Sample records for zebrafish infection model

  1. 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.

  2. 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

  3. 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

  4. 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

  5. Bioactive Molecule from Streptomyces sp. Mitigates MDR Klebsiella pneumoniae in Zebrafish Infection Model

    PubMed Central

    Cheepurupalli, Lalitha; Raman, Thiagarajan; Rathore, Sudarshan S.; Ramakrishnan, Jayapradha

    2017-01-01

    The emergence and spread of multi-drug resistant (MDR) especially carbapenem-resistant Klebsiella pneumoniae is a major emerging threat to public health, leading to excess in mortality rate as high as 50–86%. MDR K. pneumoniae manifests all broad mechanisms of drug resistance, hence development of new drugs to treat MDR K. pneumoniae infection has become a more relevant question in the scientific community. In the present study a potential Streptomyces sp. ASK2 was isolated from rhizosphere soil of medicinal plant. The multistep HPLC purification identified the active principle exhibiting antagonistic activity against MDR K. pneumoniae. The purified compound was found to be an aromatic compound with aliphatic side chain molecule having a molecular weight of 444.43 Da. FT-IR showed the presence of OH and C=O as functional groups. The bioactive compound was further evaluated for drug induced toxicity and efficacy in adult zebrafish infection model. As this is the first study on K. pneumoniae – zebrafish model, the infectious doses to manifest sub-clinical and clinical infection were optimized. Furthermore, the virulence of K. pneumoniae in planktonic and biofilm state was studied in zebrafish. The MTT assay of ex vivo culture of zebrafish liver reveals non-toxic nature of the proposed ASK2 compound at an effective dose. Moreover, significant increase in survival rate of infected zebrafish suggests that ASK2 compound from a new strain of Streptomyces sp. was potent in mitigating MDR K. pneumoniae infection. PMID:28446900

  6. 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

  7. Influenza A virus infection in zebrafish recapitulates mammalian infection and sensitivity to anti-influenza drug treatment.

    PubMed

    Gabor, Kristin A; Goody, Michelle F; Mowel, Walter K; Breitbach, Meghan E; Gratacap, Remi L; Witten, P Eckhard; Kim, Carol H

    2014-11-01

    Seasonal influenza virus infections cause annual epidemics and sporadic pandemics. These present a global health concern, resulting in substantial morbidity, mortality and economic burdens. Prevention and treatment of influenza illness is difficult due to the high mutation rate of the virus, the emergence of new virus strains and increasing antiviral resistance. Animal models of influenza infection are crucial to our gaining a better understanding of the pathogenesis of and host response to influenza infection, and for screening antiviral compounds. However, the current animal models used for influenza research are not amenable to visualization of host-pathogen interactions or high-throughput drug screening. The zebrafish is widely recognized as a valuable model system for infectious disease research and therapeutic drug testing. Here, we describe a zebrafish model for human influenza A virus (IAV) infection and show that zebrafish embryos are susceptible to challenge with both influenza A strains APR8 and X-31 (Aichi). Influenza-infected zebrafish show an increase in viral burden and mortality over time. The expression of innate antiviral genes, the gross pathology and the histopathology in infected zebrafish recapitulate clinical symptoms of influenza infections in humans. This is the first time that zebrafish embryos have been infected with a fluorescent IAV in order to visualize infection in a live vertebrate host, revealing a pattern of vascular endothelial infection. Treatment of infected zebrafish with a known anti-influenza compound, Zanamivir, reduced mortality and the expression of a fluorescent viral gene product, demonstrating the validity of this model to screen for potential antiviral drugs. The zebrafish model system has provided invaluable insights into host-pathogen interactions for a range of infectious diseases. Here, we demonstrate a novel use of this species for IAV research. This model has great potential to advance our understanding of

  8. Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses.

    PubMed

    Sullivan, Con; Jurcyzszak, Denise; Goody, Michelle F; Gabor, Kristin A; Longfellow, Jacob R; Millard, Paul J; Kim, Carol H

    2017-01-20

    Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here

  9. A Model of Superinfection of Virus-Infected Zebrafish Larvae: Increased Susceptibility to Bacteria Associated With Neutrophil Death

    PubMed Central

    Boucontet, Laurent; Passoni, Gabriella; Thiry, Valéry; Maggi, Ludovico; Herbomel, Philippe; Levraud, Jean-Pierre; Colucci-Guyon, Emma

    2018-01-01

    Enhanced susceptibility to bacterial infection in the days following an acute virus infection such as flu is a major clinical problem. Mouse models have provided major advances in understanding viral-bacterial superinfections, yet interactions of the anti-viral and anti-bacterial responses remain elusive. Here, we have exploited the transparency of zebrafish to study how viral infections can pave the way for bacterial co-infections. We have set up a zebrafish model of sequential viral and bacterial infection, using sublethal doses of Sindbis virus and Shigella flexneri bacteria. This virus induces a strong type I interferons (IFN) response, while the bacterium induces a strong IL1β and TNFα-mediated inflammatory response. We found that virus-infected zebrafish larvae showed an increased susceptibility to bacterial infection. This resulted in the death with concomitant higher bacterial burden of the co-infected fish compared to the ones infected with bacteria only. By contrast, infecting with bacteria first and virus second did not lead to increased mortality or microbial burden. By high-resolution live imaging, we showed that neutrophil survival was impaired in Sindbis-then-Shigella co-infected fish. The two types of cytokine responses were strongly induced in co-infected fish. In addition to type I IFN, expression of the anti-inflammatory cytokine IL10 was induced by viral infection before bacterial superinfection. Collectively, these observations suggest the zebrafish larva as a useful animal model to address mechanisms underlying increased bacterial susceptibility upon viral infection. PMID:29881380

  10. 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

  11. Identification of infection- and defense-related genes via a dynamic host-pathogen interaction network using a Candida albicans-zebrafish infection model.

    PubMed

    Kuo, Zong-Yu; Chuang, Yung-Jen; Chao, Chun-Cheih; Liu, Fu-Chen; Lan, Chung-Yu; Chen, Bor-Sen

    2013-01-01

    Candida albicans infections and candidiasis are difficult to treat and create very serious therapeutic challenges. In this study, based on interactive time profile microarray data of C. albicans and zebrafish during infection, the infection-related protein-protein interaction (PPI) networks of the two species and the intercellular PPI network between host and pathogen were simultaneously constructed by a dynamic interaction model, modeled as an integrated network consisting of intercellular invasion and cellular defense processes during infection. The signal transduction pathways in regulating morphogenesis and hyphal growth of C. albicans were further investigated based on significant interactions found in the intercellular PPI network. Two cellular networks were also developed corresponding to the different infection stages (adhesion and invasion), and then compared with each other to identify proteins from which we can gain more insight into the pathogenic role of hyphal development in the C. albicans infection process. Important defense-related proteins in zebrafish were predicted using the same approach. The hyphal growth PPI network, zebrafish PPI network and host-pathogen intercellular PPI network were combined to form an integrated infectious PPI network that helps us understand the systematic mechanisms underlying the pathogenicity of C. albicans and the immune response of the host, and may help improve medical therapies and facilitate the development of new antifungal drugs. Copyright © 2013 S. Karger AG, Basel.

  12. A privileged intraphagocyte niche is responsible for disseminated infection of Staphylococcus aureus in a zebrafish model

    PubMed Central

    Prajsnar, Tomasz K; Hamilton, Ruth; Garcia-Lara, Jorge; McVicker, Gareth; Williams, Alexander; Boots, Michael; Foster, Simon J; Renshaw, Stephen A

    2012-01-01

    The innate immune system is the primary defence against the versatile pathogen, Staphylococcus aureus. How this organism is able to avoid immune killing and cause infections is poorly understood. Using an established larval zebrafish infection model, we have shown that overwhelming infection is due to subversion of phagocytes by staphylococci, allowing bacteria to evade killing and found foci of disease. Larval zebrafish coinfected with two S. aureus strains carrying different fluorescent reporter gene fusions (but otherwise isogenic) had bacterial lesions, at the time of host death, containing predominantly one strain. Quantitative data using two marked strains revealed that the strain ratios, during overwhelming infection, were often skewed towards the extremes, with one strain predominating. Infection with passaged bacterial clones revealed the phenomenon not to bedue to adventitious mutations acquired by the pathogen. After infection of the host, all bacteria are internalized by phagocytes and the skewing of population ratios is absolutely dependent on the presence of phagocytes. Mathematical modelling of pathogen population dynamics revealed the data patterns are consistent with the hypothesis that a small number of infected phagocytes serve as an intracellular reservoir for S. aureus, which upon release leads to disseminated infection. Strategies to specifically alter neutrophil/macrophage numbers were used to map the potential subpopulation of phagocytes acting as a pathogen reservoir, revealing neutrophils as the likely ‘niche’. Subsequently in a murine sepsis model, S. aureus abscesses in kidneys were also found to be predominantly clonal, therefore likely founded by an individual cell, suggesting a potential mechanism analogous to the zebrafish model with few protected niches. These findings add credence to the argument that S. aureus control regimes should recognize both the intracellular as well as extracellular facets of the S. aureus life cycle

  13. Infection of zebrafish embryos with live fluorescent Streptococcus pneumoniae as a real-time pneumococcal meningitis model.

    PubMed

    Jim, Kin Ki; Engelen-Lee, JooYeon; van der Sar, Astrid M; Bitter, Wilbert; Brouwer, Matthijs C; van der Ende, Arie; Veening, Jan-Willem; van de Beek, Diederik; Vandenbroucke-Grauls, Christina M J E

    2016-08-19

    Streptococcus pneumoniae is one of the most important causes of bacterial meningitis, an infection where unfavourable outcome is driven by bacterial and host-derived toxins. In this study, we developed and characterized a pneumococcal meningitis model in zebrafish embryos that allows for real-time investigation of early host-microbe interaction. Zebrafish embryos were infected in the caudal vein or hindbrain ventricle with green fluorescent wild-type S. pneumoniae D39 or a pneumolysin-deficient mutant. The kdrl:mCherry transgenic zebrafish line was used to visualize the blood vessels, whereas phagocytic cells were visualized by staining with far red anti-L-plastin or in mpx:GFP/mpeg1:mCherry zebrafish, that have green fluorescent neutrophils and red fluorescent macrophages. Imaging was performed by fluorescence confocal and time-lapse microscopy. After infection by caudal vein, we saw focal clogging of the pneumococci in the blood vessels and migration of bacteria through the blood-brain barrier into the subarachnoid space and brain tissue. Infection with pneumolysin-deficient S. pneumoniae in the hindbrain ventricle showed attenuated growth and migration through the brain as compared to the wild-type strain. Time-lapse and confocal imaging revealed that the initial innate immune response to S. pneumoniae in the subarachnoid space mainly consisted of neutrophils and that pneumolysin-mediated cytolytic activity caused a marked reduction of phagocytes. This new meningitis model permits detailed analysis and visualization of host-microbe interaction in pneumococcal meningitis in real time and is a very promising tool to further our insights in the pathogenesis of pneumococcal meningitis.

  14. 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.

  15. Establishment of a Zebrafish Infection Model for the Study of Wild-Type and Recombinant European Sheatfish Virus.

    PubMed

    Martín, Verónica; Mavian, Carla; López Bueno, Alberto; de Molina, Antonio; Díaz, Eduardo; Andrés, Germán; Alcami, Antonio; Alejo, Alí

    2015-10-01

    Amphibian-like ranaviruses include pathogens of fish, amphibians, and reptiles that have recently evolved from a fish-infecting ancestor. The molecular determinants of host range and virulence in this group are largely unknown, and currently fish infection models are lacking. We show that European sheatfish virus (ESV) can productively infect zebrafish, causing a lethal pathology, and describe a method for the generation of recombinant ESV, establishing a useful model for the study of fish ranavirus infections. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  16. Establishment of Three Francisella Infections in Zebrafish Embryos at Different Temperatures

    PubMed Central

    Brudal, Espen; Ulanova, Lilia S.; O. Lampe, Elisabeth; Rishovd, Anne-Lise; Winther-Larsen, Hanne C.

    2014-01-01

    Francisella spp. are facultative intracellular pathogens identified in increasingly diverse hosts, including mammals. F. noatunensis subsp. orientalis and F. noatunensis subsp. noatunensis infect fish inhabiting warm and cold waters, respectively, while F. tularensis subsp. novicida is highly infectious for mice and has been widely used as a model for the human pathogen F. tularensis. Here, we established zebrafish embryo infection models of fluorescently labeled F. noatunensis subsp. noatunensis, F. noatunensis subsp. orientalis, and F. tularensis subsp. novicida at 22, 28, and 32°C, respectively. All infections led to significant bacterial growth, as shown by reverse transcription-quantitative PCR (RT-qPCR), and to a robust proinflammatory immune response, dominated by increased transcription of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). F. noatunensis subsp. orientalis was the most virulent, F. noatunensis subsp. noatunensis caused chronic infection, and F. tularensis subsp. novicida showed moderate virulence and led to formation of relatively small granuloma-like structures. The use of transgenic zebrafish strains with enhanced green fluorescent protein (EGFP)-labeled immune cells revealed their detailed interactions with Francisella species. All three strains entered preferentially into macrophages, which eventually assembled into granuloma-like structures. Entry into neutrophils was also observed, though the efficiency of this event depended on the route of infection. The results demonstrate the usefulness of the zebrafish embryo model for studying infections caused by different Francisella species at a wide range of temperatures and highlight their interactions with immune cells. PMID:24614659

  17. 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.

  18. Treatment of Francisella infections via PLGA- and lipid-based nanoparticle delivery of antibiotics in a zebrafish model.

    PubMed

    Ulanova, Lilia S; Pinheiro, Marina; Vibe, Carina; Nunes, Claudia; Misaghian, Dorna; Wilson, Steven; Zhu, Kaizheng; Fenaroli, Federico; Winther-Larsen, Hanne C; Reis, Salette; Griffiths, Gareth

    2017-06-19

    We tested the efficiency of 2 different antibiotics, rifampicin and oxolinic acid, against an established infection caused by fish pathogen Francisella noatunensis ssp. orientalis (F.n.o.) in zebrafish. The drugs were tested in the free form as well as encapsulated into biodegradable nanoparticles, either polylactic-co-glycolic acid (PLGA) nanoparticles or nanostructured lipid carriers. The most promising therapies were PLGA-rifampicin nanoparticles and free oxolinic acid; the PLGA nanoparticles significantly delayed embryo mortality while free oxolinic acid prevented it. Encapsulation of rifampicin in both PLGA and nanostructured lipid carriers enhanced its efficiency against F.n.o. infection relative to the free drug. We propose that the zebrafish model is a robust, rapid system for initial testing of different treatments of bacterial diseases important for aquaculture.

  19. 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.

  20. 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

  1. 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

  2. Transient infection of the zebrafish notochord with E. coli induces chronic inflammation

    PubMed Central

    Nguyen-Chi, Mai; Phan, Quang Tien; Gonzalez, Catherine; Dubremetz, Jean-François; Levraud, Jean-Pierre; Lutfalla, Georges

    2014-01-01

    Zebrafish embryos and larvae are now well-established models in which to study infectious diseases. Infections with non-pathogenic Gram-negative Escherichia coli induce a strong and reproducible inflammatory response. Here, we study the cellular response of zebrafish larvae when E. coli bacteria are injected into the notochord and describe the effects. First, we provide direct evidence that the notochord is a unique organ that is inaccessible to leukocytes (macrophages and neutrophils) during the early stages of inflammation. Second, we show that notochord infection induces a host response that is characterised by rapid clearance of the bacteria, strong leukocyte recruitment around the notochord and prolonged inflammation that lasts several days after bacteria clearance. During this inflammatory response, il1b is first expressed in macrophages and subsequently at high levels in neutrophils. Moreover, knock down of il1b alters the recruitment of neutrophils to the notochord, demonstrating the important role of this cytokine in the maintenance of inflammation in the notochord. Eventually, infection of the notochord induces severe defects of the notochord that correlate with neutrophil degranulation occurring around this tissue. This is the first in vivo evidence that neutrophils can degranulate in the absence of a direct encounter with a pathogen. Persistent inflammation, neutrophil infiltration and restructuring of the extracellular matrix are defects that resemble those seen in bone infection and in some chondropathies. As the notochord is a transient embryonic structure that is closely related to cartilage and bone and that contributes to vertebral column formation, we propose infection of the notochord in zebrafish larvae as a new model to study the cellular and molecular mechanisms underlying cartilage and bone inflammation. PMID:24973754

  3. Streptococcus dysgalactiae subsp. dysgalactiae isolated from milk of the bovine udder as emerging pathogens: In vitro and in vivo infection of human cells and zebrafish as biological models.

    PubMed

    Alves-Barroco, Cinthia; Roma-Rodrigues, Catarina; Raposo, Luís R; Brás, Catarina; Diniz, Mário; Caço, João; Costa, Pedro M; Santos-Sanches, Ilda; Fernandes, Alexandra R

    2018-03-25

    Streptococcus dysgalactiae subsp. dysgalactiae (SDSD) is a major cause of bovine mastitis and has been regarded as an animal-restricted pathogen, although rare infections have been described in humans. Previous studies revealed the presence of virulence genes encoded by phages of the human pathogen Group A Streptococcus pyogenes (GAS) in SDSD isolated from the milk of bovine udder with mastitis. The isolates SDSD VSD5 and VSD13 could adhere and internalize human primary keratinocyte cells, suggesting a possible human infection potential of bovine isolates. In this work, the in vitro and in vivo potential of SDSD to internalize/adhere human cells of the respiratory track and zebrafish as biological models was evaluated. Our results showed that, in vitro, bovine SDSD strains could interact and internalize human respiratory cell lines and that this internalization was dependent on an active transport mechanism and that, in vivo, SDSD are able to cause invasive infections producing zebrafish morbidity and mortality. The infectious potential of these isolates showed to be isolate-specific and appeared to be independent of the presence or absence of GAS phage-encoded virulence genes. Although the infection ability of the bovine SDSD strains was not as strong as the human pathogenic S. pyogenes in the zebrafish model, results suggested that these SDSD isolates are able to interact with human cells and infect zebrafish, a vertebrate infectious model, emerging as pathogens with zoonotic capability. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  4. Transgenic expression of omega-3 PUFA synthesis genes improves zebrafish survival during Vibrio vulnificus infection.

    PubMed

    Cheng, Chih-Lun; Huang, Shin-Jie; Wu, Chih-Lu; Gong, Hong-Yi; Ken, Chuian-Fu; Hu, Shao-Yang; Wu, Jen-Leih

    2015-11-17

    Highly desaturated n-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are synthesized by desaturases and elongase. They exert hepatoprotective effects to prevent alcoholic fatty liver syndrome or cholestatic liver injury. However, it is unclear how n-3 PUFAs improve immune function in liver. Vibrio vulnificus, a gram-negative bacterial pathogen, causes high mortality of aquaculture fishes upon infection. Humans can become infected with V. vulnificus through open wounds or by eating raw seafood, and such infections may result in systemic septicemia. Moreover, patients with liver diseases are vulnerable to infection, and are more likely than healthy persons to present with liver inflammation following infection. This study quantified n-3 PUFAs and their anti-bacterial effects in Fadsd6 and Elvol5a transgenic zebrafish. Two transgenic zebrafish strains with strong liver specific expression of Fadsd6 and Elvol5a (driven by the zebrafish Fabp10 promoter) were established using the Tol2 system. Synthesis of n-3 PUFAs in these strains were increased by 2.5-fold as compared to wild type (Wt) fish. The survival rate in 24 h following challenge with V. vulnificus was 20 % in Wt, but 70 % in the transgenic strains. In addition, the bacteria counts in transgenic fish strains were significantly decreased. The expression levels of pro-inflammatory genes, such as TNF-α, IL-1β, and NF-κB, were suppressed between 9 and 12 h after challenge. This study confirms the anti-bacterial function of n-3 PUFAs in a transgenic zebrafish model. Fadsd6 and Elvol5a transgenic zebrafish are more resistant to V. vulnificus infection, and enhance survival by diminishing the attendant inflammatory response.

  5. 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

  6. Transient infection of the zebrafish notochord with E. coli induces chronic inflammation.

    PubMed

    Nguyen-Chi, Mai; Phan, Quang Tien; Gonzalez, Catherine; Dubremetz, Jean-François; Levraud, Jean-Pierre; Lutfalla, Georges

    2014-07-01

    Zebrafish embryos and larvae are now well-established models in which to study infectious diseases. Infections with non-pathogenic Gram-negative Escherichia coli induce a strong and reproducible inflammatory response. Here, we study the cellular response of zebrafish larvae when E. coli bacteria are injected into the notochord and describe the effects. First, we provide direct evidence that the notochord is a unique organ that is inaccessible to leukocytes (macrophages and neutrophils) during the early stages of inflammation. Second, we show that notochord infection induces a host response that is characterised by rapid clearance of the bacteria, strong leukocyte recruitment around the notochord and prolonged inflammation that lasts several days after bacteria clearance. During this inflammatory response, il1b is first expressed in macrophages and subsequently at high levels in neutrophils. Moreover, knock down of il1b alters the recruitment of neutrophils to the notochord, demonstrating the important role of this cytokine in the maintenance of inflammation in the notochord. Eventually, infection of the notochord induces severe defects of the notochord that correlate with neutrophil degranulation occurring around this tissue. This is the first in vivo evidence that neutrophils can degranulate in the absence of a direct encounter with a pathogen. Persistent inflammation, neutrophil infiltration and restructuring of the extracellular matrix are defects that resemble those seen in bone infection and in some chondropathies. As the notochord is a transient embryonic structure that is closely related to cartilage and bone and that contributes to vertebral column formation, we propose infection of the notochord in zebrafish larvae as a new model to study the cellular and molecular mechanisms underlying cartilage and bone inflammation. © 2014. Published by The Company of Biologists Ltd.

  7. 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.

  8. 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.

  9. Matrix metalloproteinase-9 plays a role in protecting zebrafish from lethal infection with Listeria monocytogenes by enhancing macrophage migration.

    PubMed

    Shan, Ying; Zhang, Yikai; Zhuo, Xunhui; Li, Xiaoliang; Peng, Jinrong; Fang, Weihuan

    2016-07-01

    Zebrafish could serve as an alternative animal model for pathogenic bacteria in multiple infectious routes. Our previous study showed that immersion infection in zebrafish with Listeria monocytogenes did not cause lethality but induced transient expression of several immune response genes. We used an Affymetrix gene chip to examine the expression profiles of genes of zebrafish immersion-infected with L. monocytogenes. A total of 239 genes were up-regulated and 56 genes down-regulated compared with uninfected fish. Highest expression (>20-fold) was seen with the mmp-9 gene encoding the matrix metalloproteinase-9 (Mmp-9) known to degrade the extracellular matrix proteins. By morpholino knockdown of mmp-9, we found that the morphants showed rapid death with much higher bacterial load after intravenous or intraventricular (brain ventricle) infection with L. monocytogenes. Macrophages in mmp-9-knockdown morphants had significant defect in migrating to the brain cavity upon intraventricular infection. Decreased migration of murine macrophages with knockdown of mmp-9 and cd44 was also seen in transwell inserts with 8-μm pore polycarbonate membrane, as compared with the scrambled RNA. These findings suggest that Mmp-9 is a protective molecule against infection by L. monocytogenes by engaging in migration of zebrafish macrophages to the site of infection via a non-proteolytic role. Further work is required on the molecular mechanisms governing Mmp-9-driven macrophage migration in zebrafish. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. A Zebrafish Larval Model to Assess Virulence of Porcine Streptococcus suis Strains.

    PubMed

    Zaccaria, Edoardo; Cao, Rui; Wells, Jerry M; van Baarlen, Peter

    2016-01-01

    Streptococcus suis is an encapsulated Gram-positive bacterium, and the leading cause of sepsis and meningitis in young pigs resulting in considerable economic losses in the porcine industry. It is also considered an emerging zoonotic agent. In the environment, both avirulent and virulent strains occur in pigs, and virulent strains appear to cause disease in both humans and pigs. There is a need for a convenient, reliable and standardized animal model to assess S. suis virulence. A zebrafish (Danio rerio) larvae infection model has several advantages, including transparency of larvae, low cost, ease of use and exemption from ethical legislation up to 6 days post fertilization, but has not been previously established as a model for S. suis. Microinjection of different porcine strains of S. suis in zebrafish larvae resulted in highly reproducible dose- and strain-dependent larval death, strongly correlating with presence of the S. suis capsule and to the original virulence of the strain in pigs. Additionally we compared the virulence of the two-component system mutant of ciaRH, which is attenuated for virulence in both mice and pigs in vivo. Infection of larvae with the ΔciaRH strain resulted in significantly higher survival rate compared to infection with the S10 wild-type strain. Our data demonstrate that zebrafish larvae are a rapid and reliable model to assess the virulence of clinical porcine S. suis isolates.

  11. 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 Central

    Spagnoli, Sean; Xue, Lan; Kent, Michael L.

    2015-01-01

    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 ten minutes along with taps at 18 and 60 minutes 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 minutes, whereas exposed negative fish retained partial habituation at 18 minutes. 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

  12. Escherichia coli O78 isolated from septicemic lambs shows high pathogenicity in a zebrafish model.

    PubMed

    Kjelstrup, Cecilie K; Barber, Amelia E; Norton, J Paul; Mulvey, Matthew A; L'Abée-Lund, Trine M

    2017-01-25

    The pathogenicity of Escherichia coli O78 strain K46, originally isolated from an outbreak of septicemia in neonatal lambs, was investigated in zebrafish embryo and murine models of infection. Its biofilm potential, cellulose production, and the expression of type 1 pili and curli fimbriae were measured by in vitro assays. The strain was highly pathogenic in the zebrafish embryo model of infection, where it killed all embryos within 24 h post inoculation (hpi) at doses as low as 1000 colony forming units. Zebrafish embryos inoculated with similar doses of commensal E. coli strains showed no signs of disease, and cleared the bacteria within 24 h. E. coli K46 colonized the murine gut at the same level as the uropathogenic E. coli (UPEC) reference strain CFT073 in CBA/J mice after oral inoculation, but infected the murine bladder significantly less than CFT073 after transurethral inoculation. Type 1 pili were clearly expressed by E. coli K46, while curli fimbriae and cellulose production were weakly expressed. The ability to produce biofilm varied in different growth media, but overall E. coli K46 was a poorer biofilm producer compared to the reference strain E. coli UTI89. In conclusion, the zebrafish lethality model provides further evidence that E. coli K46 is highly pathogenic and might be useful in future studies to identify bacterial virulence factors.

  13. A Zebrafish Larval Model to Assess Virulence of Porcine Streptococcus suis Strains

    PubMed Central

    Zaccaria, Edoardo; Cao, Rui; Wells, Jerry M.; van Baarlen, Peter

    2016-01-01

    Streptococcus suis is an encapsulated Gram-positive bacterium, and the leading cause of sepsis and meningitis in young pigs resulting in considerable economic losses in the porcine industry. It is also considered an emerging zoonotic agent. In the environment, both avirulent and virulent strains occur in pigs, and virulent strains appear to cause disease in both humans and pigs. There is a need for a convenient, reliable and standardized animal model to assess S. suis virulence. A zebrafish (Danio rerio) larvae infection model has several advantages, including transparency of larvae, low cost, ease of use and exemption from ethical legislation up to 6 days post fertilization, but has not been previously established as a model for S. suis. Microinjection of different porcine strains of S. suis in zebrafish larvae resulted in highly reproducible dose- and strain-dependent larval death, strongly correlating with presence of the S. suis capsule and to the original virulence of the strain in pigs. Additionally we compared the virulence of the two-component system mutant of ciaRH, which is attenuated for virulence in both mice and pigs in vivo. Infection of larvae with the ΔciaRH strain resulted in significantly higher survival rate compared to infection with the S10 wild-type strain. Our data demonstrate that zebrafish larvae are a rapid and reliable model to assess the virulence of clinical porcine S. suis isolates. PMID:26999052

  14. 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.

  15. Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish.

    PubMed

    Huang, Cong; Niethammer, Philipp

    2018-05-15

    Tissue damage and infection are deemed likewise triggers of innate immune responses. But whereas neutrophil responses to microbes are generally protective, neutrophil recruitment into damaged tissues without infection is deleterious. Why neutrophils respond to tissue damage and not just to microbes is unknown. Is it a flaw of the innate immune system that persists because evolution did not select against it, or does it provide a selective advantage? Here we dissect the contribution of tissue damage signaling to antimicrobial immune responses in a live vertebrate. By intravital imaging of zebrafish larvae, a powerful model for innate immunity, we show that prevention of tissue damage signaling upon microbial ear infection abrogates leukocyte chemotaxis and reduces animal survival, at least in part, through suppression of cytosolic phospholipase A 2 (cPla 2 ), which integrates tissue damage- and microbe-derived cues. Thus, microbial cues are insufficient, and damage signaling is essential for antimicrobial neutrophil responses in zebrafish. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. Zebrafish as a useful model for zoonotic Vibrio parahaemolyticus pathogenicity in fish and human.

    PubMed

    Zhang, Qinghua; Dong, Xuehong; Chen, Biao; Zhang, Yonghua; Zu, Yao; Li, Weiming

    2016-02-01

    Vibrio parahaemolyticus is an important aquatic zoonotic pathogen worldwide that causes vibriosis in many marine fish, and sepsis, gastroenteritis and wound infection in humans. However, the pathogenesis of different sources of V. parahaemolyticus is not fully understood. Here, we examined the pathogenicity and histopathology of fish (V. parahaemolyticus 1.2164) and human (V. parahaemolyticus 17) strains in a zebrafish (Danio rerio). We found that different infection routes resulted in different mortality in zebrafish. Moreover, death due to V. parahaemolyticus 1.2164 infection occurred quicker than that caused by V. parahaemolyticus 17 infection. Hematoxylin-eosin staining of liver, kidney and intestine sections showed histological lesions in all three organs after infection with either strain. V. parahaemolyticus 1.2164 caused more severe damage than V. parahaemolyticus 17. In particular, V. parahaemolyticus 1.2164 treatment induced more serious hydropic degeneration and venous sinus necrosis in the liver than V. parahaemolyticus 17 treatment. The expression levels of three proinflammatory cytokines, interleukin 1β (il1β), interferon phi 1 (ifnϕ1) and tumor necrosis factor α (tnfα), as determined by quantitative real-time PCR, were upregulated in all examined tissues of infected fish. Notably, the peak levels of tnfα were significantly higher than those of il1β and ifnϕ1, suggesting, together with pathological results, that tnfα and il1β play an important role in acute sepsis. High amounts of tnfα may be related to acute liver necrosis, while ifnϕ1 may respond to V. parahaemolyticus and play an antibacterial role for chronically infected adult zebrafish. Taken together, our results suggest that the zebrafish model of V. parahaemolyticus infection is useful for studying strain differences in V. parahaemolyticus pathogenesis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Tetraodon nigroviridis as a nonlethal model of infectious spleen and kidney necrosis virus (ISKNV) infection

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

    Xu Xiaopeng; Huang Lichao; Weng Shaoping

    Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus, family Iridoviridae. We have previously established a high mortality ISKNV infection model of zebrafish (Danio rerio). In this study, a nonlethal Tetraodon nigroviridis model of ISKNV infection was established. ISKNV infection did not cause lethal disease in Tetraodon but could infect almost all the organs of this species. Electron microscopy showed ISKNV particles were present in infected tissues. Immunofluorescence and quantitative real-time PCR analysis showed that nearly all the virions and infected cells were cleared at 14 d postinfection. The expression profiles of interferon-{gamma} andmore » tumor necrosis factor-{alpha} gene in response to ISKNV infection were significantly different in Tetraodon and zebrafish. The establishment of the nonlethal Tetraodon model of ISKNV infection can offer a valuable tool complementary to the zebrafish infection model for studying megalocytivirus disease, fish immune systems, and viral tropism.« less

  3. 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.

