can be recorded with a high-speed video camera and analyzed quantitatively. This technique could be adapted to study behaviors elicited by activating any zebrafish neuron. Combining this approach with genetic or pharmacological perturbations will be a powerful way to investigate circuit formation and function. PMID:23407374

  16. Detecting Developmental Neurotoxicants Using Zebrafish Embryos

    EPA Science Inventory

    As part of EPA’s program on the screening and prioritization of chemicals for developmental neurotoxicity, a rapid, cost-effective in vivo vertebrate screen is being developed using an alternative species approach. Zebrafish (Danio rerio), a small freshwater fish with external f...

  17. Stable multilineage xenogeneic replacement of definitive hematopoiesis in adult zebrafish.

    PubMed

    Hess, Isabell; Boehm, Thomas

    2016-01-18

    Bony fishes are the most numerous and phenotypically diverse group of vertebrates inhabiting our planet, making them an ideal target for identifying general principles of tissue development and function. However, lack of suitable experimental platforms prevents the exploitation of this rich source of natural phenotypic variation. Here, we use a zebrafish strain lacking definitive hematopoiesis for interspecific analysis of hematopoietic cell development. Without conditioning prior to transplantation, hematopoietic progenitor cells from goldfish stably engraft in adult zebrafish homozygous for the c-myb(I181N) mutation. However, in competitive repopulation experiments, zebrafish hematopoietic cells exhibit an advantage over their goldfish counterparts, possibly owing to subtle species-specific functional differences in hematopoietic microenvironments resulting from over 100 million years of independent evolution. Thus, our unique animal model provides an unprecedented opportunity to genetically and functionally disentangle universal and species-specific contributions of the microenvironment to hematopoietic progenitor cell maintenance and development.

  18. The structure and timescales of heat perception in larval zebrafish.

    PubMed

    Haesemeyer, Martin; Robson, Drew N; Li, Jennifer M; Schier, Alexander F; Engert, Florian

    2015-11-25

    Avoiding temperatures outside the physiological range is critical for animal survival, but how temperature dynamics are transformed into behavioral output is largely not understood. Here, we used an infrared laser to challenge freely swimming larval zebrafish with "white-noise" heat stimuli and built quantitative models relating external sensory information and internal state to behavioral output. These models revealed that larval zebrafish integrate temperature information over a time-window of 400 ms preceding a swimbout and that swimming is suppressed right after the end of a bout. Our results suggest that larval zebrafish compute both an integral and a derivative across heat in time to guide their next movement. Our models put important constraints on the type of computations that occur in the nervous system and reveal principles of how somatosensory temperature information is processed to guide behavioral decisions such as sensitivity to both absolute levels and changes in stimulation.

  19. Fluoride caused thyroid endocrine disruption in male zebrafish (Danio rerio).

    PubMed

    Jianjie, Chen; Wenjuan, Xue; Jinling, Cao; Jie, Song; Ruhui, Jia; Meiyan, Li

    2016-02-01

    Excessive fluoride in natural water ecosystem has the potential to detrimentally affect thyroid endocrine system, but little is known of such effects or underlying mechanisms in fish. In the present study, we evaluated the effects of fluoride on growth performance, thyroid histopathology, thyroid hormone levels, and gene expressions in the HPT axis in male zebrafish (Danio rerio) exposed to different determined concentrations of 0.1, 0.9, 2.0 and 4.1 M of fluoride to investigate the effects of fluoride on thyroid endocrine system and the potential toxic mechanisms caused by fluoride. The results indicated that the growth of the male zebrafish used in the experiments was significantly inhibited, the thyroid microtrastructure was changed, and the levels of T3 and T4 were disturbed in fluoride-exposed male fish. In addition, the expressional profiles of genes in HPT axis displayed alteration. The expressions of all studied genes were significantly increased in all fluoride-exposed male fish after exposure for 45 days. The transcriptional levels of corticotrophin-releasing hormone (CRH), thyroid-stimulating hormone (TSH), thyroglobulin (TG), sodium iodide symporter (NIS), iodothyronine I (DIO1), and thyroid hormone receptor alpha (TRα) were also elevated in all fluoride-exposed male fish after 90 days of exposure, while the inconsistent expressions were found in the mRNA of iodothyronineⅡ (DIO2), UDP glucuronosyltransferase 1 family a, b (UGT1ab), transthyretin (TTR), and thyroid hormone receptor beta (TRβ). These results demonstrated that fluoride could notably inhibit the growth of zebrafish, and significantly affect thyroid endocrine system by changing the microtrastructure of thyroid, altering thyroid hormone levels and endocrine-related gene expressions in male zebrafish. All above indicated that fluoride could pose a great threat to thyroid endocrine system, thus detrimentally affected the normal function of thyroid of male zebrafish. Copyright © 2015

  20. The Zebrafish Model Organism Database: new support for human disease models, mutation details, gene expression phenotypes and searching

    PubMed Central

    Howe, Douglas G.; Bradford, Yvonne M.; Eagle, Anne; Fashena, David; Frazer, Ken; Kalita, Patrick; Mani, Prita; Martin, Ryan; Moxon, Sierra Taylor; Paddock, Holly; Pich, Christian; Ramachandran, Sridhar; Ruzicka, Leyla; Schaper, Kevin; Shao, Xiang; Singer, Amy; Toro, Sabrina; Van Slyke, Ceri; Westerfield, Monte

    2017-01-01

    The Zebrafish Model Organism Database (ZFIN; http://zfin.org) is the central resource for zebrafish (Danio rerio) genetic, genomic, phenotypic and developmental data. ZFIN curators provide expert manual curation and integration of comprehensive data involving zebrafish genes, mutants, transgenic constructs and lines, phenotypes, genotypes, gene expressions, morpholinos, TALENs, CRISPRs, antibodies, anatomical structures, models of human disease and publications. We integrate curated, directly submitted, and collaboratively generated data, making these available to zebrafish research community. Among the vertebrate model organisms, zebrafish are superbly suited for rapid generation of sequence-targeted mutant lines, characterization of phenotypes including gene expression patterns, and generation of human disease models. The recent rapid adoption of zebrafish as human disease models is making management of these data particularly important to both the research and clinical communities. Here, we describe recent enhancements to ZFIN including use of the zebrafish experimental conditions ontology, ‘Fish’ records in the ZFIN database, support for gene expression phenotypes, models of human disease, mutation details at the DNA, RNA and protein levels, and updates to the ZFIN single box search. PMID:27899582

  1. Edwardsiellosis Caused by Edwardsiella ictaluri in Laboratory Populations of Zebrafish Danio rerio

    PubMed Central

    Hawke, John P.; Kent, Michael; Rogge, Matt; Baumgartner, Wes; Wiles, Judy; Shelley, Johnny; Savolainen, L. Christine; Wagner, Robert; Murray, Katy; Peterson, Tracy S.

    2014-01-01

    We report the first cases of Edwardsiella ictaluri causing epizootics in laboratory populations of Zebrafish Danio rerio. Edwardsiella ictaluri is primarily recognized as a disease of catfish species and is known to cause an economically important bacterial disease of farm-raised catfish in the USA and abroad; however, it has been isolated on occasion from 10 other genera of nonictalurid fishes. We isolated E. ictaluri from moribund Zebrafish held in quarantine at two different universities in two states and from a research facility in a third state between February 23 and December 6, 2011. Edwardsiellosis in Zebrafish can be described as a severe systemic disease characterized by tissue necrosis and the presence of large numbers of extracellular and intracellular bacteria, often within macrophages. The kidneys (pronephros and mesonephros), spleen, nares, and forebrain were the most commonly and severely affected tissues. In outbreaks, mortality was acute and numerous fish died over a 1–2 week period. Mortality continued until the majority of the population was lost, at which time the remaining fish were euthanized. In addition to these cases, four cultures of bacteria isolated from Zebrafish by another diagnostic laboratory were submitted to the Louisiana Aquatic Diagnostic Laboratory for identification and were confirmed as E. ictaluri. In total, eight cultures of E. ictaluri from Zebrafish from Louisiana, Massachusetts, Pennsylvania, and Florida were identified. The isolates were confirmed as E. ictaluri by biochemical phenotype, API 20E (bioMérieux), and amplification and sequencing of a portion of the 16S rRNA gene. Edwardsiella ictaluri isolates from Zebrafish are believed to comprise a unique group and were differentiated from catfish isolates by exhibiting weaker motility, autoaggregation in broth, a different plasmid profile (two plasmids of 4.0 and 3.5 kb), a different API 20E code (4204000), and lack of lipopolysaccharide recognition with Mab Ed9

  2. Polysaccharides from astragali radix restore chemical-induced blood vessel loss in zebrafish

    PubMed Central

    2012-01-01

    Background Astragali Radix has been used widely for the treatment of cardiovascular and cerebrovascular diseases, and to enhance endurance and stamina in traditional Chinese medicine (TCM) for over 2000 years. The polysaccharide constituents of Astragali Radix (ARP) are considered as one of the major constituents contributing to the multiple pharmacological effects of this medicinal plant. The purpose of the study is to evaluate the vascular regenerative activities of ARPs in a chemically-induced blood vessel loss model in zebrafish. Methods Blood vessel loss was induced in both Tg(fli-1a:EGFP)y1 and Tg(fli-1a:nEGFP)y7 embryos by administration of 300 nM VEGFR tyrosine kinase inhibitor II (VRI) for 3 h at 24 hpf (hour post-fertilization). Then, the blood vessel damaged zebrafish were treated with ARPs for 21 h and 45 h after VRI withdrawal. Morphological changes in intersegmental vessels (ISVs) of zebrafish larvae were observed under the fluorescence microscope and measured quantitatively. The rescue effect of ARPs in the zebrafish models was validated by measuring the relative mRNA expressions of Kdrl, Kdr and Flt-1 using real-time PCR. Results Two polysaccharide fractions, P4 (50000 D < molecular weight & diameter < 0.1 μm) and P5 (molecular diameter > 0.1 μm), isolated from Astragali Radix by ultrafiltration, produced a significant and dose-dependent recovery in VRI-induced blood vessel loss in zebrafish. Furthermore, the down-regulation of Flk-1 and Flt-1 mRNA expression induced by VRI was reversed by treatment with P4. Conclusion The present study demonstrates that P4 isolated from Astragali Radix reduces VRI-induced blood vessel loss in zebrafish. These findings support the hypothesis that polysaccharides are one of the active constituents in Astragali Radix, contributing to its beneficial effect on treatment of diseases associated with a deficiency in angiogenesis. PMID:22357377

  3. Cell migration during heart regeneration in zebrafish.

    PubMed

    Tahara, Naoyuki; Brush, Michael; Kawakami, Yasuhiko

    2016-07-01

    Zebrafish possess the remarkable ability to regenerate injured hearts as adults, which contrasts the very limited ability in mammals. Although very limited, mammalian hearts do in fact have measurable levels of cardiomyocyte regeneration. Therefore, elucidating mechanisms of zebrafish heart regeneration would provide information of naturally occurring regeneration to potentially apply to mammalian studies, in addition to addressing this biologically interesting phenomenon in itself. Studies over the past 13 years have identified processes and mechanisms of heart regeneration in zebrafish. After heart injury, pre-existing cardiomyocytes dedifferentiate, enter the cell cycle, and repair the injured myocardium. This process requires interaction with epicardial cells, endocardial cells, and vascular endothelial cells. Epicardial cells envelope the heart, while endocardial cells make up the inner lining of the heart. They provide paracrine signals to cardiomyocytes to regenerate the injured myocardium, which is vascularized during heart regeneration. In addition, accumulating results suggest that local migration of these major cardiac cell types have roles in heart regeneration. In this review, we summarize the characteristics of various heart injury methods used in the research community and regeneration of the major cardiac cell types. Then, we discuss local migration of these cardiac cell types and immune cells during heart regeneration. Developmental Dynamics 245:774-787, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. Circadian clocks, rhythmic synaptic plasticity and the sleep-wake cycle in zebrafish.

    PubMed

    Elbaz, Idan; Foulkes, Nicholas S; Gothilf, Yoav; Appelbaum, Lior

    2013-01-01

    The circadian clock and homeostatic processes are fundamental mechanisms that regulate sleep. Surprisingly, despite decades of research, we still do not know why we sleep. Intriguing hypotheses suggest that sleep regulates synaptic plasticity and consequently has a beneficial role in learning and memory. However, direct evidence is still limited and the molecular regulatory mechanisms remain unclear. The zebrafish provides a powerful vertebrate model system that enables simple genetic manipulation, imaging of neuronal circuits and synapses in living animals, and the monitoring of behavioral performance during day and night. Thus, the zebrafish has become an attractive model to study circadian and homeostatic processes that regulate sleep. Zebrafish clock- and sleep-related genes have been cloned, neuronal circuits that exhibit circadian rhythms of activity and synaptic plasticity have been studied, and rhythmic behavioral outputs have been characterized. Integration of this data could lead to a better understanding of sleep regulation. Here, we review the progress of circadian clock and sleep studies in zebrafish with special emphasis on the genetic and neuroendocrine mechanisms that regulate rhythms of melatonin secretion, structural synaptic plasticity, locomotor activity and sleep.

  5. Induction of Social Behavior in Zebrafish: Live Versus Computer Animated Fish as Stimuli

    PubMed Central

    Qin, Meiying; Wong, Albert; Seguin, Diane

    2014-01-01

    Abstract The zebrafish offers an excellent compromise between system complexity and practical simplicity and has been suggested as a translational research tool for the analysis of human brain disorders associated with abnormalities of social behavior. Unlike laboratory rodents zebrafish are diurnal, thus visual cues may be easily utilized in the analysis of their behavior and brain function. Visual cues, including the sight of conspecifics, have been employed to induce social behavior in zebrafish. However, the method of presentation of these cues and the question of whether computer animated images versus live stimulus fish have differential effects have not been systematically analyzed. Here, we compare the effects of five stimulus presentation types: live conspecifics in the experimental tank or outside the tank, playback of video-recorded live conspecifics, computer animated images of conspecifics presented by two software applications, the previously employed General Fish Animator, and a new application Zebrafish Presenter. We report that all stimuli were equally effective and induced a robust social response (shoaling) manifesting as reduced distance between stimulus and experimental fish. We conclude that presentation of live stimulus fish, or 3D images, is not required and 2D computer animated images are sufficient to induce robust and consistent social behavioral responses in zebrafish. PMID:24575942

  6. Zebrafish zic2 controls formation of periocular neural crest and choroid fissure morphogenesis.

    PubMed

    Sedykh, Irina; Yoon, Baul; Roberson, Laura; Moskvin, Oleg; Dewey, Colin N; Grinblat, Yevgenya

    2017-09-01

    The vertebrate retina develops in close proximity to the forebrain and neural crest-derived cartilages of the face and jaw. Coloboma, a congenital eye malformation, is associated with aberrant forebrain development (holoprosencephaly) and with craniofacial defects (frontonasal dysplasia) in humans, suggesting a critical role for cross-lineage interactions during retinal morphogenesis. ZIC2, a zinc-finger transcription factor, is linked to human holoprosencephaly. We have previously used morpholino assays to show zebrafish zic2 functions in the developing forebrain, retina and craniofacial cartilage. We now report that zebrafish with genetic lesions in zebrafish zic2 orthologs, zic2a and zic2b, develop with retinal coloboma and craniofacial anomalies. We demonstrate a requirement for zic2 in restricting pax2a expression and show evidence that zic2 function limits Hh signaling. RNA-seq transcriptome analysis identified an early requirement for zic2 in periocular neural crest as an activator of alx1, a transcription factor with essential roles in craniofacial and ocular morphogenesis in human and zebrafish. Collectively, these data establish zic2 mutant zebrafish as a powerful new genetic model for in-depth dissection of cell interactions and genetic controls during craniofacial complex development. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Induction of social behavior in zebrafish: live versus computer animated fish as stimuli.

    PubMed

    Qin, Meiying; Wong, Albert; Seguin, Diane; Gerlai, Robert

    2014-06-01

    The zebrafish offers an excellent compromise between system complexity and practical simplicity and has been suggested as a translational research tool for the analysis of human brain disorders associated with abnormalities of social behavior. Unlike laboratory rodents zebrafish are diurnal, thus visual cues may be easily utilized in the analysis of their behavior and brain function. Visual cues, including the sight of conspecifics, have been employed to induce social behavior in zebrafish. However, the method of presentation of these cues and the question of whether computer animated images versus live stimulus fish have differential effects have not been systematically analyzed. Here, we compare the effects of five stimulus presentation types: live conspecifics in the experimental tank or outside the tank, playback of video-recorded live conspecifics, computer animated images of conspecifics presented by two software applications, the previously employed General Fish Animator, and a new application Zebrafish Presenter. We report that all stimuli were equally effective and induced a robust social response (shoaling) manifesting as reduced distance between stimulus and experimental fish. We conclude that presentation of live stimulus fish, or 3D images, is not required and 2D computer animated images are sufficient to induce robust and consistent social behavioral responses in zebrafish.

  8. Zebrafish Get Connected: Investigating Neurotransmission Targets and Alterations in Chemical Toxicity

    PubMed Central

    Horzmann, Katharine A.; Freeman, Jennifer L.

    2016-01-01

    Neurotransmission is the basis of neuronal communication and is critical for normal brain development, behavior, learning, and memory. Exposure to drugs and chemicals can alter neurotransmission, often through unknown pathways and mechanisms. The zebrafish (Danio rerio) model system is increasingly being used to study the brain and chemical neurotoxicity. In this review, the major neurotransmitter systems, including glutamate, GABA, dopamine, norepinephrine, serotonin, acetylcholine, histamine, and glutamate are surveyed and pathways of synthesis, transport, metabolism, and action are examined. Differences between human and zebrafish neurochemical pathways are highlighted. We also review techniques for evaluating neurological function, including the measurement of neurotransmitter levels, assessment of gene expression through transcriptomic analysis, and the recording of neurobehavior. Finally examples of chemical toxicity studies evaluating alterations in neurotransmitter systems in the zebrafish model are reviewed. PMID:28730152

  9. Adaptive Locomotor Behavior in Larval Zebrafish

    PubMed Central

    Portugues, Ruben; Engert, Florian

    2011-01-01

    In this study we report that larval zebrafish display adaptive locomotor output that can be driven by unexpected visual feedback. We develop a new assay that addresses visuomotor integration in restrained larval zebrafish. The assay involves a closed-loop environment in which the visual feedback a larva receives depends on its own motor output in a way that resembles freely swimming conditions. The experimenter can control the gain of this closed feedback loop, so that following a given motor output the larva experiences more or less visual feedback depending on whether the gain is high or low. We show that increases and decreases in this gain setting result in adaptive changes in behavior that lead to a generalized decrease or increase of motor output, respectively. Our behavioral analysis shows that both the duration and tail beat frequency of individual swim bouts can be modified, as well as the frequency with which bouts are elicited. These changes can be implemented rapidly, following an exposure to a new gain of just 175 ms. In addition, modifications in some behavioral parameters accumulate over tens of seconds and effects last for at least 30 s from trial to trial. These results suggest that larvae establish an internal representation of the visual feedback expected from a given motor output and that the behavioral modifications are driven by an error signal that arises from the discrepancy between this expectation and the actual visual feedback. The assay we develop presents a unique possibility for studying visuomotor integration using imaging techniques available in the larval zebrafish. PMID:21909325

  10. Adaptive locomotor behavior in larval zebrafish.

    PubMed

    Portugues, Ruben; Engert, Florian

    2011-01-01

    In this study we report that larval zebrafish display adaptive locomotor output that can be driven by unexpected visual feedback. We develop a new assay that addresses visuomotor integration in restrained larval zebrafish. The assay involves a closed-loop environment in which the visual feedback a larva receives depends on its own motor output in a way that resembles freely swimming conditions. The experimenter can control the gain of this closed feedback loop, so that following a given motor output the larva experiences more or less visual feedback depending on whether the gain is high or low. We show that increases and decreases in this gain setting result in adaptive changes in behavior that lead to a generalized decrease or increase of motor output, respectively. Our behavioral analysis shows that both the duration and tail beat frequency of individual swim bouts can be modified, as well as the frequency with which bouts are elicited. These changes can be implemented rapidly, following an exposure to a new gain of just 175 ms. In addition, modifications in some behavioral parameters accumulate over tens of seconds and effects last for at least 30 s from trial to trial. These results suggest that larvae establish an internal representation of the visual feedback expected from a given motor output and that the behavioral modifications are driven by an error signal that arises from the discrepancy between this expectation and the actual visual feedback. The assay we develop presents a unique possibility for studying visuomotor integration using imaging techniques available in the larval zebrafish.

  11. Disruption of Epithalamic Left-Right Asymmetry Increases Anxiety in Zebrafish.

    PubMed

    Facchin, Lucilla; Duboué, Erik R; Halpern, Marnie E

    2015-12-02

    Differences between the left and right sides of the brain are found throughout the animal kingdom, but the consequences of altered neural asymmetry are not well understood. In the zebrafish epithalamus, the parapineal is located on the left side of the brain where it influences development of the adjacent dorsal habenular (dHb) nucleus, causing the left and right dHb to differ in their organization, gene expression, and connectivity. Left-right (L-R) reversal of parapineal position and dHb asymmetry occurs spontaneously in a small percentage of the population, whereas the dHb develop symmetrically following experimental ablation of the parapineal. The habenular region was previously implicated in modulating fear in both mice and zebrafish, but the relevance of its L-R asymmetry is unclear. We now demonstrate that disrupting directionality of the zebrafish epithalamus causes reduced exploratory behavior and increased cortisol levels, indicative of enhanced anxiety. Accordingly, exposure to buspirone, an anxiolytic agent, significantly suppresses atypical behavior. Axonal projections from the parapineal to the dHb are more variable when it is located on the right side of the brain, revealing that L-R reversals do not necessarily represent a neuroanatomical mirror image. The results highlight the importance of directional asymmetry of the epithalamus in the regulation of stress responses in zebrafish. Copyright © 2015 the authors 0270-6474/15/3515847-13$15.00/0.

  12. Zebrafish as a disease model for studying human hepatocellular carcinoma.

    PubMed

    Lu, Jeng-Wei; Ho, Yi-Jung; Yang, Yi-Ju; Liao, Heng-An; Ciou, Shih-Ci; Lin, Liang-In; Ou, Da-Liang

    2015-11-14

    Liver cancer is one of the world's most common cancers and the second leading cause of cancer deaths. Hepatocellular carcinoma (HCC), a primary hepatic cancer, accounts for 90%-95% of liver cancer cases. The pathogenesis of HCC consists of a stepwise process of liver damage that extends over decades, due to hepatitis, fatty liver, fibrosis, and cirrhosis before developing fully into HCC. Multiple risk factors are highly correlated with HCC, including infection with the hepatitis B or C viruses, alcohol abuse, aflatoxin exposure, and metabolic diseases. Over the last decade, genetic alterations, which include the regulation of multiple oncogenes or tumor suppressor genes and the activation of tumorigenesis-related pathways, have also been identified as important factors in HCC. Recently, zebrafish have become an important living vertebrate model organism, especially for translational medical research. In studies focusing on the biology of cancer, carcinogen induced tumors in zebrafish were found to have many similarities to human tumors. Several zebrafish models have therefore been developed to provide insight into the pathogenesis of liver cancer and the related drug discovery and toxicology, and to enable the evaluation of novel small-molecule inhibitors. This review will focus on illustrative examples involving the application of zebrafish models to the study of human liver disease and HCC, through transgenesis, genome editing technology, xenografts, drug discovery, and drug-induced toxic liver injury.

  13. Zebrafish as a disease model for studying human hepatocellular carcinoma

    PubMed Central

    Lu, Jeng-Wei; Ho, Yi-Jung; Yang, Yi-Ju; Liao, Heng-An; Ciou, Shih-Ci; Lin, Liang-In; Ou, Da-Liang

    2015-01-01

    Liver cancer is one of the world’s most common cancers and the second leading cause of cancer deaths. Hepatocellular carcinoma (HCC), a primary hepatic cancer, accounts for 90%-95% of liver cancer cases. The pathogenesis of HCC consists of a stepwise process of liver damage that extends over decades, due to hepatitis, fatty liver, fibrosis, and cirrhosis before developing fully into HCC. Multiple risk factors are highly correlated with HCC, including infection with the hepatitis B or C viruses, alcohol abuse, aflatoxin exposure, and metabolic diseases. Over the last decade, genetic alterations, which include the regulation of multiple oncogenes or tumor suppressor genes and the activation of tumorigenesis-related pathways, have also been identified as important factors in HCC. Recently, zebrafish have become an important living vertebrate model organism, especially for translational medical research. In studies focusing on the biology of cancer, carcinogen induced tumors in zebrafish were found to have many similarities to human tumors. Several zebrafish models have therefore been developed to provide insight into the pathogenesis of liver cancer and the related drug discovery and toxicology, and to enable the evaluation of novel small-molecule inhibitors. This review will focus on illustrative examples involving the application of zebrafish models to the study of human liver disease and HCC, through transgenesis, genome editing technology, xenografts, drug discovery, and drug-induced toxic liver injury. PMID:26576090

  14. Redundant roles of PRDM family members in zebrafish craniofacial development.

    PubMed

    Ding, Hai-Lei; Clouthier, David E; Artinger, Kristin B

    2013-01-01

    PRDM proteins are evolutionary conserved Zn-Finger transcription factors that share a characteristic protein domain organization. Previous studies have shown that prdm1a is required for the specification and differentiation of neural crest cells in the zebrafish. Here we examine other members of this family, specifically prdm3, 5, and 16, in the differentiation of the zebrafish craniofacial skeleton. prdm3 and prdm16 are strongly expressed in the pharyngeal arches, while prdm5 is expressed specifically in the area of the forming neurocranium. Knockdown of prdm3 and prdm16 results in a reduction in the neural crest markers dlx2a and barx1 and defects in both the viscerocranium and the neurocranium. The knockdown of prdm3 and prdm16 in combination is additive in the neurocranium, but not in the viscerocranium. Injection of sub-optimal doses of prdm1a with prdm3 or prdm16 Morpholinos together leads to more severe phenotypes in the viscerocranium and neurocranium. prdm5 mutants have defects in the neurocranium and prdm1a and prdm5 double mutants also show more severe phenotypes. Overall, our data reveal that prdm3, 5, and 16 are involved in the zebrafish craniofacial development and that prdm1a may interact with prdm3, 5, and 16 in the formation of the craniofacial skeleton in zebrafish. Copyright © 2012 Wiley Periodicals, Inc.

  15. Redundant Roles of PRDM Family Members in Zebrafish Craniofacial Development

    PubMed Central

    Ding, Hai-Lei; Clouthier, David E.; Artinger, Kristin B.

    2014-01-01

    Background PRDM proteins are evolutionary conserved Zn-Finger transcription factors that share a characteristic protein domain organization. Previous studies have shown that prdm1a is required for the specification and differentiation of neural crest cells in the zebrafish. Results Here we examine other members of this family, specifically prdm3, 5, and 16, in the differentiation of the zebrafish craniofacial skeleton. prdm3 and prdm16 are strongly expressed in the pharyngeal arches, while prdm5 is expressed specifically in the area of the forming neurocranium. Knockdown of prdm3 and prdm16 results in a reduction in the neural crest markers dlx2a and barx1 and defects in both the viscerocranium and the neurocranium. The knockdown of prdm3 and prdm16 in combination is additive in the neurocranium, but not in the viscerocranium. Injection of sub-optimal doses of prdm1a with prdm3 or prdm16 Morpholinos together leads to more severe phenotypes in the viscerocranium and neurocranium. prdm5 mutants have defects in the neurocranium and prdm1a and prdm5 double mutants also show more severe phenotypes. Conclusions Overall, our data reveal that prdm3, 5, and 16 are involved in the zebrafish craniofacial development and that prdm1a may interact with prdm3, 5, and 16 in the formation of the craniofacial skeleton in zebrafish. PMID:23109401

  16. Live-cell imaging of Salmonella Typhimurium interaction with zebrafish larvae after injection and immersion delivery methods.

    PubMed

    Varas, Macarena; Fariña, Alonso; Díaz-Pascual, Francisco; Ortíz-Severín, Javiera; Marcoleta, Andrés E; Allende, Miguel L; Santiviago, Carlos A; Chávez, Francisco P

    2017-04-01

    The zebrafish model has been used to determine the role of vertebrate innate immunity during bacterial infections. Here, we compare the in vivo immune response induced by GFP-tagged Salmonella Typhimurium inoculated by immersion and microinjection in transgenic zebrafish larvae. Our novel infection protocols in zebrafish allow live-cell imaging of Salmonella colonization. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. BDE 49 and developmental toxicity in zebrafish

    PubMed Central

    McClain, Valerie; Stapleton, Heather M.; Gallagher, Evan

    2011-01-01

    The polybrominated diphenyl ethers (PBDEs) are a group of brominated flame retardants. Human health concerns of these agents have largely centered upon their potential to elicit reproductive and developmental effects. Of the various congeners, BDE 49 (2,2’,4,5’-tetrabromodiphenyl ether) has been poorly studied, despite the fact that it is often detected in the tissues of fish and wildlife species. Furthermore, we have previously shown that BDE 49 is a metabolic debromination product of BDE 99 hepatic metabolism in salmon, carp and trout, underscoring the need for a better understanding of biological effects. In the current study, we investigated the developmental toxicity of BDE 49 using the zebrafish (Danio rerio) embryo larval model. Embryo and larval zebrafish were exposed to BDE 49 at either 5 hours post fertilization (hpf) or 24 hpf and monitored for developmental and neurotoxicity. Exposure to BDE 49 at concentrations of 4 µM- 32 µM caused a dose-dependent loss in survivorship at 6 days post fertilization (dpf). Morphological impairments were observed prior to the onset of mortality, the most striking of which included severe dorsal curvatures of the tail. The incidence of dorsal tail curvatures was dose and time dependent. Exposure to BDE 49 caused cardiac toxicity as evidenced by a significant reduction in zebrafish heart rates at 6 dpf but not earlier, suggesting that cardiac toxicity was non-specific and associated with physiological stress. Neurobehavioral injury from BDE 49 was evidenced by an impairment of touch-escape responses observed at 5 dpf. Our results indicate that BDE 49 is a developmental toxicant in larval zebrafish that can cause morphological abnormalities and adversely affect neurobehavior. The observed toxicities from BDE 49 were similar in scope to those previously reported for the more common tetrabrominated congener, BDE 47, and also for other lower brominated PBDEs, suggest that these compounds may share similarities in risk to

  18. Making a Difference: Education at the 10th International Conference on Zebrafish Development and Genetics

    PubMed Central

    Liang, Jennifer O.; Pickart, Michael A.; Pierret, Chris; Tomasciewicz, Henry G.

    2012-01-01

    Abstract Scientists, educators, and students met at the 10th International Conference on Zebrafish Development and Genetics during the 2-day Education Workshop, chaired by Dr. Jennifer Liang and supported in part by the Genetics Society of America. The goal of the workshop was to share expertise, to discuss the challenges faced when using zebrafish in the classroom, and to articulate goals for expanding the impact of zebrafish in education. PMID:23244686

  19. Long-term drug administration in the adult zebrafish using oral gavage for cancer preclinical studies

    PubMed Central

    Dang, Michelle; Henderson, Rachel E.; Garraway, Levi A.

    2016-01-01

    ABSTRACT Zebrafish are a major model for chemical genetics, and most studies use embryos when investigating small molecules that cause interesting phenotypes or that can rescue disease models. Limited studies have dosed adults with small molecules by means of water-borne exposure or injection techniques. Challenges in the form of drug delivery-related trauma and anesthesia-related toxicity have excluded the adult zebrafish from long-term drug efficacy studies. Here, we introduce a novel anesthetic combination of MS-222 and isoflurane to an oral gavage technique for a non-toxic, non-invasive and long-term drug administration platform. As a proof of principle, we established drug efficacy of the FDA-approved BRAFV600E inhibitor, Vemurafenib, in adult zebrafish harboring BRAFV600E melanoma tumors. In the model, adult casper zebrafish intraperitoneally transplanted with a zebrafish melanoma cell line (ZMEL1) and exposed to daily sub-lethal dosing at 100 mg/kg of Vemurafenib for 2 weeks via oral gavage resulted in an average 65% decrease in tumor burden and a 15% mortality rate. In contrast, Vemurafenib-resistant ZMEL1 cell lines, generated in culture from low-dose drug exposure for 4 months, did not respond to the oral gavage treatment regimen. Similarly, this drug treatment regimen can be applied for treatment of primary melanoma tumors in the zebrafish. Taken together, we developed an effective long-term drug treatment system that will allow the adult zebrafish to be used to identify more effective anti-melanoma combination therapies and opens up possibilities for treating adult models of other diseases. PMID:27482819

  20. Short- and long-term effects of nicotine and the histone deacetylase inhibitor phenylbutyrate on novel object recognition in zebrafish.

    PubMed

    Faillace, M P; Pisera-Fuster, A; Medrano, M P; Bejarano, A C; Bernabeu, R O

    2017-03-01

    Zebrafish have a sophisticated color- and shape-sensitive visual system, so we examined color cue-based novel object recognition in zebrafish. We evaluated preference in the absence or presence of drugs that affect attention and memory retention in rodents: nicotine and the histone deacetylase inhibitor (HDACi) phenylbutyrate (PhB). The objective of this study was to evaluate whether nicotine and PhB affect innate preferences of zebrafish for familiar and novel objects after short- and long-retention intervals. We developed modified object recognition (OR) tasks using neutral novel and familiar objects in different colors. We also tested objects which differed with respect to the exploratory behavior they elicited from naïve zebrafish. Zebrafish showed an innate preference for exploring red or green objects rather than yellow or blue objects. Zebrafish were better at discriminating color changes than changes in object shape or size. Nicotine significantly enhanced or changed short-term innate novel object preference whereas PhB had similar effects when preference was assessed 24 h after training. Analysis of other zebrafish behaviors corroborated these results. Zebrafish were innately reluctant or prone to explore colored novel objects, so drug effects on innate preference for objects can be evaluated changing the color of objects with a simple geometry. Zebrafish exhibited recognition memory for novel objects with similar innate significance. Interestingly, nicotine and PhB significantly modified innate object preference.

  1. Haploinsufficiency of the genes encoding the tumor suppressor Pten predisposes zebrafish to hemangiosarcoma.

    PubMed

    Choorapoikayil, Suma; Kuiper, Raoul V; de Bruin, Alain; den Hertog, Jeroen

    2012-03-01

    PTEN is an essential tumor suppressor that antagonizes Akt/PKB signaling. The zebrafish genome encodes two Pten genes, ptena and ptenb. Here, we report that zebrafish mutants that retain a single wild-type copy of ptena or ptenb (ptena(+/-)ptenb(-/-) or ptena(-/-)ptenb(+/-)) are viable and fertile. ptena(+/-)ptenb(-/-) fish develop tumors at a relatively high incidence (10.2%) and most tumors developed close to the eye (26/30). Histopathologically, the tumor masses were associated with the retrobulbar vascular network and diagnosed as hemangiosarcomas. A single tumor was identified in 42 ptena(-/-)ptenb(+/-) fish and was also diagnosed as hemangiosarcoma. Immunohistochemistry indicated that the tumor cells in ptena(+/-)ptenb(-/-) and ptena(-/-)ptenb(+/-) fish proliferated rapidly and were of endothelial origin. Akt/PKB signaling was activated in the tumors, whereas Ptena was still detected in tumor tissue from ptena(+/-)ptenb(-/-) zebrafish. We conclude that haploinsufficiency of the genes encoding Pten predisposes to hemangiosarcoma in zebrafish.