  4. 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.

  5. The Zebrafish as a New Model for the In Vivo Study of Shigella flexneri Interaction with Phagocytes and Bacterial Autophagy

    PubMed Central

    Mostowy, Serge; Boucontet, Laurent; Mazon Moya, Maria J.; Sirianni, Andrea; Boudinot, Pierre; Hollinshead, Michael; Cossart, Pascale; Herbomel, Philippe; Levraud, Jean-Pierre; Colucci-Guyon, Emma

    2013-01-01

    Autophagy, an ancient and highly conserved intracellular degradation process, is viewed as a critical component of innate immunity because of its ability to deliver cytosolic bacteria to the lysosome. However, the role of bacterial autophagy in vivo remains poorly understood. The zebrafish (Danio rerio) has emerged as a vertebrate model for the study of infections because it is optically accessible at the larval stages when the innate immune system is already functional. Here, we have characterized the susceptibility of zebrafish larvae to Shigella flexneri, a paradigm for bacterial autophagy, and have used this model to study Shigella-phagocyte interactions in vivo. Depending on the dose, S. flexneri injected in zebrafish larvae were either cleared in a few days or resulted in a progressive and ultimately fatal infection. Using high resolution live imaging, we found that S. flexneri were rapidly engulfed by macrophages and neutrophils; moreover we discovered a scavenger role for neutrophils in eliminating infected dead macrophages and non-immune cell types that failed to control Shigella infection. We observed that intracellular S. flexneri could escape to the cytosol, induce septin caging and be targeted to autophagy in vivo. Depletion of p62 (sequestosome 1 or SQSTM1), an adaptor protein critical for bacterial autophagy in vitro, significantly increased bacterial burden and host susceptibility to infection. These results show the zebrafish larva as a new model for the study of S. flexneri interaction with phagocytes, and the manipulation of autophagy for anti-bacterial therapy in vivo. PMID:24039575

  6. A prebiotic role of Ecklonia cava improves the mortality of Edwardsiella tarda-infected zebrafish models via regulating the growth of lactic acid bacteria and pathogen bacteria.

    PubMed

    Lee, WonWoo; Oh, Jae Young; Kim, Eun-A; Kang, Nalae; Kim, Kil-Nam; Ahn, Ginnae; Jeon, You-Jin

    2016-07-01

    In this study, the beneficial prebiotic roles of Ecklonia cava (E. cava, EC) were evaluated on the growth of lactic acid bacteria (LAB) and pathogen bacteria and the mortality of pathogen-bacteria infected zebrafish model. The result showed that the original E. cava (EC) led to the highest growth effects on three LABs (Lactobacillus brevis, L. brevis; Lactobacillus pentosus, L. pentosus; Lactobacillus plantarum; L. plantarum) and it was dose-dependent manners. Also, EC, its Celluclast enzymatic (ECC) and 100% ethanol extracts (ECE) showed the anti-bacterial activities on the fish pathogenic bacteria such as (Edwardsiella tarda; E. tarda, Streptococcus iniae; S. iniae, and Vibrio harveyi; V. harveyi). Interestingly, EC induced the higher production of the secondary metabolites from L. plantarum in MRS medium. The secondary metabolites produced by EC significantly inhibited the growth of pathogen bacteria. In further in vivo study, the co-treatment of EC and L. plantarum improved the growth and mortality of E. tarda-infected zebrafish as regulating the expression of inflammatory molecules such as iNOS and COX2. Taken together, our present study suggests that the EC plays an important role as a potential prebiotic and has a protective effect against the infection caused by E. tarda injection in zebrafish. Also, our conclusion from this evidence is that EC can be used and applied as a useful prebiotic. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. 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.

  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. 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.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. A zebrafish larval model reveals early tissue-specific innate immune responses to Mucor circinelloides.

    PubMed

    Voelz, Kerstin; Gratacap, Remi L; Wheeler, Robert T

    2015-11-01

    Mucormycosis is an emerging fungal infection that is clinically difficult to manage, with increasing incidence and extremely high mortality rates. Individuals with diabetes, suppressed immunity or traumatic injury are at increased risk of developing disease. These individuals often present with defects in phagocytic effector cell function. Research using mammalian models and phagocytic effector cell lines has attempted to decipher the importance of the innate immune system in host defence against mucormycosis. However, these model systems have not been satisfactory for direct analysis of the interaction between innate immune effector cells and infectious sporangiospores in vivo. Here, we report the first real-time in vivo analysis of the early innate immune response to mucormycete infection using a whole-animal zebrafish larval model system. We identified differential host susceptibility, dependent on the site of infection (hindbrain ventricle and swim bladder), as well as differential functions of the two major phagocyte effector cell types in response to viable and non-viable spores. Larval susceptibility to mucormycete spore infection was increased upon immunosuppressant treatment. We showed for the first time that macrophages and neutrophils were readily recruited in vivo to the site of infection in an intact host and that spore phagocytosis can be observed in real-time in vivo. While exploring innate immune effector recruitment dynamics, we discovered the formation of phagocyte clusters in response to fungal spores that potentially play a role in fungal spore dissemination. Spores failed to activate pro-inflammatory gene expression by 6 h post-infection in both infection models. After 24 h, induction of a pro-inflammatory response was observed only in hindbrain ventricle infections. Only a weak pro-inflammatory response was initiated after spore injection into the swim bladder during the same time frame. In the future, the zebrafish larva as a live whole

  1. 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

  2. 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.

  3. 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

  4. Adequate Th2-Type Response Associates with Restricted Bacterial Growth in Latent Mycobacterial Infection of Zebrafish

    PubMed Central

    Hammarén, Milka Marjut; Luukinen, Bruno Vincent; Pesu, Marko; Rämet, Mika; Parikka, Mataleena

    2014-01-01

    Tuberculosis is still a major health problem worldwide. Currently it is not known what kind of immune responses lead to successful control and clearance of Mycobacterium tuberculosis. This gap in knowledge is reflected by the inability to develop sufficient diagnostic and therapeutic tools to fight tuberculosis. We have used the Mycobacterium marinum infection model in the adult zebrafish and taken advantage of heterogeneity of zebrafish population to dissect the characteristics of adaptive immune responses, some of which are associated with well-controlled latency or bacterial clearance while others with progressive infection. Differences in T cell responses between subpopulations were measured at the transcriptional level. It was discovered that a high total T cell level was usually associated with lower bacterial loads alongside with a T helper 2 (Th2)-type gene expression signature. At late time points, spontaneous reactivation with apparent symptoms was characterized by a low Th2/Th1 marker ratio and a substantial induction of foxp3 reflecting the level of regulatory T cells. Characteristic gata3/tbx21 has potential as a biomarker for the status of mycobacterial disease. PMID:24968056

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. Priming of innate antimycobacterial immunity by heat-killed Listeria monocytogenes induces sterilizing response in the adult zebrafish tuberculosis model

    PubMed Central

    Luukinen, Hanna; Vanha-aho, Leena-Maija; Svorjova, Aleksandra; Kantanen, Laura; Järvinen, Sampsa; Dufour, Eric; Rämet, Mika; Hytönen, Vesa Pekka

    2018-01-01

    ABSTRACT Mycobacterium tuberculosis remains one of the most problematic infectious agents, owing to its highly developed mechanisms to evade host immune responses combined with the increasing emergence of antibiotic resistance. Host-directed therapies aiming to optimize immune responses to improve bacterial eradication or to limit excessive inflammation are a new strategy for the treatment of tuberculosis. In this study, we have established a zebrafish-Mycobacterium marinum natural host-pathogen model system to study induced protective immune responses in mycobacterial infection. We show that priming adult zebrafish with heat-killed Listeria monocytogenes (HKLm) at 1 day prior to M. marinum infection leads to significantly decreased mycobacterial loads in the infected zebrafish. Using rag1−/− fish, we show that the protective immunity conferred by HKLm priming can be induced through innate immunity alone. At 24 h post-infection, HKLm priming leads to a significant increase in the expression levels of macrophage-expressed gene 1 (mpeg1), tumor necrosis factor α (tnfa) and nitric oxide synthase 2b (nos2b), whereas superoxide dismutase 2 (sod2) expression is downregulated, implying that HKLm priming increases the number of macrophages and boosts intracellular killing mechanisms. The protective effects of HKLm are abolished when the injected material is pretreated with nucleases or proteinase K. Importantly, HKLm priming significantly increases the frequency of clearance of M. marinum infection by evoking sterilizing immunity (25 vs 3.7%, P=0.0021). In this study, immune priming is successfully used to induce sterilizing immunity against mycobacterial infection. This model provides a promising new platform for elucidating the mechanisms underlying sterilizing immunity and to develop host-directed treatment or prevention strategies against tuberculosis. This article has an associated First Person interview with the first author of the paper. PMID:29208761

  3. 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.

  4. 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).

  5. 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.

  6. 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.

  7. Zebrafish (Danio rerio) as a screen for attenuation of Lancefield group C streptococci and a model for streptococcal pathogenesis.

    PubMed

    Borst, L B; Patterson, S K; Lanka, S; Suyemoto, M M; Maddox, C W

    2013-05-01

    Group C streptococci are highly contagious pyogenic bacteria responsible for respiratory tract, lymph node, urogenital tract, and wound infections. Wild-type strains of Streptococcus equi ssp equi (S. equi) and Streptococcus equi ssp zooepidemicus (S. zoo) as well as a commercially available modified live vaccine strain of S. equi were evaluated for virulence in zebrafish. Survival times, histologic lesions, and relative gene expression were compared among groups. Based on the intramuscular route of infection, significantly shorter survival times were observed in fish infected with wild-type strain when compared to modified live vaccine and S. zoo strains. Histologically, S. zoo-infected fish demonstrated a marked increase in inflammatory infiltrates (predominantly macrophages) at the site of infection, as well as increased cellularity in the spleen and renal interstitium. In contrast, minimal cellular immune response was observed in S. equi-injected fish with local tissue necrosis and edema predominating. Based on whole comparative genomic hybridization, increased transcription of positive acute-phase proteins, coagulation factors, and antimicrobial peptides were observed in S. equi-injected fish relative to S. zoo-injected fish, while mediators of cellular inflammation, including CXC chemokines and granulin, were upregulated in S. zoo-injected fish relative to S. equi-injected fish. In a screen of 11 clinical isolates, S. equi strains with a single nucleotide deletion in the upstream region of szp, a known virulence factor of streptococci, were found to be significantly attenuated in zebrafish. These collective findings underscore the value of the zebrafish as a model of streptococcal pathogenesis.

  8. Dithiazole thione derivative as competitive NorA efflux pump inhibitor to curtail multi drug resistant clinical isolate of MRSA in a zebrafish infection model.

    PubMed

    Lowrence, Rene Christena; Raman, Thiagarajan; Makala, Himesh V; Ulaganathan, Venkatasubramanian; Subramaniapillai, Selva Ganesan; Kuppuswamy, Ashok Ayyappa; Mani, Anisha; Chittoor Neelakantan, Sundaresan; Nagarajan, Saisubramanian

    2016-11-01

    Multi drug resistant (MDR) pathogens pose a serious threat to public health since they can easily render most potent drugs ineffective. Efflux pump inhibitors (EPI) can be used to counter the MDR phenotypes arising due to increased efflux. In the present study, a series of dithiazole thione derivatives were synthesized and checked for its antibacterial and efflux pump inhibitory (EPI) activity. Among 10 dithiazole thione derivatives, real-time efflux studies revealed that seven compounds were potent EPIs relative to CCCP. Zebrafish toxicity studies identified four non-toxic putative EPIs. Both DTT3 and DTT9 perturbed membrane potential and DTT6 was haemolytic. Among DTT6 and DTT10, the latter was less toxic as evidenced by histopathology studies. Since DTT10 was non-haemolytic, did not affect the membrane potential, and was least toxic, it was chosen further for in vivo study, wherein DTT10 potentiated effect of ciprofloxacin against clinical strain of MRSA and reduced bacterial burden in muscle and skin tissue of infected zebrafish by ~ 1.7 and 2.5 log fold respectively. Gene expression profiling of major efflux transport proteins by qPCR revealed that clinical isolate of MRSA, in the absence of antibiotic, upregulated NorA, NorB and MepA pump, whereas it downregulates NorC and MgrA relative to wild-type strain of Staphylococcus aureus. In vitro studies with NorA mutant strains and substrate profiling revealed that at higher concentrations DTT10 is likely to function as a competitive inhibitor of NorA efflux protein in S. aureus, whereas at lower concentrations it might inhibit ciprofloxacin efflux through NorB and MepA as implied by docking studies. A novel non-toxic, non-haemolytic dithiazole thione derivative (DTT10) was identified as a potent competitive inhibitor of NorA efflux pump in S. aureus using in silico, in vitro and in vivo studies. This study also underscores the importance of using zebrafish infection model to screen and evaluate putative EPI for

  9. N-hexanoyl-L-homoserine lactone-degrading Pseudomonas aeruginosa PsDAHP1 protects zebrafish against Vibrio parahaemolyticus infection.

    PubMed

    Vinoj, Gopalakrishnan; Jayakumar, Rengarajan; Chen, Jiann-Chu; Withyachumnarnkul, Boonsirm; Shanthi, Sathappan; Vaseeharan, Baskaralingam

    2015-01-01

    Four strains of N-hexanoyl-L-homoserine lactone (AHL)-degrading Pseudomonas spp., named PsDAHP1, PsDAHP2, PsDAHP3, and PsDAHP4 were isolated and identified from the intestine of Fenneropenaeus indicus. PsDAHP1 showed the highest AHL-degrading activity among the four isolates. PsDAHP1 inhibited biofilm-forming exopolysaccharide and altered cell surface hydrophobicity of virulent green fluorescent protein (GFP)-tagged Vibrio parahaemolyticus DAHV2 (GFP-VpDAHV2). Oral administration of PsDAHP1 significantly reduced zebrafish mortality caused by GFP-VpDAHV2 challenge, and inhibited colonisation of GFP-VpDAHV2 in the gills and intestine of zebrafish as evidence by confocal laser scanning microscope and selective plating. Furthermore, zebrafish receiving PsDAHP1-containing feed had increased phagocytic cells of its leucocytes, increased serum activities of superoxide dismutase and lysozyme. The results suggest that Pseudomonas aeruginosa PsDAHP1 could protect zebrafish from V. parahaemolyticus infection by inhibiting biofilm formation and enhancing defence mechanisms of the fish. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. 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...

  11. Muscles provide protection during microbial infection by activating innate immune response pathways in Drosophila and zebrafish.

    PubMed

    Chatterjee, Arunita; Roy, Debasish; Patnaik, Esha; Nongthomba, Upendra

    2016-06-01

    Muscle contraction brings about movement and locomotion in animals. However, muscles have also been implicated in several atypical physiological processes including immune response. The role of muscles in immunity and the mechanism involved has not yet been deciphered. In this paper, using Drosophila indirect flight muscles (IFMs) as a model, we show that muscles are immune-responsive tissues. Flies with defective IFMs are incapable of mounting a potent humoral immune response. Upon immune challenge, the IFMs produce anti-microbial peptides (AMPs) through the activation of canonical signaling pathways, and these IFM-synthesized AMPs are essential for survival upon infection. The trunk muscles of zebrafish, a vertebrate model system, also possess the capacity to mount an immune response against bacterial infections, thus establishing that immune responsiveness of muscles is evolutionarily conserved. Our results suggest that physiologically fit muscles might boost the innate immune response of an individual. © 2016. Published by The Company of Biologists Ltd.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. The Proprotein Convertase Subtilisin/Kexin FurinA Regulates Zebrafish Host Response against Mycobacterium marinum

    PubMed Central

    Ojanen, Markus J. T.; Turpeinen, Hannu; Cordova, Zuzet M.; Hammarén, Milka M.; Harjula, Sanna-Kaisa E.; Parikka, Mataleena; Rämet, Mika

    2015-01-01

    Tuberculosis is a chronic bacterial disease with a complex pathogenesis. An effective immunity against Mycobacterium tuberculosis requires both the innate and adaptive immune responses, including proper T helper (Th) type 1 cell function. FURIN is a proprotein convertase subtilisin/kexin (PCSK) enzyme, which is highly expressed in Th1 type cells. FURIN expression in T cells is essential for maintaining peripheral immune tolerance, but its role in the innate immunity and infections has remained elusive. Here, we utilized Mycobacterium marinum infection models in zebrafish (Danio rerio) to investigate how furin regulates host responses against mycobacteria. In steady-state furinAtd204e/+ fish reduced furinA mRNA levels associated with low granulocyte counts and elevated Th cell transcription factor expressions. Silencing furin genes reduced the survival of M. marinum-infected zebrafish embryos. A mycobacterial infection upregulated furinA in adult zebrafish, and infected furinAtd204e/+ mutants exhibited a proinflammatory phenotype characterized by elevated tumor necrosis factor a (tnfa), lymphotoxin alpha (lta) and interleukin 17a/f3 (il17a/f3) expression levels. The enhanced innate immune response in the furinAtd204e/+ mutants correlated with a significantly decreased bacterial burden in a chronic M. marinum infection model. Our data show that upregulated furinA expression can serve as a marker for mycobacterial disease, since it inhibits early host responses and consequently promotes bacterial growth in a chronic infection. PMID:25624351

  19. Zebrafish tissue injury causes upregulation of interleukin-1 and caspase-dependent amplification of the inflammatory response.

    PubMed

    Ogryzko, Nikolay V; Hoggett, Emily E; Solaymani-Kohal, Sara; Tazzyman, Simon; Chico, Timothy J A; Renshaw, Stephen A; Wilson, Heather L

    2014-02-01

    Interleukin-1 (IL-1), the 'gatekeeper' of inflammation, is the apical cytokine in a signalling cascade that drives the early response to injury or infection. Expression, processing and secretion of IL-1 are tightly controlled, and dysregulated IL-1 signalling has been implicated in a number of pathologies ranging from atherosclerosis to complications of infection. Our understanding of these processes comes from in vitro monocytic cell culture models as lines or primary isolates, in which a range and spectra of IL-1 secretion mechanisms have been described. We therefore investigated whether zebrafish embryos provide a suitable in vivo model for studying IL-1-mediated inflammation. Structurally, zebrafish IL-1β shares a β-sheet-rich trefoil structure with its human counterpart. Functionally, leukocyte expression of IL-1β was detectable only following injury, which activated leukocytes throughout zebrafish embryos. Migration of macrophages and neutrophils was attenuated by inhibitors of either caspase-1 or P2X7, which similarly inhibited the activation of NF-κB at the site of injury. Zebrafish offer a new and versatile model to study the IL-1β pathway in inflammatory disease and should offer unique insights into IL-1 biology in vivo.

  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. Pituitary adenylate cyclase-activating polypeptide (PACAP) in zebrafish models of nephrotic syndrome

    PubMed Central

    van den Heuvel, Lambertus P.; Khodaparast, Laleh; Khodaparast, Ladan; van Geet, Chris; Freson, Kathleen

    2017-01-01

    Pituitary adenylate cyclase-activating polypeptide (PACAP) is an inhibitor of megakaryopoiesis and platelet function. Recently, PACAP deficiency was observed in children with nephrotic syndrome (NS), associated with increased platelet count and aggregability and increased risk of thrombosis. To further study PACAP deficiency in NS, we used transgenic Tg(cd41:EGFP) zebrafish with GFP-labeled thrombocytes. We generated two models for congenital NS, a morpholino injected model targeting nphs1 (nephrin), which is mutated in the Finnish-type congenital NS. The second model was induced by exposure to the nephrotoxic compound adriamycin. Nephrin RNA expression was quantified and zebrafish embryos were live-screened for proteinuria and pericardial edema as evidence of renal impairment. Protein levels of PACAP and its binding-protein ceruloplasmin were measured and GFP-labeled thrombocytes were quantified. We also evaluated the effects of PACAP morpholino injection and the rescue effects of PACAP-38 peptide in both congenital NS models. Nephrin downregulation and pericardial edema were observed in both nephrin morpholino injected and adriamycin exposed congenital NS models. However, PACAP deficiency was demonstrated only in the adriamycin exposed condition. Ceruloplasmin levels and the number of GFP-labeled thrombocytes remained unchanged in both models. PACAP morpholino injections worsened survival rates and the edema phenotype in both congenital NS models while injection with human PACAP-38 could only rescue the adriamycin exposed model. We hereby report, for the first time, PACAP deficiency in a NS zebrafish model as a consequence of adriamycin exposure. However, distinct from the human congenital NS, both zebrafish models retained normal levels of ceruloplasmin and thrombocytes. We further extend the renoprotective effects of the PACAP-38 peptide against adriamycin toxicity in zebrafish. PMID:28759637

  2. 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

  3. Pseudoloma neurophilia infections in zebrafish Danio rerio: effects of stress on survival, growth, and reproduction.

    USGS Publications Warehouse

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

    2009-01-01

    Pseudoloma neurophilia (Microsporidia) is a common disease of zebrafish Danio rerio, including those used as research models. We conducted a study comprised of 4 separate experiments to determine the effects of husbandry stress on preexisting and experimental P. neurophilia infections and the subsequent effects on survival, infection onset and intensity, fish growth, and reproduction. In fish (AB strain) with preexisting infections, stress or feeding cortisol significantly increased mortality over 7 wk compared to no stress or cortisol treatment. In contrast, no mortality was observed in fish (TL strain) experimentally exposed to P. neurophilia over 10 wk. A third experiment involved experimental exposure of AB fish to P. neurophilia and exposure to crowding and handling stressors. No mortality was associated with P. neurophilia regardless of stress treatment over a period of 20 wk. However, the onset of infection occurred sooner in stress-treated fish. Stress significantly increased the mean intensity of infection (described as xenoma area/spinal cord area in histological sections) at Week 20 post-exposure (PE). In fish with preexisting infections, myositis was significantly greater in stressed and cortisol-treated fish than those not stressed. With experimental exposure of AB fish, stressed and infected groups weighed significantly less than the control group at Week 20 PE. Regarding fecundity, the number of larvae hatched at 5 d post fertilization was negatively associated with mean infection intensity among P. neurophilia-infected and stressed AB fish. These experiments are the first to show empirically that P. neurophilia can be associated with reduced weight and fecundity, and that stress can exacerbate the severity of the infection.

  4. 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

  5. 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.

  6. 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

  7. Copper toxicology, oxidative stress and inflammation using zebrafish as experimental model.

    PubMed

    Pereira, Talita Carneiro Brandão; Campos, Maria Martha; Bogo, Maurício Reis

    2016-07-01

    Copper is an essential micronutrient and a key catalytic cofactor in a wide range of enzymes. As a trace element, copper levels are tightly regulated and both its deficit and excess are deleterious to the organism. Under inflammatory conditions, serum copper levels are increased and trigger oxidative stress responses that activate inflammatory responses. Interestingly, copper dyshomeostasis, oxidative stress and inflammation are commonly present in several chronic diseases. Copper exposure can be easily modeled in zebrafish; a consolidated model in toxicology with increasing interest in immunity-related research. As a result of developmental, economical and genetic advantages, this freshwater teleost is uniquely suitable for chemical and genetic large-scale screenings, representing a powerful experimental tool for a whole-organism approach, mechanistic studies, disease modeling and beyond. Copper toxicological and more recently pro-inflammatory effects have been investigated in both larval and adult zebrafish with breakthrough findings. Here, we provide an overview of copper metabolism in health and disease and its effects on oxidative stress and inflammation responses in zebrafish models. Copper-induced inflammation is highlighted owing to its potential to easily mimic pro-oxidative and pro-inflammatory features that combined with zebrafish genetic tractability could help further in the understanding of copper metabolism, inflammatory responses and related diseases. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  8. Zebrafish tissue injury causes upregulation of interleukin-1 and caspase-dependent amplification of the inflammatory response

    PubMed Central

    Ogryzko, Nikolay V.; Hoggett, Emily E.; Solaymani-Kohal, Sara; Tazzyman, Simon; Chico, Timothy J. A.; Renshaw, Stephen A.; Wilson, Heather L.

    2014-01-01

    ABSTRACT Interleukin-1 (IL-1), the ‘gatekeeper’ of inflammation, is the apical cytokine in a signalling cascade that drives the early response to injury or infection. Expression, processing and secretion of IL-1 are tightly controlled, and dysregulated IL-1 signalling has been implicated in a number of pathologies ranging from atherosclerosis to complications of infection. Our understanding of these processes comes from in vitro monocytic cell culture models as lines or primary isolates, in which a range and spectra of IL-1 secretion mechanisms have been described. We therefore investigated whether zebrafish embryos provide a suitable in vivo model for studying IL-1-mediated inflammation. Structurally, zebrafish IL-1β shares a β-sheet-rich trefoil structure with its human counterpart. Functionally, leukocyte expression of IL-1β was detectable only following injury, which activated leukocytes throughout zebrafish embryos. Migration of macrophages and neutrophils was attenuated by inhibitors of either caspase-1 or P2X7, which similarly inhibited the activation of NF-κB at the site of injury. Zebrafish offer a new and versatile model to study the IL-1β pathway in inflammatory disease and should offer unique insights into IL-1 biology in vivo. PMID:24203886

  9. 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.

  10. Roles of inflammatory caspases during processing of zebrafish interleukin-1β in Francisella noatunensis infection

    USGS Publications Warehouse

    Vojtech, Lucia N.; Scharping, Nichole; Woodson, James C.; Hansen, John D.

    2012-01-01

    The interleukin-1 family of cytokines are essential for the control of pathogenic microbes but are also responsible for devastating autoimmune pathologies. Consequently, tight regulation of inflammatory processes is essential for maintaining homeostasis. In mammals, interleukin-1 beta (IL-1β) is primarily regulated at two levels, transcription and processing. The main pathway for processing IL-1β is the inflammasome, a multiprotein complex that forms in the cytosol and which results in the activation of inflammatory caspase (caspase 1) and the subsequent cleavage and secretion of active IL-1β. Although zebrafish encode orthologs of IL-1β and inflammatory caspases, the processing of IL-1β by activated caspase(s) has never been examined. Here, we demonstrate that in response to infection with the fish-specific bacterial pathogen Francisella noatunensis, primary leukocytes from adult zebrafish display caspase-1-like activity that results in IL-1β processing. Addition of caspase 1 or pancaspase inhibitors considerably abrogates IL-1β processing. As in mammals, this processing event is concurrent with the secretion of cleaved IL-1β into the culture medium. Furthermore, two putative zebrafish inflammatory caspase orthologs, caspase A and caspase B, are both able to cleave IL-1β, but with different specificities. These results represent the first demonstration of processing and secretion of zebrafish IL-1β in response to a pathogen, contributing to our understanding of the evolutionary processes governing the regulation of inflammation.                   

  11. [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.

  12. 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.

  13. Perspectives on zebrafish models of hallucinogenic drugs and related psychotropic compounds.

    PubMed

    Neelkantan, Nikhil; Mikhaylova, Alina; Stewart, Adam Michael; Arnold, Raymond; Gjeloshi, Visar; Kondaveeti, Divya; Poudel, Manoj K; Kalueff, Allan V

    2013-08-21

    Among different classes of psychotropic drugs, hallucinogenic agents exert one of the most prominent effects on human and animal behaviors, markedly altering sensory, motor, affective, and cognitive responses. The growing clinical and preclinical interest in psychedelic, dissociative, and deliriant hallucinogens necessitates novel translational, sensitive, and high-throughput in vivo models and screens. Primate and rodent models have been traditionally used to study cellular mechanisms and neural circuits of hallucinogenic drugs' action. The utility of zebrafish ( Danio rerio ) in neuroscience research is rapidly growing due to their high physiological and genetic homology to humans, ease of genetic manipulation, robust behaviors, and cost effectiveness. Possessing a fully characterized genome, both adult and larval zebrafish are currently widely used for in vivo screening of various psychotropic compounds, including hallucinogens and related drugs. Recognizing the growing importance of hallucinogens in biological psychiatry, here we discuss hallucinogenic-induced phenotypes in zebrafish and evaluate their potential as efficient preclinical models of drug-induced states in humans.

  14. Perspectives on Zebrafish Models of Hallucinogenic Drugs and Related Psychotropic Compounds

    PubMed Central

    2013-01-01

    Among different classes of psychotropic drugs, hallucinogenic agents exert one of the most prominent effects on human and animal behaviors, markedly altering sensory, motor, affective, and cognitive responses. The growing clinical and preclinical interest in psychedelic, dissociative, and deliriant hallucinogens necessitates novel translational, sensitive, and high-throughput in vivo models and screens. Primate and rodent models have been traditionally used to study cellular mechanisms and neural circuits of hallucinogenic drugs’ action. The utility of zebrafish (Danio rerio) in neuroscience research is rapidly growing due to their high physiological and genetic homology to humans, ease of genetic manipulation, robust behaviors, and cost effectiveness. Possessing a fully characterized genome, both adult and larval zebrafish are currently widely used for in vivo screening of various psychotropic compounds, including hallucinogens and related drugs. Recognizing the growing importance of hallucinogens in biological psychiatry, here we discuss hallucinogenic-induced phenotypes in zebrafish and evaluate their potential as efficient preclinical models of drug-induced states in humans. PMID:23883191

  15. Toward developmental models of psychiatric disorders in zebrafish

    PubMed Central

    Norton, William H. J.