  2. The Prx1 limb enhancers: targeted gene expression in developing zebrafish pectoral fins.

    PubMed

    Hernández-Vega, Amayra; Minguillón, Carolina

    2011-08-01

    Limbs represent an excellent model to study the induction, growth, and patterning of several organs. A breakthrough to study gene function in various tissues has been the characterization of regulatory elements that allow tissue-specific interference of gene function. The mouse Prx1 promoter has been used to generate limb-specific mutants and overexpress genes in tetrapod limbs. Although zebrafish possess advantages that favor their use to study limb morphogenesis, there is no driver described suitable for specifically interfering with gene function in developing fins. We report the generation of zebrafish lines that express enhanced green fluorescent protein (EGFP) driven by the mouse Prx1 enhancer in developing pectoral fins. We also describe the expression pattern of the zebrafish prrx1 genes and identify three conserved non-coding elements (CNEs) that we use to generate fin-specific EGFP reporter lines. Finally, we show that the mouse and zebrafish regulatory elements may be used to modify gene function in pectoral fins. Copyright © 2011 Wiley-Liss, Inc.

  3. Effect of gametes aging on their activation and fertilizability in zebrafish (Danio rerio).

    PubMed

    Cardona-Costa, Jose; Pérez-Camps, Mireia; García-Ximénez, Fernando; Espinós, Francisco J

    2009-03-01

    The zebrafish represents an important model organism for biological research. In this context, in vitro collection and fertilization of zebrafish gametes are basic and widely used techniques for many topical research works. In this work, the fertilization ability and normal embryo development of gold-type zebrafish sperm and eggs were re-evaluated after being stored for different times at 8 degrees C in a modified medium (Hanks' saline supplemented with 1.5 g BSA and 0.1 g ClNa; 320 mOsm, pH 7.4). Results obtained indicated that the temporal limits usually recommended for zebrafish sperm to fertilize fresh eggs (2 h) could be extended for up to 24 h without significant differences compared with fresh sperm. In contrast, the rapid egg aging observed (even less than 1 h) recommends minimizing as far as possible the egg storage time before fertilization. These results suggest a possible strain difference in the fertilization response.

  4. Report of Workshop on Euthanasia for Zebrafish-A Matter of Welfare and Science.

    PubMed

    Köhler, Almut; Collymore, Chereen; Finger-Baier, Karin; Geisler, Robert; Kaufmann, Larissa; Pounder, Kieran C; Schulte-Merker, Stefan; Valentim, Ana; Varga, Zoltan M; Weiss, Jürgen; Strähle, Uwe

    2017-12-01

    The increasing importance of zebrafish as a biomedical model organism is reflected by the steadily growing number of publications and laboratories working with this species. Regulatory recommendations for euthanasia as issued in Directive 2010/63/EU are, however, based on experience with fish species used for food production and do not take the small size and specific physiology of zebrafish into account. Consequently, the currently recommended methods of euthanasia in the Directive 2010/63/EU are either not applicable or may interfere with research goals. An international workshop was held in Karlsruhe, Germany, March 9, 2017, to discuss and propose alternative methods for euthanasia of zebrafish. The aim was to identify methods that adequately address the physiology of zebrafish and its use as a biomedical research model, follow the principles of the 3Rs (Replacement, Reduction, and Refinement) in animal experimentation and consider animal welfare during anesthesia and euthanasia. The results of the workshop are summarized here in the form of a white paper.

  5. Knockdown of zebrafish Fancd2 causes developmental abnormalities via p53-dependent apoptosis.

    PubMed

    Liu, Ting Xi; Howlett, Niall G; Deng, Min; Langenau, David M; Hsu, Karl; Rhodes, Jennifer; Kanki, John P; D'Andrea, Alan D; Look, A Thomas

    2003-12-01

    Mechanisms underlying the multiple developmental defects observed in Fanconi anemia (FA) patients are not well defined. We have identified the zebrafish homolog of human FANCD2, which encodes a nuclear effector protein that is monoubiquitinated in response to DNA damage, targeting it to nuclear foci where it preserves chromosomal integrity. Fancd2-deficient zebrafish embryos develop defects similar to those found in children with FA, including shortened body length, microcephaly, and microophthalmia, which are due to extensive cellular apoptosis. Developmental defects and increased apoptosis in Fancd2-deficient zebrafish were corrected by injection of human FANCD2 or zebrafish bcl2 mRNA, or by knockdown of p53, indicating that in the absence of Fancd2, developing tissues spontaneously undergo p53-dependent apoptosis. Thus, Fancd2 is essential during embryogenesis to prevent inappropriate apoptosis in neural cells and other tissues undergoing high levels of proliferative expansion, implicating this mechanism in the congenital abnormalities observed in human infants with FA.

  6. Comparative toxicity of several metal oxide nanoparticle aqueous suspensions to Zebrafish (Danio rerio) early developmental stage.

    PubMed

    Zhu, Xiaoshan; Zhu, Lin; Duan, Zhenghua; Qi, Ruiqi; Li, Yan; Lang, Yupeng

    2008-02-15

    With the emergence of manufactured nanomaterials, it is urgent to carry out researches on their potential environmental impacts and biological effects. To better understand the potential ecotoxicological impacts of metal oxide nanoparticles released to aquatic environments, the zebrafish 96-h embryo-larval bioassay was used to assess and compare the developmental toxicities of nanoscale zinc oxide (nZnO), titanium dioxide (nTiO(2)) and alumina (nAl(2)O(3)) aqueous suspensions. Toxicological endpoints such as zebrafish embryos or larvae survival, hatching rate and malformation were noted and described within 96 h of exposure. Meanwhile, a comparative experiment with their bulk counterparts (i.e., ZnO/bulk, TiO(2)/bulk and Al(2)O(3)/bulk) was conducted to understand the effect of particle size on their toxicities. The results showed that: (i) both nZnO and ZnO/bulk aqueous suspensions delayed zebrafish embryo and larva development, decreased their survival and hatching rates, and caused tissue damage. The 96-h LC(50) of nZnO and ZnO/bulk aqueous suspensions on the zebrafish survival are 1.793 mg/L and 1.550 mg/L respectively; and the 84-h EC(50) on the zebrafish embryo hatching rate are 2.065 mg/L and 2.066 mg/L respectively. Serious tissue ulceration was found on zebrafish larvae exposed to nZnO and ZnO/bulk aqueous suspensions. (ii) In contrast, neither nTiO(2) and TiO(2)/bulk nor nAl(2)O(3) and Al(2)O(3)/bulk showed any toxicity to zebrafish embryos and larvae under the same experimental condition. It revealed that the metal oxide nanoparticles with different chemical composition have different zebrafish developmental toxicities. (iii) Exposures of nTiO(2), nZnO and nAl(2)O(3) produced toxic effects on zebrafish embryos and larvae, which was not different from the effects caused by exposing to their bulk counterparts. This is the first study about the developmental toxicity of metal oxide nanoparticles, and the results demonstrate that nZnO is very toxic to

  7. Zebrafish in Brazilian Science: Scientific Production, Impact, and Collaboration.

    PubMed

    Gheno, Ediane Maria; Rosemberg, Denis Broock; Souza, Diogo Onofre; Calabró, Luciana

    2016-06-01

    By means of scientometric indicators, this study investigated the characteristics of scientific production and research collaboration involving zebrafish (Danio rerio) in Brazilian Science indexed by the Web of Science (WoS). Citation data were collected from the WoS and data regarding Impact Factor (IF) were gathered from journals in the Journal Citation Reports. Collaboration was evaluated according to coauthorship data, creating representative nets with VOSviewer. Zebrafish has attained remarkable importance as an experimental model organism in recent years and an increase in scientific production with zebrafish is observed in Brazil and around the world. The citation impact of the worldwide scientific production is superior when compared to the Brazilian scientific production. However, the citation impact of the Brazilian scientific production is consistently increasing. Brazil does not follow the international trends with regard to publication research fields. The state of Rio Grande do Sul has the greatest number of articles and the institution with the largest number of publications is Pontifícia Universidade Católica do Rio Grande do Sul. Journals' average IF is higher in Brazilian publications with international coauthorship, and around 90% of articles are collaborative. The Brazilian institutions presenting the greatest number of collaborations are Pontifícia Universidade Católica do Rio Grande do Sul, Universidade Federal do Rio Grande do Sul, Fundação Universidade Federal de Rio Grande, and Universidade de São Paulo. These data indicate that Brazilian research using zebrafish presents a growth in terms of number of publications, citation impact, and collaborative work.

  8. Chlorpyrifos-Oxon Disrupts Zebrafish Axonal Growth and Motor Behavior

    PubMed Central

    Yang, Dongren; Lauridsen, Holly; Buels, Kalmia; Chi, Lai-Har; La Du, Jane; Bruun, Donald A.; Olson, James R.; Tanguay, Robert L.; Lein, Pamela J.

    2011-01-01

    Axonal morphology is a critical determinant of neuronal connectivity, and perturbation of the rate or extent of axonal growth during development has been linked to neurobehavioral deficits in animal models and humans. We previously demonstrated that the organophosphorus pesticide (OP) chlorpyrifos (CPF) inhibits axonal growth in cultured neurons. In this study, we used a zebrafish model to determine whether CPF, its oxon metabolite (CPFO), or the excreted metabolite trichloro-2-pyridinol (TCPy) alter spatiotemporal patterns of axonal growth in vivo. Static waterborne exposure to CPFO, but not CPF or TCPy, at concentrations ≥ 0.03μM from 24- to 72-h post fertilization significantly inhibited acetylcholinesterase, and high-performance liquid chromatography detected significantly more TCPy in zebrafish exposed to 0.1μM CPFO versus 1.0μM CPF. These data suggest that zebrafish lack the metabolic enzymes to activate CPF during these early developmental stages. Consistent with this, CPFO, but not CPF, significantly inhibited axonal growth of sensory neurons, primary motoneurons, and secondary motoneurons at concentrations ≥ 0.1μM. Secondary motoneurons were the most sensitive to axonal growth inhibition by CPFO, which was observed at concentrations that did not cause mortality, gross developmental defects, or aberrant somatic muscle differentiation. CPFO effects on axonal growth correlated with adverse effects on touch-induced swimming behavior, suggesting the functional relevance of these structural changes. These data suggest that altered patterns of neuronal connectivity contribute to the developmental neurotoxicity of CPF and demonstrate the relevance of zebrafish as a model for studying OP developmental neurotoxicity. PMID:21346248

  9. Zebrafish in Brazilian Science: Scientific Production, Impact, and Collaboration

    PubMed Central

    Gheno, Ediane Maria; Rosemberg, Denis Broock; Souza, Diogo Onofre

    2016-01-01

    Abstract By means of scientometric indicators, this study investigated the characteristics of scientific production and research collaboration involving zebrafish (Danio rerio) in Brazilian Science indexed by the Web of Science (WoS). Citation data were collected from the WoS and data regarding Impact Factor (IF) were gathered from journals in the Journal Citation Reports. Collaboration was evaluated according to coauthorship data, creating representative nets with VOSviewer. Zebrafish has attained remarkable importance as an experimental model organism in recent years and an increase in scientific production with zebrafish is observed in Brazil and around the world. The citation impact of the worldwide scientific production is superior when compared to the Brazilian scientific production. However, the citation impact of the Brazilian scientific production is consistently increasing. Brazil does not follow the international trends with regard to publication research fields. The state of Rio Grande do Sul has the greatest number of articles and the institution with the largest number of publications is Pontifícia Universidade Católica do Rio Grande do Sul. Journals' average IF is higher in Brazilian publications with international coauthorship, and around 90% of articles are collaborative. The Brazilian institutions presenting the greatest number of collaborations are Pontifícia Universidade Católica do Rio Grande do Sul, Universidade Federal do Rio Grande do Sul, Fundação Universidade Federal de Rio Grande, and Universidade de São Paulo. These data indicate that Brazilian research using zebrafish presents a growth in terms of number of publications, citation impact, and collaborative work. PMID:27045850

  10. Simple, economical heat-shock devices for zebrafish housing racks.

    PubMed

    Duszynski, Robert J; Topczewski, Jacek; LeClair, Elizabeth E

    2011-12-01

    One reason for the popularity of the zebrafish (Danio rerio) as a model vertebrate is the ability to manipulate gene expression in this organism. A common method is to induce gene expression transiently under control of a heat-shock promoter (e.g., hsp70l). By making simple mechanical adjustments to small aquarium heaters (25-50W), we were able to produce consistent and reliable heat-shock conditions within a conventional zebrafish housing system. Up to two heat-shock intervals per day (>37°C) could be maintained under conditions of continuous flow (5-25 mL/min). Temperature logging every 30 s indicated rapid warm up times, consistent heat-shock lengths, and accurate and precise peak water temperatures (mean±SD=38°C±0.2°C). The biological effects of these heat-shock treatments were confirmed by observing inducible expression of enhanced green fluorescent protein (EGFP) and inhibition of caudal fin regeneration in a transgenic fish line expressing a dominant negative fibroblast growth factor receptor (Tg(hsp70l:dnfgfr1-EGFP)(pd1)). These devices are inexpensive, easily modified, and can be calibrated to accommodate a variety of experimental designs. After setup on a programmable timer, the heaters require no intervention to produce consistent daily heat shocks, and all other standard care protocols can be followed in the fish facility. The simplicity and stability of these devices make them suitable for long-term heat shocks at any stage of the zebrafish lifecycle (>7 days postfertilization), and useful for both laboratory and classroom experiments on transgenic zebrafish.

  11. Neurobehavioral impairments produced by developmental lead exposure persisted for generations in zebrafish (Danio rerio).

    PubMed

    Xu, Xiaojuan; Weber, Daniel; Burge, Rebekah; VanAmberg, Kelsey

    2016-01-01

    The zebrafish has become a useful animal model for studying the effects of environmental contaminants on neurobehavioral development due to its ease of breeding, high number of eggs per female, short generation times, and a well-established avoidance conditioning paradigm. Using avoidance conditioning as the behavioral paradigm, the present study investigated the effects of embryonic exposure to lead (Pb) on learning in adult zebrafish and the third (F3) generation of those fish. In Experiment 1, adult zebrafish that were developmentally exposed to 0.0, 0.1, 1.0 or 10.0μM Pb (2-24h post fertilization) as embryos were trained and tested for avoidance responses. The results showed that adult zebrafish hatched from embryos exposed to 0.0 or 0.1μM Pb learned avoidance responses during training and displayed significantly increased avoidance responses during testing, while those hatched from embryos exposed to 1.0 or 10.0μM Pb displayed no significant increases in avoidance responses from training to testing. In Experiment 2, the F3 generation of zebrafish that were developmentally exposed to an identical exposure regimen as in Experiment 1 were trained and tested for avoidance responses. The results showed that the F3 generation of zebrafish developmentally exposed as embryos to 0.0 or 0.1μM Pb learned avoidance responses during training and displayed significantly increased avoidance responses during testing, while the F3 generation of zebrafish developmentally exposed as embryos to 1.0 or 10.0μM Pb displayed no significant changes in avoidance responses from training to testing. Thus, developmental Pb exposure produced learning impairments that persisted for at least three generations, demonstrating trans-generational effects of embryonic exposure to Pb. Copyright © 2015. Published by Elsevier B.V.

  12. Exposure to tributyltin induces endoplasmic reticulum stress and the unfolded protein response in zebrafish.

    PubMed

    Komoike, Yuta; Matsuoka, Masato

    2013-10-15

    Tributyltin (TBT) is a major marine contaminant and causes endocrine disruption, hepatotoxicity, immunotoxicity, and neurotoxicity. However, the molecular mechanisms underlying the toxicity of TBT have not been fully elucidated. We examined whether exposure to TBT induces the endoplasmic reticulum (ER) stress response in zebrafish, a model organism. Zebrafish-derived BRF41 fibroblast cells were exposed to 0.5 or 1 μM TBT for 0.5-16 h and subsequently lysed and immunoblotted to detect ER stress-related proteins. Zebrafish embryos, grown until 32 h post fertilization (hpf), were exposed to 1 μM TBT for 16 h and used in whole mount in situ hybridization and immunohistochemistry to visualize the expression of ER chaperones and an ER stress-related apoptosis factor. Exposure of the BRF41 cells to TBT caused phosphorylation of the zebrafish homolog of protein kinase RNA-activated-like ER kinase (PERK), eukaryotic translation initiation factor 2 alpha (eIF2α), and inositol-requiring enzyme 1 (IRE1), characteristic splicing of X-box binding protein 1 (XBP1) mRNA, and enhanced expression of activating transcription factor 4 (ATF4) protein. In TBT-exposed zebrafish embryos, ectopic expression of the gene encoding zebrafish homolog of the 78 kDa glucose-regulating protein (GRP78) and gene encoding CCAAT/enhancer-binding protein homologous protein (CHOP) was detected in the precursors of the neuromast, which is a sensory organ for detecting water flow and vibration. Our in vitro and in vivo studies revealed that exposure of zebrafish to TBT induces the ER stress response via activation of both the PERK-eIF2α and IRE1-XBP1 pathways of the unfolded protein response (UPR) in an organ-specific manner. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Optimal Anesthetic Regime for Motionless Three-Dimensional Image Acquisition During Longitudinal Studies of Adult Nonpigmented Zebrafish.

    PubMed

    Lockwood, Nicola; Parker, Jennifer; Wilson, Carole; Frankel, Paul

    2017-04-01

    With many live imaging techniques, it is crucial that a deep level of anesthesia is reached and maintained throughout image acquisition without reducing zebrafish viability. This is particularly true for three-dimensional tomographic imaging modalities. Currently, the most commonly used anesthetic in the zebrafish community, MS-222 (tricaine methanesulfonate), does not allow this. We show, using a combination of both MS-222 and isoflurane, that we can significantly improve the anesthetic regime required for motionless image acquisition of live adult zebrafish. We have benchmarked this against the requirements of our novel quantitative imaging platform, compressive sensing optical projection tomography. Using nonpigmented transgenic zebrafish, we show that a combination of 175 ppm of both anesthetics improves the maintenance of deep anesthesia for prolonged periods of time and it can be used repeatedly to enable longitudinal imaging. Importantly, it does not affect the health or viability of the adult zebrafish. We also show that nonpigmented fish, with a mutated form of the gene transparent, took significantly longer to reach deep anesthesia. The anesthetic regime presented in this study should lead to significant improvements in accuracy and information achievable from imaging live adult zebrafish and in its application to longitudinal studies.

  14. The synthetic substance hypoxanthine 3-N-oxide elicits alarm reactions in zebrafish (Danio rerio)

    PubMed Central

    Parra, Kevin V.; Adrian, James C.; Gerlai, Robert

    2009-01-01

    Zebrafish, one of the preferred study species of geneticists, is gaining increasing popularity in behavioral neuroscience. This small and prolific species may be an excellent tool with which the biological mechanisms of vertebrate brain function and behavior are investigated. Zebrafish has been proposed as a model organism in the analysis of fear responses and human anxiety disorders. Species-specific cues signaling the presence of predators have been successfully utilized in such research. Zebrafish has been shown to respond to its natural alarm substance with species-typical fear reactions. However, the extraction of this alarm substance and ascertaining its consistent dosing has been problematic. A synthetic substance with a known chemical identity and molecular weight would allow precise dosing and experimental control. Previously, the chemical component, hypoxanthine 3-N-oxide, common to several fish alarm substances has been identified and has been shown to elicit alarm reactions in fish species belonging to the Osteriophysan superorder. In the current study we investigate the effect of hypoxanthine 3-N-oxide by exposing zebrafish to three different concentrations of this synthetic substance. Our results show that the substance efficaciously induces species-typical fear reactions increasing the number of erratic movement episodes and jumps in zebrafish. We discuss the translational relevance of our findings and conclude that hypoxanthine 3-N-oxide will have utility to elicit fear responses in the laboratory in a precisely controlled manner in zebrafish. PMID:19583985

  15. The Zebrafish Xenograft Platform: Evolution of a Novel Cancer Model and Preclinical Screening Tool.

    PubMed

    Wertman, Jaime; Veinotte, Chansey J; Dellaire, Graham; Berman, Jason N

    2016-01-01

    Animal xenografts of human cancers represent a key preclinical tool in the field of cancer research. While mouse xenografts have long been the gold standard, investigators have begun to use zebrafish (Danio rerio) xenotransplantation as a relatively rapid, robust and cost-effective in vivo model of human cancers. There are several important methodological considerations in the design of an informative and efficient zebrafish xenotransplantation experiment. Various transgenic fish strains have been created that facilitate microscopic observation, ranging from the completely transparent casper fish to the Tg(fli1:eGFP) fish that expresses fluorescent GFP protein in its vascular tissue. While human cancer cell lines have been used extensively in zebrafish xenotransplantation studies, several reports have also used primary patient samples as the donor material. The zebrafish is ideally suited for transplanting primary patient material by virtue of the relatively low number of cells required for each embryo (between 50 and 300 cells), the absence of an adaptive immune system in the early zebrafish embryo, and the short experimental timeframe (5-7 days). Following xenotransplantation into the fish, cells can be tracked using in vivo or ex vivo measures of cell proliferation and migration, facilitated by fluorescence or human-specific protein expression. Importantly, assays have been developed that allow for the reliable detection of in vivo human cancer cell growth or inhibition following administration of drugs of interest. The zebrafish xenotransplantation model is a unique and effective tool for the study of cancer cell biology.

  16. Distinct structure and activity of monoamine oxidase in the brain of zebrafish (Danio rerio).

    PubMed

    Anichtchik, Oleg; Sallinen, Ville; Peitsaro, Nina; Panula, Pertti

    2006-10-10

    Monoamine oxidase (MAO) is a mitochondrial flavoprotein involved in the metabolism of, e.g., aminergic neurotransmitters and the parkinsonism-inducing neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). We have reported earlier MPTP-related alterations of brain catecholaminergic system in zebrafish (Danio rerio) brain. Here we describe the structural and functional properties of zebrafish MAO and the distribution of MAO mRNA and activity in zebrafish brain. The gene is located in chromosome 9 and consists of 15 exons. The amino acid composition of the active center resembles both human MAO-A and MAO-B. The enzyme displayed the highest substrate specificity for tyramine, followed by serotonin, phenylethylamine, MPTP, and dopamine; isoform-specific antagonists blocked the activity of the enzyme with equal potency. Zebrafish MAO mRNA, which was present in several tissues, and enzyme displayed differential distribution in the brain; dopaminergic cell clusters had low to moderate levels of MAO activity, whereas the highest levels of MAO activity were detected in noradrenergic and serotonergic cell groups and the habenulointerpeduncular pathway, including its caudal projection to the medial ventral rhombencephalon. The results of this study confirm the presence of functionally active MAO in zebrafish brain and other tissues and characterize the neural systems that express MAO and areas of intense activity in the brain. They also suggest that MPTP toxicity not related to MAO may affect the zebrafish brain.

  17. Satellite-like cells contribute to pax7-dependent skeletal muscle repair in adult zebrafish

    PubMed Central

    Berberoglu, Michael A.; Gallagher, Thomas L.; Morrow, Zachary T.; Talbot, Jared C.; Hromowyk, Kimberly J.; Tenente, Inês M.; Langenau, David M.; Amacher, Sharon L.

    2017-01-01

    Satellite cells, also known as muscle stem cells, are responsible for skeletal muscle growth and repair in mammals. Pax7 and Pax3 transcription factors are established satellite cell markers required for muscle development and regeneration, and there is great interest in identifying additional factors that regulate satellite cell proliferation, differentiation, and/or skeletal muscle regeneration. Due to the powerful regenerative capacity of many zebrafish tissues, even in adults, we are exploring the regenerative potential of adult zebrafish skeletal muscle. Here, we show that adult zebrafish skeletal muscle contains cells similar to mammalian satellite cells. Adult zebrafish satellite-like cells have dense heterochromatin, express Pax7 and Pax3, proliferate in response to injury, and show peak myogenic responses 4–5 days post-injury (dpi). Furthermore, using a pax7a-driven GFP reporter, we present evidence implicating satellite-like cells as a possible source of new muscle. In lieu of central nucleation, which distinguishes regenerating myofibers in mammals, we describe several characteristics that robustly identify newly-forming myofibers from surrounding fibers in injured adult zebrafish muscle. These characteristics include partially overlapping expression in satellite cells and regenerating myofibers of two RNA-binding proteins Rbfox2 and Rbfoxl1, known to regulate embryonic muscle development and function. Finally, by analyzing pax7a; pax7b double mutant zebrafish, we show that Pax7 is required for adult skeletal muscle repair, as it is in the mouse. PMID:28279710

  18. Advanced Echocardiography in Adult Zebrafish Reveals Delayed Recovery of Heart Function after Myocardial Cryoinjury

    PubMed Central

    Kossack, Mandy; Juergensen, Lonny; Fuchs, Dieter; Katus, Hugo A.; Hassel, David

    2015-01-01

    Translucent zebrafish larvae represent an established model to analyze genetics of cardiac development and human cardiac disease. More recently adult zebrafish are utilized to evaluate mechanisms of cardiac regeneration and by benefiting from recent genome editing technologies, including TALEN and CRISPR, adult zebrafish are emerging as a valuable in vivo model to evaluate novel disease genes and specifically validate disease causing mutations and their underlying pathomechanisms. However, methods to sensitively and non-invasively assess cardiac morphology and performance in adult zebrafish are still limited. We here present a standardized examination protocol to broadly assess cardiac performance in adult zebrafish by advancing conventional echocardiography with modern speckle-tracking analyses. This allows accurate detection of changes in cardiac performance and further enables highly sensitive assessment of regional myocardial motion and deformation in high spatio-temporal resolution. Combining conventional echocardiography measurements with radial and longitudinal velocity, displacement, strain, strain rate and myocardial wall delay rates after myocardial cryoinjury permitted to non-invasively determine injury dimensions and to longitudinally follow functional recovery during cardiac regeneration. We show that functional recovery of cryoinjured hearts occurs in three distinct phases. Importantly, the regeneration process after cryoinjury extends far beyond the proposed 45 days described for ventricular resection with reconstitution of myocardial performance up to 180 days post-injury (dpi). The imaging modalities evaluated here allow sensitive cardiac phenotyping and contribute to further establish adult zebrafish as valuable cardiac disease model beyond the larval developmental stage. PMID:25853735

  19. Fgf signaling is required for zebrafish tooth development.

    PubMed

    Jackman, William R; Draper, Bruce W; Stock, David W

    2004-10-01

    We have investigated fibroblast growth factor (FGF) signaling during the development of the zebrafish pharyngeal dentition with the goal of uncovering novel roles for FGFs in tooth development as well as phylogenetic and topographic diversity in the tooth developmental pathway. We found that the tooth-related expression of several zebrafish genes is similar to that of their mouse orthologs, including both epithelial and mesenchymal markers. Additionally, significant differences in gene expression between zebrafish and mouse teeth are indicated by the apparent lack of fgf8 and pax9 expression in zebrafish tooth germs. FGF receptor inhibition with SU5402 at 32 h blocked dental epithelial morphogenesis and tooth mineralization. While the pharyngeal epithelium remained intact as judged by normal pitx2 expression, not only was the mesenchymal expression of lhx6 and lhx7 eliminated as expected from mouse studies, but the epithelial expression of dlx2a, dlx2b, fgf3, and fgf4 was as well. This latter result provides novel evidence that the dental epithelium is a target of FGF signaling. However, the failure of SU5402 to block localized expression of pitx2 suggests that the earliest steps of tooth initiation are FGF-independent. Investigations of specific FGF ligands with morpholino antisense oligonucleotides revealed only a mild tooth shape phenotype following fgf4 knockdown, while fgf8 inhibition revealed only a subtle down-regulation of dental dlx2b expression with no apparent effect on tooth morphology. Our results suggest redundant FGF signals target the dental epithelium and together are required for dental morphogenesis. Further work will be required to elucidate the nature of these signals, particularly with respect to their origins and whether they act through the mesenchyme.

  20. Transcriptomic characterization of cold acclimation in larval zebrafish

    PubMed Central

    2013-01-01

    Background Temperature is one of key environmental parameters that affect the whole life of fishes and an increasing number of studies have been directed towards understanding the mechanisms of cold acclimation in fish. However, the adaptation of larvae to cold stress and the cold-specific transcriptional alterations in fish larvae remain largely unknown. In this study, we characterized the development of cold-tolerance in zebrafish larvae and investigated the transcriptional profiles under cold stress using RNA-seq. Results Pre-exposure of 96 hpf zebrafish larvae to cold stress (16°C) for 24 h significantly increased their survival rates under severe cold stress (12°C). RNA-seq generated 272 million raw reads from six sequencing libraries and about 92% of the processed reads were mapped to the reference genome of zebrafish. Differential expression analysis identified 1,431 up- and 399 down-regulated genes. Gene ontology enrichment analysis of cold-induced genes revealed that RNA splicing, ribosome biogenesis and protein catabolic process were the most highly overrepresented biological processes. Spliceosome, proteasome, eukaryotic ribosome biogenesis and RNA transport were the most highly enriched pathways for genes up-regulated by cold stress. Moreover, alternative splicing of 197 genes and promoter switching of 64 genes were found to be regulated by cold stress. A shorter isoform of stk16 that lacks 67 amino acids at the N-terminus was specifically generated by skipping the second exon in cold-treated larvae. Alternative promoter usage was detected for per3 gene under cold stress, which leading to a highly up-regulated transcript encoding a truncated protein lacking the C-terminal domains. Conclusions These findings indicate that zebrafish larvae possess the ability to build cold-tolerance under mild low temperature and transcriptional and post-transcriptional regulations are extensively involved in this acclimation process. PMID:24024969

  1. An open-source method to analyze optokinetic reflex responses in larval zebrafish.

    PubMed

    Scheetz, Seth D; Shao, Enhua; Zhou, Yangzhong; Cario, Clinton L; Bai, Qing; Burton, Edward A

    2018-01-01

    Optokinetic reflex (OKR) responses provide a convenient means to evaluate oculomotor, integrative and afferent visual function in larval zebrafish models, which are commonly used to elucidate molecular mechanisms underlying development, disease and repair of the vertebrate nervous system. We developed an open-source MATLAB-based solution for automated quantitative analysis of OKR responses in larval zebrafish. The package includes applications to: (i) generate sinusoidally-transformed animated grating patterns suitable for projection onto a cylindrical screen to elicit the OKR; (ii) determine and record the angular orientations of the eyes in each frame of a video recording showing the OKR response; and (iii) analyze angular orientation data from the tracking program to yield a set of parameters that quantify essential elements of the OKR. The method can be employed without modification using the operating manual provided. In addition, annotated source code is included, allowing users to modify or adapt the software for other applications. We validated the algorithms and measured OKR responses in normal larval zebrafish, showing good agreement with published quantitative data, where available. We provide the first open-source method to elicit and analyze the OKR in larval zebrafish. The wide range of parameters that are automatically quantified by our algorithms significantly expands the scope of quantitative analysis previously reported. Our method for quantifying OKR responses will be useful for numerous applications in neuroscience using the genetically- and chemically-tractable zebrafish model. Published by Elsevier B.V.

  2. Quantitative assessment of cypermethrin induced behavioural and biochemical anomalies in adult zebrafish.

    PubMed

    Nema, Shubham; Bhargava, Yogesh

    2018-05-23

    Cypermethrin is one of the top five pesticides used globally. Although the effect of cypermethrin on the embryonic stages of zebrafish is well characterized, its toxic effect on the behaviour of adult zebrafish is largely unknown. Here we used videogram and automated tracking approach to quantitatively assess behavioural toxicity induced by the short exposure of cypermethrin to adult zebrafish. We observed that cypermethrin at 25 ppb level induced behavioural toxicity in adult zebrafish. Motor activity of the treated group was significantly retarded which affected their overall exploratory behaviour including their visit to the central arena of the open-field test. Furthermore, the treated group showed erratic movements (covered less distance per unit time) without affecting their angle based behavioural endpoints. In contrast to the control group, the cypermethrin exposed group showed frequent freezing behaviour. However, their freezing episodes were characterized by constant drift-like movement caused by the loss of their voluntary control over the motor coordination. These behavioural changes are similar to typical anxiety-like behaviour. Though, cypermethrin exposure at ppb level for just half an hour was sufficient to induce behavioural toxicity, it failed to alter brain superoxide dismutase and acetylcholine esterase enzyme activity. Our data indicates that acute short-term exposure of cypermethrin induces behavioural anomalies in adult zebrafish through a mechanism distinct from alteration of brain superoxide dismutase and the acetylcholine esterase activity. Copyright © 2018 Elsevier Inc. All rights reserved.

  3. MK-801 increases locomotor activity in a context-dependent manner in zebrafish.

    PubMed

    Tran, Steven; Muraleetharan, Arrujyan; Fulcher, Niveen; Chatterjee, Diptendu; Gerlai, Robert

    2016-01-01

    Zebrafish have become a popular animal model for behavioral neuroscience with an increasing number of studies examining the effects of pharmacological compounds targeting the brain. Exposure to MK-801, a non-competitive N-methyl-d-aspartate receptor antagonist has been shown to increase locomotor activity in zebrafish. However, others have failed to replicate this finding as several contradicting studies report no changes in locomotor activity following exposure to similar doses. In the current study we reconcile these behavioral reports by demonstrating that zebrafish do not exhibit changes in locomotor activity during exposure to non-sedative doses of MK-801. Interestingly, zebrafish do exhibit significant increases in locomotion if pre-treated with MK-801 followed by subsequent testing in a novel environment, which suggests the effects of MK-801 are context-dependent. In addition, we examine the potential role of the dopaminergic system in mediating MK-801's locomotor stimulant effect by quantifying the levels of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the brains of zebrafish following a 30 min exposure to 10 μM of MK-801 (the dose found to induce the largest increase in locomotor activity). Our findings indicate that the MK-801-induced increase in locomotor activity is not accompanied by changes in whole-brain levels of dopamine or DOPAC. Overall, our results suggest that MK-801's context-dependent locomotor stimulant effect may be independent of whole-brain dopaminergic activation. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Novel Insights into the Genetic Controls of Primitive and Definitive Hematopoiesis from Zebrafish Models

    PubMed Central

    Sood, Raman; Liu, Paul

    2012-01-01

    Hematopoiesis is a dynamic process where initiation and maintenance of hematopoietic stem cells, as well as their differentiation into erythroid, myeloid and lymphoid lineages, are tightly regulated by a network of transcription factors. Understanding the genetic controls of hematopoiesis is crucial as perturbations in hematopoiesis lead to diseases such as anemia, thrombocytopenia, or cancers, including leukemias and lymphomas. Animal models, particularly conventional and conditional knockout mice, have played major roles in our understanding of the genetic controls of hematopoiesis. However, knockout mice for most of the hematopoietic transcription factors are embryonic lethal, thus precluding the analysis of their roles during the transition from embryonic to adult hematopoiesis. Zebrafish are an ideal model organism to determine the function of a gene during embryonic-to-adult transition of hematopoiesis since bloodless zebrafish embryos can develop normally into early larval stage by obtaining oxygen through diffusion. In this review, we discuss the current status of the ontogeny and regulation of hematopoiesis in zebrafish. By providing specific examples of zebrafish morphants and mutants, we have highlighted the contributions of the zebrafish model to our overall understanding of the roles of transcription factors in regulation of primitive and definitive hematopoiesis. PMID:22888355

  5. In vivo cell biology in zebrafish – providing insights into vertebrate development and disease

    PubMed Central

    Vacaru, Ana M.; Unlu, Gokhan; Spitzner, Marie; Mione, Marina; Knapik, Ela W.; Sadler, Kirsten C.