    2013-01-01

    Psychiatric disorders are a diverse set of diseases that affect all aspects of mental function including social interaction, thinking, feeling, and mood. Although psychiatric disorders place a large economic burden on society, the drugs available to treat them are often palliative with variable efficacy and intolerable side-effects. The development of novel drugs has been hindered by a lack of knowledge about the etiology of these diseases. It is thus necessary to further investigate psychiatric disorders using a combination of human molecular genetics, gene-by-environment studies, in vitro pharmacological and biochemistry experiments, animal models, and investigation of the non-biological basis of these diseases, such as environmental effects. Many psychiatric disorders, including autism spectrum disorder, attention-deficit/hyperactivity disorder, mental retardation, and schizophrenia can be triggered by alterations to neural development. The zebrafish is a popular model for developmental biology that is increasingly used to study human disease. Recent work has extended this approach to examine psychiatric disorders as well. However, since psychiatric disorders affect complex mental functions that might be human specific, it is not possible to fully model them in fish. In this review, I will propose that the suitability of zebrafish for developmental studies, and the genetic tools available to manipulate them, provide a powerful model to study the roles of genes that are linked to psychiatric disorders during neural development. The relative speed and ease of conducting experiments in zebrafish can be used to address two areas of future research: the contribution of environmental factors to disease onset, and screening for novel therapeutic compounds. PMID:23637652

  16. 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

  17. Effect of microalgae on intestinal inflammation triggered by soybean meal and bacterial infection in zebrafish

    PubMed Central

    Solís, Camila J.; Ulloa, Pilar E.; Hedrera, Manuel; Pizarro-Guajardo, Marjorie; Paredes-Sabja, Daniel

    2017-01-01

    Soybean meal has been used in many commercial diets for farm fish; despite this component inducing intestinal inflammation. On the other hand, microalgae have increasingly been used as dietary supplements in fish feed. Nevertheless, the vast quantity of microalgae species means that many remain under- or unstudied, thus limiting wide scale commercial application. In this work, we evaluated the effects to zebrafish (Danio rerio) of including Tetraselmis sp (Ts); Phaeodactylum tricornutum (Pt); Chlorella sp (Ch); Nannochloropsis oculata (No); or Nannochloropsis gaditana (Ng) as additives in a soybean meal-based diet on intestinal inflammation and survival after Edwardsiella tarda infection. In larvae fed a soybean meal diet supplemented with Ts, Pt, Ch, or Ng, the quantity of neutrophils present in the intestine drastically decreased as compared to larvae fed only the soybean meal diet. Likewise, Ts or Ch supplements in soybean meal or fishmeal increased zebrafish survival by more than 20% after being challenged. In the case of Ts, the observed effect correlated with an increased number of neutrophils present at the infection site. These results suggest that the inclusion of Ts or Ch in fish diets could allow the use of SBM and at the same time improve performance against pathogen. PMID:29117213

  18. 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.

  19. 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

  20. Zebrafish models for functional and toxicological screening of nanoscale drug delivery systems: promoting preclinical applications

    PubMed Central

    Lee, Keon Yong; Jang, Gun Hyuk; Byun, Cho Hyun; Jeun, Minhong

    2017-01-01

    Preclinical screening with animal models is an important initial step in clinical translation of new drug delivery systems. However, establishing efficacy, biodistribution, and biotoxicity of complex, multicomponent systems in small animal models can be expensive and time-consuming. Zebrafish models represent an alternative for preclinical studies for nanoscale drug delivery systems. These models allow easy optical imaging, large sample size, and organ-specific studies, and hence an increasing number of preclinical studies are employing zebrafish models. In this review, we introduce various models and discuss recent studies of nanoscale drug delivery systems in zebrafish models. Also in the end, we proposed a guideline for the preclinical trials to accelerate the progress in this field. PMID:28515222

  1. Zebrafish models for functional and toxicological screening of nanoscale drug delivery systems: promoting preclinical applications.

    PubMed

    Lee, Keon Yong; Jang, Gun Hyuk; Byun, Cho Hyun; Jeun, Minhong; Searson, Peter C; Lee, Kwan Hyi

    2017-06-30

    Preclinical screening with animal models is an important initial step in clinical translation of new drug delivery systems. However, establishing efficacy, biodistribution, and biotoxicity of complex, multicomponent systems in small animal models can be expensive and time-consuming. Zebrafish models represent an alternative for preclinical studies for nanoscale drug delivery systems. These models allow easy optical imaging, large sample size, and organ-specific studies, and hence an increasing number of preclinical studies are employing zebrafish models. In this review, we introduce various models and discuss recent studies of nanoscale drug delivery systems in zebrafish models. Also in the end, we proposed a guideline for the preclinical trials to accelerate the progress in this field. © 2017 The Author(s).

  2. 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

  3. A zebrafish model of chordoma initiated by notochord-driven expression of HRASV12

    PubMed Central

    Burger, Alexa; Vasilyev, Aleksandr; Tomar, Ritu; Selig, Martin K.; Nielsen, G. Petur; Peterson, Randall T.; Drummond, Iain A.; Haber, Daniel A.

    2014-01-01

    Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer. PMID:24311731

  4. 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

  5. Zebrafish as a systems toxicology model for developmental neurotoxicity testing.

    PubMed

    Nishimura, Yuhei; Murakami, Soichiro; Ashikawa, Yoshifumi; Sasagawa, Shota; Umemoto, Noriko; Shimada, Yasuhito; Tanaka, Toshio

    2015-02-01

    The developing brain is extremely sensitive to many chemicals. Exposure to neurotoxicants during development has been implicated in various neuropsychiatric and neurological disorders, including autism spectrum disorder, attention deficit hyperactive disorder, schizophrenia, Parkinson's disease, and Alzheimer's disease. Although rodents have been widely used for developmental neurotoxicity testing, experiments using large numbers of rodents are time-consuming, expensive, and raise ethical concerns. Using alternative non-mammalian animal models may relieve some of these pressures by allowing testing of large numbers of subjects while reducing expenses and minimizing the use of mammalian subjects. In this review, we discuss some of the advantages of using zebrafish in developmental neurotoxicity testing, focusing on central nervous system development, neurobehavior, toxicokinetics, and toxicodynamics in this species. We also describe some important examples of developmental neurotoxicity testing using zebrafish combined with gene expression profiling, neuroimaging, or neurobehavioral assessment. Zebrafish may be a systems toxicology model that has the potential to reveal the pathways of developmental neurotoxicity and to provide a sound basis for human risk assessments. © 2014 Japanese Teratology Society.

  6. Susceptibility of zebrafish (Danio rerio) to a model pathogen, spring viremia of carp virus

    USGS Publications Warehouse

    Sanders, George E.; Batts, William N.; Winton, James R.

    2003-01-01

    To improve our understanding of the genetic basis of fish disease, we developed a pathogen model, using zebrafish (Danio rerio) and spring virema of carp virus (SVCV). Replicate groups of 10 fish were acclimated to 20 or 24°C, then were exposed to SVCV concentrations of 103 to 105 plaque-forming units per milliliter (PFU/ml) of water and observed daily. In a second trial, fish were acclimated to 15°C, and replicate groups of 10 fish were exposed to SVCV at a concentration of 105 PFU/ml; however, the temperature was raised 1°C/wk. Moribund fish were collected for histologic examination, and dead fish were assayed for virus by use of cell culture and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Mortality exceeded 50% in fish exposed to 105 PFU of SVCV/ml at the lower temperatures. Clinical signs of disease became evident seven days after viral exposure and were observed most consistently in fish of the 105 PFU/ml groups. Affected zebrafish were anorectic and listless, with epidermal petechial hemorrhages followed by death. Use of plaque assays and RT-PCR analysis confirmed presence of SVCV at titers ≥ 104 PFU/g of tissue. Histologic lesions included multifocal brachial necrosis and melanomacrophage proliferation in gills, liver, and kidneys. These results indicate that zebrafish are susceptible to infection by SVCV under conditions that mimic a natural route of exposure.

  7. ZEBRAFISH AS AN IN VIVO MODEL FOR SUSTAINABLE CHEMICAL DESIGN.

    PubMed

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

    2016-12-21

    Heightened public awareness about the many thousands of chemicals in use and present as persistent contaminants in the environment has increased the demand for safer chemicals and more rigorous toxicity testing. There is a growing recognition that the use of traditional test models and empirical approaches is impractical for screening for toxicity the many thousands of chemicals in the environment and the hundreds of new chemistries introduced each year. These realities coupled with the green chemistry movement have prompted efforts to implement more predictive-based approaches to evaluate chemical toxicity early in product development. While used for many years in environmental toxicology and biomedicine, zebrafish use has accelerated more recently in genetic toxicology, high throughput screening (HTS), and behavioral testing. This review describes major advances in these testing methods that have positioned the zebrafish as a highly applicable model in chemical safety evaluations and sustainable chemistry efforts. Many toxic responses have been shown to be shared among fish and mammals owing to their generally well-conserved development, cellular networks, and organ systems. These shared responses have been observed for chemicals that impair endocrine functioning, development, and reproduction, as well as those that elicit cardiotoxicity and carcinogenicity, among other diseases. HTS technologies with zebrafish enable screening large chemical libraries for bioactivity that provide opportunities for testing early in product development. A compelling attribute of the zebrafish centers on being able to characterize toxicity mechanisms across multiple levels of biological organization from the genome to receptor interactions and cellular processes leading to phenotypic changes such as developmental malformations. Finally, there is a growing recognition of the links between human and wildlife health and the need for approaches that allow for assessment of real world

  8. ZEBRAFISH AS AN IN VIVO MODEL FOR SUSTAINABLE CHEMICAL DESIGN

    PubMed Central

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

    2016-01-01

    Heightened public awareness about the many thousands of chemicals in use and present as persistent contaminants in the environment has increased the demand for safer chemicals and more rigorous toxicity testing. There is a growing recognition that the use of traditional test models and empirical approaches is impractical for screening for toxicity the many thousands of chemicals in the environment and the hundreds of new chemistries introduced each year. These realities coupled with the green chemistry movement have prompted efforts to implement more predictive-based approaches to evaluate chemical toxicity early in product development. While used for many years in environmental toxicology and biomedicine, zebrafish use has accelerated more recently in genetic toxicology, high throughput screening (HTS), and behavioral testing. This review describes major advances in these testing methods that have positioned the zebrafish as a highly applicable model in chemical safety evaluations and sustainable chemistry efforts. Many toxic responses have been shown to be shared among fish and mammals owing to their generally well-conserved development, cellular networks, and organ systems. These shared responses have been observed for chemicals that impair endocrine functioning, development, and reproduction, as well as those that elicit cardiotoxicity and carcinogenicity, among other diseases. HTS technologies with zebrafish enable screening large chemical libraries for bioactivity that provide opportunities for testing early in product development. A compelling attribute of the zebrafish centers on being able to characterize toxicity mechanisms across multiple levels of biological organization from the genome to receptor interactions and cellular processes leading to phenotypic changes such as developmental malformations. Finally, there is a growing recognition of the links between human and wildlife health and the need for approaches that allow for assessment of real world

  9. Control of Mucosal Candidiasis in the Zebrafish Swim Bladder Depends on Neutrophils That Block Filament Invasion and Drive Extracellular-Trap Production

    PubMed Central

    Gratacap, Remi L.; Scherer, Allison K.; Seman, Brittany G.

    2017-01-01

    ABSTRACT Candida albicans is a ubiquitous mucosal commensal that is normally prevented from causing acute or chronic invasive disease. Neutrophils contribute to protection in oral infection but exacerbate vulvovaginal candidiasis. To dissect the role of neutrophils during mucosal candidiasis, we took advantage of a new, transparent zebrafish swim bladder infection model. Intravital microscopic tracking of individual animals revealed that the blocking of neutrophil recruitment leads to rapid mortality in this model through faster disease progression. Conversely, artificial recruitment of neutrophils during early infection reduces disease pressure. Noninvasive longitudinal tracking showed that mortality is a consequence of C. albicans breaching the epithelial barrier and invading surrounding tissues. Accordingly, we found that a hyperfilamentous C. albicans strain breaches the epithelial barrier more frequently and causes mortality in immunocompetent zebrafish. A lack of neutrophils at the infection site is associated with less fungus-associated extracellular DNA and less damage to fungal filaments, suggesting that neutrophil extracellular traps help to protect the epithelial barrier from C. albicans breach. We propose a homeostatic model where C. albicans disease pressure is balanced by neutrophil-mediated damage of fungi, maintaining this organism as a commensal while minimizing the risk of damage to host tissue. The unequaled ability to dissect infection dynamics at a high spatiotemporal resolution makes this zebrafish model a unique tool for understanding mucosal host-pathogen interactions. PMID:28607100

  10. 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

  11. Surface tension and modeling of cellular intercalation during zebrafish gastrulation.

    PubMed

    Calmelet, Colette; Sepich, Diane

    2010-04-01

    In this paper we discuss a model of zebrafish embryo notochord development based on the effect of surface tension of cells at the boundaries. We study the process of interaction of mesodermal cells at the boundaries due to adhesion and cortical tension, resulting in cellular intercalation. From in vivo experiments, we obtain cell outlines of time-lapse images of cell movements during zebrafish embryo development. Using Cellular Potts Model, we calculate the total surface energy of the system of cells at different time intervals at cell contacts. We analyze the variations of total energy depending on nature of cell contacts. We demonstrate that our model can be viable by calculating the total surface energy value for experimentally observed configurations of cells and showing that in our model these configurations correspond to a decrease in total energy values in both two and three dimensions.

  12. A zebrafish model of chordoma initiated by notochord-driven expression of HRASV12.

    PubMed

    Burger, Alexa; Vasilyev, Aleksandr; Tomar, Ritu; Selig, Martin K; Nielsen, G Petur; Peterson, Randall T; Drummond, Iain A; Haber, Daniel A

    2014-07-01

    Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer. © 2014. Published by The Company of Biologists Ltd.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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 ...

  18. Diving into the world of alcohol teratogenesis: a review of zebrafish models of fetal alcohol spectrum disorder.

    PubMed

    Fernandes, Yohaan; Buckley, Desire M; Eberhart, Johann K

    2018-04-01

    The term fetal alcohol spectrum disorder (FASD) refers to the entire suite of deleterious outcomes resulting from embryonic exposure to alcohol. Along with other reviews in this special issue, we provide insight into how animal models, specifically the zebrafish, have informed our understanding of FASD. We first provide a brief introduction to FASD. We discuss the zebrafish as a model organism and its strengths for alcohol research. We detail how zebrafish has been used to model some of the major defects present in FASD. These include behavioral defects, such as social behavior as well as learning and memory, and structural defects, disrupting organs such as the brain, sensory organs, heart, and craniofacial skeleton. We provide insights into how zebrafish research has aided in our understanding of the mechanisms of ethanol teratogenesis. We end by providing some relatively recent advances that zebrafish has provided in characterizing gene-ethanol interactions that may underlie FASD.

  19. 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.

  20. Analyzing notochord segmentation and intervertebral disc formation using the twhh:gfp transgenic zebrafish model.

    PubMed

    Haga, Yutaka; Dominique, Vincent J; Du, Shao Jun

    2009-10-01

    To characterize the process of vertebral segmentation and disc formation in living animals, we analyzed tiggy-winkle hedgehog (twhh):green fluorescent protein (gfp) and sonic hedgehog (shh):gfp transgenic zebrafish models that display notochord-specific GFP expression. We found that they showed distinct patterns of expression in the intervertebral discs of late stage fish larvae and adult zebrafish. A segmented pattern of GFP expression was detected in the intervertebral disc of twhh:gfp transgenic fish. In contrast, little GFP expression was found in the intervertebral disc of shh:gfp transgenic fish. Treating twhh:gfp transgenic zebrafish larvae with exogenous retinoic acid (RA), a teratogenic factor on normal development, resulted in disruption of notochord segmentation and formation of oversized vertebrae. Histological analysis revealed that the oversized vertebrae are likely due to vertebral fusion. These studies demonstrate that the twhh:gfp transgenic zebrafish is a useful model for studying vertebral segmentation and disc formation, and moreover, that RA signaling may play a role in this process.

  1. A DN-mda5 transgenic zebrafish model demonstrates that Mda5 plays an important role in snakehead rhabdovirus resistance.

    PubMed

    Gabor, K A; Charette, J R; Pietraszewski, M J; Wingfield, D J; Shim, J S; Millard, P J; Kim, C H

    2015-08-01

    Melanoma Differentiation-Associated protein 5 (MDA5) is a member of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) family, which is a cytosolic pattern recognition receptor that detects viral nucleic acids. Here we show an Mda5-dependent response to rhabdovirus infection in vivo using a dominant-negative mda5 transgenic zebrafish. Dominant-negative mda5 zebrafish embryos displayed an impaired antiviral immune response compared to wild-type counterparts that can be rescued by recombinant full-length Mda5. To our knowledge, we have generated the first dominant-negative mda5 transgenic zebrafish and demonstrated a critical role for Mda5 in the antiviral response to rhabdovirus. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. 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.

  3. Zebrafish as a model for acetylcholinesterase-inhibiting organophosphorus agent exposure and oxime reactivation

    PubMed Central

    Koenig, Jeffrey A.; Dao, Thuy L.; Kan, Robert K.; Shih, Tsung-Ming

    2016-01-01

    The current research progression efforts for investigating novel treatments for exposure to organophosphorus (OP) compounds that inhibit acetylcholinesterase (AChE), including pesticides and chemical warfare nerve agents (CWNAs), rely solely on in vitro cell assays and in vivo rodent models. The zebrafish (Danio rerio) is a popular, well-established vertebrate model in biomedical research that offers high-throughput capabilities and genetic manipulation not readily available with rodents. A number of research studies have investigated the effects of subacute developmental exposure to OP pesticides in zebrafish, observing detrimental effects on gross morphology, neuronal development, and behavior. Few studies, however, have utilized this model to evaluate treatments, such as oxime reactivators, anticholinergics, or anticonvulsants, following acute exposure. Preliminary work has investigated the effects of CWNA exposure. The results clearly demonstrated relative toxicity and oxime efficacy similar to that reported for the rodent model. This review surveys the current literature utilizing zebrafish as a model for OP exposure and highlights its potential use as a high-throughput system for evaluating AChE reactivator antidotal treatments to acute pesticide and CWNA exposure. PMID:27123828

  4. 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.

  5. 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.

  6. Macrophage-expressed perforins mpeg1 and mpeg1.2 have an anti-bacterial function in zebrafish.

    PubMed

    Benard, Erica L; Racz, Peter I; Rougeot, Julien; Nezhinsky, Alexander E; Verbeek, Fons J; Spaink, Herman P; Meijer, Annemarie H

    2015-01-01

    Macrophage-expressed gene 1 (MPEG1) encodes an evolutionarily conserved protein with a predicted membrane attack complex/perforin domain associated with host defence against invading pathogens. In vertebrates, MPEG1/perforin-2 is an integral membrane protein of macrophages, suspected to be involved in the killing of intracellular bacteria by pore-forming activity. Zebrafish have 3 copies of MPEG1; 2 are expressed in macrophages, whereas the third could be a pseudogene. The mpeg1 and mpeg1.2 genes show differential regulation during infection of zebrafish embryos with the bacterial pathogens Mycobacterium marinum and Salmonella typhimurium. While mpeg1 is downregulated during infection with both pathogens, mpeg1.2 is infection inducible. Upregulation of mpeg1.2 is partially dependent on the presence of functional Mpeg1 and requires the Toll-like receptor adaptor molecule MyD88 and the transcription factor NFκB. Knockdown of mpeg1 alters the immune response to M. marinum infection and results in an increased bacterial burden. In Salmonella typhimurium infection, both mpeg1 and mpeg1.2 knockdown increase the bacterial burdens, but mpeg1 morphants show increased survival times. The combined results of these two in vivo infection models support the anti-bacterial function of the MPEG1/perforin-2 family and indicate that the intricate cross-regulation of the two mpeg1 copies aids the zebrafish host in combatting infection of various pathogens. © 2014 S. Karger AG, Basel.

  7. 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.

  8. 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.

  9. 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

  10. Modeling consequences of prolonged strong unpredictable stress in zebrafish: Complex effects on behavior and physiology.

    PubMed

    Song, Cai; Liu, Bai-Ping; Zhang, Yong-Ping; Peng, Zhilan; Wang, JiaJia; Collier, Adam D; Echevarria, David J; Savelieva, Katerina V; Lawrence, Robert F; Rex, Christopher S; Meshalkina, Darya A; Kalueff, Allan V

    2018-02-02

    Chronic stress is the major pathogenetic factor of human anxiety and depression. Zebrafish (Danio rerio) have become a novel popular model species for neuroscience research and CNS drug discovery. The utility of zebrafish for mimicking human affective disorders is also rapidly growing. Here, we present a new zebrafish model of clinically relevant, prolonged unpredictable strong chronic stress (PUCS). The 5-week PUCS induced overt anxiety-like and motor retardation-like behaviors in adult zebrafish, also elevating whole-body cortisol and proinflammatory cytokines - interleukins IL-1β and IL-6. PUCS also elevated whole-body levels of the anti-inflammatory cytokine IL-10 and increased the density of dendritic spines in zebrafish telencephalic neurons. Chronic treatment of fish with an antidepressant fluoxetine (0.1mg/L for 8days) normalized their behavioral and endocrine phenotypes, as well as corrected stress-elevated IL-1β and IL-6 levels, similar to clinical and rodent data. The CNS expression of the bdnf gene, the two genes of its receptors (trkB, p75), and the gfap gene of glia biomarker, the glial fibrillary acidic protein, was unaltered in all three groups. However, PUCS elevated whole-body BDNF levels and the telencephalic dendritic spine density (which were corrected by fluoxetine), thereby somewhat differing from the effects of chronic stress in rodents. Together, these findings support zebrafish as a useful in-vivo model of chronic stress, also calling for further cross-species studies of both shared/overlapping and distinct neurobiological responses to chronic stress. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Editor's Highlight: Transgenic Zebrafish Reporter Lines as Alternative In Vivo Organ Toxicity Models.

    PubMed

    Poon, Kar Lai; Wang, Xingang; Lee, Serene G P; Ng, Ashley S; Goh, Wei Huang; Zhao, Zhonghua; Al-Haddawi, Muthafar; Wang, Haishan; Mathavan, Sinnakaruppan; Ingham, Philip W; McGinnis, Claudia; Carney, Tom J

    2017-03-01

    Organ toxicity, particularly liver toxicity, remains one of the major reasons for the termination of drug candidates in the development pipeline as well as withdrawal or restrictions of marketed drugs. A screening-amenable alternative in vivo model such as zebrafish would, therefore, find immediate application in the early prediction of unacceptable organ toxicity. To identify highly upregulated genes as biomarkers of toxic responses in the zebrafish model, a set of well-characterized reference drugs that cause drug-induced liver injury (DILI) in the clinic were applied to zebrafish larvae and adults. Transcriptome microarray analysis was performed on whole larvae or dissected adult livers. Integration of data sets from different drug treatments at different stages identified common upregulated detoxification pathways. Within these were candidate biomarkers which recurred in multiple treatments. We prioritized 4 highly upregulated genes encoding enzymes acting in distinct phases of the drug metabolism pathway. Through promoter isolation and fosmid recombineering, eGFP reporter transgenic zebrafish lines were generated and evaluated for their response to DILI drugs. Three of the 4 generated reporter lines showed a dose and time-dependent induction in endodermal organs to reference drugs and an expanded drug set. In conclusion, through integrated transcriptomics and transgenic approaches, we have developed parallel independent zebrafish in vivo screening platforms able to predict organ toxicities of preclinical drugs. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  12. A zebrafish model of diabetes mellitus and metabolic memory.

    PubMed

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

    2013-02-28

    Diabetes mellitus currently affects 346 million individuals and this is projected to increase to 400 million by 2030. Evidence from both the laboratory and large scale clinical trials has revealed that diabetic complications progress unimpeded via the phenomenon of metabolic memory even when glycemic control is pharmaceutically achieved. Gene expression can be stably altered through epigenetic changes which not only allow cells and organisms to quickly respond to changing environmental stimuli but also confer the ability of the cell to "memorize" these encounters once the stimulus is removed. As such, the roles that these mechanisms play in the metabolic memory phenomenon are currently being examined. We have recently reported the development of a zebrafish model of type I diabetes mellitus and characterized this model to show that diabetic zebrafish not only display the known secondary complications including the changes associated with diabetic retinopathy, diabetic nephropathy and impaired wound healing but also exhibit impaired caudal fin regeneration. This model is unique in that the zebrafish is capable to regenerate its damaged pancreas and restore a euglycemic state similar to what would be expected in post-transplant human patients. Moreover, multiple rounds of caudal fin amputation allow for the separation and study of pure epigenetic effects in an in vivo system without potential complicating factors from the previous diabetic state. Although euglycemia is achieved following pancreatic regeneration, the diabetic secondary complication of fin regeneration and skin wound healing persists indefinitely. In the case of impaired fin regeneration, this pathology is retained even after multiple rounds of fin regeneration in the daughter fin tissues. These observations point to an underlying epigenetic process existing in the metabolic memory state. Here we present the methods needed to successfully generate the diabetic and metabolic memory groups of fish and

  13. Operant models of relapse in zebrafish (Danio rerio): Resurgence, renewal, and reinstatement.

    PubMed

    Kuroda, Toshikazu; Mizutani, Yuto; Cançado, Carlos R X; Podlesnik, Christopher A

    2017-09-29

    Zebrafish are a widely used animal model in biomedical research, as an alternative to mammals, for having features such as a fully sequenced genome, high fecundity, and low-cost maintenance, but behavioral research with these fish remains scarce. The present study investigated whether zebrafish could be a new animal model for studies on the relapse of behavior (e.g., addiction and overeating) after the behavior has been extinguished. Specifically, we examined whether zebrafish would show three different types of relapse commonly studied with other species: resurgence, renewal, and reinstatement. For resurgence, a target response (i.e., approaching a sensor) was established by presenting a reinforcer (i.e., shrimp eggs) contingent upon the response in Phase 1; the target response was extinguished while introducing reinforcement for an alternative response in Phase 2; neither response produced the reinforcer in Phase 3. For renewal, a target response was established under Context A in Phase 1 and was extinguished under Context B in Phase 2; the fish were placed back in Context A in Phase 3, where extinction remained in effect. For reinstatement, a target response was established in Phase 1 and was extinguished in Phase 2; the reinforcer was presented independently of responding in Phase 3. Each type of relapse occurred in Phase 3. These results replicate and extend previous findings on relapse to a new species and suggest that zebrafish can be a useful animal model for studying the interactions of biological and environmental factors that lead to relapse. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Zebrafish as a Model for Epilepsy-Induced Cognitive Dysfunction: A Pharmacological, Biochemical and Behavioral Approach

    PubMed Central

    Kundap, Uday P.; Kumari, Yatinesh; Othman, Iekhsan; Shaikh, Mohd. Farooq

    2017-01-01

    Epilepsy is a neuronal disorder allied with distinct neurological and behavioral alterations characterized by recurrent spontaneous epileptic seizures. Impairment of the cognitive performances such as learning and memory is frequently observed in epileptic patients. Anti-epileptic drugs (AEDs) are efficient to the majority of patients. However, 30% of this population seems to be refractory to the drug treatment. These patients are not seizure-free and frequently they show impaired cognitive functions. Unfortunately, as a side effect, some AEDs could contribute to such impairment. The major problem associated with conducting studies on epilepsy-related cognitive function is the lack of easy, rapid, specific and sensitive in vivo testing models. However, by using a number of different techniques and parameters in the zebrafish, we can incorporate the unique feature of specific disorder to study the molecular and behavior basis of this disease. In the view of current literature, the goal of the study was to develop a zebrafish model of epilepsy induced cognitive dysfunction. In this study, the effect of AEDs on locomotor activity and seizure-like behavior was tested against the pentylenetetrazole (PTZ) induced seizures in zebrafish and epilepsy associated cognitive dysfunction was determined using T-maze test followed by neurotransmitter estimation and gene expression analysis. It was observed that all the AEDs significantly reversed PTZ induced seizure in zebrafish, but had a negative impact on cognitive functions of zebrafish. AEDs were found to modulate neurotransmitter levels, especially GABA, glutamate, and acetylcholine and gene expression in the drug treated zebrafish brains. Therefore, combination of behavioral, neurochemical and genenetic information, makes this model a useful tool for future research and discovery of newer and safer AEDs. PMID:28824436

  15. 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.

  16. 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

  17. Whole plant based treatment of hypercholesterolemia with Crataegus laevigata in a zebrafish model

    PubMed Central

    2012-01-01

    Background Consumers are increasingly turning to plant-based complementary and alternative medicines to treat hypercholesterolemia. Many of these treatments are untested and their efficacy is unknown. This multitude of potential remedies necessitates a model system amenable to testing large numbers of organisms that maintains similarity to humans in both mode of drug administration and overall physiology. Here we develop the larval zebrafish (4–30 days post fertilization) as a vertebrate model of dietary plant-based treatment of hypercholesterolemia and test the effects of Crataegus laevigata in this model. Methods Larval zebrafish were fed high cholesterol diets infused with fluorescent sterols and phytomedicines. Plants were ground with mortar and pestle into a fine powder before addition to food. Fluorescent sterols were utilized to optically quantify relative difference in intravascular cholesterol levels between groups of fish. We utilized the Zeiss 7-Live Duo high-speed confocal platform in order to both quantify intravascular sterol fluorescence and to capture video of the heart beat for determination of cardiac output. Results In this investigation we developed and utilized a larval zebrafish model to investigate dietary plant-based intervention of the pathophysiology of hypercholesterolemia. We found BODIPY-cholesterol effectively labels diet-introduced intravascular cholesterol levels (P < 0.05, Student’s t-test). We also established that zebrafish cardiac output declines as cholesterol dose increases (difference between 0.1% and 8% (w/w) high cholesterol diet-treated cardiac output significant at P < 0.05, 1-way ANOVA). Using this model, we found hawthorn leaves and flowers significantly reduce intravascular cholesterol levels (P < 0.05, 1-way ANOVA) and interact with cholesterol to impact cardiac output in hypercholesterolemic fish (2-way ANOVA, P < 0.05 for interaction effect). Conclusions The results of this study demonstrate

  18. Whole plant based treatment of hypercholesterolemia with Crataegus laevigata in a zebrafish model.

    PubMed

    Littleton, Robert M; Miller, Matthew; Hove, Jay R

    2012-07-23

    Consumers are increasingly turning to plant-based complementary and alternative medicines to treat hypercholesterolemia. Many of these treatments are untested and their efficacy is unknown. This multitude of potential remedies necessitates a model system amenable to testing large numbers of organisms that maintains similarity to humans in both mode of drug administration and overall physiology. Here we develop the larval zebrafish (4-30 days post fertilization) as a vertebrate model of dietary plant-based treatment of hypercholesterolemia and test the effects of Crataegus laevigata in this model. Larval zebrafish were fed high cholesterol diets infused with fluorescent sterols and phytomedicines. Plants were ground with mortar and pestle into a fine powder before addition to food. Fluorescent sterols were utilized to optically quantify relative difference in intravascular cholesterol levels between groups of fish. We utilized the Zeiss 7-Live Duo high-speed confocal platform in order to both quantify intravascular sterol fluorescence and to capture video of the heart beat for determination of cardiac output. In this investigation we developed and utilized a larval zebrafish model to investigate dietary plant-based intervention of the pathophysiology of hypercholesterolemia. We found BODIPY-cholesterol effectively labels diet-introduced intravascular cholesterol levels (P < 0.05, Student's t-test). We also established that zebrafish cardiac output declines as cholesterol dose increases (difference between 0.1% and 8% (w/w) high cholesterol diet-treated cardiac output significant at P < 0.05, 1-way ANOVA). Using this model, we found hawthorn leaves and flowers significantly reduce intravascular cholesterol levels (P < 0.05, 1-way ANOVA) and interact with cholesterol to impact cardiac output in hypercholesterolemic fish (2-way ANOVA, P < 0.05 for interaction effect). The results of this study demonstrate that the larval zebrafish has the potential to become a powerful

  19. The zebrafish as a gerontology model in nervous system aging, disease, and repair.

    PubMed

    Van Houcke, Jessie; De Groef, Lies; Dekeyster, Eline; Moons, Lieve

    2015-11-01

    Considering the increasing number of elderly in the world's population today, developing effective treatments for age-related pathologies is one of the biggest challenges in modern medical research. Age-related neurodegeneration, in particular, significantly impacts important sensory, motor, and cognitive functions, seriously constraining life quality of many patients. Although our understanding of the causal mechanisms of aging has greatly improved in recent years, animal model systems still have much to tell us about this complex process. Zebrafish (Danio rerio) have gained enormous popularity for this research topic over the past decade, since their life span is relatively short but, like humans, they are still subject to gradual aging. In addition, the extensive characterization of its well-conserved molecular and cellular physiology makes the zebrafish an excellent model to unravel the underlying mechanisms of aging, disease, and repair. This review provides a comprehensive overview of the progress made in zebrafish gerontology, with special emphasis on nervous system aging. We review the evidence that classic hallmarks of aging can also be recognized within this small vertebrate, both at the molecular and cellular level. Moreover, we illustrate the high level of similarity with age-associated human pathologies through a survey of the functional deficits that arise as zebrafish age. Copyright © 2015 Elsevier B.V. All rights reserved.

  20. Zebrafish Embryo as an In Vivo Model for Behavioral and Pharmacological Characterization of Methylxanthine Drugs.

    PubMed

    Basnet, Ram Manohar; Guarienti, Michela; Memo, Maurizio

    2017-03-09

    Zebrafish embryo is emerging as an important tool for behavior analysis as well as toxicity testing. In this study, we compared the effect of nine different methylxanthine drugs using zebrafish embryo as a model. We performed behavioral analysis, biochemical assay and Fish Embryo Toxicity (FET) test in zebrafish embryos after treatment with methylxanthines. Each drug appeared to behave in different ways and showed a distinct pattern of results. Embryos treated with seven out of nine methylxanthines exhibited epileptic-like pattern of movements, the severity of which varied with drugs and doses used. Cyclic AMP measurement showed that, despite of a significant increase in cAMP with some compounds, it was unrelated to the observed movement behavior changes. FET test showed a different pattern of toxicity with different methylxanthines. Each drug could be distinguished from the other based on its effect on mortality, morphological defects and teratogenic effects. In addition, there was a strong positive correlation between the toxic doses (TC 50 ) calculated in zebrafish embryos and lethal doses (LD 50 ) in rodents obtained from TOXNET database. Taken together, all these findings elucidate the potentiality of zebrafish embryos as an in vivo model for behavioral and toxicity testing of methylxanthines and other related compounds.

  1. Automated analysis of brain activity for seizure detection in zebrafish models of epilepsy.

    PubMed

    Hunyadi, Borbála; Siekierska, Aleksandra; Sourbron, Jo; Copmans, Daniëlle; de Witte, Peter A M

    2017-08-01

    Epilepsy is a chronic neurological condition, with over 30% of cases unresponsive to treatment. Zebrafish larvae show great potential to serve as an animal model of epilepsy in drug discovery. Thanks to their high fecundity and relatively low cost, they are amenable to high-throughput screening. However, the assessment of seizure occurrences in zebrafish larvae remains a bottleneck, as visual analysis is subjective and time-consuming. For the first time, we present an automated algorithm to detect epileptic discharges in single-channel local field potential (LFP) recordings in zebrafish. First, candidate seizure segments are selected based on their energy and length. Afterwards, discriminative features are extracted from each segment. Using a labeled dataset, a support vector machine (SVM) classifier is trained to learn an optimal feature mapping. Finally, this SVM classifier is used to detect seizure segments in new signals. We tested the proposed algorithm both in a chemically-induced seizure model and a genetic epilepsy model. In both cases, the algorithm delivered similar results to visual analysis and found a significant difference in number of seizures between the epileptic and control group. Direct comparison with multichannel techniques or methods developed for different animal models is not feasible. Nevertheless, a literature review shows that our algorithm outperforms state-of-the-art techniques in terms of accuracy, precision and specificity, while maintaining a reasonable sensitivity. Our seizure detection system is a generic, time-saving and objective method to analyze zebrafish LPF, which can replace visual analysis and facilitate true high-throughput studies. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Gene knockdown by morpholino-modified oligonucleotides in the zebrafish model: applications for developmental toxicology

    PubMed Central

    Timme-Laragy, Alicia R.; Karchner, Sibel I.; Hahn, Mark E.

    2014-01-01

    Summary The zebrafish (Danio rerio) has long been used as a model for developmental biology, making it an excellent model to use also in developmental toxicology. The many advantages of zebrafish include their small size, prolific spawning, rapid development, and transparent embryos. They can be easily manipulated genetically through the use of transgenic technology and gene knock-down via morpholino-modified antisense oligonucleotides (MOs). Knocking down specific genes to assess their role in the response to toxicant exposure provides a way to further our knowledge of how developmental toxicants work on a molecular and mechanistic level, while establishing a relationship between these molecular events and morphological, behavioral, and/or physiological effects (i.e. phenotypic anchoring). In this chapter we address important considerations for using MOs to study developmental toxicology in zebrafish embryos and provide a protocol for their use. PMID:22669659

  3. Zebrafish (Danio rerio) Models To Assess Acute, Developmental, And Neurodevelopmental Toxicity

    EPA Science Inventory

    Zebrafish (Danio rerio) acute, developmental, and neurodevelopmental model systems have been developed to assess both known and unknown environmental contaminants. Developmental toxicity is assessed using death and dysmorphology as endpoints, whereas neurodevelopmental toxicity ...

  4. Developing an Experimental Model of Vascular Toxicity in Embryonic Zebrafish

    EPA Science Inventory

    Developing an Experimental Model of Vascular Toxicity in Embryonic Zebrafish Tamara Tal, Integrated Systems Toxicology Division, U.S. EPA Background: There are tens of thousands of chemicals that have yet to be fully evaluated for their toxicity by validated in vivo testing ...

  5. A transgenic zebrafish model for monitoring xbp1 splicing and endoplasmic reticulum stress in vivo.

    PubMed

    Li, Junling; Chen, Zhiliang; Gao, Lian-Yong; Colorni, Angelo; Ucko, Michal; Fang, Shengyun; Du, Shao Jun

    2015-08-01

    Accumulation of misfolded or unfolded proteins in the endoplasmic reticulum (ER) triggers ER stress that initiates unfolded protein response (UPR). XBP1 is a transcription factor that mediates one of the key signaling pathways of UPR to cope with ER stress through regulating gene expression. Activation of XBP1 involves an unconventional mRNA splicing catalyzed by IRE1 endonuclease that removes an internal 26 nucleotides from xbp1 mRNA transcripts in the cytoplasm. Researchers have taken advantage of this unique activation mechanism to monitor XBP1 activation, thereby UPR, in cell culture and transgenic models. Here we report a Tg(ef1α:xbp1δ-gfp) transgenic zebrafish line to monitor XBP1 activation using GFP as a reporter especially in zebrafish oocytes and developing embryos. The Tg(ef1α:xbp1δ-gfp) transgene was constructed using part of the zebrafish xbp1 cDNA containing the splicing element. ER stress induced splicing results in the cDNA encoding a GFP-tagged partial XBP1 without the transactivation activation domain (XBP1Δ-GFP). The results showed that xbp1 transcripts mainly exist as the spliced active isoform in unfertilized oocytes and zebrafish embryos prior to zygotic gene activation at 3 hours post fertilization. A strong GFP expression was observed in unfertilized oocytes, eyes, brain and skeletal muscle in addition to a weak expression in the hatching gland. Incubation of transgenic zebrafish embryos with (dithiothreitol) DTT significantly induced XBP1Δ-GFP expression. Collectively, these studies unveil the presence of maternal xbp1 splicing in zebrafish oocytes, fertilized eggs and early stage embryos. The Tg(ef1α:xbp1δ-gfp) transgenic zebrafish provides a useful model for in vivo monitoring xbp1 splicing during development and under ER stress conditions. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  6. Comparison of cannabinoids with known analgesics using a novel high throughput zebrafish larval model of nociception.

    PubMed

    Ellis, L D; Berrue, F; Morash, M; Achenbach, J C; Hill, J; McDougall, J J

    2018-01-30

    It has been established that both adult and larval zebrafish are capable of showing nociceptive responses to noxious stimuli; however, the use of larvae to test novel analgesics has not been fully explored. Zebrafish larvae represent a low-cost, high-throughput alternative to traditional mammalian models for the assessment of product efficacy during the initial stages of drug development. In the current study, a novel model of nociception using zebrafish larvae is described. During the recovery from an acute exposure to low levels of acetic acid, larvae display innate changes in behaviour that may be indicative of nociception. To assess the usefulness of this model for testing potential analgesics, three known synthetic pain medications were assessed (ibuprofen, acetaminophen and tramadol) along with three naturally occurring products (honokiol, tetrahydrocannabinol and cannabidiol). When the effect of each compound on both the acetic acid recovery and control activity was compared there appeared to be both similarities and differences between the compounds. One of the most interesting effects was found for cannabidiol which appeared to oppose the activity change during the recovery period of AA exposed larvae while having a nominal effect on control activity. This would appear to be in line with current research that has demonstrated the nociceptive properties of cannabidiol. Here we have provided a novel model that will complement existing zebrafish models and will expand on the potential use of zebrafish larvae for studying both nociception and new analgesics. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  7. 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.

  8. Use of TSHβ:EGFP transgenic zebrafish as a rapid in vivo model for assessing thyroid-disrupting chemicals

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

    Ji, Cheng; Graduate University of Chinese Academy of Sciences, Beijing; Jin, Xia

    Accumulating evidence indicates that a wide range of chemicals have the ability to interfere with the hypothalamic–pituitary–thyroid (HPT) axis. Novel endpoints should be evaluated in addition to existing methods in order to effectively assess the effects of these chemicals on the HPT axis. Thyroid-stimulating hormone subunit β (TSHβ) plays central regulatory roles in the HPT system. We identified the regulatory region that determines the expression level of zebrafish TSHβ in the anterior pituitary. In the transgenic zebrafish with EGFP driven by the TSHβ promoter, the similar responsive patterns between the expression levels of TSHβ:EGFP and endogenous TSHβ mRNA in themore » pituitary are observed following treatments with goitrogen chemicals and exogenous thyroid hormones (THs). These results suggest that the TSHβ:EGFP transgenic reporter zebrafish may be a useful alternative in vivo model for the assessment of chemicals interfering with the HPT system. Highlights: ► The promoter of zebrafish TSHβ gene has been identified. ► The stable TSHβ:EGFP transgenic zebrafish reporter germline has been generated. ► The EGFP in the transgenic fish recapitulated the pattern of pituitary TSHβ mRNA. ► The transgenic zebrafish may be an in vivo model for EDC assessment.« less

  9. Safety evaluation of the temporary consolidant based on a zebrafish embryo model.

    PubMed

    Zhang, Lu; Zhang, Xiao Yan; Hu, Yulan; Zhang, Bing Jian

    2018-05-07

    Temporary consolidants, such as cyclododecane, menthol, coumarin, and ethyl maltol, are proved to be effective for urgent conservation in archaeological field. However, the safety of these temporary consolidants is still unknown although they are always heavily used in archaeological field. Thus reports on the toxicity of these temporary consolidants are limited. Here, a zebrafish model was used for safety evaluation of these four temporary consolidants. In the present study, four temporary consolidants, including cyclododecane, menthol, coumarin, and ethyl maltol, at different concentrations were incubated with zebrafish embryos, and their biological toxic effects were firstly evaluated. It was shown that embryo exposure to temporary consolidants resulted in an increased mortality and malformation rate, and a decreased hatching rate. The order of embryo toxicity of the four types of temporary consolidants tested was menthol > coumarin > ethyl maltol > cyclododecane. Although the embryo toxicity of cyclododecane was minimal, some studies have indicated that this temporary consolidant could be bioaccumulated. The results also suggested that the zebrafish embryos can serve as a reliable model for the evaluation of embryo toxicity of temporary consolidants, as this model could offer the possibility to perform the rapid, medium throughput, cost-effective analyses. Copyright © 2018 Elsevier Ltd. All rights reserved.

  10. Fluoride exposure abates pro-inflammatory response and induces in vivo apoptosis rendering zebrafish (Danio rerio) susceptible to bacterial infections.

    PubMed

    Singh, Rashmi; Khatri, Preeti; Srivastava, Nidhi; Jain, Shruti; Brahmachari, Vani; Mukhopadhyay, Asish; Mazumder, Shibnath

    2017-04-01

    The present study describes the immunotoxic effect of chronic fluoride exposure on adult zebrafish (Danio rerio). Zebrafish were exposed to fluoride (71.12 mg/L; 1/10 LC 50 ) for 30 d and the expression of selected genes studied. We observed significant elevation in the detoxification pathway gene cyp1a suggesting chronic exposure to non-lethal concentration of fluoride is indeed toxic to fish. Fluoride mediated pro-oxidative stress is implicated with the downregulation in superoxide dismutase 1 and 2 (sod1/2) genes. Fluoride affected DNA repair machinery by abrogating the expression of the DNA repair gene rad51 and growth arrest and DNA damage inducible beta a gene gadd45ba. The upregulated expression of casp3a coupled with altered Bcl-2 associated X protein/B-cell lymphoma 2 ratio (baxa/bcl2a) clearly suggested chronic fluoride exposure induced the apoptotic cascade in zebrafish. Fluoride-exposed zebrafish when challenged with non-lethal dose of fish pathogen A. hydrophila revealed gross histopathology in spleen, bacterial persistence and significant mortality. We report that fluoride interferes with system-level output of pro-inflammatory cytokines tumour necrosis factor-α, interleukin-1β and interferon-γ, as a consequence, bacteria replicate efficiently causing significant fish mortality. We conclude, chronic fluoride exposure impairs the redox balance, affects DNA repair machinery with pro-apoptotic implications and suppresses pro-inflammatory cytokines expression abrogating host immunity to bacterial infections. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Tolerance and efficacy of emamectin benzoate and ivermectin for the treatment of Pseudocapillaria tomentosa in laboratory zebrafish (Danio rerio).

    PubMed

    Collymore, Chereen; Watral, Virginia; White, Julie R; Colvin, Michael E; Rasmussen, Skye; Tolwani, Ravi J; Kent, Michael L

    2014-10-01

    Tolerance of adult zebrafish and efficacy of emamectin benzoate and ivermectin in eliminating Pseudocapillaria tomentosa infection were evaluated. In the tolerance study, behavioral changes, fecundity, histopathology, and mortality were evaluated for in-feed administration of emamectin (0.05, 0.10, and 0.25 mg/kg) and ivermectin (0.05 and 0.10 mg/kg). All doses of emamectin were well tolerated. Ivermectin 0.05 mg/kg administration resulted in mild behavioral changes and a transient decrease in fecundity. Ivermectin 0.10 mg/kg administration resulted in severe behavioral changes and some mortality. In the efficacy study, emamectin (0.05 and 0.25 mg/kg) and ivermectin (0.05 mg/kg) were evaluated for their efficacy in eliminating P. tomentosa infection. Emamectin reduced parasite burden in infected zebrafish, and ivermectin eliminated intestinal nematode infections. Despite a small margin of safety, ivermectin 0.05 mg/kg was effective at eliminating P. tomentosa infection in adult zebrafish. Higher doses or a longer course of treatment may be needed for complete elimination of P. tomentosa infection using emamectin. In this study, we propose two possible treatments for intestinal nematode infections in zebrafish.

  12. Tolerance and Efficacy of Emamectin Benzoate and Ivermectin for the Treatment of Pseudocapillaria tomentosa in Laboratory Zebrafish (Danio rerio)

    PubMed Central

    Collymore, Chereen; Watral, Virginia; White, Julie R.; Colvin, Michael E.; Rasmussen, Skye; Tolwani, Ravi J.

    2014-01-01

    Abstract Tolerance of adult zebrafish and efficacy of emamectin benzoate and ivermectin in eliminating Pseudocapillaria tomentosa infection were evaluated. In the tolerance study, behavioral changes, fecundity, histopathology, and mortality were evaluated for in-feed administration of emamectin (0.05, 0.10, and 0.25 mg/kg) and ivermectin (0.05 and 0.10 mg/kg). All doses of emamectin were well tolerated. Ivermectin 0.05 mg/kg administration resulted in mild behavioral changes and a transient decrease in fecundity. Ivermectin 0.10 mg/kg administration resulted in severe behavioral changes and some mortality. In the efficacy study, emamectin (0.05 and 0.25 mg/kg) and ivermectin (0.05 mg/kg) were evaluated for their efficacy in eliminating P. tomentosa infection. Emamectin reduced parasite burden in infected zebrafish, and ivermectin eliminated intestinal nematode infections. Despite a small margin of safety, ivermectin 0.05 mg/kg was effective at eliminating P. tomentosa infection in adult zebrafish. Higher doses or a longer course of treatment may be needed for complete elimination of P. tomentosa infection using emamectin. In this study, we propose two possible treatments for intestinal nematode infections in zebrafish. PMID:25237985

  13. 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.

  14. 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.

  15. From Omics to Drug Metabolism and High Content Screen of Natural Product in Zebrafish: A New Model for Discovery of Neuroactive Compound

    PubMed Central

    Hung, Ming Wai; Zhang, Zai Jun; Li, Shang; Lei, Benson; Yuan, Shuai; Cui, Guo Zhen; Man Hoi, Pui; Chan, Kelvin; Lee, Simon Ming Yuen

    2012-01-01

    The zebrafish (Danio rerio) has recently become a common model in the fields of genetics, environmental science, toxicology, and especially drug screening. Zebrafish has emerged as a biomedically relevant model for in vivo high content drug screening and the simultaneous determination of multiple efficacy parameters, including behaviour, selectivity, and toxicity in the content of the whole organism. A zebrafish behavioural assay has been demonstrated as a novel, rapid, and high-throughput approach to the discovery of neuroactive, psychoactive, and memory-modulating compounds. Recent studies found a functional similarity of drug metabolism systems in zebrafish and mammals, providing a clue with why some compounds are active in zebrafish in vivo but not in vitro, as well as providing grounds for the rationales supporting the use of a zebrafish screen to identify prodrugs. Here, we discuss the advantages of the zebrafish model for evaluating drug metabolism and the mode of pharmacological action with the emerging omics approaches. Why this model is suitable for identifying lead compounds from natural products for therapy of disorders with multifactorial etiopathogenesis and imbalance of angiogenesis, such as Parkinson's disease, epilepsy, cardiotoxicity, cerebral hemorrhage, dyslipidemia, and hyperlipidemia, is addressed. PMID:22919414

  16. Host immune response and acute disease in a zebrafish model of francisella pathogenesis

    USGS Publications Warehouse

    Vojtech, L.N.; Sanders, G.E.; Conway, C.; Ostland, V.; Hansen, J.D.

    2009-01-01

    Members of the bacterial genus Francisella are highly virulent and infectious pathogens. New models to study Francisella pathogenesis in evolutionarily distinct species are needed to provide comparative insight, as the mechanisms of host resistance and pathogen virulence are not well understood. We took advantage of the recent discovery of a novel species of Francisella to establish a zebrafish/Francisella comparative model of pathogenesis and host immune response. Adult zebraflsh were susceptible to acute Francisella-induced disease and suffered mortality in a dose-dependent manner. Using immunohistochemical analysis, we localized bacterial antigens primarily to lymphoid tissues and livers of zebraflsh following infection by intraperitoneal injection, which corresponded to regions of local cellular necrosis. Francisella sp. bacteria replicated rapidly in these tissues beginning 12 h postinfection, and bacterial titers rose steadily, leveled off, and then decreased by 7 days postinfection. Zebraflsh mounted a significant tissue-specific proinflammatory response to infection as measured by the upregulation of interleukin-l?? (IL-1??), gamma interferon, and tumor necrosis factor alpha mRNA beginning by 6 h postinfection and persisting for up to 7 days postinfection. In addition, exposure of zebraflsh to heat-killed bacteria demonstrated that the significant induction of IL-?? was highly specific to live bacteria. Taken together, the pathology and immune response to acute Francisella infection in zebraflsh share many features with those in mammals, highlighting the usefulness of this new model system for addressing both general and specific questions about Francisella host-pathogen interactions via an evolutionary approach. Copyright ?? 2009, American Society for Microbiology. All Rights Reserved.

  17. Zebrafish as an Alternative Vertebrate Model for Investigating Developmental Toxicity—The Triadimefon Example

    PubMed Central

    Zoupa, Maria; Machera, Kyriaki

    2017-01-01

    Triadimefon is a widely used triazole fungicide known to cause severe developmental defects in several model organisms and in humans. The present study evaluated in detail the developmental effects seen in zebrafish embryos exposed to triadimefon, confirmed and expanded upon previous phenotypic findings and compared them to those observed in other traditional animal models. In order to do this, we exposed embryos to 2 and 4 µg/mL triadimefon and evaluated growth until 120 h post-fertilization (hpf) through gross morphology examination. Our analysis revealed significant developmental defects at the highest tested concentration including somite deformities, severe craniofacial defects, a cleft phenotype along the three primary neural divisions, a rigorously hypoplastic or even absent mandible and a hypoplastic morphology of the pharyngeal arches. Interestingly, massive pericardial edemas, abnormal shaped hearts, brachycardia and inhibited or absent blood circulation were also observed. Our results revealed that the presented zebrafish phenotypes are comparable to those seen in other organism models and those derived from human observations as a result of triadimefon exposure. We therefore demonstrated that zebrafish provide an excellent system for study of compounds with toxic significance and can be used as an alternative model for developmental toxicity studies to predict effects in mammals. PMID:28417904

  18. PAX3-FOXO1 transgenic zebrafish models identify HES3 as a mediator of rhabdomyosarcoma tumorigenesis.

    PubMed

    Kendall, Genevieve C; Watson, Sarah; Xu, Lin; LaVigne, Collette A; Murchison, Whitney; Rakheja, Dinesh; Skapek, Stephen X; Tirode, Franck; Delattre, Olivier; Amatruda, James F

    2018-06-05

    Alveolar rhabdomyosarcoma is a pediatric soft-tissue sarcoma caused by PAX3/7-FOXO1 fusion oncogenes and is characterized by impaired skeletal muscle development. We developed human PAX3-FOXO1 -driven zebrafish models of tumorigenesis and found that PAX3-FOXO1 exhibits discrete cell lineage susceptibility and transformation. Tumors developed by 1.6-19 months and were primitive neuroectodermal tumors or rhabdomyosarcoma. We applied this PAX3-FOXO1 transgenic zebrafish model to study how PAX3-FOXO1 leverages early developmental pathways for oncogenesis and found that her3 is a unique target. Ectopic expression of the her3 human ortholog, HES3 , inhibits myogenesis in zebrafish and mammalian cells, recapitulating the arrested muscle development characteristic of rhabdomyosarcoma. In patients, HES3 is overexpressed in fusion-positive versus fusion-negative tumors. Finally, HES3 overexpression is associated with reduced survival in patients in the context of the fusion. Our novel zebrafish rhabdomyosarcoma model identifies a new PAX3-FOXO1 target, her3 / HES3 , that contributes to impaired myogenic differentiation and has prognostic significance in human disease. © 2018, Kendall et al.

  19. A zebrafish transgenic model of Ewing's sarcoma reveals conserved mediators of EWS-FLI1 tumorigenesis.

    PubMed

    Leacock, Stefanie W; Basse, Audrey N; Chandler, Garvin L; Kirk, Anne M; Rakheja, Dinesh; Amatruda, James F

    2012-01-01

    Ewing's sarcoma, a malignant bone tumor of children and young adults, is a member of the small-round-blue-cell tumor family. Ewing's sarcoma family tumors (ESFTs), which include peripheral primitive neuroectodermal tumors (PNETs), are characterized by chromosomal translocations that generate fusions between the EWS gene and ETS-family transcription factors, most commonly FLI1. The EWS-FLI1 fusion oncoprotein represents an attractive therapeutic target for treatment of Ewing's sarcoma. The cell of origin of ESFT and the molecular mechanisms by which EWS-FLI1 mediates tumorigenesis remain unknown, and few animal models of Ewing's sarcoma exist. Here, we report the use of zebrafish as a vertebrate model of EWS-FLI1 function and tumorigenesis. Mosaic expression of the human EWS-FLI1 fusion protein in zebrafish caused the development of tumors with histology strongly resembling that of human Ewing's sarcoma. The incidence of tumors increased in a p53 mutant background, suggesting that the p53 pathway suppresses EWS-FLI1-driven tumorigenesis. Gene expression profiling of the zebrafish tumors defined a set of genes that might be regulated by EWS-FLI1, including the zebrafish ortholog of a crucial EWS-FLI1 target gene in humans. Stable zebrafish transgenic lines expressing EWS-FLI1 under the control of the heat-shock promoter exhibit altered embryonic development and defective convergence and extension, suggesting that EWS-FLI1 interacts with conserved developmental pathways. These results indicate that functional targets of EWS-FLI1 that mediate tumorigenesis are conserved from zebrafish to human and provide a novel context in which to study the function of this fusion oncogene.

  20. Zebrafish as a Vertebrate Model System to Evaluate Effects of Environmental Toxicants on Cardiac Development and Function.

    PubMed

    Sarmah, Swapnalee; Marrs, James A

    2016-12-16

    Environmental pollution is a serious problem of the modern world that possesses a major threat to public health. Exposure to environmental pollutants during embryonic development is particularly risky. Although many pollutants have been verified as potential toxicants, there are new chemicals in the environment that need assessment. Heart development is an extremely sensitive process, which can be affected by environmentally toxic molecule exposure during embryonic development. Congenital heart defects are the most common life-threatening global health problems, and the etiology is mostly unknown. The zebrafish has emerged as an invaluable model to examine substance toxicity on vertebrate development, particularly on cardiac development. The zebrafish offers numerous advantages for toxicology research not found in other model systems. Many laboratories have used the zebrafish to study the effects of widespread chemicals in the environment on heart development, including pesticides, nanoparticles, and various organic pollutants. Here, we review the uses of the zebrafish in examining effects of exposure to external molecules during embryonic development in causing cardiac defects, including chemicals ubiquitous in the environment and illicit drugs. Known or potential mechanisms of toxicity and how zebrafish research can be used to provide mechanistic understanding of cardiac defects are discussed.

  1. Zebrafish as a Vertebrate Model System to Evaluate Effects of Environmental Toxicants on Cardiac Development and Function

    PubMed Central

    Sarmah, Swapnalee; Marrs, James A.

    2016-01-01

    Environmental pollution is a serious problem of the modern world that possesses a major threat to public health. Exposure to environmental pollutants during embryonic development is particularly risky. Although many pollutants have been verified as potential toxicants, there are new chemicals in the environment that need assessment. Heart development is an extremely sensitive process, which can be affected by environmentally toxic molecule exposure during embryonic development. Congenital heart defects are the most common life-threatening global health problems, and the etiology is mostly unknown. The zebrafish has emerged as an invaluable model to examine substance toxicity on vertebrate development, particularly on cardiac development. The zebrafish offers numerous advantages for toxicology research not found in other model systems. Many laboratories have used the zebrafish to study the effects of widespread chemicals in the environment on heart development, including pesticides, nanoparticles, and various organic pollutants. Here, we review the uses of the zebrafish in examining effects of exposure to external molecules during embryonic development in causing cardiac defects, including chemicals ubiquitous in the environment and illicit drugs. Known or potential mechanisms of toxicity and how zebrafish research can be used to provide mechanistic understanding of cardiac defects are discussed. PMID:27999267

  2. 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.

  3. 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

  4. The Texas-Indiana Virtual STAR Center: Zebrafish Models for Developmental Toxicity Screening

    EPA Pesticide Factsheets

    The Texas-Indiana Virtual STAR Center: Zebrafish Models for Developmental Toxicity Screening (Presented by Maria Bondesson Bolin, Ph.D, University of Houston, Center for Nuclear Receptors and Cell Signaling) (3/22/2012)

  5. 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.

  6. 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

  7. Etiology and functional validation of gastrointestinal motility dysfunction in a zebrafish model of CHARGE syndrome.

    PubMed

    Cloney, Kellie; Steele, Shelby L; Stoyek, Matthew R; Croll, Roger P; Smith, Frank M; Prykhozhij, Sergey V; Brown, Mary M; Midgen, Craig; Blake, Kim; Berman, Jason N

    2018-06-01

    CHARGE syndrome is linked to autosomal-dominant mutations in the CHD7 gene and results in a number of physiological and structural abnormalities, including heart defects, hearing and vision loss, and gastrointestinal (GI) problems. Of these challenges, GI problems have a profound impact throughout an individual's life, resulting in increased morbidity and mortality. A homolog of CHD7 has been identified in the zebrafish, the loss of which recapitulates many of the features of the human disease. Using a morpholino chd7 knockdown model complemented by a chd7 null mutant zebrafish line, we examined GI structure, innervation, and motility in larval zebrafish. Loss of chd7 resulted in physically smaller GI tracts with normal epithelial and muscular histology, but decreased and disorganized vagal projections, particularly in the foregut. chd7 morphant larvae had significantly less ability to empty their GI tract of gavaged fluorescent beads, and this condition was only minimally improved by the prokinetic agents, domperidone and erythromycin, in keeping with mixed responses to these agents in patients with CHARGE syndrome. The conserved genetics and transparency of the zebrafish have provided new insights into the consequences of chd7 gene dysfunction on the GI system and cranial nerve patterning. These findings highlight the opportunity of the zebrafish to serve as a preclinical model for studying compounds that may improve GI motility in individuals with CHARGE syndrome. © 2018 Federation of European Biochemical Societies.