    2014-01-01

    ABSTRACT Over the past decades, studies using zebrafish have significantly advanced our understanding of the cellular basis for development and human diseases. Zebrafish have rapidly developing transparent embryos that allow comprehensive imaging of embryogenesis combined with powerful genetic approaches. However, forward genetic screens in zebrafish have generated unanticipated findings that are mirrored by human genetic studies: disruption of genes implicated in basic cellular processes, such as protein secretion or cytoskeletal dynamics, causes discrete developmental or disease phenotypes. This is surprising because many processes that were assumed to be fundamental to the function and survival of all cell types appear instead to be regulated by cell-specific mechanisms. Such discoveries are facilitated by experiments in whole animals, where zebrafish provides an ideal model for visualization and manipulation of organelles and cellular processes in a live vertebrate. Here, we review well-characterized mutants and newly developed tools that underscore this notion. We focus on the secretory pathway and microtubule-based trafficking as illustrative examples of how studying cell biology in vivo using zebrafish has broadened our understanding of the role fundamental cellular processes play in embryogenesis and disease. PMID:24481493

  6. Impact of CdSe/ZnS quantum dots on the development of zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Lei, Yong; Xiao, Qi; Huang, Shan; Xu, Wansu; Zhang, Zhe; He, Zhike; Liu, Yi; Deng, Fengjiao

    2011-12-01

    Due to their unique fluorescent characteristics, quantum dots (QDs) have been successfully applied in the fields of biotechnology and medicine, but there is very limited information regarding their biodistribution and chronic toxicity in vivo. In this article, the biological behavior and toxic effects of mercaptoacetic acid-CdSe/ZnS QDs (MAA-QDs) in developing zebrafish embryos were investigated by in vivo tests. The MAA-QDs were introduced into zebrafish through microinjection at early stage. The results showed that the MAA-QDs at certain concentrations influenced the survival of zebrafish embryos, but treated embryos without developmental defects were also observed. MAA-QDs injected into the cytoplasm at the one-cell stage were allocated to progeny blastoderm cells during proliferation and almost never entered the yolk. The formation of notochord and primordial germ cells with normal morphologies was detected in the treated embryos by whole-mount in situ hybridization. Furthermore, traces of the element cadmium were mainly discovered in the tissue of liver and kidney of 3-month-old-treated zebrafish by quantitative assessment with inductively coupled plasma mass spectrometry. Thus, we hypothesized that low concentration MAA-QDs have chronic toxicities when they were delivered into zebrafish organs.

  7. Properties of the Visible Light Phototaxis and UV Avoidance Behaviors in the Larval Zebrafish.

    PubMed

    Guggiana-Nilo, Drago A; Engert, Florian

    2016-01-01

    For many organisms, color is an essential source of information from visual scenes. The larval zebrafish has the potential to be a model for the study of this topic, given its tetrachromatic retina and high dependence on vision. In this study we took a step toward understanding how the larval zebrafish might use color sensing. To this end, we used a projector-based paradigm to force a choice of a color stimulus at every turn of the larva. The stimuli used spanned most of the larval spectral range, including activation of its Ultraviolet (UV) cone, which has not been described behaviorally before. We found that zebrafish larvae swim toward visible wavelengths (>400 nm) when choosing between them and darkness, as has been reported with white light. However, when presented with UV light and darkness zebrafish show an intensity dependent avoidance behavior. This UV avoidance does not interact cooperatively with phototaxis toward longer wavelengths, but can compete against it in an intensity dependent manner. Finally, we show that the avoidance behavior depends on the presence of eyes with functional UV cones. These findings open future avenues for studying the neural circuits that underlie color sensing in the larval zebrafish.

  8. Stab wound injury of the zebrafish adult telencephalon: a method to investigate vertebrate brain neurogenesis and regeneration.

    PubMed

    Schmidt, Rebecca; Beil, Tanja; Strähle, Uwe; Rastegar, Sepand

    2014-08-04

    Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury. In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

  9. Mechanistic insights into the effect of nanoparticles on zebrafish hatch.

    PubMed

    Ong, Kimberly Jessica; Zhao, Xinxin; Thistle, Maria E; Maccormack, Tyson J; Clark, Rhett J; Ma, Guibin; Martinez-Rubi, Yadienka; Simard, Benoit; Loo, Joachim Say Chye; Veinot, Jonathan G C; Goss, Greg G

    2014-05-01

    Aquatic organisms are susceptible to waterborne nanoparticles (NP) and there is only limited understanding of the mechanisms by which these emerging contaminants may affect biological processes. This study used silicon (nSi), cadmium selenide (nCdSe), silver (nAg) and zinc NPs (nZnO) as well as single-walled carbon nanotubes (SWCNT) to assess NP effects on zebrafish (Danio rerio) hatch. Exposure of 10 mg/L nAg and nCdSe delayed zebrafish hatch and 100 mg/L of nCdSe as well as 10 and 100 mg/L of uncoated nZnO completely inhibited hatch and the embryos died within the chorion. Both the morphology and the movement of the embryos were not affected, and it was determined that the main mechanism of hatch inhibition by NPs is likely through the interaction of NPs with the zebrafish hatching enzyme. Furthermore, it was concluded that the observed effects arose from the NPs themselves and not their dissolved metal components.

  10. Toxic effects of brominated indoles and phenols on zebrafish embryos.

    PubMed

    Kammann, U; Vobach, M; Wosniok, W

    2006-07-01

    Organobromine compounds in the marine environment have been the focus of growing attention in past years. In contrast to anthropogenic brominated flame retardants, other brominated compounds are produced naturally, e.g., by common polychaete worms and algae. Brominated phenols and indoles assumed to be of biogenic origin have been detected in water and sediment extracts from the German Bight. These substances as well as some of their isomers have been tested with the zebrafish embryo test and were found to cause lethal as well as nonlethal malformations. The zebrafish test was able to detect a log K(OW)-related toxicity for bromophenols, suggesting nonpolar narcosis as a major mode of action. Different effect patterns could be observed for brominated indoles and bromophenols. The comparison of effective concentrations in the zebrafish embryo test with the concentrations determined in water samples suggests the possibility that brominated indoles may affect early life stages of marine fish species in the North Sea.

  11. Effects of piracetam on behavior and memory in adult zebrafish.

    PubMed

    Grossman, Leah; Stewart, Adam; Gaikwad, Siddharth; Utterback, Eli; Wu, Nadine; Dileo, John; Frank, Kevin; Hart, Peter; Howard, Harry; Kalueff, Allan V

    2011-04-25

    Piracetam, a derivative of γ-aminobutyric acid, exerts memory-enhancing and mild anxiolytic effects in human and rodent studies. To examine the drug's behavioral profile further, we assessed its effects on behavioral and endocrine (cortisol) responses of adult zebrafish (Danio rerio)--a novel model species rapidly gaining popularity in neurobehavioral research. Overall, acute piracetam did not affect zebrafish novel tank and light-dark box behavior at mild doses (25-400mg/L), but produced nonspecific behavioral inhibition at 700mg/L. No effects on cortisol levels or inter-/intra-session habituation in the novel tank test were observed for acute or chronic mild non-sedative dose of 200mg/L. In contrast, fish exposed to chronic piracetam at this dose performed significantly better in the cued learning plus-maze test. This observation parallels clinical and rodent literature on the behavioral profile of piracetam, supporting the utility of zebrafish paradigms for testing nootropic agents. Copyright © 2011 Elsevier Inc. All rights reserved.

  12. Dietary Strontium Increases Bone Mineral Density in Intact Zebrafish (Danio rerio): A Potential Model System for Bone Research

    PubMed Central

    Padgett-Vasquez, Steve; Garris, Heath W.; Nagy, Tim R.; D'Abramo, Louis R.; Watts, Stephen A.

    2010-01-01

    Abstract Zebrafish (Danio rerio) skeletal bone possesses properties similar to human bone, which suggests that they may be used as a model to study mineralization characteristics of the human Haversian system, as well as human bone diseases. One prerequisite for the use of zebrafish as an alternative osteoporotic bone model is to determine whether their bone displays functional plasticity similar to that observed in other bone models. Strontium citrate was supplemented into a laboratory-prepared diet (45% crude protein) to produce dietary strontium levels of 0%, 0.63%, 1.26%, 1.89%, and 2.43% and fed ad libitum twice daily for 12 weeks to 28-day-old intact zebrafish. Length was determined at 4-week intervals, and both weight and length were recorded at 12 weeks. At 12 weeks, seven zebrafish from each dietary level were analyzed for total bone mineral density by microcomputed tomography. Dietary strontium citrate supplementation significantly (p < 0.05) increased zebrafish whole-body and spinal column bone mineral density. In addition, trace amounts of strontium were incorporated into the scale matrix in those zebrafish that consumed strontium-supplemented diets. These findings suggest that zebrafish bone displays plasticity similar to that reported for other bone models (i.e., rat, mouse, and monkey) that received supplements of strontium compounds and zebrafish should be viewed as an increasingly valuable bone model. PMID:20874492

  13. Hedgehog signaling is required at multiple stages of zebrafish tooth development.

    PubMed

    Jackman, William R; Yoo, James J; Stock, David W

    2010-11-30

    The accessibility of the developing zebrafish pharyngeal dentition makes it an advantageous system in which to study many aspects of tooth development from early initiation to late morphogenesis. In mammals, hedgehog signaling is known to be essential for multiple stages of odontogenesis; however, potential roles for the pathway during initiation of tooth development or in later morphogenesis are incompletely understood. We have identified mRNA expression of the hedgehog ligands shha and the receptors ptc1 and ptc2 during zebrafish pharyngeal tooth development. We looked for, but did not detect, tooth germ expression of the other known zebrafish hedgehog ligands shhb, dhh, ihha, or ihhb, suggesting that as in mammals, only Shh participates in zebrafish tooth development. Supporting this idea, we found that morphological and gene expression evidence of tooth initiation is eliminated in shha mutant embryos, and that morpholino antisense oligonucleotide knockdown of shha, but not shhb, function prevents mature tooth formation. Hedgehog pathway inhibition with the antagonist compound cyclopamine affected tooth formation at each stage in which we applied it: arresting development at early stages and disrupting mature tooth morphology when applied later. These results suggest that hedgehog signaling is required continuously during odontogenesis. In contrast, over-expression of shha had no effect on the developing dentition, possibly because shha is normally extensively expressed in the zebrafish pharyngeal region. We have identified previously unknown requirements for hedgehog signaling for early tooth initiation and later morphogenesis. The similarity of our results with data from mouse and other vertebrates suggests that despite gene duplication and changes in the location of where teeth form, the roles of hedgehog signaling in tooth development have been largely conserved during evolution.

  14. Developmental exposure to acetaminophen does not induce hyperactivity in zebrafish larvae.

    PubMed

    Reuter, Isabel; Knaup, Sabine; Romanos, Marcel; Lesch, Klaus-Peter; Drepper, Carsten; Lillesaar, Christina

    2016-08-01

    First line pain relief medication during pregnancy relies nearly entirely on the over-the-counter analgesic acetaminophen, which is generally considered safe to use during gestation. However, recent epidemiological studies suggest a risk of developing attention-deficit/hyperactivity disorder (ADHD)-like symptoms in children if mothers use acetaminophen during pregnancy. Currently, there are no experimental proofs that prenatal acetaminophen exposure causes developmental brain alterations of progeny. Exposure to high acetaminophen concentrations causes liver toxicity, which is well investigated in different model organisms. However, sub-liver-toxic concentrations have not been experimentally investigated with respect to ADHD endophenotypes such as hyperactivity. We used zebrafish to investigate the potential impact of acetaminophen exposure on locomotor activity levels, and compared it to the established zebrafish Latrophilin 3 (Lphn3) ADHD-model. We determined the sub-liver-toxic concentration of acetaminophen in zebrafish larvae and treated wild-type and lphn3.1 knockdown larvae with increasing concentrations of acetaminophen. We were able to confirm that lphn3.1 knockdown alone causes hyperactivity, strengthening the implication of Lphn3 dysfunction as an ADHD risk factor. Neither acute nor chronic exposure to acetaminophen at sub-liver-toxic concentrations in wild-type or lphn3.1 knock-downs increases locomotor activity levels. Together our findings show that embryonic to larval exposure to acetaminophen does not cause hyperactivity in zebrafish larvae. Furthermore, there are no additive and/or synergistic effects of acetaminophen exposure in a susceptible background induced by knock-down of lphn3.1. Our experimental study suggests that there is, at least in zebrafish larvae, no direct link between embryonic acetaminophen exposure and hyperactivity. Further work is necessary to clarify this issue in humans.

  15. 3D Finite Element Electrical Model of Larval Zebrafish ECG Signals

    PubMed Central

    Crowcombe, James; Dhillon, Sundeep Singh; Hurst, Rhiannon Mary; Egginton, Stuart; Müller, Ferenc; Sík, Attila; Tarte, Edward

    2016-01-01

    Assessment of heart function in zebrafish larvae using electrocardiography (ECG) is a potentially useful tool in developing cardiac treatments and the assessment of drug therapies. In order to better understand how a measured ECG waveform is related to the structure of the heart, its position within the larva and the position of the electrodes, a 3D model of a 3 days post fertilisation (dpf) larval zebrafish was developed to simulate cardiac electrical activity and investigate the voltage distribution throughout the body. The geometry consisted of two main components; the zebrafish body was modelled as a homogeneous volume, while the heart was split into five distinct regions (sinoatrial region, atrial wall, atrioventricular band, ventricular wall and heart chambers). Similarly, the electrical model consisted of two parts with the body described by Laplace’s equation and the heart using a bidomain ionic model based upon the Fitzhugh-Nagumo equations. Each region of the heart was differentiated by action potential (AP) parameters and activation wave conduction velocities, which were fitted and scaled based on previously published experimental results. ECG measurements in vivo at different electrode recording positions were then compared to the model results. The model was able to simulate action potentials, wave propagation and all the major features (P wave, R wave, T wave) of the ECG, as well as polarity of the peaks observed at each position. This model was based upon our current understanding of the structure of the normal zebrafish larval heart. Further development would enable us to incorporate features associated with the diseased heart and hence assist in the interpretation of larval zebrafish ECGs in these conditions. PMID:27824910

  16. Triphasic low-dose response in zebrafish embryos irradiated by microbeam protons.

    PubMed

    Choi, Viann Wing Yan; Yum, Emily Hoi Wa; Konishi, Teruaki; Oikawa, Masakazu; Cheng, Shuk Han; Yu, Kwan Ngok

    2012-01-01

    The microbeam irradiation system (Single-Particle Irradiation System to Cell, acronym as SPICE) at the National Institute of Radiological Sciences (NIRS), Japan, was employed to irradiate dechorionated zebrafish embryos at the 2-cell stage at 0.75 h post fertilization (hpf) by microbeam protons. Either one or both of the cells of the embryos were irradiated with 10, 20, 40, 50, 80, 100, 160, 200, 300 and 2000 protons each with an energy of 3.37 MeV. The embryos were then returned back to the incubator until 24 hpf for analyses. The levels of apoptosis in zebrafish embryos at 25 hpf were quantified through terminal dUTP transferase-mediated nick end-labeling (TUNEL) assay, with the apoptotic signals captured by a confocal microscope. The results revealed a triphasic dose-response for zebrafish embryos with both cells irradiated at the 2-cell stage, namely, (1) increase in apoptotic signals for < 200 protons (< 30 mGy), (2) hormesis to reduce the apoptotic signals below the spontaneous number for 200-400 protons (at doses of 30-60 mGy), and (3) increase in apoptotic signals again for > 600 protons (at doses > 90 mGy). The dose response for zebrafish embryos with only one cell irradiated at the 2-cell stage was also likely a triphasic one, but the apoptotic signals in the first zone (< 200 protons or < 30 mGy) did not have significant differences from those of the background. At the same time, the experimental data were in line with induction of radiation-induced bystander effect as well as rescue effect in the zebrafish embryos, particular in those embryos with unirradiated cells.

  17. The role of Fanconi anemia/BRCA genes in zebrafish sex determination.

    PubMed

    Rodríguez-Marí, Adriana; Postlethwait, John H

    2011-01-01

    Fanconi anemia (FA) is a human disease of bone marrow failure, leukemia, squamous cell carcinoma, and developmental anomalies, including hypogonadism and infertility. Bone marrow transplants improve hematopoietic phenotypes but do not prevent other cancers. FA arises from mutation in any of the 15 FANC genes that cooperate to repair double stranded DNA breaks by homologous recombination. Zebrafish has a single ortholog of each human FANC gene and unexpectedly, mutations in at least two of them (fancl and fancd1(brca2)) lead to female-to-male sex reversal. Investigations show that, as in human, zebrafish fanc genes are required for genome stability and for suppressing apoptosis in tissue culture cells, in embryos treated with DNA damaging agents, and in meiotic germ cells. The sex reversal phenotype requires the action of Tp53 (p53), an activator of apoptosis. These results suggest that in normal sex determination, zebrafish oocytes passing through meiosis signal the gonadal soma to maintain expression of aromatase, an enzyme that converts androgen to estrogen, thereby feminizing the gonad and the individual. According to this model, normal male and female zebrafish differ in genetic factors that control the strength of the late meiotic oocyte-derived signal, probably by regulating the number of meiotic oocytes, which environmental factors can also alter. Transcripts from fancd1(brca2) localize at the animal pole of the zebrafish oocyte cytoplasm and are required for normal oocyte nuclear architecture, for normal embryonic development, and for preventing ovarian tumors. Embryonic DNA repair and sex reversal phenotypes provide assays for the screening of small molecule libraries for therapeutic substances for FA. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Using visual lateralization to model learning and memory in zebrafish larvae

    PubMed Central

    Andersson, Madelene Åberg; Ek, Fredrik; Olsson, Roger

    2015-01-01

    Impaired learning and memory are common symptoms of neurodegenerative and neuropsychiatric diseases. Present, there are several behavioural test employed to assess cognitive functions in animal models, including the frequently used novel object recognition (NOR) test. However, although atypical functional brain lateralization has been associated with neuropsychiatric conditions, spanning from schizophrenia to autism, few animal models are available to study this phenomenon in learning and memory deficits. Here we present a visual lateralization NOR model (VLNOR) in zebrafish larvae as an assay that combines brain lateralization and NOR. In zebrafish larvae, learning and memory are generally assessed by habituation, sensitization, or conditioning paradigms, which are all representatives of nondeclarative memory. The VLNOR is the first model for zebrafish larvae that studies a memory similar to the declarative memory described for mammals. We demonstrate that VLNOR can be used to study memory formation, storage, and recall of novel objects, both short and long term, in 10-day-old zebrafish. Furthermore we show that the VLNOR model can be used to study chemical modulation of memory formation and maintenance using dizocilpine (MK-801), a frequently used non-competitive antagonist of the NMDA receptor, used to test putative antipsychotics in animal models. PMID:25727677

  19. Using Zebrafish to Test the Genetic Basis of Human Craniofacial Diseases.

    PubMed

    Machado, R Grecco; Eames, B Frank

    2017-10-01

    Genome-wide association studies (GWASs) opened an innovative and productive avenue to investigate the molecular basis of human craniofacial disease. However, GWASs identify candidate genes only; they do not prove that any particular one is the functional villain underlying disease or just an unlucky genomic bystander. Genetic manipulation of animal models is the best approach to reveal which genetic loci identified from human GWASs are functionally related to specific diseases. The purpose of this review is to discuss the potential of zebrafish to resolve which candidate genetic loci are mechanistic drivers of craniofacial diseases. Many anatomic, embryonic, and genetic features of craniofacial development are conserved among zebrafish and mammals, making zebrafish a good model of craniofacial diseases. Also, the ability to manipulate gene function in zebrafish was greatly expanded over the past 20 y, enabling systems such as Gateway Tol2 and CRISPR-Cas9 to test gain- and loss-of-function alleles identified from human GWASs in coding and noncoding regions of DNA. With the optimization of genetic editing methods, large numbers of candidate genes can be efficiently interrogated. Finding the functional villains that underlie diseases will permit new treatments and prevention strategies and will increase understanding of how gene pathways operate during normal development.