  8. Zebrafish and Medaka: new model organisms for modern biomedical research.

    PubMed

    Lin, Cheng-Yung; Chiang, Cheng-Yi; Tsai, Huai-Jen

    2016-01-28

    Although they are primitive vertebrates, zebrafish (Danio rerio) and medaka (Oryzias latipes) have surpassed other animals as the most used model organisms based on their many advantages. Studies on gene expression patterns, regulatory cis-elements identification, and gene functions can be facilitated by using zebrafish embryos via a number of techniques, including transgenesis, in vivo transient assay, overexpression by injection of mRNAs, knockdown by injection of morpholino oligonucleotides, knockout and gene editing by CRISPR/Cas9 system and mutagenesis. In addition, transgenic lines of model fish harboring a tissue-specific reporter have become a powerful tool for the study of biological sciences, since it is possible to visualize the dynamic expression of a specific gene in the transparent embryos. In particular, some transgenic fish lines and mutants display defective phenotypes similar to those of human diseases. Therefore, a wide variety of fish model not only sheds light on the molecular mechanisms underlying disease pathogenesis in vivo but also provides a living platform for high-throughput screening of drug candidates. Interestingly, transgenic model fish lines can also be applied as biosensors to detect environmental pollutants, and even as pet fish to display beautiful fluorescent colors. Therefore, transgenic model fish possess a broad spectrum of applications in modern biomedical research, as exampled in the following review.

  9. 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.

  10. 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.

  11. A larval zebrafish model of bipolar disorder as a screening platform for neuro-therapeutics.

    PubMed

    Ellis, Lee David; Soanes, Kelly Howard

    2012-08-01

    Modelling neurological diseases has proven extraordinarily difficult due to the phenotypic complexity of each disorder. The zebrafish has become a useful model system with which to study abnormal neurological and behavioural activity and holds promise as a model of human disease. While most of the disease modelling using zebrafish has made use of adults, larvae hold tremendous promise for the high-throughput screening of potential therapeutics. The further development of larval disease models will strengthen their ability to contribute to the drug screening process. Here we have used zebrafish larvae to model the symptoms of bipolar disorder by treating larvae with sub-convulsive concentrations of the GABA antagonist pentylenetetrazol (PTZ). A number of therapeutics that act on different targets, in addition to those that have been used to treat bipolar disorder, were tested against this model to assess its predictive value. Carbamazepine, valproic acid, baclofen and honokiol, were found to oppose various aspects of the PTZ-induced changes in activity. Lidocaine and haloperidol exacerbated the PTZ-induced activity changes and sulpiride had no effect. By comparing the degree of phenotypic rescue with the mechanism of action of each therapeutic we have shown that the low-concentration PTZ model can produce a number of intermediate phenotypes that model symptoms of bipolar disorder, may be useful in modelling other disease states, and will help predict the efficacy of novel therapeutics. Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

  12. Embryonic Zebrafish Model - A Well-Established Method for Rapidly Assessing the Toxicity of Homeopathic Drugs: - Toxicity Evaluation of Homeopathic Drugs Using Zebrafish Embryo Model.

    PubMed

    Gupta, Himanshu R; Patil, Yogesh; Singh, Dipty; Thakur, Mansee

    2016-12-01

    Advancements in nanotechnology have led to nanoparticle (NP) use in various fields of medicine. Although the potential of NPs is promising, the lack of documented evidence on the toxicological effects of NPs is concerning. A few studies have documented that homeopathy uses NPs. Unfortunately, very few sound scientific studies have explored the toxic effects of homeopathic drugs. Citing this lack of high-quality scientific evidence, regulatory agencies have been reluctant to endorse homeopathic treatment as an alternative or adjunct treatment. This study aimed to enhance our insight into the impact of commercially-available homeopathic drugs, to study the presence of NPs in those drugs and any deleterious effects they might have, and to determine the distribution pattern of NPs in zebrafish embryos ( Danio rerio ). Homeopathic dilutions were studied using high-resolution transmission electron microscopy with selected area electron diffraction (SAED). For the toxicity assessment on Zebrafish, embryos were exposed to a test solution from 4 - 6 hours post-fertilization, and embryos/larvae were assessed up to 5 days post-fertilization (dpf) for viability and morphology. Toxicity was recorded in terms of mortality, hatching delay, phenotypic defects and metal accumulation. Around 5 dpf was found to be the optimum developmental stage for evaluation. The present study aimed to conclusively prove the presence of NPs in all high dilutions of homeopathic drugs. Embryonic zebrafish were exposed to three homeopathic drugs with two potencies (30CH, 200CH) during early embryogenesis. The resulting morphological and cellular responses were observed. Exposure to these potencies produced no visibly significant malformations, pericardial edema, and mortality and no necrotic and apoptotic cellular death. Our findings clearly demonstrate that no toxic effects were observed for these three homeopathic drugs at the potencies and exposure times used in this study. The embryonic zebrafish

  13. Gene expression changes in a zebrafish model of drug dependency suggest conservation of neuro-adaptation pathways.

    PubMed

    Kily, Layla J M; Cowe, Yuka C M; Hussain, Osman; Patel, Salma; McElwaine, Suzanne; Cotter, Finbarr E; Brennan, Caroline H

    2008-05-01

    Addiction is a complex psychiatric disorder considered to be a disease of the brain's natural reward reinforcement system. Repeated stimulation of the 'reward' pathway leads to adaptive changes in gene expression and synaptic organization that reinforce drug taking and underlie long-term changes in behaviour. The primitive nature of reward reinforcement pathways and the near universal ability of abused drugs to target the same system allow drug-associated reward and reinforcement to be studied in non-mammalian species. Zebrafish have proved to be a valuable model system for the study of vertebrate development and disease. Here we demonstrate that adult zebrafish show a dose-dependent acute conditioned place preference (CPP) reinforcement response to ethanol or nicotine. Repeated exposure of adult zebrafish to either nicotine or ethanol leads to a robust CPP response that persists following 3 weeks of abstinence and in the face of adverse stimuli, a behavioural indicator of the establishment of dependence. Microarray analysis using whole brain samples from drug-treated and control zebrafish identified 1362 genes that show a significant change in expression between control and treated individuals. Of these genes, 153 are common to both ethanol- and nicotine-treated animals. These genes include members of pathways and processes implicated in drug dependence in mammalian models, revealing conservation of neuro-adaptation pathways between zebrafish and mammals.

  14. Validation of visualized transgenic zebrafish as a high throughput model to assay bradycardia related cardio toxicity risk candidates.

    PubMed

    Wen, Dingsheng; Liu, Aiming; Chen, Feng; Yang, Julin; Dai, Renke

    2012-10-01

    Drug-induced QT prolongation usually leads to torsade de pointes (TdP), thus for drugs in the early phase of development this risk should be evaluated. In the present study, we demonstrated a visualized transgenic zebrafish as an in vivo high-throughput model to assay the risk of drug-induced QT prolongation. Zebrafish larvae 48 h post-fertilization expressing green fluorescent protein in myocardium were incubated with compounds reported to induce QT prolongation or block the human ether-a-go-go-related gene (hERG) K⁺ current. The compounds sotalol, indapaminde, erythromycin, ofoxacin, levofloxacin, sparfloxacin and roxithromycin were additionally administrated by microinjection into the larvae yolk sac. The ventricle heart rate was recorded using the automatic monitoring system after incubation or microinjection. As a result, 14 out of 16 compounds inducing dog QT prolongation caused bradycardia in zebrafish. A similar result was observed with 21 out of 26 compounds which block hERG current. Among the 30 compounds which induced human QT prolongation, 25 caused bradycardia in this model. Thus, the risk of compounds causing bradycardia in this transgenic zebrafish correlated with that causing QT prolongation and hERG K⁺ current blockage in established models. The tendency that high logP values lead to high risk of QT prolongation in this model was indicated, and non-sensitivity of this model to antibacterial agents was revealed. These data suggest application of this transgenic zebrafish as a high-throughput model to screen QT prolongation-related cardio toxicity of the drug candidates. Copyright © 2012 John Wiley & Sons, Ltd.

  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. Transforming growth factor-β signalling controls human breast cancer metastasis in a zebrafish xenograft model.

    PubMed

    Drabsch, Yvette; He, Shuning; Zhang, Long; Snaar-Jagalska, B Ewa; ten Dijke, Peter

    2013-11-07

    The transforming growth factor beta (TGF-β) signalling pathway is known to control human breast cancer invasion and metastasis. We demonstrate that the zebrafish xenograft assay is a robust and dependable animal model for examining the role of pharmacological modulators and genetic perturbation of TGF-β signalling in human breast tumour cells. We injected cancer cells into the embryonic circulation (duct of cuvier) and examined their invasion and metastasis into the avascular collagenous tail. Various aspects of the TGF-β signalling pathway were blocked by chemical inhibition, small interfering RNA (siRNA), or small hairpin RNA (shRNA). Analysis was conducted using fluorescent microscopy. Breast cancer cells with different levels of malignancy, according to in vitro and in vivo mouse studies, demonstrated invasive and metastatic properties within the embryonic zebrafish model that nicely correlated with their differential tumourigenicity in mouse models. Interestingly, MCF10A M2 and M4 cells invaded into the caudal hematopoietic tissue and were visible as a cluster of cells, whereas MDA MB 231 cells invaded into the tail fin and were visible as individual cells. Pharmacological inhibition with TGF-β receptor kinase inhibitors or tumour specific Smad4 knockdown disturbed invasion and metastasis in the zebrafish xenograft model and closely mimicked the results we obtained with these cells in a mouse metastasis model. Inhibition of matrix metallo proteinases, which are induced by TGF-β in breast cancer cells, blocked invasion and metastasis of breast cancer cells. The zebrafish-embryonic breast cancer xenograft model is applicable for the mechanistic understanding, screening and development of anti-TGF-β drugs for the treatment of metastatic breast cancer in a timely and cost-effective manner.

  17. Zebrafish: A Model for the Study of Toxicants Affecting Muscle Development and Function

    PubMed Central

    Dubińska-Magiera, Magda; Daczewska, Małgorzata; Lewicka, Anna; Migocka-Patrzałek, Marta; Niedbalska-Tarnowska, Joanna; Jagla, Krzysztof

    2016-01-01

    The rapid progress in medicine, agriculture, and allied sciences has enabled the development of a large amount of potentially useful bioactive compounds, such as drugs and pesticides. However, there is another side of this phenomenon, which includes side effects and environmental pollution. To avoid or minimize the uncontrollable consequences of using the newly developed compounds, researchers seek a quick and effective means of their evaluation. In achieving this goal, the zebrafish (Danio rerio) has proven to be a highly useful tool, mostly because of its fast growth and development, as well as the ability to absorb the molecules diluted in water through its skin and gills. In this review, we focus on the reports concerning the application of zebrafish as a model for assessing the impact of toxicants on skeletal muscles, which share many structural and functional similarities among vertebrates, including zebrafish and humans. PMID:27869769

  18. Zebrafish – As an Integrative Model for Twenty-first Century Toxicity Testing

    EPA Science Inventory

    The zebrafish embryo is a useful small model for investigating vertebrate development because of its transparency, low cost, transgenic and morpholino capabilities, conservation of cell signaling, and concordance with mammalian developmental phenotypes. From these advantages, the...

  19. Model of voluntary ethanol intake in zebrafish: Effect on behavior and hypothalamic orexigenic peptides

    PubMed Central

    Sterling, M.E.; Karatayev, O.; Chang, G.-Q.; Algava, D.B.; Leibowitz, S.F

    2014-01-01

    Recent studies in zebrafish have shown that exposure to ethanol in tank water affects various behaviors, including locomotion, anxiety and aggression, and produces changes in brain neurotransmitters, such as serotonin and dopamine. Building on these investigations, the present study had two goals: first, to develop a method for inducing voluntary ethanol intake in individual zebrafish, which can be used as a model in future studies to examine how this behavior is affected by various manipulations, and second, to characterize the effects of this ethanol intake on different behaviors and the expression of hypothalamic orexigenic peptides, galanin (GAL) and orexin (OX), which are known in rodents to stimulate consumption of ethanol and alter behaviors associated with alcohol abuse. Thus, we first developed a new model of voluntary intake of ethanol in fish by presenting this ethanol mixed with gelatin, which they readily consume. Using this model, we found that individual zebrafish can be trained in a short period of time to consume stable levels of 10% or 20% ethanol (v/v) mixed with gelatin and that their intake of this ethanol-gelatin mixture leads to pharmacologically-relevant blood ethanol concentrations which are strongly, positively correlated with the amount ingested. Intake of this ethanol-gelatin mixture increased locomotion, reduced anxiety, and stimulated aggressive behavior, while increasing expression of GAL and OX in specific hypothalamic areas. These findings, confirming results in rats, provide a method in zebrafish for investigating with forward genetics and pharmacological techniques the role of different brain mechanisms in controlling ethanol intake. PMID:25257106

  20. 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.

  1. Stab wound injury of the zebrafish telencephalon: a model for comparative analysis of reactive gliosis.

    PubMed

    Baumgart, Emily Violette; Barbosa, Joana S; Bally-Cuif, Laure; Götz, Magdalena; Ninkovic, Jovica

    2012-03-01

    Reactive glia, including astroglia and oligodendrocyte progenitors (OPCs) are at the core of the reaction to injury in the mammalian brain with initially beneficial and later partially adverse functions such as scar formation. Given the different glial composition in the adult zebrafish brain with radial ependymoglia but no parenchymal astrocytes, we examined the glial response to an invasive stab wound injury model in the adult zebrafish telencephalon. Strikingly, already a few days after injury the wound was closed without any scar tissue. Similar to mammals, microglia cells reacted first and accumulated close to the injury site, while neither GFAP+ radial ependymoglia nor adult OPCs were recruited to the injury site. Moreover, OPCs failed to increase their proliferation after this injury, while the number of proliferating GFAP+ glia was increased until 7 days after injury. Importantly, neurogenesis was also increased after injury, generating additional neurons recruited to the parenchyma which survived for several months. Thus, these data suggest that the specific glial environment in the adult zebrafish telencephalon is not only permissive for long-term neuronal survival, but avoids scar formation. Invasive injury in the adult zebrafish telencephalon may therefore provide a useful model to untangle the molecular mechanisms involved in these beneficial glial reactions. Copyright © 2011 Wiley Periodicals, Inc.

  2. 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.

  3. 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.

  4. Modeling mixtures of thyroid gland function disruptors in a vertebrate alternative model, the zebrafish eleutheroembryo

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

    Thienpont, Benedicte; Barata, Carlos; Raldúa, Demetrio, E-mail: drpqam@cid.csic.es

    2013-06-01

    Maternal thyroxine (T4) plays an essential role in fetal brain development, and even mild and transitory deficits in free-T4 in pregnant women can produce irreversible neurological effects in their offspring. Women of childbearing age are daily exposed to mixtures of chemicals disrupting the thyroid gland function (TGFDs) through the diet, drinking water, air and pharmaceuticals, which has raised the highest concern for the potential additive or synergic effects on the development of mild hypothyroxinemia during early pregnancy. Recently we demonstrated that zebrafish eleutheroembryos provide a suitable alternative model for screening chemicals impairing the thyroid hormone synthesis. The present study usedmore » the intrafollicular T4-content (IT4C) of zebrafish eleutheroembryos as integrative endpoint for testing the hypotheses that the effect of mixtures of TGFDs with a similar mode of action [inhibition of thyroid peroxidase (TPO)] was well predicted by a concentration addition concept (CA) model, whereas the response addition concept (RA) model predicted better the effect of dissimilarly acting binary mixtures of TGFDs [TPO-inhibitors and sodium-iodide symporter (NIS)-inhibitors]. However, CA model provided better prediction of joint effects than RA in five out of the six tested mixtures. The exception being the mixture MMI (TPO-inhibitor)-KClO{sub 4} (NIS-inhibitor) dosed at a fixed ratio of EC{sub 10} that provided similar CA and RA predictions and hence it was difficult to get any conclusive result. There results support the phenomenological similarity criterion stating that the concept of concentration addition could be extended to mixture constituents having common apical endpoints or common adverse outcomes. - Highlights: • Potential synergic or additive effect of mixtures of chemicals on thyroid function. • Zebrafish as alternative model for testing the effect of mixtures of goitrogens. • Concentration addition seems to predict better the effect

  5. Gene knockdown by morpholino-modified oligonucleotides in the zebrafish (Danio rerio) model: applications for developmental toxicology.

    PubMed

    Timme-Laragy, Alicia R; Karchner, Sibel I; Hahn, Mark E

    2012-01-01

    The zebrafish (Danio rerio) has long been used as a model for developmental biology, making it an excellent model to use also in developmental toxicology. The many advantages of zebrafish include their small size, prolific spawning, rapid development, and transparent embryos. They can be easily manipulated genetically through the use of transgenic technology and gene knockdown via morpholino-modified antisense oligonucleotides (MOs). Knocking down specific genes to assess their role in the response to toxicant exposure provides a way to further our knowledge of how developmental toxicants work on a molecular and mechanistic level while establishing a relationship between these molecular events and morphological, behavioral, and/or physiological effects (i.e., phenotypic anchoring). In this chapter, we address important considerations for using MOs to study developmental toxicology in zebrafish embryos and provide a protocol for their use.

  6. Orthosiphon stamineus Leaf Extract Affects TNF-α and Seizures in a Zebrafish Model

    PubMed Central

    Choo, Brandon Kar Meng; Kundap, Uday P.; Kumari, Yatinesh; Hue, Seow-Mun; Othman, Iekhsan; Shaikh, Mohd Farooq

    2018-01-01

    Epileptic seizures result from abnormal brain activity and can affect motor, autonomic and sensory function; as well as, memory, cognition, behavior, or emotional state. Effective anti-epileptic drugs (AEDs) are available but have tolerability issues due to their side effects. The Malaysian herb Orthosiphon stamineus, is a traditional epilepsy remedy and possesses anti-inflammatory, anti-oxidant and free-radical scavenging abilities, all of which are known to protect against seizures. This experiment thus aimed to explore if an ethanolic leaf extract of O. stamineus has the potential to be a novel symptomatic treatment for epileptic seizures in a zebrafish model; and the effects of the extract on the expression levels of several genes in the zebrafish brain which are associated with seizures. The results of this study indicate that O. stamineus has the potential to be a novel symptomatic treatment for epileptic seizures as it is pharmacologically active against seizures in a zebrafish model. The anti-convulsive effect of this extract is also comparable to that of diazepam at higher doses and can surpass diazepam in certain cases. Treatment with the extract also counteracts the upregulation of NF-κB, NPY and TNF-α as a result of a Pentylenetetrazol (PTZ) treated seizure. The anti-convulsive action for this extract could be at least partially due to its downregulation of TNF-α. Future work could include the discovery of the active anti-convulsive compound, as well as determine if the extract does not cause cognitive impairment in zebrafish. PMID:29527169

  7. Evaluating Different Virulence Traits of Klebsiella pneumoniae Using Dictyostelium discoideum and Zebrafish Larvae as Host Models

    PubMed Central

    Marcoleta, Andrés E.; Varas, Macarena A.; Ortiz-Severín, Javiera; Vásquez, Leonardo; Berríos-Pastén, Camilo; Sabag, Andrea V.; Chávez, Francisco P.; Allende, Miguel L.; Santiviago, Carlos A.; Monasterio, Octavio; Lagos, Rosalba

    2018-01-01

    Multiresistant and invasive hypervirulent Klebsiella pneumoniae strains have become one of the most urgent bacterial pathogen threats. Recent analyses revealed a high genomic plasticity of this species, harboring a variety of mobile genetic elements associated with virulent strains, encoding proteins of unknown function whose possible role in pathogenesis have not been addressed. K. pneumoniae virulence has been studied mainly in animal models such as mice and pigs, however, practical, financial, ethical and methodological issues limit the use of mammal hosts. Consequently, the development of simple and cost-effective experimental approaches with alternative host models is needed. In this work we described the use of both, the social amoeba and professional phagocyte Dictyostelium discoideum and the fish Danio rerio (zebrafish) as surrogate host models to study K. pneumoniae virulence. We compared three K. pneumoniae clinical isolates evaluating their resistance to phagocytosis, intracellular survival, lethality, intestinal colonization, and innate immune cells recruitment. Optical transparency of both host models permitted studying the infective process in vivo, following the Klebsiella-host interactions through live-cell imaging. We demonstrated that K. pneumoniae RYC492, but not the multiresistant strains 700603 and BAA-1705, is virulent to both host models and elicits a strong immune response. Moreover, this strain showed a high resistance to phagocytosis by D. discoideum, an increased ability to form biofilms and a more prominent and irregular capsule. Besides, the strain 700603 showed the unique ability to replicate inside amoeba cells. Genomic comparison of the K. pneumoniae strains showed that the RYC492 strain has a higher overall content of virulence factors although no specific genes could be linked to its phagocytosis resistance, nor to the intracellular survival observed for the 700603 strain. Our results indicate that both zebrafish and D. discoideum

  8. Beyond the zebrafish: diverse fish species for modeling human disease

    PubMed Central

    Schartl, Manfred

    2014-01-01

    ABSTRACT In recent years, zebrafish, and to a lesser extent medaka, have become widely used small animal models for human diseases. These organisms have convincingly demonstrated the usefulness of fish for improving our understanding of the molecular and cellular mechanisms leading to pathological conditions, and for the development of new diagnostic and therapeutic tools. Despite the usefulness of zebrafish and medaka in the investigation of a wide spectrum of traits, there is evidence to suggest that other fish species could be better suited for more targeted questions. With the emergence of new, improved sequencing technologies that enable genomic resources to be generated with increasing efficiency and speed, the potential of non-mainstream fish species as disease models can now be explored. A key feature of these fish species is that the pathological condition that they model is often related to specific evolutionary adaptations. By exploring these adaptations, new disease-causing and disease-modifier genes might be identified; thus, diverse fish species could be exploited to better understand the complexity of disease processes. In addition, non-mainstream fish models could allow us to study the impact of environmental factors, as well as genetic variation, on complex disease phenotypes. This Review will discuss the opportunities that such fish models offer for current and future biomedical research. PMID:24271780

  9. 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

  10. Modeling Zebrafish Developmental Toxicity using a Concurrent In vitro Assay Battery (SOT)

    EPA Science Inventory

    We describe the development of computational models that predict activity in a repeat-dose zebrafish embryo developmental toxicity assay using a combination of physico-chemical parameters and in vitro (human) assay measurements. The data set covered 986 chemicals including pestic...

  11. 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

  12. Breaking symmetry: the zebrafish as a model for understanding left-right asymmetry in the developing brain.

    PubMed

    Roussigne, Myriam; Blader, Patrick; Wilson, Stephen W

    2012-03-01

    How does left-right asymmetry develop in the brain and how does the resultant asymmetric circuitry impact on brain function and lateralized behaviors? By enabling scientists to address these questions at the levels of genes, neurons, circuitry and behavior,the zebrafish model system provides a route to resolve the complexity of brain lateralization. In this review, we present the progress made towards characterizing the nature of the gene networks and the sequence of morphogenetic events involved in the asymmetric development of zebrafish epithalamus. In an attempt to integrate the recent extensive knowledge into a working model and to identify the future challenges,we discuss how insights gained at a cellular/developmental level can be linked to the data obtained at a molecular/genetic level. Finally, we present some evolutionary thoughts and discuss how significant discoveries made in zebrafish should provide entry points to better understand the evolutionary origins of brain lateralization.

  13. Novel degenerative and developmental defects in a zebrafish model of mucolipidosis type IV

    PubMed Central

    Li, Huiqing; Pei, Wuhong; Vergarajauregui, Sivia; Zerfas, Patricia M.; Raben, Nina; Burgess, Shawn M.; Puertollano, Rosa

    2017-01-01

    Abstract Mucolipidosis type IV (MLIV) is a lysosomal storage disease characterized by neurologic and ophthalmologic abnormalities. There is currently no effective treatment. MLIV is caused by mutations in MCOLN1, a lysosomal cation channel from the transient receptor potential (TRP) family. In this study, we used genome editing to knockout the two mcoln1 genes present in Danio rerio (zebrafish). Our model successfully reproduced the retinal and neuromuscular defects observed in MLIV patients, indicating that this model is suitable for studying the disease pathogenesis. Importantly, our model revealed novel insights into the origins and progression of the MLIV pathology, including the contribution of autophagosome accumulation to muscle dystrophy and the role of mcoln1 in embryonic development, hair cell viability and cellular maintenance. The generation of a MLIV model in zebrafish is particularly relevant given the suitability of this organism for large-scale in vivo drug screening, thus providing unprecedented opportunities for therapeutic discovery. PMID:28449103

  14. 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.

  15. 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.

  16. Development of a transgenic zebrafish model expressing GFP in the notochord, somite and liver directed by the hfe2 gene promoter.

    PubMed

    Bian, Yue-Hong; Xu, Cheng; Li, Junling; Xu, Jin; Zhang, Hongwei; Du, Shao Jun

    2011-08-01

    Hemojuvelin, also known as RGMc, is encoded by hfe2 gene that plays an important role in iron homeostasis. hfe2 is specifically expressed in the notochord, developing somite and skeletal muscles during development. The molecular regulation of hfe2 expression is, however, not clear. We reported here the characterization of hfe2 gene expression and the regulation of its tissue-specific expression in zebrafish embryos. We demonstrated that the 6 kb 5'-flanking sequence upstream of the ATG start codon in the zebrafish hfe2 gene could direct GFP specific expression in the notochord, somites, and skeletal muscle of zebrafish embryos, recapitulating the expression pattern of the endogenous gene. However, the Tg(hfe2:gfp) transgene is also expressed in the liver of fish embryos, which did not mimic the expression of the endogenous hfe2 at the early stage. Nevertheless, the Tg(hfe2:gfp) transgenic zebrafish provides a useful model to study liver development. Treating Tg(hfe2:gfp) transgenic zebrafish embryos with valproic acid, a liver development inhibitor, significantly inhibited GFP expression in zebrafish. Together, these data indicate that the tissue specific expression of hfe2 in the notochord, somites and muscles is regulated by regulatory elements within the 6 kb 5'-flanking sequence of the hfe2 gene. Moreover, the Tg(hfe2:gfp) transgenic zebrafish line provides a useful model system for analyzing liver development in zebrafish.

  17. Zebrafish Heart Failure Models for the Evaluation of Chemical Probes and Drugs

    PubMed Central

    Monte, Aaron; Cook, James M.; Kabir, Mohd Shahjahan; Peterson, Karl P.

    2013-01-01

    Abstract Heart failure is a complex disease that involves genetic, environmental, and physiological factors. As a result, current medication and treatment for heart failure produces limited efficacy, and better medication is in demand. Although mammalian models exist, simple and low-cost models will be more beneficial for drug discovery and mechanistic studies of heart failure. We previously reported that aristolochic acid (AA) caused cardiac defects in zebrafish embryos that resemble heart failure. Here, we showed that cardiac troponin T and atrial natriuretic peptide were expressed at significantly higher levels in AA-treated embryos, presumably due to cardiac hypertrophy. In addition, several human heart failure drugs could moderately attenuate the AA-induced heart failure by 10%–40%, further verifying the model for drug discovery. We then developed a drug screening assay using the AA-treated zebrafish embryos and identified three compounds. Mitogen-activated protein kinase kinase inhibitor (MEK-I), an inhibitor for the MEK-1/2 known to be involved in cardiac hypertrophy and heart failure, showed nearly 60% heart failure attenuation. C25, a chalcone derivative, and A11, a phenolic compound, showed around 80% and 90% attenuation, respectively. Time course experiments revealed that, to obtain 50% efficacy, these compounds were required within different hours of AA treatment. Furthermore, quantitative polymerase chain reaction showed that C25, not MEK-I or A11, strongly suppressed inflammation. Finally, C25 and MEK-I, but not A11, could also rescue the doxorubicin-induced heart failure in zebrafish embryos. In summary, we have established two tractable heart failure models for drug discovery and three potential drugs have been identified that seem to attenuate heart failure by different mechanisms. PMID:24351044

  18. 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

  19. 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.

  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. Adult zebrafish intestine resection: a novel model of short bowel syndrome, adaptation, and intestinal stem cell regeneration.

    PubMed

    Schall, K A; Holoyda, K A; Grant, C N; Levin, D E; Torres, E R; Maxwell, A; Pollack, H A; Moats, R A; Frey, M R; Darehzereshki, A; Al Alam, D; Lien, C; Grikscheit, T C

    2015-08-01

    Loss of significant intestinal length from congenital anomaly or disease may lead to short bowel syndrome (SBS); intestinal failure may be partially offset by a gain in epithelial surface area, termed adaptation. Current in vivo models of SBS are costly and technically challenging. Operative times and survival rates have slowed extension to transgenic models. We created a new reproducible in vivo model of SBS in zebrafish, a tractable vertebrate model, to facilitate investigation of the mechanisms of intestinal adaptation. Proximal intestinal diversion at segment 1 (S1, equivalent to jejunum) was performed in adult male zebrafish. SBS fish emptied distal intestinal contents via stoma as in the human disease. After 2 wk, S1 was dilated compared with controls and villus ridges had increased complexity, contributing to greater villus epithelial perimeter. The number of intervillus pockets, the intestinal stem cell zone of the zebrafish increased and contained a higher number of bromodeoxyuridine (BrdU)-labeled cells after 2 wk of SBS. Egf receptor and a subset of its ligands, also drivers of adaptation, were upregulated in SBS fish. Igf has been reported as a driver of intestinal adaptation in other animal models, and SBS fish exposed to a pharmacological inhibitor of the Igf receptor failed to demonstrate signs of intestinal adaptation, such as increased inner epithelial perimeter and BrdU incorporation. We describe a technically feasible model of human SBS in the zebrafish, a faster and less expensive tool to investigate intestinal stem cell plasticity as well as the mechanisms that drive intestinal adaptation. Copyright © 2015 the American Physiological Society.

  2. Adult zebrafish intestine resection: a novel model of short bowel syndrome, adaptation, and intestinal stem cell regeneration

    PubMed Central

    Schall, K. A.; Holoyda, K. A.; Grant, C. N.; Levin, D. E.; Torres, E. R.; Maxwell, A.; Pollack, H. A.; Moats, R. A.; Frey, M. R.; Darehzereshki, A.; Al Alam, D.; Lien, C.

    2015-01-01

    Loss of significant intestinal length from congenital anomaly or disease may lead to short bowel syndrome (SBS); intestinal failure may be partially offset by a gain in epithelial surface area, termed adaptation. Current in vivo models of SBS are costly and technically challenging. Operative times and survival rates have slowed extension to transgenic models. We created a new reproducible in vivo model of SBS in zebrafish, a tractable vertebrate model, to facilitate investigation of the mechanisms of intestinal adaptation. Proximal intestinal diversion at segment 1 (S1, equivalent to jejunum) was performed in adult male zebrafish. SBS fish emptied distal intestinal contents via stoma as in the human disease. After 2 wk, S1 was dilated compared with controls and villus ridges had increased complexity, contributing to greater villus epithelial perimeter. The number of intervillus pockets, the intestinal stem cell zone of the zebrafish increased and contained a higher number of bromodeoxyuridine (BrdU)-labeled cells after 2 wk of SBS. Egf receptor and a subset of its ligands, also drivers of adaptation, were upregulated in SBS fish. Igf has been reported as a driver of intestinal adaptation in other animal models, and SBS fish exposed to a pharmacological inhibitor of the Igf receptor failed to demonstrate signs of intestinal adaptation, such as increased inner epithelial perimeter and BrdU incorporation. We describe a technically feasible model of human SBS in the zebrafish, a faster and less expensive tool to investigate intestinal stem cell plasticity as well as the mechanisms that drive intestinal adaptation. PMID:26089336

  3. Evaluation in zebrafish model of the toxicity of rhodamine B-conjugated crotamine, a peptide potentially useful for diagnostics and therapeutics.

    PubMed

    Chan, Judy Yuet-Wa; Zhou, Hefeng; Kwan, Yiu Wa; Chan, Shun Wan; Radis-Baptista, Gandhi; Lee, Simon Ming-Yuen

    2017-11-01

    Crotamine is defensin-like cationic peptide from rattlesnake venom that possesses anticancer, antimicrobial, and antifungal properties. Despite these promising biological activities, toxicity is a major concern associated with the development of venom-derived peptides as therapeutic agents. In the present study, we used zebrafish as a system model to evaluate the toxicity of rhodamine B-conjugated (RhoB) crotamine derivative. The lethal toxic concentration of RhoB-crotamine was as low as 4 μM, which effectively kill zebrafish larvae in less than 10 min. With non-lethal concentrations (<1 μM), crotamine caused malformation in zebrafish embryos, delayed or completely halted hatching, adversely affected embryonic developmental programming, decreased the cardiac functions, and attenuated the swimming distance of zebrafish. The RhoB-crotamine translocated across vitelline membrane and accumulated in zebrafish yolk sac. These results demonstrate the sensitive responsivity of zebrafish to trial crotamine analogues for the development of novel therapeutic peptides with improved safety, bioavailability, and efficacy profiles. © 2017 Wiley Periodicals, Inc.

  4. 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.

  5. 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.

  6. 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

  7. The zebrafish embryo model in toxicology and teratology, September 2–3, 2010, Karlsruhe, Germany.

    PubMed

    Busch, Wibke; Duis, Karen; Fenske, Martina; Maack, Gerd; Legler, Juliette; Padilla, Stephanie; Strähle, Uwe; Witters, Hilda; Scholz, Stefan

    2011-05-01

    The use of fish embryos is gaining popularity for research in the area of toxicology and teratology. Particularly embryos of the zebrafish offer an array of different applications ranging from regulatory testing to mechanistic research. For this reason a consortium of two research centres and a company with the support of the COST Action EuFishBiomed has organised the Workshop “The zebrafish embryo model in toxicology and teratology”, in Karlsruhe, Germany, 2nd–3rd September 2010. The workshop aimed at bringing together experts from different areas of toxicology using the (zebra)fish embryo and stimulating networking between scientists and representatives from regulatory bodies, research institutions and industry. Recent findings, presented in various platform presentations in the area of regulatory toxicity, high throughput screening, toxicogenomics, as well as environmental and human risk assessment are highlighted in this meeting report. Furthermore, the constraints and possibilities of the model as discussed at the workshop are described. A follow up-meeting was appreciated by the about 120 participants and is planned for 2012.

  8. 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

  9. 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

  10. Zebrafish: an exciting model for investigating the spatio-temporal pattern of enteric nervous system development.

    PubMed

    Doodnath, Reshma; Dervan, Adrian; Wride, Michael A; Puri, Prem

    2010-12-01

    Recently, the zebrafish (Danio rerio) has been shown to be an excellent model for human paediatric research. Advantages over other models include its small size, externally visually accessible development and ease of experimental manipulation. The enteric nervous system (ENS) consists of neurons and enteric glia. Glial cells permit cell bodies and processes of neurons to be arranged and maintained in a proper spatial arrangement, and are essential in the maintenance of basic physiological functions of neurons. Glial fibrillary acidic protein (GFAP) is expressed in astrocytes, but also expressed outside of the central nervous system. The aim of this study was to investigate the spatio-temporal pattern of GFAP expression in developing zebrafish ENS from 24 h post-fertilization (hpf), using transgenic fish that express green fluorescent protein (GFP). Zebrafish embryos were collected from transgenic GFP Tg(GFAP:GFP)(mi2001) adult zebrafish from 24 to 120 hpf, fixed and processed for whole mount immunohistochemistry. Antibodies to Phox2b were used to identify enteric neurons. Specimens were mounted on slides and imaging was performed using a fluorescent laser confocal microscope. GFAP:GFP labelling outside the spinal cord was identified in embryos from 48 hpf. The patterning was intracellular and consisted of elongated profiles that appeared to migrate away from the spinal cord into the periphery. At 72 and 96 hpf, GFAP:GFP was expressed dorsally and ventrally to the intestinal tract. At 120 hpf, GFAP:GFP was expressed throughout the intestinal wall, and clusters of enteric neurons were identified using Phox2b immunofluorescence along the pathway of GFAP:GFP positive processes, indicative of a migratory pathway of ENS precursors from the spinal cord into the intestine. The pattern of migration of GFAP:GFP expressing cells outside the spinal cord suggests an organized, early developing migratory pathway to the ENS. This shows for the first time that Tg(GFAP:GFP)(mi2001

  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. 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.

  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. 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.

  15. 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

  16. Model-free information-theoretic approach to infer leadership in pairs of zebrafish.

    PubMed

    Butail, Sachit; Mwaffo, Violet; Porfiri, Maurizio

    2016-04-01

    Collective behavior affords several advantages to fish in avoiding predators, foraging, mating, and swimming. Although fish schools have been traditionally considered egalitarian superorganisms, a number of empirical observations suggest the emergence of leadership in gregarious groups. Detecting and classifying leader-follower relationships is central to elucidate the behavioral and physiological causes of leadership and understand its consequences. Here, we demonstrate an information-theoretic approach to infer leadership from positional data of fish swimming. In this framework, we measure social interactions between fish pairs through the mathematical construct of transfer entropy, which quantifies the predictive power of a time series to anticipate another, possibly coupled, time series. We focus on the zebrafish model organism, which is rapidly emerging as a species of choice in preclinical research for its genetic similarity to humans and reduced neurobiological complexity with respect to mammals. To overcome experimental confounds and generate test data sets on which we can thoroughly assess our approach, we adapt and calibrate a data-driven stochastic model of zebrafish motion for the simulation of a coupled dynamical system of zebrafish pairs. In this synthetic data set, the extent and direction of the coupling between the fish are systematically varied across a wide parameter range to demonstrate the accuracy and reliability of transfer entropy in inferring leadership. Our approach is expected to aid in the analysis of collective behavior, providing a data-driven perspective to understand social interactions.

  17. Model-free information-theoretic approach to infer leadership in pairs of zebrafish

    NASA Astrophysics Data System (ADS)

    Butail, Sachit; Mwaffo, Violet; Porfiri, Maurizio

    2016-04-01

    Collective behavior affords several advantages to fish in avoiding predators, foraging, mating, and swimming. Although fish schools have been traditionally considered egalitarian superorganisms, a number of empirical observations suggest the emergence of leadership in gregarious groups. Detecting and classifying leader-follower relationships is central to elucidate the behavioral and physiological causes of leadership and understand its consequences. Here, we demonstrate an information-theoretic approach to infer leadership from positional data of fish swimming. In this framework, we measure social interactions between fish pairs through the mathematical construct of transfer entropy, which quantifies the predictive power of a time series to anticipate another, possibly coupled, time series. We focus on the zebrafish model organism, which is rapidly emerging as a species of choice in preclinical research for its genetic similarity to humans and reduced neurobiological complexity with respect to mammals. To overcome experimental confounds and generate test data sets on which we can thoroughly assess our approach, we adapt and calibrate a data-driven stochastic model of zebrafish motion for the simulation of a coupled dynamical system of zebrafish pairs. In this synthetic data set, the extent and direction of the coupling between the fish are systematically varied across a wide parameter range to demonstrate the accuracy and reliability of transfer entropy in inferring leadership. Our approach is expected to aid in the analysis of collective behavior, providing a data-driven perspective to understand social interactions.

  18. The Nordic countries meeting on the zebrafish as a model for development and disease 2012.

    PubMed

    Andersson Lendahl, Monika; Zetterberg, Henrik

    2013-03-01

    The first Nordic Countries Meeting on the Zebrafish as a Model for Development and Disease took place at Karolinska Institutet in Stockholm, November 21-23, 2012. The meeting gathered 130 scientists, students, and company representatives from Iceland, Finland, Norway, Denmark, and Sweden, as well as invited guests and keynote speakers from England, Scotland, Germany, Poland, The Netherlands, Singapore, Japan, and the United States. Presentations covered a wide range of topics, including developmental biology, genetics, evolutionary biology, toxicology, behavioral studies, and disease mechanisms. The need for formal guidance and training in zebrafish housing, husbandry, and health monitoring was recognized, and the meeting expressed its support for the joint working group of the FELASA/COST action BM0804 EuFishBioMed. The decision was made to turn the Nordic meeting into an annual event and create a Nordic network of zebrafish researchers.

  19. 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

  20. 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.

  1. 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.

  2. Cell Fate of Müller Cells During Photoreceptor Regeneration in an N-Methyl-N-nitrosourea-Induced Retinal Degeneration Model of Zebrafish.

    PubMed

    Ogai, Kazuhiro; Hisano, Suguru; Sugitani, Kayo; Koriyama, Yoshiki; Kato, Satoru

    2016-01-01

    Zebrafish can regenerate several organs such as the tail fin, heart, central nervous system, and photoreceptors. Very recently, a study has demonstrated the photoreceptor regeneration in the alkylating agent N-methyl-N-nitrosourea (MNU)-induced retinal degeneration (RD) zebrafish model, in which whole photoreceptors are lost within a week after MNU treatment and then regenerated within a month. The research has also shown massive proliferation of Müller cells within a week. To address the question of whether proliferating Müller cells are the source of regenerating photoreceptors, which remains unknown in the MNU-induced zebrafish RD model, we employed a BrdU pulse-chase technique to label the proliferating cells within a week after MNU treatment. As a result of the BrdU pulse-chase technique, a number of BrdU(+) cells were observed in the outer nuclear layer as well as the inner nuclear layer. This implies that regenerating photoreceptors are derived from proliferating Müller cells in the zebrafish MNU-induced RD model.

  3. 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.

  4. Importance of Toxicokinetics to Assess the Utility of Zebrafish Larvae as Model for Psychoactive Drug Screening Using Meta-Chlorophenylpiperazine (mCPP) as Example

    PubMed Central

    Kirla, Krishna Tulasi; Groh, Ksenia J.; Poetzsch, Michael; Banote, Rakesh Kumar; Stadnicka-Michalak, Julita; Eggen, Rik I. L.; Schirmer, Kristin; Kraemer, Thomas

    2018-01-01

    The number of new psychoactive substances (NPS) increases rapidly, harming society and fuelling the need for alternative testing strategies. These should allow the ever-increasing number of drugs to be tested more effectively for their toxicity and psychoactive effects. One proposed strategy is to complement rodent models with zebrafish (Danio rerio) larvae. Yet, our understanding of the toxicokinetics in this model, owing to the waterborne drug exposure and the distinct physiology of the fish, is incomplete. We here explore the toxicokinetics and behavioral effects of an NPS, meta-chlorophenylpiperazine (mCPP), in zebrafish larvae. Uptake kinetics of mCPP, supported by toxicokinetic modeling, strongly suggested the existence of active transport processes. Internal distribution showed a dominant accumulation in the eye, implying that in zebrafish, like in mammals, melanin could serve as a binding site for basic drugs. We confirmed this by demonstrating significantly lower drug accumulation in two types of hypo-pigmented fish. Comparison of the elimination kinetics between mCPP and previously characterized cocaine demonstrated that drug affinities to melanin in zebrafish vary depending on the structure of the test compound. As expected from mCPP-elicited responses in rodents and humans, zebrafish larvae displayed hypoactive behavior. However, significant differences were seen between zebrafish and rodents with regard to the concentration-dependency of the behavioral response and the comparability of tissue levels, corroborating the need to consider the organism-internal distribution of the chemical to allow appropriate dose modeling while evaluating effects and concordance between zebrafish and mammals. Our results highlight commonalities and differences of mammalian versus the fish model in need of further exploration. PMID:29755353

  5. Importance of Toxicokinetics to Assess the Utility of Zebrafish Larvae as Model for Psychoactive Drug Screening Using Meta-Chlorophenylpiperazine (mCPP) as Example.

    PubMed

    Kirla, Krishna Tulasi; Groh, Ksenia J; Poetzsch, Michael; Banote, Rakesh Kumar; Stadnicka-Michalak, Julita; Eggen, Rik I L; Schirmer, Kristin; Kraemer, Thomas

    2018-01-01

    The number of new psychoactive substances (NPS) increases rapidly, harming society and fuelling the need for alternative testing strategies. These should allow the ever-increasing number of drugs to be tested more effectively for their toxicity and psychoactive effects. One proposed strategy is to complement rodent models with zebrafish ( Danio rerio ) larvae. Yet, our understanding of the toxicokinetics in this model, owing to the waterborne drug exposure and the distinct physiology of the fish, is incomplete. We here explore the toxicokinetics and behavioral effects of an NPS, meta-chlorophenylpiperazine (mCPP), in zebrafish larvae. Uptake kinetics of mCPP, supported by toxicokinetic modeling, strongly suggested the existence of active transport processes. Internal distribution showed a dominant accumulation in the eye, implying that in zebrafish, like in mammals, melanin could serve as a binding site for basic drugs. We confirmed this by demonstrating significantly lower drug accumulation in two types of hypo-pigmented fish. Comparison of the elimination kinetics between mCPP and previously characterized cocaine demonstrated that drug affinities to melanin in zebrafish vary depending on the structure of the test compound. As expected from mCPP-elicited responses in rodents and humans, zebrafish larvae displayed hypoactive behavior. However, significant differences were seen between zebrafish and rodents with regard to the concentration-dependency of the behavioral response and the comparability of tissue levels, corroborating the need to consider the organism-internal distribution of the chemical to allow appropriate dose modeling while evaluating effects and concordance between zebrafish and mammals. Our results highlight commonalities and differences of mammalian versus the fish model in need of further exploration.

  6. 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.

  7. 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

  8. Zebrafish is a predictive model for identifying compounds that protect against brain toxicity in severe acute organophosphorus intoxication.

    PubMed

    Faria, Melissa; Prats, Eva; Padrós, Francesc; Soares, Amadeu M V M; Raldúa, Demetrio

    2017-04-01

    Acute organophosphorus (OP) intoxication is a worldwide clinical and public health problem. In addition to cholinergic crisis, neurodegeneration and brain damage are hallmarks of the severe form of this toxidrome. Recently, we generated a chemical model of severe acute OP intoxication in zebrafish that is characterized by altered head morphology and brain degeneration. The pathophysiological pathways resulting in brain toxicity in this model are similar to those described in humans. The aim of this study was to assess the predictive power of this zebrafish model by testing the effect of a panel of drugs that provide protection in mammalian models. The selected drugs included "standard therapy" drugs (atropine and pralidoxime), reversible acetylcholinesterase inhibitors (huperzine A, galantamine, physostigmine and pyridostigmine), N-methyl-D-aspartate (NMDA) receptor antagonists (MK-801 and memantine), dual-function NMDA receptor and acetylcholine receptor antagonists (caramiphen and benactyzine) and anti-inflammatory drugs (dexamethasone and ibuprofen). The effects of these drugs on zebrafish survival and the prevalence of abnormal head morphology in the larvae exposed to 4 µM chlorpyrifos oxon [1 × median lethal concentration (LC 50 )] were determined. Moreover, the neuroprotective effects of pralidoxime, memantine, caramiphen and dexamethasone at the gross morphological level were confirmed by histopathological and transcriptional analyses. Our results demonstrated that the zebrafish model for severe acute OP intoxication has a high predictive value and can be used to identify new compounds that provide neuroprotection against severe acute OP intoxication.

  9. 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.

  10. Large-Scale Phenotype-Based Antiepileptic Drug Screening in a Zebrafish Model of Dravet Syndrome1,2,3

    PubMed Central

    Dinday, Matthew T.

    2015-01-01

    Abstract Mutations in a voltage-gated sodium channel (SCN1A) result in Dravet Syndrome (DS), a catastrophic childhood epilepsy. Zebrafish with a mutation in scn1Lab recapitulate salient phenotypes associated with DS, including seizures, early fatality, and resistance to antiepileptic drugs. To discover new drug candidates for the treatment of DS, we screened a chemical library of ∼1000 compounds and identified 4 compounds that rescued the behavioral seizure component, including 1 compound (dimethadione) that suppressed associated electrographic seizure activity. Fenfluramine, but not huperzine A, also showed antiepileptic activity in our zebrafish assays. The effectiveness of compounds that block neuronal calcium current (dimethadione) or enhance serotonin signaling (fenfluramine) in our zebrafish model suggests that these may be important therapeutic targets in patients with DS. Over 150 compounds resulting in fatality were also identified. We conclude that the combination of behavioral and electrophysiological assays provide a convenient, sensitive, and rapid basis for phenotype-based drug screening in zebrafish mimicking a genetic form of epilepsy. PMID:26465006

  11. 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.

  12. A characterization of the ZFL cell line and primary hepatocytes as in vitro liver cell models for the zebrafish (Danio rerio).

    PubMed

    Eide, Marta; Rusten, Marte; Male, Rune; Jensen, Knut Helge Midtbø; Goksøyr, Anders

    2014-02-01

    The zebrafish (Danio rerio) is a widely used model species in biomedical research. The ZFL cell line, established from zebrafish liver, and freshly isolated primary hepatocytes from zebrafish have been used in several toxicological studies. However, no previous report has compared and characterized these two systems at the level of gene expression. The aim of this study was to evaluate the ZFL cell line in comparison to primary hepatocytes as in vitro models for studying effects of environmental contaminants in zebrafish liver. Using quantitative real-time PCR, the basal level and transcriptional induction potential of key genes involved in toxic responses in the ZFL cell line, primary hepatocytes and whole liver from zebrafish were compared. The study showed that the ZFL cells have lower levels of mRNA of most selected genes compared to zebrafish liver. The induced gene transcription following exposure to ligand was much lower in ZFL cells compared to zebrafish primary hepatocytes at the doses tested. Importantly, oestrogen receptor and vitellogenin genes showed low basal transcription and no induction response in the ZFL cell line. In conclusion, it appears that primary hepatocytes are well suited for studying environmental contaminants including xenoestrogens, but may show large sex-dependent differences in gene transcription. The ZFL cell line shows potential in toxicological studies involving the aryl hydrocarbon receptor pathway. However, low potential for transcriptional induction of genes in general should be expected, especially notable when studying estrogenic responses. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. 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.

  14. Cross-species pharmacological characterization of the allylglycine seizure model in mice and larval zebrafish.

    PubMed

    Leclercq, Karine; Afrikanova, Tatiana; Langlois, Melanie; De Prins, An; Buenafe, Olivia E; Rospo, Chiara C; Van Eeckhaut, Ann; de Witte, Peter A M; Crawford, Alexander D; Smolders, Ilse; Esguerra, Camila V; Kaminski, Rafal M

    2015-04-01

    Treatment-resistant seizures affect about a third of patients suffering from epilepsy. To fulfill the need for new medications targeting treatment-resistant seizures, a number of rodent models offer the opportunity to assess a variety of potential treatment approaches. The use of such models, however, has proven to be time-consuming and labor-intensive. In this study, we performed pharmacological characterization of the allylglycine (AG) seizure model, a simple in vivo model for which we demonstrated a high level of treatment resistance. (d,l)-Allylglycine inhibits glutamic acid decarboxylase (GAD) - the key enzyme in γ-aminobutyric acid (GABA) biosynthesis - leading to GABA depletion, seizures, and neuronal damage. We performed a side-by-side comparison of mouse and zebrafish acute AG treatments including biochemical, electrographic, and behavioral assessments. Interestingly, seizure progression rate and GABA depletion kinetics were comparable in both species. Five mechanistically diverse antiepileptic drugs (AEDs) were used. Three out of the five AEDs (levetiracetam, phenytoin, and topiramate) showed only a limited protective effect (mainly mortality delay) at doses close to the TD50 (dose inducing motor impairment in 50% of animals) in mice. The two remaining AEDs (diazepam and sodium valproate) displayed protective activity against AG-induced seizures. Experiments performed in zebrafish larvae revealed behavioral AED activity profiles highly analogous to those obtained in mice. Having demonstrated cross-species similarities and limited efficacy of tested AEDs, we propose the use of AG in zebrafish as a convenient and high-throughput model of treatment-resistant seizures. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. The Nordic Countries Meeting on the Zebrafish as a Model for Development and Disease 2012

    PubMed Central

    Zetterberg, Henrik

    2013-01-01

    Abstract The first Nordic Countries Meeting on the Zebrafish as a Model for Development and Disease took place at Karolinska Institutet in Stockholm, November 21–23, 2012. The meeting gathered 130 scientists, students, and company representatives from Iceland, Finland, Norway, Denmark, and Sweden, as well as invited guests and keynote speakers from England, Scotland, Germany, Poland, The Netherlands, Singapore, Japan, and the United States. Presentations covered a wide range of topics, including developmental biology, genetics, evolutionary biology, toxicology, behavioral studies, and disease mechanisms. The need for formal guidance and training in zebrafish housing, husbandry, and health monitoring was recognized, and the meeting expressed its support for the joint working group of the FELASA/COST action BM0804 EuFishBioMed. The decision was made to turn the Nordic meeting into an annual event and create a Nordic network of zebrafish researchers. PMID:23590403

  2. Distinct innate immune phagocyte responses to Aspergillus fumigatus conidia and hyphae in zebrafish larvae.

    PubMed

    Knox, Benjamin P; Deng, Qing; Rood, Mary; Eickhoff, Jens C; Keller, Nancy P; Huttenlocher, Anna

    2014-10-01

    Aspergillus fumigatus is the most common filamentous fungal pathogen of immunocompromised hosts, resulting in invasive aspergillosis (IA) and high mortality rates. Innate immunity is known to be the predominant host defense against A. fumigatus; however, innate phagocyte responses to A. fumigatus in an intact host and their contributions to host survival remain unclear. Here, we describe a larval zebrafish A. fumigatus infection model amenable to real-time imaging of host-fungal interactions in live animals. Following infection with A. fumigatus, innate phagocyte populations exhibit clear preferences for different fungal morphologies: macrophages rapidly phagocytose conidia and form aggregates around hyphae, while the neutrophil response is dependent upon the presence of hyphae. Depletion of macrophages rendered host larvae susceptible to invasive disease. Moreover, a zebrafish model of human leukocyte adhesion deficiency with impaired neutrophil function also resulted in invasive disease and impaired host survival. In contrast, macrophage-deficient but not neutrophil-deficient larvae exhibited attenuated disease following challenge with a less virulent (ΔlaeA) strain of A. fumigatus, which has defects in secondary metabolite production. Taking these results together, we have established a new vertebrate model for studying innate immune responses to A. fumigatus that reveals distinct roles for neutrophils and macrophages in mediating host defense against IA. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  3. Zebrafish fin immune responses during high mortality infections with viral haemorrhagic septicemia rhabdovirus. A proteomic and transcriptomic approach.

    PubMed

    Encinas, Paloma; Rodriguez-Milla, Miguel A; Novoa, Beatriz; Estepa, Amparo; Figueras, Antonio; Coll, Julio

    2010-09-27

    Despite rhabdoviral infections being one of the best known fish diseases, the gene expression changes induced at the surface tissues after the natural route of infection (infection-by-immersion) have not been described yet. This work describes the differential infected versus non-infected expression of proteins and immune-related transcripts in fins and organs of zebrafish Danio rerio shortly after infection-by-immersion with viral haemorrhagic septicemia virus (VHSV). Two-dimensional differential gel electrophoresis detected variations on the protein levels of the enzymes of the glycolytic pathway and cytoskeleton components but it detected very few immune-related proteins. Differential expression of immune-related gene transcripts estimated by quantitative polymerase chain reaction arrays and hybridization to oligo microarrays showed that while more transcripts increased in fins than in organs (spleen, head kidney and liver), more transcripts decreased in organs than in fins. Increased differential transcript levels in fins detected by both arrays corresponded to previously described infection-related genes such as complement components (c3b, c8 and c9) or class I histocompatibility antigens (mhc1) and to newly described genes such as secreted immunoglobulin domain (sid4), macrophage stimulating factor (mst1) and a cluster differentiation antigen (cd36). The genes described would contribute to the knowledge of the earliest molecular events occurring in the fish surfaces at the beginning of natural rhabdoviral infections and/or might be new candidates to be tested as adjuvants for fish vaccines.

  4. Costimulatory Function of Cd58/Cd2 Interaction in Adaptive Humoral Immunity in a Zebrafish Model.

    PubMed

    Shao, Tong; Shi, Wei; Zheng, Jia-Yu; Xu, Xiao-Xiao; Lin, Ai-Fu; Xiang, Li-Xin; Shao, Jian-Zhong

    2018-01-01

    CD58 and CD2 have long been known as a pair of reciprocal adhesion molecules involved in the immune modulations of CD8 + T and NK-mediated cellular immunity in humans and several other mammals. However, the functional roles of CD58 and CD2 in CD4 + T-mediated adaptive humoral immunity remain poorly defined. Moreover, the current functional observations of CD58 and CD2 were mainly acquired from in vitro assays, and in vivo investigation is greatly limited due to the absence of a Cd58 homology in murine models. In this study, we identified cd58 and cd2 homologs from the model species zebrafish ( Danio rerio ). These two molecules share conserved structural features to their mammalian counterparts. Functionally, cd58 and cd2 were significantly upregulated on antigen-presenting cells and Cd4 + T cells upon antigen stimulation. Blockade or knockdown of Cd58 and Cd2 dramatically impaired the activation of antigen-specific Cd4 + T and mIgM + B cells, followed by the inhibition of antibody production and host defense against bacterial infections. These results indicate that CD58/CD2 interaction was required for the full activation of CD4 + T-mediated adaptive humoral immunity. The interaction of Cd58 with Cd2 was confirmed by co-immunoprecipitation and functional competitive assays by introducing a soluble Cd2 protein. This study highlights a new costimulatory mechanism underlying the regulatory network of adaptive immunity and makes zebrafish an attractive model organism for the investigation of CD58/CD2-mediated immunology and disorders. It also provides a cross-species understanding of the evolutionary history of costimulatory signals from fish to mammals as a whole.

  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. 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

  7. 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.

  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. 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.

  10. 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

  11. 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.

  12. Osteosarcoma Models: From Cell Lines to Zebrafish

    PubMed Central

    Mohseny, Alexander B.; Hogendoorn, Pancras C. W.; Cleton-Jansen, Anne-Marie

    2012-01-01

    High-grade osteosarcoma is an aggressive tumor most commonly affecting adolescents. The early age of onset might suggest genetic predisposition; however, the vast majority of the tumors are sporadic. Early onset, most often lack of a predisposing condition or lesion, only infrequent (<2%) prevalence of inheritance, extensive genomic instability, and a wide histological heterogeneity are just few factors to mention that make osteosarcoma difficult to study. Therefore, it is sensible to design and use models representative of the human disease. Here we summarize multiple osteosarcoma models established in vitro and in vivo, comment on their utilities, and highlight newest achievements, such as the use of zebrafish embryos. We conclude that to gain a better understanding of osteosarcoma, simplification of this extremely complex tumor is needed. Therefore, we parse the osteosarcoma problem into parts and propose adequate models to study them each separately. A better understanding of osteosarcoma provides opportunities for discovering and assaying novel effective treatment strategies. “Sometimes the model is more interesting than the original disease” PJ Hoedemaeker (1937–2007). PMID:22566751

  13. Longitudinal visualization of vascular occlusion, reperfusion, and remodeling in a zebrafish model of retinal vascular leakage using OCT angiography

    NASA Astrophysics Data System (ADS)

    Spitz, Kathleen; Bozic, Ivan; Desai, Vineet; Rao, Gopikrishna M.; Pollock, Lana M.; Anand-Apte, Bela; Tao, Yuankai K.

    2017-02-01

    Diabetic retinopathy (DR) and age-related macular degeneration (AMD) are two of the leading causes of blindness and visual impairment in the world. Neovascularization results in severe vision loss in DR and AMD and, thus, there is an unmet need to identify mechanisms of pathogenesis and novel anti-angiogenic therapies. Zebrafish is a leading model organism for studying human disease pathogenesis, and the highly conserved drug activity between zebrafish and humans and their ability to readily absorb small molecules dissolved in water has benefited pharmaceutical discovery. Here, we use optical coherence tomography (OCT) and OCT angiography (OCT-A) to perform noninvasive, in vivo retinal imaging in a zebrafish model of vascular leakage. Zebrafish were treated with diethylaminobenzaldehyde (DEAB) to induce vascular leakage and imaged with OCT and OCT-A at six time points over two weeks: baseline one day before treatment and one, three, six, eight, and ten days post treatment. Longitudinal functional imaging showed significant vascular response immediately after DEAB treatment. Observed vascular changes included partial or complete vascular occlusion immediately after treatment and reperfusion during a two-week period. Increased vascular tortuosity several days post treatment indicated remodeling, and bifurcations and collateral vessel formation were also observed. In addition, significant treatment response variabilities were observed in the contralateral eye of the same animal. Anatomical and functional normalization was observed in most animals by ten days post treatment. These preliminary results motivate potential applications of OCT-A as a tool for studying pathogenesis and therapeutic screening in zebrafish models of retinal vascular disease.

  14. Mind the fish: zebrafish as a model in cognitive social neuroscience

    PubMed Central

    Oliveira, Rui F.