  20. Zebrafish Cardiac Muscle Thick Filaments: Isolation Technique and Three-Dimensional Structure

    PubMed Central

    González-Solá, Maryví; AL-Khayat, Hind A.; Behra, Martine; Kensler, Robert W.

    2014-01-01

    To understand how mutations in thick filament proteins such as cardiac myosin binding protein-C or titin, cause familial hypertrophic cardiomyopathies, it is important to determine the structure of the cardiac thick filament. Techniques for the genetic manipulation of the zebrafish are well established and it has become a major model for the study of the cardiovascular system. Our goal is to develop zebrafish as an alternative system to the mammalian heart model for the study of the structure of the cardiac thick filaments and the proteins that form it. We have successfully isolated thick filaments from zebrafish cardiac muscle, using a procedure similar to those for mammalian heart, and analyzed their structure by negative-staining and electron microscopy. The isolated filaments appear well ordered with the characteristic 42.9 nm quasi-helical repeat of the myosin heads expected from x-ray diffraction. We have performed single particle image analysis on the collected electron microscopy images for the C-zone region of these filaments and obtained a three-dimensional reconstruction at 3.5 nm resolution. This reconstruction reveals structure similar to the mammalian thick filament, and demonstrates that zebrafish may provide a useful model for the study of the changes in the cardiac thick filament associated with disease processes. PMID:24739166

  1. Combined toxicity of silica nanoparticles and methylmercury on cardiovascular system in zebrafish (Danio rerio) embryos.

    PubMed

    Duan, Junchao; Hu, Hejing; Li, Qiuling; Jiang, Lizhen; Zou, Yang; Wang, Yapei; Sun, Zhiwei

    2016-06-01

    This study was to investigate the combined toxicity of silica nanoparticles (SiNPs) and methylmercury (MeHg) on cardiovascular system in zebrafish (Danio rerio) embryos. Ultraviolet absorption analysis showed that the co-exposure system had high absorption and stability. The dosages used in this study were based on the NOAEL level. Zebrafish embryos exposed to the co-exposure of SiNPs and MeHg did not show any cardiovascular malformation or atrioventricular block, but had an inhibition effect on bradycardia. Using o-Dianisidine for erythrocyte staining, the cardiac output of zebrafish embryos was decreased gradually in SiNPs, MeHg, co-exposure groups, respectively. Co-exposure of SiNPs and MeHg enhanced the vascular endothelial damage in Tg(fli-1:EGFP) transgenic zebrafish line. Moreover, the co-exposure significantly activated the oxidative stress and inflammatory response in neutrophils-specific Tg(mpo:GFP) transgenic zebrafish line. This study suggested that the combined toxic effects of SiNPs and MeHg on cardiovascular system had more severe toxicity than the single exposure alone. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Smoc2 modulates embryonic myelopoiesis during zebrafish development.

    PubMed

    Mommaerts, Hendrik; Esguerra, Camila V; Hartmann, Ursula; Luyten, Frank P; Tylzanowski, Przemko

    2014-11-01

    SMOC2 is a member of the BM-40 (SPARC) family of matricellular proteins, reported to influence signaling in the extracellular compartment. In mice, Smoc2 is expressed in many different tissues and was shown to enhance the response to angiogenic growth factors, mediate cell adhesion, keratinocyte migration, and metastasis. Additionally, SMOC2 is associated with vitiligo and craniofacial and dental defects. The function of Smoc2 during early zebrafish development has not been determined to date. In pregastrula zebrafish embryos, smoc2 is expressed ubiquitously. As development progresses, the expression pattern becomes more anteriorly restricted. At the onset of blood cell circulation, smoc2 morphants presented a mild ventralization of posterior structures. Molecular analysis of the smoc2 morphants indicated myelopoietic defects in the rostral blood islands during segmentation stages. Hemangioblast development and further specification of the myeloid progenitor cells were shown to be impaired. Additional experiments indicated that Bmp target genes were down-regulated in smoc2 morphants. Our findings reveal that Smoc2 is an essential player in the development of myeloid cells of the anterior lateral plate mesoderm during embryonic zebrafish development. Furthermore, our data show that Smoc2 affects the transcription of Bmp target genes without affecting initial dorsoventral patterning or mesoderm development. Copyright © 2014 Wiley Periodicals, Inc.

  3. The common neural parasite Pseudoloma neurophilia is associated with altered startle response habituation in adult zebrafish (Danio rerio): Implications for the zebrafish as a model organism.

    PubMed

    Spagnoli, Sean; Xue, Lan; Kent, Michael L

    2015-09-15

    The zebrafish's potential as a model for human neurobehavioral research appears nearly limitless despite its relatively recent emergence as an experimental organism. Since the zebrafish has only been part of the research community for a handful of decades, pathogens from its commercial origins continue to plague laboratory stocks. One such pathogen is Pseudoloma neurophilia, a common microparasite in zebrafish laboratories world-wide that generally produces subclinical infections. Given its high prevalence, its predilection for the host's brain and spinal cord, and the delicate nature of neurobehavioral research, the behavioral consequences of subclinical P. neurophilia infection must be explored. Fish infected via cohabitation were tested for startle response habituation in parallel with controls in a device that administered ten taps over 10 min along with taps at 18 and 60 min to evaluate habituation extinction. After testing, fish were euthanized and evaluated for infection via histopathology. Infected fish had a significantly smaller reduction in startle velocity during habituation compared to uninfected tankmates and controls. Habituation was eliminated in infected and control fish at 18 min, whereas exposed negative fish retained partial habituation at 18 min. Infection was also associated with enhanced capture evasion: Despite the absence of external symptoms, infected fish tended to be caught later than uninfected fish netted from the same tank. The combination of decreased overall habituation, early extinction of habituation compared to uninfected cohorts, and enhanced netting evasion indicates that P. neurophilia infection is associated with a behavioral phenotype distinct from that of controls and uninfected cohorts. Because of its prevalence in zebrafish facilities, P. neurophilia has the potential to insidiously influence a wide range of neurobehavioral studies if these associations are causative. Rigorous health screening is therefore vital to the

  4. The DVR-1 (Vg1) transcript of zebrafish is maternally supplied and distributed throughout the embryo.

    PubMed

    Helde, K A; Grunwald, D J

    1993-10-01

    It is not known how region- or tissue-specific differences are generated in the zebrafish embryo. To look at the potential role of maternal transcripts in generating cell diversity, we have isolated and characterized the zebrafish homologue of Xenopus DVR-1 (Vg1), a maternally supplied RNA that encodes a member of the transforming growth factor-beta superfamily. The zebrafish DVR-1 RNA is maternally supplied and its protein product shares a high degree of sequence identity with Xenopus DVR-1. These conserved features indicate that DVR-1 is likely to have an essential function in early embryogenesis. However, unlike the frog transcript, which is restricted to vegetal cells, DVR-1 RNA is distributed equally among all zebrafish blastomeres. We suggest that the ubiquitous distribution of DVR-1 RNA reflects a significant aspect of the developmental strategy of the zebrafish in which each blastomere retains an equivalent developmental potential throughout the cleavage period.

  5. Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota.

    PubMed

    Bates, Jennifer M; Akerlund, Janie; Mittge, Erika; Guillemin, Karen

    2007-12-13

    Vertebrates harbor abundant lipopolysaccharide (LPS) in their gut microbiota. Alkaline phosphatases can dephosphorylate and detoxify the endotoxin component of LPS. Here, we show that expression of the zebrafish intestinal alkaline phosphatase (Iap), localized to the intestinal lumen brush border, is induced during establishment of the gut microbiota. Iap-deficient zebrafish are hypersensitive to LPS toxicity and exhibit the excessive intestinal neutrophil influx characteristic of wild-type zebrafish exposed to LPS. Both of these Iap mutant phenotypes are dependent on Myd88 and Tumor Necrosis Factor Receptor (Tnfr), proteins also involved in LPS sensitivity in mammals. When reared germ-free, the intestines of Iap-deficient zebrafish are devoid of neutrophils. Together, these findings demonstrate that the endogenous microbiota establish the normal homeostatic level of neutrophils in the zebrafish intestine through a process involving Iap, Myd88, and Tnfr. Thus, by preventing inflammatory responses, Iap plays a crucial role in promoting mucosal tolerance to resident gut bacteria.

  6. E2F4 Promotes Neuronal Regeneration and Functional Recovery after Spinal Cord Injury in Zebrafish

    PubMed Central

    Sasagawa, Shota; Nishimura, Yuhei; Hayakawa, Yuka; Murakami, Soichiro; Ashikawa, Yoshifumi; Yuge, Mizuki; Okabe, Shiko; Kawaguchi, Koki; Kawase, Reiko; Tanaka, Toshio

    2016-01-01

    Mammals exhibit poor recovery after spinal cord injury (SCI), whereas non-mammalian vertebrates exhibit significant spontaneous recovery after SCI. The mechanisms underlying this difference have not been fully elucidated; therefore, the purpose of this study was to investigate these mechanisms. Using comparative transcriptome analysis, we demonstrated that genes related to cell cycle were significantly enriched in the genes specifically dysregulated in zebrafish SCI. Most of the cell cycle-related genes dysregulated in zebrafish SCI were down-regulated, possibly through activation of e2f4. Using a larval zebrafish model of SCI, we demonstrated that the recovery of locomotive function and neuronal regeneration after SCI were significantly inhibited in zebrafish treated with an E2F4 inhibitor. These results suggest that activation of e2f4 after SCI may be responsible, at least in part, for the significant recovery in zebrafish. This provides novel insight into the lack of recovery after SCI in mammals and informs potential therapeutic strategies. PMID:27242526

  7. Primary Spinal OPC Culture System from Adult Zebrafish to Study Oligodendrocyte Differentiation In Vitro.

    PubMed

    Kroehne, Volker; Tsata, Vasiliki; Marrone, Lara; Froeb, Claudia; Reinhardt, Susanne; Gompf, Anne; Dahl, Andreas; Sterneckert, Jared; Reimer, Michell M

    2017-01-01

    Endogenous oligodendrocyte progenitor cells (OPCs) are a promising target to improve functional recovery after spinal cord injury (SCI) by remyelinating denuded, and therefore vulnerable, axons. Demyelination is the result of a primary insult and secondary injury, leading to conduction blocks and long-term degeneration of the axons, which subsequently can lead to the loss of their neurons. In response to SCI, dormant OPCs can be activated and subsequently start to proliferate and differentiate into mature myelinating oligodendrocytes (OLs). Therefore, researchers strive to control OPC responses, and utilize small molecule screening approaches in order to identify mechanisms of OPC activation, proliferation, migration and differentiation. In zebrafish, OPCs remyelinate axons of the optic tract after lysophosphatidylcholine (LPC)-induced demyelination back to full thickness myelin sheaths. In contrast to zebrafish, mammalian OPCs are highly vulnerable to excitotoxic stress, a cause of secondary injury, and remyelination remains insufficient. Generally, injury induced remyelination leads to shorter internodes and thinner myelin sheaths in mammals. In this study, we show that myelin sheaths are lost early after a complete spinal transection injury, but are re-established within 14 days after lesion. We introduce a novel, easy-to-use, inexpensive and highly reproducible OPC culture system based on dormant spinal OPCs from adult zebrafish that enables in vitro analysis. Zebrafish OPCs are robust, can easily be purified with high viability and taken into cell culture. This method enables to examine why zebrafish OPCs remyelinate better than their mammalian counterparts, identify cell intrinsic responses, which could lead to pro-proliferating or pro-differentiating strategies, and to test small molecule approaches. In this methodology paper, we show efficient isolation of OPCs from adult zebrafish spinal cord and describe culture conditions that enable analysis up to 10

  8. Development of an opioid self-administration assay to study drug seeking in zebrafish.

    PubMed

    Bossé, Gabriel D; Peterson, Randall T

    2017-09-29

    The zebrafish (Danio rerio) has become an excellent tool to study mental health disorders, due to its physiological and genetic similarity to humans, ease of genetic manipulation, and feasibility of small molecule screening. Zebrafish have been shown to exhibit characteristics of addiction to drugs of abuse in non-contingent assays, including conditioned place preference, but contingent assays have been limited to a single assay for alcohol consumption. Using inexpensive electronic, mechanical, and optical components, we developed an automated opioid self-administration assay for zebrafish, enabling us to measure drug seeking and gain insight into the underlying biological pathways. Zebrafish trained in the assay for five days exhibited robust self-administration, which was dependent on the function of the μ-opioid receptor. In addition, a progressive ratio protocol was used to test conditioned animals for motivation. Furthermore, conditioned fish continued to seek the drug despite an adverse consequence and showed signs of stress and anxiety upon withdrawal of the drug. Finally, we validated our assay by confirming that self-administration in zebrafish is dependent on several of the same molecular pathways as in other animal models. Given the ease and throughput of this assay, it will enable identification of important biological pathways regulating drug seeking and could lead to the development of new therapeutic molecules to treat addiction. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. simUfish: An Interactive Application to Teach K-12 Students About Zebrafish Behavior.

    PubMed

    Mwaffo, Violet; Korneyeva, Veronika; Porfiri, Maurizio

    2017-10-01

    As the zebrafish is rapidly becoming a species of choice in preclinical research, several efforts are being placed toward creating educational programs for K-12 students based on this promising model organism. However, as any other model organisms, the use of zebrafish in classroom settings requires additional experimental resources and poses ethical challenges related to animal use. To mitigate these factors, we have developed an application (app), simUfish, which implements a mathematical model of zebrafish behavior for generating multiple fish trajectories and animating their body undulations. simUfish is developed using a multiplatform game engine and is expected to promote the knowledge of zebrafish behavior to both K-12 students and the general public. Specifically, it demonstrates basic principles of fish individual and social behaviors, including environment interaction; fear response toward a predator; shoaling; and attraction toward a stimulus, which can be a food source or simply a finger placed on the touch screen. The effectiveness of the app as an accessible experimental tool for learning was tested in an outreach activity on middle school students from the New York City school system. The results from this activity show an immediate, tangible improvement of students' satisfaction and willingness to learn about key concepts on zebrafish behavior, accompanied by high level of interest in life sciences.

  10. Glucocorticoid activity detected by in vivo zebrafish assay and in vitro glucocorticoid receptor bioassay at environmental relevant concentrations.

    PubMed

    Chen, Qiyu; Jia, Ai; Snyder, Shane A; Gong, Zhiyuan; Lam, Siew Hong

    2016-02-01

    Glucocorticoids are pharmaceutical contaminants of emerging concern due to their incomplete removal during wastewater treatment, increased presence in aquatic environment and their biological potency. The zebrafish is a popular model for aquatic toxicology and environmental risk assessment. This study aimed to determine if glucocorticoids at environmental concentrations would perturb expression of selected glucocorticoid-responsive genes in zebrafish and to investigate their potentials as an in vivo zebrafish assay in complementing in vitro glucocorticoid receptor bioassay. The relative expression of eleven glucocorticoid-responsive genes in zebrafish larvae and liver of adult male zebrafish exposed to three representative glucocorticoids (dexamethasone, prednisolone and triamcinolone) was determined. The expression of pepck, baiap2 and pxr was up-regulated in zebrafish larvae and the expression of baiap2, pxr and mmp-2 was up-regulated in adult zebrafish exposed to glucocorticoids at concentrations equivalent to total glucocorticoids reported in environmental samples. The responsiveness of the specific genes were sufficiently robust in zebrafish larvae exposed to a complex environmental sample detected with in vitro glucocorticoid activity equivalent to 478 pM dexamethasone (DEX-EQ) and confirmed to contain low concentration (0.2 ng/L or less) of the targeted glucocorticoids, and possibly other glucocorticoid-active compounds. The findings provided in vivo relevance to the in vitro glucocorticoid activity and suggested that the environmental sample can perturb glucocorticoid-responsive genes in its original, or half the diluted, concentration as may be found in the environment. The study demonstrated the important complementary roles of in vivo zebrafish and in vitro bioassays coupled with analytical chemistry in monitoring environmental glucocorticoid contaminants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Report of the Second European Zebrafish Principal Investigator Meeting in Karlsruhe, Germany, March 21-24, 2012.

    PubMed

    Cavodeassi, Florencia; Del Bene, Filippo; Fürthauer, Maximilian; Grabher, Clemens; Herzog, Wiebke; Lehtonen, Sanna; Linker, Claudia; Mercader, Nadia; Mikut, Ralf; Norton, William; Strähle, Uwe; Tiso, Natascia; Foulkes, Nicholas S

    2013-03-01

    The second European Zebrafish Principal Investigator (PI) Meeting was held in March, 2012, in Karlsruhe, Germany. It brought together PIs from all over Europe who work with fish models such as zebrafish and medaka to discuss their latest results, as well as to resolve strategic issues faced by this research community. Scientific discussion ranged from the development of new technologies for working with fish models to progress in various fields of research such as injury and repair, disease models, and cell polarity and dynamics. This meeting also marked the establishment of the European Zebrafish Resource Centre (EZRC) at Karlsruhe that in the future will serve as an important focus and community resource for zebrafish- and medaka-based research.