    2013-01-01

    Understanding how the brain implements social behavior on one hand, and how social processes feedback on the brain to promote fine-tuning of behavioral output according to changes in the social environment is a major challenge in contemporary neuroscience. A critical step to take this challenge successfully is finding the appropriate level of analysis when relating social to biological phenomena. Given the enormous complexity of both the neural networks of the brain and social systems, the use of a cognitive level of analysis (in an information processing perspective) is proposed here as an explanatory interface between brain and behavior. A conceptual framework for a cognitive approach to comparative social neuroscience is proposed, consisting of the following steps to be taken across different species with varying social systems: (1) identification of the functional building blocks of social skills; (2) identification of the cognitive mechanisms underlying the previously identified social skills; and (3) mapping these information processing mechanisms onto the brain. Teleost fish are presented here as a group of choice to develop this approach, given the diversity of social systems present in closely related species that allows for planned phylogenetic comparisons, and the availability of neurogenetic tools that allows the visualization and manipulation of selected neural circuits in model species such as the zebrafish. Finally, the state-of-the art of zebrafish social cognition and of the tools available to map social cognitive abilities to neural circuits in zebrafish are reviewed. PMID:23964204

  15. Mind the fish: zebrafish as a model in cognitive social neuroscience.

    PubMed

    Oliveira, Rui F

    2013-01-01

    Understanding how the brain implements social behavior on one hand, and how social processes feedback on the brain to promote fine-tuning of behavioral output according to changes in the social environment is a major challenge in contemporary neuroscience. A critical step to take this challenge successfully is finding the appropriate level of analysis when relating social to biological phenomena. Given the enormous complexity of both the neural networks of the brain and social systems, the use of a cognitive level of analysis (in an information processing perspective) is proposed here as an explanatory interface between brain and behavior. A conceptual framework for a cognitive approach to comparative social neuroscience is proposed, consisting of the following steps to be taken across different species with varying social systems: (1) identification of the functional building blocks of social skills; (2) identification of the cognitive mechanisms underlying the previously identified social skills; and (3) mapping these information processing mechanisms onto the brain. Teleost fish are presented here as a group of choice to develop this approach, given the diversity of social systems present in closely related species that allows for planned phylogenetic comparisons, and the availability of neurogenetic tools that allows the visualization and manipulation of selected neural circuits in model species such as the zebrafish. Finally, the state-of-the art of zebrafish social cognition and of the tools available to map social cognitive abilities to neural circuits in zebrafish are reviewed.

  16. Muscle dysfunction in a zebrafish model of Duchenne muscular dystrophy.

    PubMed

    Widrick, Jeffrey J; Alexander, Matthew S; Sanchez, Benjamin; Gibbs, Devin E; Kawahara, Genri; Beggs, Alan H; Kunkel, Louis M

    2016-11-01

    Sapje zebrafish lack the protein dystrophin and are the smallest vertebrate model of Duchenne muscular dystrophy (DMD). Their small size makes them ideal for large-scale drug discovery screens. However, the extent that sapje mimic the muscle dysfunction of higher vertebrate models of DMD is unclear. We used an optical birefringence assay to differentiate affected dystrophic sapje larvae from their unaffected siblings and then studied trunk muscle contractility at 4-7 days postfertilization. Preparation cross-sectional area (CSA) was similar for affected and unaffected larvae, yet tetanic forces of affected preparations were only 30-60% of normal. ANCOVA indicated that the linear relationship observed between tetanic force and CSA for unaffected preparations was absent in the affected population. Consequently, the average force/CSA of affected larvae was depressed 30-70%. Disproportionate reductions in twitch vs. tetanic force, and a slowing of twitch tension development and relaxation, indicated that the myofibrillar disorganization evident in the birefringence assay could not explain the entire force loss. Single eccentric contractions, in which activated preparations were lengthened 5-10%, resulted in tetanic force deficits in both groups of larvae. However, deficits of affected preparations were three- to fivefold greater at all strains and ages, even after accounting for any recovery. Based on these functional assessments, we conclude that the sapje mutant zebrafish is a phenotypically severe model of DMD. The severe contractile deficits of sapje larvae represent novel physiological endpoints for therapeutic drug screening. Copyright © 2016 the American Physiological Society.

  17. 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

  18. 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.

  19. Dietary sodium propionate affects mucosal immune parameters, growth and appetite related genes expression: Insights from zebrafish model.

    PubMed

    Hoseinifar, Seyed Hossein; Safari, Roghieh; Dadar, Maryam

    2017-03-01

    Propionate is a short-chain fatty acid (SCFA) that improves physiological and pathophysiological properties. However, there is limited information available about the effects of SCFAs on mucosal immune parameters as well as growth and appetite related genes expression. The aim of the present study was to evaluate the effect of sodium propionate (SP) intake on the mucosal immune parameters, growth and appetite related genes expression using zebrafish (Danio rerio) as model organism. Zebrafish fed control or diet supplemented with different levels (0.5, 1 and 2%) of SP for 8weeks. At the end of feeding trial, the expression of the key genes related to growth and appetite (GH, IGF1, MYSTN and Ghrl) was evaluated. Also, mucosal immune parameters (Total Ig, lysozyme and protease activity) were studied in skin mucus of zebrafish. The results showed that dietary administration of SP significantly (P<0.05) up-regulated the expression of GH, IGF1 and down-regulated MYSTN gene. Also, feeding zebrafish with SP supplemented diet significantly increased appetite related gene expression (P<0.05) with a more pronounced effect in higher inclusion levels. Compared with control group, the expression of appetite related gene (Ghrl) was remarkably (P<0.05) higher in SP fed zebrafish. Also, elevated mucosal immune parameters was observed in zebrafish fed SP supplemented diet. The present results revealed beneficial effects of dietary SP on mucosal immune response and growth and appetite related genes expression. These results also highlighted the potential use of SP as additive in human diets. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Unique and potent effects of acute ibogaine on zebrafish: the developing utility of novel aquatic models for hallucinogenic drug research.

    PubMed

    Cachat, Jonathan; Kyzar, Evan J; Collins, Christopher; Gaikwad, Siddharth; Green, Jeremy; Roth, Andrew; El-Ounsi, Mohamed; Davis, Ari; Pham, Mimi; Landsman, Samuel; Stewart, Adam Michael; Kalueff, Allan V

    2013-01-01

    An indole alkaloid, ibogaine is the principal psychoactive component of the iboga plant, used by indigenous peoples in West Africa for centuries. Modulating multiple neurotransmitter systems, the drug is a potent hallucinogen in humans, although its psychotropic effects remain poorly understood. Expanding the range of model species is an important strategy for translational neuroscience research. Here we exposed adult zebrafish (Danio rerio) to 10 and 20mg/L of ibogaine, testing them in the novel tank, light-dark box, open field, mirror stimulation, social preference and shoaling tests. In the novel tank test, the zebrafish natural diving response (geotaxis) was reversed by ibogaine, inducing initial top swimming followed by bottom dwelling. Ibogaine also attenuated the innate preference for dark environments (scototaxis) in the light-dark box test. While it did not exert overt locomotor or thigmotaxic responses in the open field test, the drug altered spatiotemporal exploration of novel environment, inducing clear preference of some areas over others. Ibogaine also promoted 'mirror' exploration in the mirror stimulation test, disrupted group cohesion in the shoaling test, and evoked strong coloration responses due to melanophore aggregation, but did not alter brain c-fos expression or whole-body cortisol levels. Overall, our results support the complex pharmacological profile of ibogaine and its high sensitivity in zebrafish models, dose-dependently affecting multiple behavioral domains. While future investigations in zebrafish may help elucidate the mechanisms underlying these unique behavioral effects, our study strongly supports the developing utility of aquatic models in hallucinogenic drug research. High sensitivity of three-dimensional phenotyping approaches applied here to behavioral effects of ibogaine in zebrafish provides further evidence of how 3D reconstructions of zebrafish swimming paths may be useful for high-throughput pharmacological screening

  1. A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish.

    PubMed

    Radev, Zlatko; Hermel, Jean-Michel; Elipot, Yannick; Bretaud, Sandrine; Arnould, Sylvain; Duchateau, Philippe; Ruggiero, Florence; Joly, Jean-Stéphane; Sohm, Frédéric

    2015-01-01

    Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders.

  2. A TALEN-Exon Skipping Design for a Bethlem Myopathy Model in Zebrafish

    PubMed Central

    Elipot, Yannick; Bretaud, Sandrine; Arnould, Sylvain; Duchateau, Philippe; Ruggiero, Florence; Joly, Jean-Stéphane; Sohm, Frédéric

    2015-01-01

    Presently, human collagen VI-related diseases such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) remain incurable, emphasizing the need to unravel their etiology and improve their treatments. In UCMD, symptom onset occurs early, and both diseases aggravate with ageing. In zebrafish fry, morpholinos reproduced early UCMD and BM symptoms but did not allow to study the late phenotype. Here, we produced the first zebrafish line with the human mutation frequently found in collagen VI-related disorders such as UCMD and BM. We used a transcription activator-like effector nuclease (TALEN) to design the col6a1ama605003-line with a mutation within an essential splice donor site, in intron 14 of the col6a1 gene, which provoke an in-frame skipping of exon 14 in the processed mRNA. This mutation at a splice donor site is the first example of a template-independent modification of splicing induced in zebrafish using a targetable nuclease. This technique is readily expandable to other organisms and can be instrumental in other disease studies. Histological and ultrastructural analyzes of homozygous and heterozygous mutant fry and 3 months post-fertilization (mpf) fish revealed co-dominantly inherited abnormal myofibers with disorganized myofibrils, enlarged sarcoplasmic reticulum, altered mitochondria and misaligned sarcomeres. Locomotion analyzes showed hypoxia-response behavior in 9 mpf col6a1 mutant unseen in 3 mpf fish. These symptoms worsened with ageing as described in patients with collagen VI deficiency. Thus, the col6a1ama605003-line is the first adult zebrafish model of collagen VI-related diseases; it will be instrumental both for basic research and drug discovery assays focusing on this type of disorders. PMID:26221953

  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. The zebrafish as a novel animal model to study the molecular mechanisms of mechano-electrical feedback in the heart

    PubMed Central

    Werdich, Andreas A; Brzezinski, Anna; Jeyaraj, Darwin; Ficker, Eckhard; Wan, Xiaoping; McDermott, Brian M; Sabeh, M Khaled; MacRae, Calum A; Rosenbaum, David S

    2013-01-01

    Altered mechanical loading of the heart leads to hypertrophy, decompensated heart failure and fatal arrhythmias. However, the molecular mechanisms that link mechanical and electrical dysfunction remain poorly understood. Growing evidence suggest that ventricular electrical remodeling (VER) is a process that can be induced by altered mechanical stress, creating persistent electrophysiological changes that predispose the heart to life-threatening arrhythmias. While VER is clearly a physiological property of the human heart, as evidenced by “T wave memory”, it is also thought to occur in a variety of pathological states associated with altered ventricular activation such as bundle branch block, myocardial infarction, and cardiac pacing. Animal models that are currently being used for investigating stretch-induced VER have significant limitations. The zebrafish has recently emerged as an attractive animal model for studying cardiovascular disease and could overcome some of these limitations. Owing to its extensively sequenced genome, high conservation of gene function, and the comprehensive genetic resources that are available in this model, the zebrafish may provide new insights into the molecular mechanisms that drive detrimental electrical remodeling in response to stretch. Here, we have established a zebrafish model to study mechano-electrical feedback in the heart, which combines efficient genetic manipulation with high-precision stretch and high-resolution electrophysiology. In this model, only ninety minutes of ventricular stretch caused VER and recapitulated key features of VER found previously in the mammalian heart. Our data suggest that the zebrafish model is a powerful platform for investigating the molecular mechanisms underlying mechano-electrical feedback and VER in the heart. PMID:22835662

  5. 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.

  6. 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

  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. 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.

  9. Expression pattern of cdkl5 during zebrafish early development: implications for use as model for atypical Rett syndrome.

    PubMed

    Vitorino, Marta; Cunha, Nídia; Conceição, Natércia; Cancela, M Leonor

    2018-05-11

    Atypical Rett syndrome is a child neurodevelopmental disorder induced by mutations in CDKL5 gene and characterized by a progressive regression in development with loss of purposeful use of the hands, slowed brain and head growth, problems with walking, seizures, and intellectual disability. At the moment, there is no cure for this pathology and little information is available concerning animal models capable of mimicking its phenotypes, thus the development of additional animal models should be of interest to gain more knowledge about the disease. Zebrafish has been used successfully as model organism for many human genetic diseases; however, no information is available concerning the spatial and temporal expression of cdkl5 orthologous in this organism. In the present study, we identified the developmental expression patterns of cdkl5 in zebrafish by quantitative PCR and whole-mount in situ hybridization. cdkl5 is expressed maternally at low levels during the first 24 h of development. After that the expression of the gene increases significantly and it starts to be expressed mainly in the nervous system and in several brain structures, such as telencephalon, mesencephalon and diencephalon. The expression patterns of cdkl5 in zebrafish is in accordance with the tissues known to be affected in humans and associated to symptoms and deficits observed in Rett syndrome patients thus providing the first evidence that zebrafish could be an alternative model to study the molecular pathways of this disease as well as to test possible therapeutic approaches capable of rescuing the phenotype.

  10. 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.

  11. Fetal Origins of Life Stage Disease: A Zebrafish Model for the ...

    EPA Pesticide Factsheets

    In the U.S., childhood obesity has more than doubled in children and quadrupled in adolescents in the past 30 years, affects 35% of adults, and costs the U.S. healthcare industry >$200 billion annually. The chemical environment in the womb may cause susceptibility to different life-stage and life-long metabolic diseases including obesity. The challenge is to understand if exposures during developmentally sensitive windows impact life-stage disease, such as obesity, by increasing adipose tissue mass. In vitro models lack the integrated systems approach needed to assess adipose development, while mammalian models are impractical in a screen of thousands of chemicals. Therefore, an obesogen screening method was developed to interrogate bioactivity using a full systems approach, in a vertebrate zebrafish model with complete metabolic activity, at a time when the full signaling repertoire is expressed and active, to optimally examine how chemical dose and duration impact life-stage adipose mass. A time-line for adipose depot formation was mapped in zebrafish 6−14 days post fertilization (dpf) using the lipophilic dye, Nile Red, in combination with fluorescent microscopy. Those time points were then used to investigate the impact of embryonic tributyltin chloride (TBT, a known obesogen) exposure (10nM daily renewal, 0−5dpf) on adipose mass. Fluorescent microscopy revealed adipose depots that were larger and appeared 2 days earlier in TBT treated compared to contro

  12. Chronic Unpredictable Stress (CUS)-Induced Anxiety and Related Mood Disorders in a Zebrafish Model: Altered Brain Proteome Profile Implicates Mitochondrial Dysfunction

    PubMed Central

    Chakravarty, Sumana; Reddy, Bommana R.; Sudhakar, Sreesha R.; Saxena, Sandeep; Das, Tapatee; Meghah, Vuppalapaty; Brahmendra Swamy, Cherukuvada V.; Kumar, Arvind; Idris, Mohammed M.

    2013-01-01

    Anxiety and depression are major chronic mood disorders, and the etiopathology for each appears to be repeated exposure to diverse unpredictable stress factors. Most of the studies on anxiety and related mood disorders are performed in rodents, and a good model is chronic unpredictable stress (CUS). In this study, we have attempted to understand the molecular basis of the neuroglial and behavioral changes underlying CUS-induced mood disorders in the simplest vertebrate model, the zebrafish, Danio rerio. Zebrafish were subjected to a CUS paradigm in which two different stressors were used daily for 15 days, and thorough behavioral analyses were performed to assess anxiety and related mood disorder phenotypes using the novel tank test, shoal cohesion and scototaxis. Fifteen days of exposure to chronic stressors appears to induce an anxiety and related mood disorder phenotype. Decreased neurogenesis, another hallmark of anxiety and related disorders in rodents, was also observed in this zebrafish model. The common molecular markers of rodent anxiety and related disorders, corticotropin-releasing factor (CRF), calcineurin (ppp3r1a) and phospho cyclic AMP response element binding protein (pCREB), were also replicated in the fish model. Finally, using 2DE FTMS/ITMSMS proteomics analyses, 18 proteins were found to be deregulated in zebrafish anxiety and related disorders. The most affected process was mitochondrial function, 4 of the 18 differentially regulated proteins were mitochondrial proteins: PHB2, SLC25A5, VDAC3 and IDH2, as reported in rodent and clinical samples. Thus, the zebrafish CUS model and proteomics can facilitate not only uncovering new molecular targets of anxiety and related mood disorders but also the routine screening of compounds for drug development. PMID:23691016

  13. 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...

  14. Identification of DEAD-Box RNA Helicase DDX41 as a Trafficking Protein That Involves in Multiple Innate Immune Signaling Pathways in a Zebrafish Model.

    PubMed

    Ma, Jun-Xia; Li, Jiang-Yuan; Fan, Dong-Dong; Feng, Wei; Lin, Ai-Fu; Xiang, Li-Xin; Shao, Jian-Zhong

    2018-01-01

    DDX41 is an important sensor for host recognition of DNA viruses and initiation of nuclear factor-κB (NF-κB) and IFN signaling pathways in mammals. However, its occurrence and functions in other vertebrates remain poorly defined. Here, a DDX41 ortholog [ Danio rerio DDX41 ( Dr DDX41)] with various conserved structural features to its mammalian counterparts was identified from a zebrafish model. This Dr DDX41 was found to be a trafficking protein distributed in the nucleus of resting cells but transported into the cytoplasm under DNA stimulation. Two nuclear localization signal motifs were localized beside the coiled-coil domain, whereas one nuclear export signal motif existed in the DEADc domain. Dr DDX41 acts as an initiator for the activation of NF-κB and IFN signaling pathways in a Danio rerio STING ( Dr STING)-dependent manner through its DEADc domain, which is a typical performance of mammalian DDX41. These observations suggested the conservation of DDX41 proteins throughout the vertebrate evolution, making zebrafish an alternative model in understanding DDX41-mediated immunology. With this model system, we found that Dr DDX41 contributes to Dr STING- Danio rerio STAT6 ( Dr STAT6)-mediated chemokine ( Danio rerio CCL20) production through its DEADc domain. To the best of our knowledge, this work is the first report showing that DDX41 is an upstream initiator in this newly identified signaling pathway. The Dr DDX41-mediated signaling pathways play important roles in innate antibacterial immunity because knockdown of either Dr DDX41 or Dr STING/ Dr STAT6 significantly reduced the survival of zebrafish under Aeromonas hydrophilia or Edwardsiella tarda infection. Our findings would enrich the current knowledge of DDX41-mediated immunology and the evolutionary history of the DDX41 family.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. Generation of Demyelination Models by Targeted Ablation of Oligodendrocytes in the Zebrafish CNS

    PubMed Central

    Chung, Ah-Young; Kim, Pan-Soo; Kim, Suhyun; Kim, Eunmi; Kim, Dohyun; Jeong, Inyoung; Kim, Hwan-Ki; Ryu, Jae-Ho; Kim, Cheol-Hee; Choi, June; Seo, Jin-Ho; Park, Hae-Chul

    2013-01-01

    Demyelination is the pathological process by which myelin sheaths are lost from around axons, and is usually caused by a direct insult targeted at the oligodendrocytes in the vertebrate central nervous system (CNS). A demyelinated CNS is usually remyelinated by a population of oligodendrocyte progenitor cells, which are widely distributed throughout the adult CNS. However, myelin disruption and remyelination failure affect the normal function of the nervous system, causing human diseases such as multiple sclerosis. In spite of numerous studies aimed at understanding the remyelination process, many questions still remain unanswered. Therefore, to study remyelination mechanisms in vivo, a demyelination animal model was generated using a transgenic zebrafish system in which oligodendrocytes are conditionally ablated in the larval and adult CNS. In this transgenic system, bacterial nitroreductase enzyme (NTR), which converts the prodrug metronidazole (Mtz) into a cytotoxic DNA cross-linking agent, is expressed in oligodendrocyte lineage cells under the control of the mbp and sox10 promoter. Exposure of transgenic zebrafish to Mtz-containing media resulted in rapid ablation of oligodendrocytes and CNS demyelination within 48 h, but removal of Mtz medium led to efficient remyelination of the demyelinated CNS within 7 days. In addition, the demyelination and remyelination processes could be easily observed in living transgenic zebrafish by detecting the fluorescent protein, mCherry, indicating that this transgenic system can be used as a valuable animal model to study the remyelination process in vivo, and to conduct high-throughput primary screens for new drugs that facilitate remyelination. PMID:23807048

  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. 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

  2. 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.

  3. 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.

  4. 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.

  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. 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...

  7. Interaction of mercury and selenium in the larval stage zebrafish vertebrate model.

    PubMed

    MacDonald, Tracy C; Korbas, Malgorzata; James, Ashley K; Sylvain, Nicole J; Hackett, Mark J; Nehzati, Susan; Krone, Patrick H; George, Graham N; Pickering, Ingrid J

    2015-08-01

    The compounds of mercury can be more toxic than those of any other non-radioactive heavy element. Despite this, environmental mercury pollution and human exposure to mercury are widespread, and are increasing. While the unusual ability of selenium to cancel the toxicity of mercury compounds has been known for nearly five decades, only recently have some aspects of the molecular mechanisms begun to be understood. We report herein a study of the interaction of mercury and selenium in the larval stage zebrafish, a model vertebrate system, using X-ray fluorescence imaging. Exposure of larval zebrafish to inorganic mercury shows nano-scale structures containing co-localized mercury and selenium. No such co-localization is seen with methylmercury exposure under similar conditions. Micro X-ray absorption spectra support the hypothesis that the co-localized deposits are most likely comprised of highly insoluble mixed chalcogenide HgSxSe(1-x) where x is 0.4-0.9, probably with the cubic zincblende structure.

  8. Rac2 Functions in Both Neutrophils and Macrophages To Mediate Motility and Host Defense in Larval Zebrafish.

    PubMed

    Rosowski, Emily E; Deng, Qing; Keller, Nancy P; Huttenlocher, Anna

    2016-12-15

    Leukocyte motility is required for host defense responses. Rac-family Rho GTPases are implicated in leukocyte function; however, the distinct roles of different Rac isoforms in host defense in vivo have remained unclear. In this study, we generated Rac2-deficient zebrafish using transcription activator-like effector nucleases to directly compare the role of Rac2 in vivo in neutrophils and macrophages in motility and the response to infection. This zebrafish larval model is highly amenable to live imaging of leukocyte behavior, and we report that in rac2 -/- larvae both neutrophils and macrophages are defective in basic motility, leading to impaired responses to localized wounds or infections. rac2 -/- larvae are highly susceptible to infection with Pseudomonas aeruginosa, which can be almost fully rescued by ectopic expression of either Rac2 or Rac1 specifically in neutrophils, indicating that these isoforms have partially overlapping functions in vivo. Rescue of Rac2 expression specifically in macrophages also confers resistance to Pseudomonas infection, highlighting an important role for Rac2 in this leukocyte population as well. Surprisingly, in contrast to neutrophils expressing a Rac2 dominant inhibitory human disease mutation, rac2 -/- neutrophils do not have altered polarity or mobilization from hematopoietic tissue, suggesting that a different Rac isoform, such as Rac1, also contributes to these phenotypes in vivo. Copyright © 2016 by The American Association of Immunologists, Inc.

  9. Phenotypic and biomarker evaluation of zebrafish larvae as an alternative model to predict mammalian hepatotoxicity.

    PubMed

    Verstraelen, Sandra; Peers, Bernard; Maho, Walid; Hollanders, Karen; Remy, Sylvie; Berckmans, Pascale; Covaci, Adrian; Witters, Hilda

    2016-09-01

    Zebrafish phenotypic assays have shown promise to assess human hepatotoxicity, though scoring of liver morphology remains subjective and difficult to standardize. Liver toxicity in zebrafish larvae at 5 days was assessed using gene expression as the biomarker approach, complementary to phenotypic analysis and analytical data on compound uptake. This approach aimed to contribute to improved hepatotoxicity prediction, with the goal of identifying biomarker(s) as a step towards the development of transgenic models for prioritization. Morphological effects of hepatotoxic compounds (acetaminophen, amiodarone, coumarin, methapyrilene and myclobutanil) and saccharin as the negative control were assessed after exposure in zebrafish larvae. The hepatotoxic compounds induced the expected zebrafish liver degeneration or changes in size, whereas saccharin did not have any phenotypic adverse effect. Analytical methods based on liquid chromatography-mass spectrometry were optimized to measure stability of selected compounds in exposure medium and internal concentration in larvae. All compounds were stable, except amiodarone for which precipitation was observed. There was a wide variation between the levels of compound in the zebrafish larvae with a higher uptake of amiodarone, methapyrilene and myclobutanil. Detection of hepatocyte markers (CP, CYP3A65, GC and TF) was accomplished by in situ hybridization of larvae to coumarin and myclobutanil and confirmed by real-time reverse transcription-quantitative polymerase chain reaction. Experiments showed decreased expression of all markers. Next, other liver-specific biomarkers (i.e. FABP10a and NR1H4) and apoptosis (i.e. CASP-3 A and TP53) or cytochrome P450-related (CYP2K19) and oxidoreductase activity-related (ZGC163022) genes, were screened. Links between basic mechanisms of liver injury and results of biomarker responses are described. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  10. Zebrafish as a model for apolipoprotein biology: comprehensive expression analysis and a role for ApoA-IV in regulating food intake.

    PubMed

    Otis, Jessica P; Zeituni, Erin M; Thierer, James H; Anderson, Jennifer L; Brown, Alexandria C; Boehm, Erica D; Cerchione, Derek M; Ceasrine, Alexis M; Avraham-Davidi, Inbal; Tempelhof, Hanoch; Yaniv, Karina; Farber, Steven A

    2015-03-01

    Improved understanding of lipoproteins, particles that transport lipids throughout the circulation, is vital to developing new treatments for the dyslipidemias associated with metabolic syndrome. Apolipoproteins are a key component of lipoproteins. Apolipoproteins are proteins that structure lipoproteins and regulate lipid metabolism through control of cellular lipid exchange. Constraints of cell culture and mouse models mean that there is a need for a complementary model that can replicate the complex in vivo milieu that regulates apolipoprotein and lipoprotein biology. Here, we further establish the utility of the genetically tractable and optically clear larval zebrafish as a model of apolipoprotein biology. Gene ancestry analyses were implemented to determine the closest human orthologs of the zebrafish apolipoprotein A-I (apoA-I), apoB, apoE and apoA-IV genes and therefore ensure that they have been correctly named. Their expression patterns throughout development were also analyzed, by whole-mount mRNA in situ hybridization (ISH). The ISH results emphasized the importance of apolipoproteins in transporting yolk and dietary lipids: mRNA expression of all apolipoproteins was observed in the yolk syncytial layer, and intestinal and liver expression was observed from 4-6 days post-fertilization (dpf). Furthermore, real-time PCR confirmed that transcription of three of the four zebrafish apoA-IV genes was increased 4 hours after the onset of a 1-hour high-fat feed. Therefore, we tested the hypothesis that zebrafish ApoA-IV performs a conserved role to that in rat in the regulation of food intake by transiently overexpressing ApoA-IVb.1 in transgenic larvae and quantifying ingestion of co-fed fluorescently labeled fatty acid during a high-fat meal as an indicator of food intake. Indeed, ApoA-IVb.1 overexpression decreased food intake by approximately one-third. This study comprehensively describes the expression and function of eleven zebrafish apolipoproteins and

  11. 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.

  12. 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.

  13. 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

  14. 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

  15. The Zebrafish Brain in Research and Teaching: A Simple in Vivo and in Vitro Model for the Study of Spontaneous Neural Activity

    ERIC Educational Resources Information Center

    Vargas, R.; Johannesdottir, I. P.; Sigurgeirsson, B.; Porsteinsson, H.; Karlsson, K. AE.

    2011-01-01

    Recently, the zebrafish ("Danio rerio") has been established as a key animal model in neuroscience. Behavioral, genetic, and immunohistochemical techniques have been used to describe the connectivity of diverse neural circuits. However, few studies have used zebrafish to understand the function of cerebral structures or to study neural circuits.…

  16. Up-regulation of autophagy-related gene 5 (ATG5) protects dopaminergic neurons in a zebrafish model of Parkinson's disease.

    PubMed

    Hu, Zhan-Ying; Chen, Bo; Zhang, Jing-Pu; Ma, Yuan-Yuan

    2017-11-03

    Parkinson's disease (PD) is one of the most epidemic neurodegenerative diseases and is characterized by movement disorders arising from loss of midbrain dopaminergic (DA) neurons. Recently, the relationship between PD and autophagy has received considerable attention, but information about the mechanisms involved is lacking. Here, we report that autophagy-related gene 5 ( ATG5 ) is potentially important in protecting dopaminergic neurons in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model in zebrafish. Using analyses of zebrafish swimming behavior, in situ hybridization, immunofluorescence, and expressions of genes and proteins related to PD and autophagy, we found that the ATG5 expression level was decreased and autophagy flux was blocked in this model. The ATG5 down-regulation led to the upgrade of PD-associated proteins, such as β-synuclein, Parkin, and PINK1, aggravation of MPTP-induced PD-mimicking pathological locomotor behavior, DA neuron loss labeled by tyrosine hydroxylase (TH) or dopamine transporter (DAT), and blocked autophagy flux in the zebrafish model. ATG5 overexpression alleviated or reversed these PD pathological features, rescued DA neuron cells as indicated by elevated TH/DAT levels, and restored autophagy flux. The role of ATG5 in protecting DA neurons was confirmed by expression of the human atg5 gene in the zebrafish model. Our findings reveal that ATG5 has a role in neuroprotection, and up-regulation of ATG5 may serve as a goal in the development of drugs for PD prevention and management. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. 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

  18. Zebrafish as a Model for Systems Medicine R&D: Rethinking the Metabolic Effects of Carrier Solvents and Culture Buffers Determined by (1)H NMR Metabolomics.

    PubMed

    Akhtar, Muhammad T; Mushtaq, Mian Y; Verpoorte, Robert; Richardson, Michael K; Choi, Young H

    2016-01-01

    Zebrafish is a frequently employed model organism in systems medicine and biomarker discovery. A crosscutting fundamental question, and one that has been overlooked in the field, is the "system-wide" (omics) effects induced in zebrafish by metabolic solvents and culture buffers. Indeed, any bioactivity or toxicity test requires that the target compounds are dissolved in an appropriate nonpolar solvent or aqueous media. It is important to know whether the solvent or the buffer itself has an effect on the zebrafish model organism. We evaluated the effects of two organic carrier solvents used in research with zebrafish, as well as in drug screening: dimethyl sulfoxide (DMSO) and ethanol, and two commonly used aqueous buffers (egg water and Hank's balanced salt solution). The effects of three concentrations (0.01, 0.1, and 1%) of DMSO and ethanol were tested in the 5-day-old zebrafish embryo using proton nuclear magnetic resonance ((1)H NMR) based metabolomics. DMSO (1% and 0.1%, but not 0.01%) exposure significantly decreased the levels of adenosine triphosphate (ATP), betaine, alanine, histidine, lactate, acetate, and creatine (p < 0.05). By contrast, ethanol exposure did not alter the embryos' metabolome at any concentration tested. The two different aqueous media noted above impacted the zebrafish embryo metabolome as evidenced by changes in valine, alanine, lactate, acetate, betaine, glycine, glutamate, adenosine triphosphate, and histidine. These results show that DMSO has greater effects on the embryo metabolome than ethanol, and thus is used with caution as a carrier solvent in zebrafish biomarker research and oral medicine. Moreover, the DMSO concentration should not be higher than 0.01%. Careful attention is also warranted for the use of the buffers egg water and Hank's balanced salt solution in zebrafish. In conclusion, as zebrafish is widely used as a model organism in life sciences, metabolome changes induced by solvents and culture buffers warrant further

  19. 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

  20. Effects of the wood extractive betulinol and 17beta-oestradiol on reproduction in zebrafish, Danio rerio (Hamilton)--complications due to a bacterial infection.