  12. In Vivo Cardiotoxicity Induced by Sodium Aescinate in Zebrafish Larvae.

    PubMed

    Liang, Jinfeng; Jin, Wangdong; Li, Hongwen; Liu, Hongcui; Huang, Yanfeng; Shan, Xiaowen; Li, Chunqi; Shan, Letian; Efferth, Thomas

    2016-02-23

    Sodium aescinate (SA) is a widely-applied triterpene saponin product derived from horse chestnut seeds, possessing vasoactive and organ-protective activities with oral or injection administration in the clinic. To date, no toxicity or adverse events in SA have been reported, by using routine models (in vivo or in vitro), which are insufficient to predict all aspects of its pharmacological and toxicological actions. In this study, taking advantage of transparent zebrafish larvae (Danio rerio), we evaluated cardiovascular toxicity of SA at doses of 1/10 MNLC, 1/3 MNLC, MNLC and LC10 by yolk sac microinjection. The qualitative and quantitative cardiotoxicity in zebrafish was assessed at 48 h post-SA treatment, using specific phenotypic endpoints: heart rate, heart rhythm, heart malformation, pericardial edema, circulation abnormalities, thrombosis and hemorrhage. The results showed that SA at 1/10 MNLC and above doses could induce obvious cardiac and pericardial malformations, whilst 1/3 MNLC and above doses could induce significant cardiac malfunctions (heart rate and circulation decrease/absence), as compared to untreated or vehicle-treated control groups. Such cardiotoxic manifestations occurred in more than 50% to 100% of all zebrafish treated with SA at MNLC and LC10. Our findings have uncovered the potential cardiotoxicity of SA for the first time, suggesting more attention to the risk of its clinical application. Such a time- and cost-saving zebrafish cardiotoxicity assay is very valid and reliable for rapid prediction of compound toxicity during drug research and development.

  13. Toxicity of chlorine to zebrafish embryos

    PubMed Central

    Kent, Michael L.; Buchner, Cari; Barton, Carrie; Tanguay, Robert L.

    2014-01-01

    Surface disinfection of fertilized fish eggs is widely used in aquaculture to reduce extraovum pathogens that may be released from brood fish during spawning, and this is routinely used in zebrafish (Danio rerio) research laboratories. Most laboratories use approximately 25 – 50 ppm unbuffered chlorine solution for 5 – 10 min. Treatment of embryos with chlorine has significant germicidal effects for many Gram-negative bacteria, viruses, and trophozoite stages of protozoa, it has reduced efficacy against cyst or spore stages of protozoa and certain Mycobacterium spp. Therefore, we evaluated the toxicity of unbufferred and buffered chlorine solution to embryos exposed at 6 or 24 hours post-fertilization (hpf) to determine if higher concentrations can be used for treating zebrafish embryos. Most of our experiments entailed using an outbred line (5D), with both mortality and malformations as endpoints. We found that 6 hpf embryos consistently were more resistant than 24 hpf embryos to the toxic effects of chlorine. Chlorine is more toxic and germicidal at lower pHs, and chlorine causes elevated pH. Consistent with this, we found that unbufferred chlorine solutions (pH ca 8–9) were less toxic at corresponding concentrations than solutions buffered to pH 7. Based on our findings here, we recommend treating 6 hpf embryos for 10 min and 24 hpf for 5 min with unbuffered chlorine solution at 100 ppm. One trial indicated that AB fish, a popular outbred line, are more susceptible to toxicity than 5Ds. This suggests that variability between zebrafish lines occurs, and researchers should evaluate each line or strain under their particular laboratory conditions for selection of the optimum chlorine treatment procedure. PMID:24429474

  14. Ecological risk study on subacute toxicology experiment of streptomycin wastewater for Zebrafish

    NASA Astrophysics Data System (ADS)

    Shi, Qing; Shen, Hongyan

    2017-08-01

    An exposure experiment was conducted to study the effect of different volume fraction of effluent streptomycin wastewater on the activity of the peroxidase (POD) activity and the malondialdehyde (MDA) content in muscles of Zebrafish for 20 days. The results show that POD activity is significantly induced on the eighth day. POD activities in the muscles of Zebrafish exposed to the streptomycin wastewater of 20% exposure group were significantly different (0.01Zebrafish that exposed streptomycin wastewater ranged from 10.42 to 54.26, and the wastewater of 10% exposure group induced the maximum value on the twelve day. The study indicates that low concentration streptomycin wastewater has impacts on the antioxidant defense system and antioxidant ability of Zebrafish.

  15. Fishing for Nature's Hits: Establishment of the Zebrafish as a Model for Screening Antidiabetic Natural Products.

    PubMed

    Tabassum, Nadia; Tai, Hongmei; Jung, Da-Woon; Williams, Darren R

    2015-01-01

    Diabetes mellitus affects millions of people worldwide and significantly impacts their quality of life. Moreover, life threatening diseases, such as myocardial infarction, blindness, and renal disorders, increase the morbidity rate associated with diabetes. Various natural products from medicinal plants have shown potential as antidiabetes agents in cell-based screening systems. However, many of these potential "hits" fail in mammalian tests, due to issues such as poor pharmacokinetics and/or toxic side effects. To address this problem, the zebrafish (Danio rerio) model has been developed as a "bridge" to provide an experimentally convenient animal-based screening system to identify drug candidates that are active in vivo. In this review, we discuss the application of zebrafish to drug screening technologies for diabetes research. Specifically, the discovery of natural product-based antidiabetes compounds using zebrafish will be described. For example, it has recently been demonstrated that antidiabetic natural compounds can be identified in zebrafish using activity guided fractionation of crude plant extracts. Moreover, the development of fluorescent-tagged glucose bioprobes has allowed the screening of natural product-based modulators of glucose homeostasis in zebrafish. We hope that the discussion of these advances will illustrate the value and simplicity of establishing zebrafish-based assays for antidiabetic compounds in natural products-based laboratories.

  16. Protective effects of edaravone against cisplatin-induced hair cell damage in zebrafish.

    PubMed

    Hong, Seok Jin; Im, Gi Jung; Chang, Jiwon; Chae, Sung Won; Lee, Seung Hoon; Kwon, Soon Young; Jung, Hak Hyun; Chung, Ah Young; Park, Hae Chul; Choi, June

    2013-06-01

    Edaravone is known to have a potent free radical scavenging effect. The objective of the present study was to evaluate the effects of edaravone on cisplatin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP). Five day post-fertilization zebrafish larvae were exposed to 1000 μM cisplatin and 50 μM, 100 μM, 250 μM, 500 μM, 750 μM, and 1000 μM concentrations of edaravone for 4h. Hair cells within neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed by fluorescence microscopy and confocal microscopy (n=10). Hair cell survival was calculated as a percentage of the hair cells in the control group that were not exposed to cisplatin. Ultrastructural changes were evaluated using scanning electron microscopy and transmission electron microscopy. Edaravone protected cisplatin-induced hair cell loss of neuromasts (edaravone 750 μM: 8.7 ± 1.5 cells, cisplatin 1000 μM only: 3.7 ± 0.9 cells; n=10, p<0.0001) and decreased the Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) reaction. Structures of mitochondria and hair cell within neuromasts in ultrastructural analysis were preserved in zebrafish exposed to 1000 μM cisplatin and 750 μM edaravone for 4h. Edaravone attenuated cisplatin-induced hair cell damage in zebrafish. The results of the current study suggest that cisplatin induces apoptosis, and the apoptotic cell death can be prevented by treatment with edaravone in zebrafish. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  17. Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

    PubMed Central

    Bernut, Audrey; Dupont, Christian; Sahuquet, Alain; Herrmann, Jean-Louis; Lutfalla, Georges; Kremer, Laurent

    2015-01-01

    Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions. The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model. In this respect, the present manuscript describes the use of zebrafish as an in vivo model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate. PMID:26382225

  18. An automatic method to calculate heart rate from zebrafish larval cardiac videos.

    PubMed

    Kang, Chia-Pin; Tu, Hung-Chi; Fu, Tzu-Fun; Wu, Jhe-Ming; Chu, Po-Hsun; Chang, Darby Tien-Hao

    2018-05-09

    Zebrafish is a widely used model organism for studying heart development and cardiac-related pathogenesis. With the ability of surviving without a functional circulation at larval stages, strong genetic similarity between zebrafish and mammals, prolific reproduction and optically transparent embryos, zebrafish is powerful in modeling mammalian cardiac physiology and pathology as well as in large-scale high throughput screening. However, an economical and convenient tool for rapid evaluation of fish cardiac function is still in need. There have been several image analysis methods to assess cardiac functions in zebrafish embryos/larvae, but they are still improvable to reduce manual intervention in the entire process. This work developed a fully automatic method to calculate heart rate, an important parameter to analyze cardiac function, from videos. It contains several filters to identify the heart region, to reduce video noise and to calculate heart rates. The proposed method was evaluated with 32 zebrafish larval cardiac videos that were recording at three-day post-fertilization. The heart rate measured by the proposed method was comparable to that determined by manual counting. The experimental results show that the proposed method does not lose accuracy while largely reducing the labor cost and uncertainty of manual counting. With the proposed method, researchers do not have to manually select a region of interest before analyzing videos. Moreover, filters designed to reduce video noise can alleviate background fluctuations during the video recording stage (e.g. shifting), which makes recorders generate usable videos easily and therefore reduce manual efforts while recording.

  19. Selectivity of natural, synthetic and environmental estrogens for zebrafish estrogen receptors

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pinto, Caroline; Grimaldi, Marina; Boulahtouf, Abdelhay

    2014-10-01

    Zebrafish, Danio rerio, is increasingly used as an animal model to study the effects of pharmaceuticals and environmental estrogens. As most of these estrogens have only been tested on human estrogen receptors (ERs), it is necessary to measure their effects on zebrafish ERs. In humans there are two distinct nuclear ERs (hERα and hERβ), whereas the zebrafish genome encodes three ERs, zfERα and two zfERβs (zfERβ1 and zfERβ2). In this study, we established HeLa-based reporter cell lines stably expressing each of the three zfERs. We first reported that estrogens more efficiently activate the zfERs at 28 °C as compared tomore » 37 °C, thus reflecting the physiological temperature of zebrafish in wildlife. We then showed significant differences in the ability of agonist and antagonist estrogens to modulate activation of the three zfER isotypes in comparison to hERs. Environmental compounds (bisphenol A, alkylphenols, mycoestrogens) which are hER panagonists and hERβ selective agonists displayed greater potency for zfERα as compared to zfERβs. Among hERα selective synthetic agonists, PPT did not activate zfERα while 16α-LE2 was the most zfERα selective compound. Altogether, these results confirm that all hER ligands control in a similar manner the transcriptional activity of zfERs although significant differences in selectivity were observed among subtypes. The zfER subtype selective ligands that we identified thus represent new valuable tools to dissect the physiological roles of the different zfERs. Finally, our work also points out that care has to be taken in transposing the results obtained using the zebrafish as a model for human physiopathology. - Highlights: • Zebrafish is increasingly used to study the effects of estrogens. • We assessed the activity of pharmaceutical and environmental estrogens on zfERs. • Environmental estrogens displayed greater potency for zfERα compared to zfERβs. • hERβ selective agonists displayed greater

  20. Next generation mothers: Maternal control of germline development in zebrafish.

    PubMed

    Dosch, Roland

    2015-01-01

    In many animals, factors deposited by the mother into the egg control the earliest events in development of the zygote. These maternal RNAs and proteins play critical roles in oocyte development and the earliest steps of embryogenesis such as fertilization, cell division and embryonic patterning. Here, this article summarizes recent discoveries made on the maternal control of germline specification in zebrafish. Moreover, this review will discuss the major gaps remaining in our understanding of this process and highlight recent technical innovations in zebrafish, which allow tackling some of these questions in the near future.

  1. The neural basis of visual behaviors in the larval zebrafish

    PubMed Central

    Portugues, Ruben; Engert, Florian

    2015-01-01

    We review visually guided behaviors in larval zebrafish and summarise what is known about the neural processing that results in these behaviors, paying particular attention to the progress made in the last 2 years. Using the examples of the optokinetic reflex, the optomotor response, prey tracking and the visual startle response, we illustrate how the larval zebrafish presents us with a very promising model vertebrate system that allows neurocientists to integrate functional and behavioral studies and from which we can expect illuminating insights in the near future. PMID:19896836

  2. The neural basis of visual behaviors in the larval zebrafish.

    PubMed

    Portugues, Ruben; Engert, Florian

    2009-12-01

    We review visually guided behaviors in larval zebrafish and summarise what is known about the neural processing that results in these behaviors, paying particular attention to the progress made in the last 2 years. Using the examples of the optokinetic reflex, the optomotor response, prey tracking and the visual startle response, we illustrate how the larval zebrafish presents us with a very promising model vertebrate system that allows neurocientists to integrate functional and behavioral studies and from which we can expect illuminating insights in the near future. Copyright 2009 Elsevier Ltd. All rights reserved.

  3. Neurobehavioral impairments caused by developmental imidacloprid exposure in zebrafish.

    PubMed

    Crosby, Emily B; Bailey, Jordan M; Oliveri, Anthony N; Levin, Edward D

    2015-01-01

    Neonicotinoid insecticides are becoming more widely applied as organophosphate (OP) insecticides are decreasing in use. Because of their relative specificity to insect nicotinic receptors, they are thought to have reduced risk of neurotoxicity in vertebrates. However, there is scant published literature concerning the neurobehavioral effects of developmental exposure of vertebrates to neonicotinoids. Using zebrafish, we investigated the neurobehavioral effects of developmental exposure to imidacloprid, a prototypic neonicotinoid pesticide. Nicotine was also administered for comparison. Zebrafish were exposed via immersion in aqueous solutions containing 45 μM or 60 μM of imidacloprid or nicotine (or vehicle control) from 4h to 5d post fertilization. The functional effects of developmental exposure to both imidacloprid and nicotine were assessed in larvae using an activity assay and during adolescence and adulthood using a battery of neurobehavioral assays, including assessment of sensorimotor response and habituation in a tactile startle test, novel tank swimming, and shoaling behavior. In larvae, developmental imidacloprid exposure at both doses significantly decreased swimming activity. The 5D strains of zebrafish were more sensitive to both nicotine and imidacloprid than the AB* strain. In adolescent and adult fish, developmental exposure to imidacloprid significantly decreased novel tank exploration and increased sensorimotor response to startle stimuli. While nicotine did not affect novel tank swimming, it increased sensorimotor response to startle stimuli at the low dose. No effects of either compound were found on shoaling behavior or habituation to a startling stimulus. Early developmental exposure to imidacloprid has both early-life and persisting effects on neurobehavioral function in zebrafish. Its developmental neurotoxicity should be further investigated. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Forkhead transcription factor foxe1 regulates chondrogenesis in zebrafish.

    PubMed

    Nakada, Chisako; Iida, Atsumi; Tabata, Yoko; Watanabe, Sumiko

    2009-12-15

    Forkhead transcription factor (Fox) e1 is a causative gene for Bamforth-Lazarus syndrome, which is characterized by hypothyroidism and cleft palate. Applying degenerate polymerase chain reaction using primers specific for the conserved forkhead domain, we identified zebrafish foxe1 (foxe1). Foxe1 is expressed in the thyroid, pharynx, and pharyngeal skeleton during development; strongly expressed in the gill and weakly expressed in the brain, eye, and heart in adult zebrafish. A loss of function of foxe1 by morpholino antisense oligo (MO) exhibited abnormal craniofacial development, shortening of Meckel's cartilage and the ceratohyals, and suppressed chondrycytic proliferation. However, at 27 hr post fertilization, the foxe1 MO-injected embryos showed normal dlx2, hoxa2, and hoxb2 expression, suggesting that the initial steps of pharyngeal skeletal development, including neural crest migration and specification of the pharyngeal arch occurred normally. In contrast, at 2 dpf, a severe reduction in the expression of sox9a, colIIaI, and runx2b, which play roles in chondrocytic proliferation and differentiation, was observed. Interestingly, fgfr2 was strongly upregulated in the branchial arches of the foxe1 MO-injected embryos. Unlike Foxe1-null mice, normal thyroid development in terms of morphology and thyroid-specific marker expression was observed in foxe1 MO-injected zebrafish embryos. Taken together, our results indicate that Foxe1 plays an important role in chondrogenesis during development of the pharyngeal skeleton in zebrafish, probably through regulation of fgfr2 expression. Furthermore, the roles reported for FOXE1 in mammalian thyroid development may have been acquired during evolution. (c) 2009 Wiley-Liss, Inc.

  5. Neurobehavioral Impairments Caused by Developmental Imidacloprid Exposure in Zebrafish

    PubMed Central

    Crosby, Emily B.; Bailey, Jordan M.; Oliveri, Anthony N.; Levin, Edward D.

    2015-01-01

    BACKGROUND Neonicotinoid insecticides are becoming more widely applied as organophosphate (OP) insecticides are decreasing in use. Because of their relative specificity to insect nicotinic receptors, they are thought to have reduced risk of neurotoxicity in vertebrates. However, there is scant published literature concerning the neurobehavioral effects of developmental exposure of vertebrates to neonicotinoids. METHODS Using zebrafish, we investigated the neurobehavioral effects of developmental exposure to imidacloprid, a prototypic neonicotinoid pesticide. Nicotine was also administered for comparison. Zebrafish were exposed via immersion in aqueous solutions containing 45 μM or 60 μM of imidacloprid or nicotine (or vehicle control) from 4 h to 5 d post fertilization. The functional effects of developmental exposure to both imidacloprid and nicotine were assessed in larvae using an activity assay and during adolescence and adulthood using a battery of neurobehavioral assays, including assessment of sensorimotor response and habituation in a tactile startle test, novel tank swimming, and shoaling behavior. RESULTS In larvae, developmental imidacloprid exposure at both doses significantly decreased swimming activity. The 5D strain of zebrafish were more sensitive to both nicotine and imidacloprid than the AB* strain. In adolescent and adult fish, developmental exposure to imidacloprid significantly decreased novel tank exploration and increased sensorimotor response to startle stimuli. While nicotine did not affect novel tank swimming, it increased sensorimotor response to startle stimuli at the low dose. No effects of either compound were found on shoaling behavior or habituation to a startling stimulus. DISCUSSION Early developmental exposure to imidacloprid has both early-life and persisting effects on neurobehavioral function in zebrafish. Its developmental neurotoxicity should be further investigated. PMID:25944383

  6. Urban stormwater runoff negatively impacts lateral line development in larval zebrafish and salmon embryos.

    PubMed

    Young, Alexander; Kochenkov, Valentin; McIntyre, Jenifer K; Stark, John D; Coffin, Allison B

    2018-02-12

    After a storm, water often runs off of impervious urban surfaces directly into aquatic ecosystems. This stormwater runoff is a cocktail of toxicants that have serious effects on the ecological integrity of aquatic habitats. Zebrafish that develop in stormwater runoff suffer from cardiovascular toxicity and impaired growth, but the effects of stormwater on fish sensory systems are not understood. Our study investigated the effect of stormwater on hair cells of the lateral line in larval zebrafish and coho salmon. Our results showed that although toxicants in stormwater did not kill zebrafish hair cells, these cells did experience damage. Zebrafish developing in stormwater also experienced impaired growth, fewer neuromasts in the lateral line, and fewer hair cells per neuromast. A similar reduction in neuromast number was observed in coho salmon reared in stormwater. Bioretention treatment, intended to filter out harmful constituents of stormwater, rescued the lateral line defects in zebrafish but not in coho salmon, suggesting that not all of the harmful constituents were removed by the filtration media and that salmonids are particularly sensitive to aquatic toxicants. Collectively, these data demonstrate that sub-lethal exposure to stormwater runoff negatively impacts a fish sensory system, which may have consequences for organismal fitness.

  7. Properties of the Visible Light Phototaxis and UV Avoidance Behaviors in the Larval Zebrafish

    PubMed Central

    Guggiana-Nilo, Drago A.; Engert, Florian

    2016-01-01

    For many organisms, color is an essential source of information from visual scenes. The larval zebrafish has the potential to be a model for the study of this topic, given its tetrachromatic retina and high dependence on vision. In this study we took a step toward understanding how the larval zebrafish might use color sensing. To this end, we used a projector-based paradigm to force a choice of a color stimulus at every turn of the larva. The stimuli used spanned most of the larval spectral range, including activation of its Ultraviolet (UV) cone, which has not been described behaviorally before. We found that zebrafish larvae swim toward visible wavelengths (>400 nm) when choosing between them and darkness, as has been reported with white light. However, when presented with UV light and darkness zebrafish show an intensity dependent avoidance behavior. This UV avoidance does not interact cooperatively with phototaxis toward longer wavelengths, but can compete against it in an intensity dependent manner. Finally, we show that the avoidance behavior depends on the presence of eyes with functional UV cones. These findings open future avenues for studying the neural circuits that underlie color sensing in the larval zebrafish. PMID:27594828

  8. Characterization of Na+ and Ca2+ Channels in Zebrafish Dorsal Root Ganglion Neurons

    PubMed Central

    Won, Yu-Jin; Ono, Fumihito; Ikeda, Stephen R.

    2012-01-01

    Background Dorsal root ganglia (DRG) somata from rodents have provided an excellent model system to study ion channel properties and modulation using electrophysiological investigation. As in other vertebrates, zebrafish (Danio rerio) DRG are organized segmentally and possess peripheral axons that bifurcate into each body segment. However, the electrical properties of zebrafish DRG sensory neurons, as compared with their mammalian counterparts, are relatively unexplored because a preparation suitable for electrophysiological studies has not been available. Methodology/Principal Findings We show enzymatically dissociated DRG neurons from juvenile zebrafish expressing Isl2b-promoter driven EGFP were easily identified with fluorescence microscopy and amenable to conventional whole-cell patch-clamp studies. Two kinetically distinct TTX-sensitive Na+ currents (rapidly- and slowly-inactivating) were discovered. Rapidly-inactivating INa were preferentially expressed in relatively large neurons, while slowly-inactivating INa was more prevalent in smaller DRG neurons. RT-PCR analysis suggests zscn1aa/ab, zscn8aa/ab, zscn4ab and zscn5Laa are possible candidates for these INa components. Voltage-gated Ca2+ currents (ICa) were primarily (87%) comprised of a high-voltage activated component arising from ω-conotoxin GVIA-sensitive CaV2.2 (N-type) Ca2+ channels. A few DRG neurons (8%) displayed a miniscule low-voltage-activated component. ICa in zebrafish DRG neurons were modulated by neurotransmitters via either voltage-dependent or -independent G-protein signaling pathway with large cell-to-cell response variability. Conclusions/Significance Our present results indicate that, as in higher vertebrates, zebrafish DRG neurons are heterogeneous being composed of functionally distinct subpopulations that may correlate with different sensory modalities. These findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and thus provide a basis for

  9. Wild Sex in Zebrafish: Loss of the Natural Sex Determinant in Domesticated Strains

    PubMed Central

    Wilson, Catherine A.; High, Samantha K.; McCluskey, Braedan M.; Amores, Angel; Yan, Yi-lin; Titus, Tom A.; Anderson, Jennifer L.; Batzel, Peter; Carvan, Michael J.; Schartl, Manfred; Postlethwait, John H.

    2014-01-01

    Sex determination can be robustly genetic, strongly environmental, or genetic subject to environmental perturbation. The genetic basis of sex determination is unknown for zebrafish (Danio rerio), a model for development and human health. We used RAD-tag population genomics to identify sex-linked polymorphisms. After verifying this “RAD-sex” method on medaka (Oryzias latipes), we studied two domesticated zebrafish strains (AB and TU), two natural laboratory strains (WIK and EKW), and two recent isolates from nature (NA and CB). All four natural strains had a single sex-linked region at the right tip of chromosome 4, enabling sex genotyping by PCR. Genotypes for the single nucleotide polymorphism (SNP) with the strongest statistical association to sex suggested that wild zebrafish have WZ/ZZ sex chromosomes. In natural strains, “male genotypes” became males and some “female genotypes” also became males, suggesting that the environment or genetic background can cause female-to-male sex reversal. Surprisingly, TU and AB lacked detectable sex-linked loci. Phylogenomics rooted on D. nigrofasciatus verified that all strains are monophyletic. Because AB and TU branched as a monophyletic clade, we could not rule out shared loss of the wild sex locus in a common ancestor despite their independent domestication. Mitochondrial DNA sequences showed that investigated strains represent only one of the three identified zebrafish haplogroups. Results suggest that zebrafish in nature possess a WZ/ZZ sex-determination mechanism with a major determinant lying near the right telomere of chromosome 4 that was modified during domestication. Strains providing the zebrafish reference genome lack key components of the natural sex-determination system but may have evolved variant sex-determining mechanisms during two decades in laboratory culture. PMID:25233988

  10. Characterizing the mechanical behavior of the zebrafish germ layers

    NASA Astrophysics Data System (ADS)

    Kealhofer, David; Serwane, Friedhelm; Mongera, Alessandro; Rowghanian, Payam; Lucio, Adam; Campàs, Otger

    Organ morphogenesis and the development of the animal body plan involve complex spatial and temporal control of tissue- and cell-level mechanics. A prime example is the generation of stresses by individual cells to reorganize the tissue. These processes have remained poorly understood due to a lack of techniques to characterize the local constitutive law of the material, which relates local cellular forces to the resulting tissue flows. We have developed a method for quantitative, local in vivo study of material properties in living tissue using magnetic droplet probes. We use this technique to study the material properties of the different zebrafish germ layers using aggregates of zebrafish mesendodermal and ectodermal cells as a model system. These aggregates are ideal for controlled studies of the mechanics of individual germ layers because of the homogeneity of the cell type and the simple spherical geometry. Furthermore, the numerous molecular tools and transgenic lines already developed for this model organism can be applied to these aggregates, allowing us to characterize the contributions of cell cortex tension and cell adhesion to the mechanical properties of the zebrafish germ layers.

  11. Hepassocin is required for hepatic outgrowth during zebrafish hepatogenesis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gao, Ming; Beijing Institute of Radiation Medicine, Beijing 100850; Yan, Hui

    2015-07-31

    Background & aims: Hepassocin (HPS) is a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage. In this paper, zebrafish were used to investigate the role of HPS in liver development. Methods and results: During zebrafish development, HPS expression is enriched in liver throughout hepatogenesis. Knockdown of HPS using its specific morpholino leads to a smaller liver phenotype. Further results showed that the HPS knockdown has no effect on the expression of the early endoderm marker gata6 and early hepatic marker hhex. In addition, results showed that the smaller-liver phenotype in HPS morphants wasmore » caused by suppression of cell proliferation, not induction of cell apoptosis. Conclusions: Current findings indicated that HPS is essential to the later stages of development in vertebrate liver organogenesis. - Highlights: • HPS is enriched in zebrafish liver and has strong similarities with other species. • Knocking down HPS with MOs results in small liver phenotype. • HPS depletion regulates liver outgrowth but not liver specification and budding. • HPS depletion causes hepatocyte proliferation arrest but not apoptosis induction.« less

  12. SENSORY HAIR CELL REGENERATION IN THE ZEBRAFISH LATERAL LINE

    PubMed Central

    Lush, Mark E.; Piotrowski, Tatjana

    2014-01-01

    Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. PMID:25045019

  13. Sensory hair cell regeneration in the zebrafish lateral line.

    PubMed

    Lush, Mark E; Piotrowski, Tatjana

    2014-10-01

    Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling, and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. Copyright © 2014 Wiley Periodicals, Inc.

  14. Advancing biology through a deeper understanding of zebrafish ecology and evolution.

    PubMed

    Parichy, David M

    2015-03-25

    Over the last two decades, the zebrafish has joined the ranks of premier model organisms for biomedical research, with a full suite of tools and genomic resources. Yet we still know comparatively little about its natural history. Here I review what is known about the natural history of the zebrafish, where significant gaps in our knowledge remain, and how a fuller appreciation of this organism's ecology and behavior, population genetics, and phylogeny can inform a variety of research endeavors.

  15. Light directs zebrafish period2 expression via conserved D and E boxes.

    PubMed

    Vatine, Gad; Vallone, Daniela; Appelbaum, Lior; Mracek, Philipp; Ben-Moshe, Zohar; Lahiri, Kajori; Gothilf, Yoav; Foulkes, Nicholas S

    2009-10-01

    For most species, light represents the principal environmental signal for entraining the endogenous circadian clock. The zebrafish is a fascinating vertebrate model for studying this process since unlike mammals, direct exposure of most of its tissues to light leads to local clock entrainment. Importantly, light induces the expression of a set of genes including certain clock genes in most zebrafish cell types in vivo and in vitro. However, the mechanism linking light to gene expression remains poorly understood. To elucidate this key mechanism, here we focus on how light regulates transcription of the zebrafish period2 (per2) gene. Using transgenic fish and stably transfected cell line-based assays, we define a Light Responsive Module (LRM) within the per2 promoter. The LRM lies proximal to the transcription start site and is both necessary and sufficient for light-driven gene expression and also for a light-dependent circadian clock regulation. Curiously, the LRM sequence is strongly conserved in other vertebrate per2 genes, even in species lacking directly light-sensitive peripheral clocks. Furthermore, we reveal that the human LRM can substitute for the zebrafish LRM to confer light-regulated transcription in zebrafish cells. The LRM contains E- and D-box elements that are critical for its function. While the E-box directs circadian clock regulation by mediating BMAL/CLOCK activity, the D-box confers light-driven expression. The zebrafish homolog of the thyrotroph embryonic factor binds efficiently to the LRM D-box and transactivates expression. We demonstrate that tef mRNA levels are light inducible and that knock-down of tef expression attenuates light-driven transcription from the per2 promoter in vivo. Together, our results support a model where a light-dependent crosstalk between E- and D-box binding factors is a central determinant of per2 expression. These findings extend the general understanding of the mechanism whereby the clock is entrained by light

  16. Phototoxicity of TiO2 nanoparticles to zebrafish (Danio rerio) is dependent on life stage.

    PubMed

    Ma, Hongbo; Diamond, Stephen A

    2013-09-01

    Zebrafish embryos have been used increasingly to evaluate nanomaterial toxicity. The present study compared phototoxicity of TiO2 nanoparticles with zebrafish at 4 life stages (embryos, yolk-sac larvae, free-swimming larvae, and juvenile) under simulated sunlight using the 96-h standard toxicity assay. Yolk-sac larvae were found to be the most sensitive to TiO2 phototoxicity, suggesting that the widely used zebrafish embryo test may not fully or accurately predict hazard and risk of these nanoparticles to small fish. Copyright © 2013 SETAC.

  17. Essential role for fibrillin-2 in zebrafish notochord and vascular morphogenesis.

    PubMed

    Gansner, John M; Madsen, Erik C; Mecham, Robert P; Gitlin, Jonathan D

    2008-10-01

    Recent studies demonstrate that lysyl oxidase cuproenzymes are critical for zebrafish notochord formation, but the molecular mechanisms of copper-dependent notochord morphogenesis are incompletely understood. We, therefore, conducted a forward genetic screen for zebrafish mutants that exhibit notochord sensitivity to lysyl oxidase inhibition, yielding a mutant with defects in notochord and vascular morphogenesis, puff daddygw1 (pfdgw1). Meiotic mapping and cloning reveal that the pfdgw1 phenotype results from disruption of the gene encoding the extracellular matrix protein fibrillin-2, and the spatiotemporal expression of fibrillin-2 is consistent with the pfdgw1 phenotype. Furthermore, each aspect of the pfdgw1 phenotype is recapitulated by morpholino knockdown of fibrillin-2. Taken together, the data reveal a genetic interaction between fibrillin-2 and the lysyl oxidases in notochord formation and demonstrate the importance of fibrillin-2 in specific early developmental processes in zebrafish. Copyright (c) 2008 Wiley-Liss, Inc.

  18. Haploinsufficiency of the genes encoding the tumor suppressor Pten predisposes zebrafish to hemangiosarcoma

    PubMed Central

    Choorapoikayil, Suma; Kuiper, Raoul V.; de Bruin, Alain; den Hertog, Jeroen

    2012-01-01

    SUMMARY PTEN is an essential tumor suppressor that antagonizes Akt/PKB signaling. The zebrafish genome encodes two Pten genes, ptena and ptenb. Here, we report that zebrafish mutants that retain a single wild-type copy of ptena or ptenb (ptena+/−ptenb−/− or ptena−/−ptenb+/−) are viable and fertile. ptena+/−ptenb−/− fish develop tumors at a relatively high incidence (10.2%) and most tumors developed close to the eye (26/30). Histopathologically, the tumor masses were associated with the retrobulbar vascular network and diagnosed as hemangiosarcomas. A single tumor was identified in 42 ptena−/−ptenb+/− fish and was also diagnosed as hemangiosarcoma. Immunohistochemistry indicated that the tumor cells in ptena+/−ptenb−/− and ptena−/−ptenb+/− fish proliferated rapidly and were of endothelial origin. Akt/PKB signaling was activated in the tumors, whereas Ptena was still detected in tumor tissue from ptena+/−ptenb−/− zebrafish. We conclude that haploinsufficiency of the genes encoding Pten predisposes to hemangiosarcoma in zebrafish. PMID:22071262

  19. Detection of blob objects in microscopic zebrafish images based on gradient vector diffusion.

    PubMed

    Li, Gang; Liu, Tianming; Nie, Jingxin; Guo, Lei; Malicki, Jarema; Mara, Andrew; Holley, Scott A; Xia, Weiming; Wong, Stephen T C

    2007-10-01

    The zebrafish has become an important vertebrate animal model for the study of developmental biology, functional genomics, and disease mechanisms. It is also being used for drug discovery. Computerized detection of blob objects has been one of the important tasks in quantitative phenotyping of zebrafish. We present a new automated method that is able to detect blob objects, such as nuclei or cells in microscopic zebrafish images. This method is composed of three key steps. The first step is to produce a diffused gradient vector field by a physical elastic deformable model. In the second step, the flux image is computed on the diffused gradient vector field. The third step performs thresholding and nonmaximum suppression based on the flux image. We report the validation and experimental results of this method using zebrafish image datasets from three independent research labs. Both sensitivity and specificity of this method are over 90%. This method is able to differentiate closely juxtaposed or connected blob objects, with high sensitivity and specificity in different situations. It is characterized by a good, consistent performance in blob object detection.

  20. Fishing for Fetal Alcohol Spectrum Disorders: Zebrafish as a Model for Ethanol Teratogenesis.

    PubMed

    Lovely, Charles Ben; Fernandes, Yohaan; Eberhart, Johann K

    2016-10-01

    Fetal Alcohol Spectrum Disorders (FASD) describes a wide array of ethanol-induced developmental defects, including craniofacial dysmorphology and cognitive impairments. It affects ∼1 in 100 children born in the United States each year. Due to the pleiotropic effects of ethanol, animal models have proven critical in characterizing the mechanisms of ethanol teratogenesis. In this review, we focus on the utility of zebrafish in characterizing ethanol-induced developmental defects. A growing number of laboratories have focused on using zebrafish to examine ethanol-induced defects in craniofacial, cardiac, ocular, and neural development, as well as cognitive and behavioral impairments. Growing evidence supports that genetic predisposition plays a role in these ethanol-induced defects, yet little is understood about these gene-ethanol interactions. With a high degree of genetic amenability, zebrafish is at the forefront of identifying and characterizing the gene-ethanol interactions that underlie FASD. Because of the conservation of gene function between zebrafish and humans, these studies will directly translate to studies of candidate genes in human populations and allow for better diagnosis and treatment of FASD.