    PubMed

    Christianson-Heiska, I; Wahteristo, P; Kastilan, E-L; Bergelin, E; Bylund, G; Isomaa, B

    2004-05-01

    Zebrafish were exposed to the wood extractive betulinol (5 microg L(-1)) and to 17beta-oestradiol (E2, 0.27 microg L(-1)) for 8 weeks in an attempt to study the possible endocrine-disrupting activity of betulinol. Females exposed to betulinol showed increased spawning intensity, while males exposed to betulinol and E2 had increased incidences of structural alterations in the testes. However, histological examination of the fish revealed that they were infected by acid-fast bacteria suspected to be Mycobacterium sp. despite a careful examination of their health state prior to the onset of the experiment. Fish exposed to betulinol and E2 showed more serious consequences of the bacterial infection than control fish indicating that the test chemicals had weakened the immune defence of the fish. When the exposure was repeated with healthy fish, an increase in the proportion of spermatogonia was seen in the testes of betulinol-treated males. A similar alteration, although not statistically significant, was also seen in the first experiment. However, no increase in the incidences of structural alterations in the testes was seen in betulinol- and E2-treated fish in the second experiment. Our study indicates that betulinol might have an endocrine-disrupting effect in zebrafish, but the increase in incidences of structural alterations in the testes might have been caused by a synergistic action between the test compounds and the bacterial infection. Our study stresses the importance of carefully checking the health of experimental fish, not only prior to the onset of an experiment but also upon termination of the experiment, in order to avoid misinterpretation of the results.

  1. Endotoxin molecule lipopolysaccharide-induced zebrafish inflammation model: a novel screening method for anti-inflammatory drugs.

    PubMed

    Yang, Li-Ling; Wang, Guo-Quan; Yang, Li-Mei; Huang, Zhi-Bing; Zhang, Wen-Qing; Yu, Lin-Zhong

    2014-02-21

    Lipopolysaccharide (LPS), an endotoxin molecule, has been used to induce inflammatory responses. In this study, LPS was used to establish an in vivo inflammation model in zebrafish for drug screening. We present an experimental method that conveniently and rapidly assesses the anti-inflammatory properties of drugs. The yolks of 3-day post-fertilization (dpf) larvae were injected with 0.5 mg/mL LPS to induce fatal inflammation. After LPS stimulation, macrophages were tracked by NR and SB staining and neutrophil migration was observed using the MPO:GFP line. Larval mortality was used as the primary end-point. Expression levels of key cytokines involved in the inflammatory response including IL-1β, IL-6, and TNF-α, were measured using quantitative reverse transcription polymerase chain reaction (RT-PCR). Macrophages and neutrophils were both recruited to the LPS-injected site during the inflammatory response. Mortality was increased by LPS in a dose-dependent manner within 48 h. Analyses of IL-1β, IL-6, and TNF-α expression levels revealed the upregulation of the inflammatory response in the LPS-injected larvae. Further, the anti-inflammatory activity of chlorogenic acid (CA) was evaluated in this zebrafish model to screen for anti-inflammatory drugs. A preliminary result showed that CA revealed a similar effect as the corticosteroid dexamethasone (DEX), which was used as a positive control, by inhibiting macrophage and neutrophil recruitment to the LPS site and improving survival. Our results suggest that this zebrafish screening model could be applied to study inflammation-mediated diseases. Moreover, the Traditional Chinese Medicine CA displays potential anti-inflammatory activity.

  2. 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.

  3. 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.

  4. Zebrafish, a Novel Model System to Study Uremic Toxins: The Case for the Sulfur Amino Acid Lanthionine.

    PubMed

    Perna, Alessandra F; Anishchenko, Evgeniya; Vigorito, Carmela; Zacchia, Miriam; Trepiccione, Francesco; D'Aniello, Salvatore; Ingrosso, Diego

    2018-04-29

    The non-proteinogenic amino acid lanthionine is a byproduct of hydrogen sulfide biosynthesis: the third endogenous vasodilator gas, after nitric oxide and carbon monoxide. While hydrogen sulfide is decreased in uremic patients on hemodialysis, lanthionine is increased and has been proposed as a new uremic toxin, since it is able to impair hydrogen sulfide production in hepatoma cells. To characterize lanthionine as a uremic toxin, we explored its effects during the early development of the zebrafish ( Danio rerio ), a widely used model to study the organ and tissue alterations induced by xenobiotics. Lanthionine was employed at concentrations reproducing those previously detected in uremia. Light-induced visual motor response was also studied by means of the DanioVision system. Treatment of zebrafish embryos with lanthionine determined acute phenotypical alterations, on heart organogenesis (disproportion in cardiac chambers), increased heart beating, and arrhythmia. Lanthionine also induced locomotor alterations in zebrafish embryos. Some of these effects could be counteracted by glutathione. Lanthionine exerted acute effects on transsulfuration enzymes and the expression of genes involved in inflammation and metabolic regulation, and modified microRNA expression in a way comparable with some alterations detected in uremia. Lanthionine meets the criteria for classification as a uremic toxin. Zebrafish can be successfully used to explore uremic toxin effects.

  5. 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.

  6. 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

  7. Identification of compounds with anti-convulsant properties in a zebrafish model of epileptic seizures

    PubMed Central

    Baxendale, Sarah; Holdsworth, Celia J.; Meza Santoscoy, Paola L.; Harrison, Michael R. M.; Fox, James; Parkin, Caroline A.; Ingham, Philip W.; Cunliffe, Vincent T.

    2012-01-01

    SUMMARY The availability of animal models of epileptic seizures provides opportunities to identify novel anticonvulsants for the treatment of people with epilepsy. We found that exposure of 2-day-old zebrafish embryos to the convulsant agent pentylenetetrazole (PTZ) rapidly induces the expression of synaptic-activity-regulated genes in the CNS, and elicited vigorous episodes of calcium (Ca2+) flux in muscle cells as well as intense locomotor activity. We then screened a library of ∼2000 known bioactive small molecules and identified 46 compounds that suppressed PTZ-inducedtranscription of the synaptic-activity-regulated gene fos in 2-day-old (2 dpf) zebrafish embryos. Further analysis of a subset of these compounds, which included compounds with known and newly identified anticonvulsant properties, revealed that they exhibited concentration-dependent inhibition of both locomotor activity and PTZ-induced fos transcription, confirming their anticonvulsant characteristics. We conclude that this in situ hybridisation assay for fos transcription in the zebrafish embryonic CNS is a robust, high-throughput in vivo indicator of the neural response to convulsant treatment and lends itself well to chemical screening applications. Moreover, our results demonstrate that suppression of PTZ-induced fos expression provides a sensitive means of identifying compounds with anticonvulsant activities. PMID:22730455

  8. 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

  9. 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.

  10. 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

  11. 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.

  12. Mortality Caused by Bath Exposure of Zebrafish (Danio rerio) Larvae to Nervous Necrosis Virus Is Limited to the Fourth Day Postfertilization

    PubMed Central

    Morick, Danny; Faigenbaum, Or; Smirnov, Margarita; Fellig, Yakov; Inbal, Adi

    2015-01-01

    Nervous necrosis virus (NNV) is a member of the Betanodavirus genus that causes fatal diseases in over 40 species of fish worldwide. Mortality among NNV-infected fish larvae is almost 100%. In order to elucidate the mechanisms responsible for the susceptibility of fish larvae to NNV, we exposed zebrafish larvae to NNV by bath immersion at 2, 4, 6, and 8 days postfertilization (dpf). Here, we demonstrate that developing zebrafish embryos are resistant to NNV at 2 dpf due to the protection afforded by the egg chorion and, to a lesser extent, by the perivitelline fluid. The zebrafish larvae succumbed to NNV infection during a narrow time window around the 4th dpf, while 6- and 8-day-old larvae were much less sensitive, with mortalities of 24% and 28%, respectively. PMID:25746990

  13. 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.

  14. 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

  15. 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.

  16. 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

  17. Innovative approaches to establish and characterize primary cultures: an ex vivo 3D system and the zebrafish model.

    PubMed

    Liverani, Chiara; La Manna, Federico; Groenewoud, Arwin; Mercatali, Laura; Van Der Pluijm, Gabri; Pieri, Federica; Cavaliere, Davide; De Vita, Alessandro; Spadazzi, Chiara; Miserocchi, Giacomo; Bongiovanni, Alberto; Recine, Federica; Riva, Nada; Amadori, Dino; Tasciotti, Ennio; Snaar-Jagalska, Ewa; Ibrahim, Toni

    2017-02-15

    Patient-derived specimens are an invaluable resource to investigate tumor biology. However, in vivo studies on primary cultures are often limited by the small amount of material available, while conventional in vitro systems might alter the features and behavior that characterize cancer cells. We present our data obtained on primary dedifferentiated liposarcoma cells cultured in a 3D scaffold-based system and injected into a zebrafish model. Primary cells were characterized in vitro for their morphological features, sensitivity to drugs and biomarker expression, and in vivo for their engraftment and invasiveness abilities. The 3D culture showed a higher enrichment in cancer cells than the standard monolayer culture and a better preservation of liposarcoma-associated markers. We also successfully grafted primary cells into zebrafish, showing their local migratory and invasive abilities. Our work provides proof of concept of the ability of 3D cultures to maintain the original phenotype of ex vivo cells, and highlights the potential of the zebrafish model to provide a versatile in vivo system for studies with limited biological material. Such models could be used in translational research studies for biomolecular analyses, drug screenings and tumor aggressiveness assays. © 2016. Published by The Company of Biologists Ltd.

  18. Animals devoid of pulmonary system as infection models in the study of lung bacterial pathogens

    PubMed Central

    López Hernández, Yamilé; Yero, Daniel; Pinos-Rodríguez, Juan M.; Gibert, Isidre

    2015-01-01

    Biological disease models can be difficult and costly to develop and use on a routine basis. Particularly, in vivo lung infection models performed to study lung pathologies use to be laborious, demand a great time and commonly are associated with ethical issues. When infections in experimental animals are used, they need to be refined, defined, and validated for their intended purpose. Therefore, alternative and easy to handle models of experimental infections are still needed to test the virulence of bacterial lung pathogens. Because non-mammalian models have less ethical and cost constraints as a subjects for experimentation, in some cases would be appropriated to include these models as valuable tools to explore host–pathogen interactions. Numerous scientific data have been argued to the more extensive use of several kinds of alternative models, such as, the vertebrate zebrafish (Danio rerio), and non-vertebrate insects and nematodes (e.g., Caenorhabditis elegans) in the study of diverse infectious agents that affect humans. Here, we review the use of these vertebrate and non-vertebrate models in the study of bacterial agents, which are considered the principal causes of lung injury. Curiously none of these animals have a respiratory system as in air-breathing vertebrates, where respiration takes place in lungs. Despite this fact, with the present review we sought to provide elements in favor of the use of these alternative animal models of infection to reveal the molecular signatures of host–pathogen interactions. PMID:25699030

  19. 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

  20. 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.

  1. 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

  2. Swimming behavior of zebrafish is accurately classified by direct modeling and behavioral space analysis

    NASA Astrophysics Data System (ADS)

    Feng, Ruopei; Chemla, Yann; Gruebele, Martin

    Larval zebrafish is a popular organism in the search for the correlation between locomotion behavior and neural pathways because of their highly stereotyped and temporally episodic swimming motion. This correlation is usually investigated using electrophysiological recordings of neural activities in partially immobilized fish. Seeking for a way to study animal behavior without constraints or intruding electrodes, which can in turn modify their behavior, our lab has introduced a parameter-free approach which allows automated classification of the locomotion behaviors of freely swimming fish. We looked into several types of swimming bouts including free swimming and two modes of escape responses and established a new classification of these behaviors. Combined with a neurokinematic model, our analysis showed the capability to probe intrinsic properties of the underlying neural pathways of freely swimming larval zebrafish by inspecting swimming movies only.

  3. 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.

  4. 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

  5. A zebrafish model for uremic toxicity: role of the complement pathway.

    PubMed

    Berman, Nathaniel; Lectura, Melisa; Thurman, Josh; Reinecke, James; Raff, Amanda C; Melamed, Michal L; Reinecke, James; Quan, Zhe; Evans, Todd; Meyer, Timothy W; Hostetter, Thomas H

    2013-01-01

    Many organic solutes accumulate in end-stage renal disease (ESRD) and some are poorly removed with urea-based prescriptions for hemodialysis. However, their toxicities have been difficult to assess. We have employed an animal model, the zebrafish embryo, to test the toxicity of uremic serum compared to control. Serum was obtained from stable ESRD patients predialysis or from normal subjects. Zebrafish embryos 24 h postfertilization were exposed to experimental media at a water:human serum ratio of 3:1. Those exposed to serum from uremic subjects had significantly reduced survival at 8 h (19 ± 18 vs. 94 ± 6%, p < 0.05, uremic serum vs. control, respectively). Embryos exposed to serum from ESRD subjects fractionated at 50 kDa showed significantly greater toxicity with the larger molecular weight fraction (83 ± 11 vs. 7 ± 17% survival, p < 0.05, <50 vs. >50 kDa, respectively). Heating serum abrogated its toxicity. EDTA, a potent inhibitor of complement by virtue of calcium chelation, reduced the toxicity of uremic serum compared to untreated uremic serum (96 ± 5 vs. 28 ± 20% survival, p < 0.016, chelated vs. nonchelated serum, respectively). Anti-factor B, a specific inhibitor of the alternative complement pathway, reduced the toxicity of uremic serum, compared to untreated uremic serum (98 ± 6 vs. 3 ± 9% survival, p < 0.016, anti-factor B treated vs. nontreated, respectively). Uremic serum is thus more toxic to zebrafish embryos than normal serum. Furthermore, this toxicity is associated with a fraction of large size, is inactivated by heat, and is reduced by both specific and nonspecific inhibitors of complement activation. Together these data lend support to the hypothesis that at least some uremic toxicities may be mediated by complement. Copyright © 2013 S. Karger AG, Basel.

  6. A Zebrafish Model for Uremic Toxicity: Role of the Complement Pathway

    PubMed Central

    Thurman, Josh; Reinecke, James; Raff, Amanda C.; Melamed, Michal L.; Reinecke, James; Quan, Zhe; Evans, Todd; Meyer, Timothy W.; Hostetter, Thomas H

    2016-01-01

    Many organic solutes accumulate in ESRD and some are poorly removed removed with urea based prescriptions for hemodialysis. However, their toxicities have been difficult to assess. We have employed an animal model, the zebrafish embryo, to test the toxicity of uremic serum compared to control. Serum was obtained from stable ESRD patients pre-dialysis or from normal subjects. Zebrafish embryos 24 hours post fertilization were exposed to experimental media at a ratio of 3:1 water:human serum. Those exposed to serum from uremic subjects had significantly reduced survival at 8 hours (19% +/− 18% vs. 94% +/− 6%; p < 0.05, uremic serum vs control, respectively). Embryos exposed to serum from ESRD subjects fractionated at 50kD showed significantly greater toxicity with the larger molecular weight fraction (83% +/− 11% vs 7% +/−17% survival, p < 0.05, <50kD vs >50 kD, respectively). Heating serum abrogated its toxicity. EDTA, a potent inhibitor of complement by virtue of calcium chelation, reduced the toxicity of uremic serum compared to untreated uremic serum (96%+/− 5% vs 28%+/− 20% survival, p < 0.016, chelated vs non chelated serum respectively). Anti- factor B, a specific inhibitor of the alternative complement pathway, reduced the toxicity of uremic serum, compared to untreated uremic serum (98% +/− 6% vs. 3% +/− 9% survival, p < 0.016, anti- factor B treated vs non treated, respectively).Uremic serum is thus more toxic to zebrafish embryos than normal serum. Furthermore, this toxicity is associated with a fraction of large size, is inactivated by heat, and is reduced by both specific and non-specific inhibitors of complement activation. Together these data lend support to the hypothesis that at least some uremic toxicities may be mediated by complement. PMID:23689420

  7. 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

  8. 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.

  9. 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.

  10. 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

  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. Mortality Caused by Bath Exposure of Zebrafish (Danio rerio) Larvae to Nervous Necrosis Virus Is Limited to the Fourth Day Postfertilization.

    PubMed

    Morick, Danny; Faigenbaum, Or; Smirnov, Margarita; Fellig, Yakov; Inbal, Adi; Kotler, Moshe

    2015-05-15

    Nervous necrosis virus (NNV) is a member of the Betanodavirus genus that causes fatal diseases in over 40 species of fish worldwide. Mortality among NNV-infected fish larvae is almost 100%. In order to elucidate the mechanisms responsible for the susceptibility of fish larvae to NNV, we exposed zebrafish larvae to NNV by bath immersion at 2, 4, 6, and 8 days postfertilization (dpf). Here, we demonstrate that developing zebrafish embryos are resistant to NNV at 2 dpf due to the protection afforded by the egg chorion and, to a lesser extent, by the perivitelline fluid. The zebrafish larvae succumbed to NNV infection during a narrow time window around the 4th dpf, while 6- and 8-day-old larvae were much less sensitive, with mortalities of 24% and 28%, respectively. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  13. 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 ...

  14. 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

  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. 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.

  17. Mutation of zebrafish dihydrolipoamide branched-chain transacylase E2 results in motor dysfunction and models maple syrup urine disease

    PubMed Central

    Friedrich, Timo; Lambert, Aaron M.; Masino, Mark A.; Downes, Gerald B.

    2012-01-01

    SUMMARY Analysis of zebrafish mutants that demonstrate abnormal locomotive behavior can elucidate the molecular requirements for neural network function and provide new models of human disease. Here, we show that zebrafish quetschkommode (que) mutant larvae exhibit a progressive locomotor defect that culminates in unusual nose-to-tail compressions and an inability to swim. Correspondingly, extracellular peripheral nerve recordings show that que mutants demonstrate abnormal locomotor output to the axial muscles used for swimming. Using positional cloning and candidate gene analysis, we reveal that a point mutation disrupts the gene encoding dihydrolipoamide branched-chain transacylase E2 (Dbt), a component of a mitochondrial enzyme complex, to generate the que phenotype. In humans, mutation of the DBT gene causes maple syrup urine disease (MSUD), a disorder of branched-chain amino acid metabolism that can result in mental retardation, severe dystonia, profound neurological damage and death. que mutants harbor abnormal amino acid levels, similar to MSUD patients and consistent with an error in branched-chain amino acid metabolism. que mutants also contain markedly reduced levels of the neurotransmitter glutamate within the brain and spinal cord, which probably contributes to their abnormal spinal cord locomotor output and aberrant motility behavior, a trait that probably represents severe dystonia in larval zebrafish. Taken together, these data illustrate how defects in branched-chain amino acid metabolism can disrupt nervous system development and/or function, and establish zebrafish que mutants as a model to better understand MSUD. PMID:22046030

  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. Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology

    PubMed Central

    Zabinyakov, Nikita; Bullivant, Garrett; Cao, Feng; Fernandez Ojeda, Matilde; Jia, Zheng Ping; Wen, Xiao-Yan; Dowling, James J.; Salomons, Gajja S.

    2017-01-01

    Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease. PMID:29053735

  20. Characterization of the first knock-out aldh7a1 zebrafish model for pyridoxine-dependent epilepsy using CRISPR-Cas9 technology.

    PubMed

    Zabinyakov, Nikita; Bullivant, Garrett; Cao, Feng; Fernandez Ojeda, Matilde; Jia, Zheng Ping; Wen, Xiao-Yan; Dowling, James J; Salomons, Gajja S; Mercimek-Andrews, Saadet

    2017-01-01

    Pyridoxine dependent epilepsy (PDE) is caused by likely pathogenic variants in ALDH7A1 (PDE-ALDH7A1) and inherited autosomal recessively. Neurotoxic alpha-amino adipic semialdehyde (alpha-AASA), piperideine 6-carboxylate and pipecolic acid accumulate in body fluids. Neonatal or infantile onset seizures refractory to anti-epileptic medications are clinical features. Treatment with pyridoxine, arginine and lysine-restricted diet does not normalize neurodevelopmental outcome or accumulation of neurotoxic metabolites. There is no animal model for high throughput drug screening. For this reason, we developed and characterized the first knock-out aldh7a1 zebrafish model using CRISPR-Cas9 technology. Zebrafish aldh7a1 mutants were generated by using a vector free method of CRISPR-Cas9 mutagenesis. Genotype analysis of aldh7a1 knock-out zebrafish was performed by high resolution melt analysis, direct sequencing and QIAxcel system. Electroencephalogram was performed. Alpha-AASA, piperideine 6-carboxylate and pipecolic acid, were measured by liquid chromatography-tandem mass spectrometry. Our knock-out aldh7a1 zebrafish has homozygous 5 base pair (bp) mutation in ALDH7A1. Knock-out aldh7a1 embryos have spontaneous rapid increase in locomotion and a rapid circling swim behavior earliest 8-day post fertilization (dpf). Electroencephalogram revealed large amplitude spike discharges compared to wild type. Knock-out aldh7a1 embryos have elevated alpha-AASA, piperideine 6-carboxylate and pipecolic acid compared to wild type embryos at 3 dpf. Knock-out aldh7a1 embryos showed no aldh7a1 protein by western blot compared to wild type. Our knock-out aldh7a1 zebrafish is a well characterized model for large-scale drug screening using behavioral and biochemical features and accurately recapitulates the human PDE-ALDH7A1 disease.

  1. The zebrafish mutant vps18 as a model for vesicle-traffic related hypopigmentation diseases.

    PubMed

    Maldonado, Ernesto; Hernandez, Fabiola; Lozano, Carlos; Castro, Marta E; Navarro, Rosa E

    2006-08-01

    Hypopigmentation is a characteristic of several diseases associated with vesicle traffic defects, like the Hermansky-Pudlak, Chediak-Higashi, and Griscelli syndromes. Hypopigmentation is also a characteristic of the zebrafish mutant vps18(hi2499A), which is affected in the gene vps18, a component of the homotypic fusion and protein sorting complex that is involved in tethering during vesicular traffic. Vps18, as part of this complex, participates in the formation of early endosomes, late endosomes, and lysosomes. Here, we show that Vps18 is also involved in the formation of melanosomes. In the zebrafish mutant vps18(hi2499A) the retroviral insertion located at exon 4 of vps18, leads to the formation of two abnormal splicing variants lacking the coding sequence for the clathrin repeat and the RING finger conserved domains. A deficiency of Vps18 in zebrafish larvae results in hepatomegaly and skin hypopigmentation. We also observed a drastic reduction in the number of melanosomes in the eye's retinal pigmented epithelium along with the accumulation of immature melanosomes. A significant reduction in the vps18(hi2499A) larvae visual system capacity was found using the optokinetic response assay. We propose that the insertional mutant vps18(hi2499A) can be used as a model for studying hypopigmentation diseases in which vesicle traffic problems exist.

  2. 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.

  3. 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

  4. Toxicogenomics to Evaluate Endocrine Disrupting Effects of Environmental Chemicals Using the Zebrafish Model

    PubMed Central

    Caballero-Gallardo, Karina; Olivero-Verbel, Jesus; Freeman, Jennifer L.

    2016-01-01

    The extent of our knowledge on the number of chemical compounds related to anthropogenic activities that can cause damage to the environment and to organisms is increasing. Endocrine disrupting chemicals (EDCs) are one group of potentially hazardous substances that include natural and synthetic chemicals and have the ability to mimic endogenous hormones, interfering with their biosynthesis, metabolism, and normal functions. Adverse effects associated with EDC exposure have been documented in aquatic biota and there is widespread interest in the characterization and understanding of their modes of action. Fish are considered one of the primary risk organisms for EDCs. Zebrafish (Danio rerio) are increasingly used as an animal model to study the effects of endocrine disruptors, due to their advantages compared to other model organisms. One approach to assess the toxicity of a compound is to identify those patterns of gene expression found in a tissue or organ exposed to particular classes of chemicals, through new technologies in genomics (toxicogenomics), such as microarrays or whole-genome sequencing. Application of these technologies permit the quantitative analysis of thousands of gene expression changes simultaneously in a single experiment and offer the opportunity to use transcript profiling as a tool to predict toxic outcomes of exposure to particular compounds. The application of toxicogenomic tools for identification of chemicals with endocrine disrupting capacity using the zebrafish model system is reviewed. PMID:28217008

  5. 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.

  6. 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

  7. 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

  8. Effect of Gabapentin/Memantine on the Infantile Nystagmus Syndrome in the Zebrafish Model: Implications for the Therapy of Ocular Motor Diseases.

    PubMed

    Bögli, Stefan Yu; Afthinos, Maresa; Huang, Melody Ying-Yu

    2017-06-01

    Infantile nystagmus syndrome (INS) is a disorder characterized by typical horizontal eye oscillations. Due to the uncertain etiology of INS, developing specific treatments remains difficult. Single reports demonstrated, on limited measures, alleviating effects of gabapentin and memantine. In the current study, we employed the zebrafish INS model belladonna (bel) to conduct an in-depth study of how gabapentin and memantine interventions alleviate INS signs, which may further restore visual conditions in affected subjects. Moreover, we described the influence of both medications on ocular motor functions in healthy zebrafish, evaluating possible iatrogenic effects. Ocular motor function and INS characteristics were assessed by eliciting optokinetic response, spontaneous nystagmus, and spontaneous saccades in light and in dark, in 5- to 6-day postfertilization bel larvae and heterozygous siblings. Single larvae were recorded before and after a 1-hour drug treatment (200 mM gabapentin/0.2 mM memantine). Both interventions significantly reduced nystagmus intensity (gabapentin: 59.98%, memantine: 39.59%). However, while the application of gabapentin affected all tested ocular motor functions, memantine specifically reduced nystagmus amplitude and intensity, and thus left controls completely unaffected. Finally, both drug treatments resulted in specific changes in nystagmus waveform and velocity. Our study provides deeper insight into gabapentin and memantine treatment effect in the zebrafish INS model. Moreover, this study should establish zebrafish as a pharmacologic animal model for treating nystagmus and ocular motor disease, serving as a basis for future large-scale drug screenings.

  9. 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.

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. A zebrafish model of manganism reveals reversible and treatable symptoms that are independent of neurotoxicity

    PubMed Central

    Bakthavatsalam, Subha; Das Sharma, Shreya; Sonawane, Mahendra; Thirumalai, Vatsala; Datta, Ankona

    2014-01-01

    Manganese (manganese ion; referred to as Mn) is essential for neuronal function, yet it is toxic at high concentrations. Environmental and occupational exposure to high concentrations of Mn causes manganism, a well-defined movement disorder in humans, with symptoms resembling Parkinson’s disease (PD). However, manganism is distinct from PD and the neural basis of its pathology is poorly understood. To address this issue, we generated a zebrafish model of manganism by incubating larvae in rearing medium containing Mn. We find that Mn-treated zebrafish larvae exhibit specific postural and locomotor defects. Larvae begin to float on their sides, show a curved spine and swim in circles. We discovered that treatment with Mn causes postural defects by interfering with mechanotransduction at the neuromasts. Furthermore, we find that the circling locomotion could be caused by long-duration bursting in the motor neurons, which can lead to long-duration tail bends in the Mn-treated larvae. Mn-treated larvae also exhibited fewer startle movements. Additionally, we show that the intensity of tyrosine hydroxylase immunoreactivity is reversibly reduced after Mn-treatment. This led us to propose that reduced dopamine neuromodulation drives the changes in startle movements. To test this, when we supplied an external source of dopamine to Mn-treated larvae, the larvae exhibited a normal number of startle swims. Taken together, these results indicate that Mn interferes with neuronal function at the sensory, motor and modulatory levels, and open avenues for therapeutically targeted studies on the zebrafish model of manganism. PMID:25261567

  18. 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.

  19. Use of the Zebrafish Larvae as a Model to Study Cigarette Smoke Condensate Toxicity

    PubMed Central

    Ellis, Lee D.; Soo, Evelyn C.; Achenbach, John C.; Morash, Michael G.; Soanes, Kelly H.

    2014-01-01

    The smoking of tobacco continues to be the leading cause of premature death worldwide and is linked to the development of a number of serious illnesses including heart disease, respiratory diseases, stroke and cancer. Currently, cell line based toxicity assays are typically used to gain information on the general toxicity of cigarettes and other tobacco products. However, they provide little information regarding the complex disease-related changes that have been linked to smoking. The ethical concerns and high cost associated with mammalian studies have limited their widespread use for in vivo toxicological studies of tobacco. The zebrafish has emerged as a low-cost, high-throughput, in vivo model in the study of toxicology. In this study, smoke condensates from 2 reference cigarettes and 6 Canadian brands of cigarettes with different design features were assessed for acute, developmental, cardiac, and behavioural toxicity (neurotoxicity) in zebrafish larvae. By making use of this multifaceted approach we have developed an in vivo model with which to compare the toxicity profiles of smoke condensates from cigarettes with different design features. This model system may provide insights into the development of smoking related disease and could provide a cost-effective, high-throughput platform for the future evaluation of tobacco products. PMID:25526262

  20. Use of the zebrafish larvae as a model to study cigarette smoke condensate toxicity.

    PubMed

    Ellis, Lee D; Soo, Evelyn C; Achenbach, John C; Morash, Michael G; Soanes, Kelly H

    2014-01-01

    The smoking of tobacco continues to be the leading cause of premature death worldwide and is linked to the development of a number of serious illnesses including heart disease, respiratory diseases, stroke and cancer. Currently, cell line based toxicity assays are typically used to gain information on the general toxicity of cigarettes and other tobacco products. However, they provide little information regarding the complex disease-related changes that have been linked to smoking. The ethical concerns and high cost associated with mammalian studies have limited their widespread use for in vivo toxicological studies of tobacco. The zebrafish has emerged as a low-cost, high-throughput, in vivo model in the study of toxicology. In this study, smoke condensates from 2 reference cigarettes and 6 Canadian brands of cigarettes with different design features were assessed for acute, developmental, cardiac, and behavioural toxicity (neurotoxicity) in zebrafish larvae. By making use of this multifaceted approach we have developed an in vivo model with which to compare the toxicity profiles of smoke condensates from cigarettes with different design features. This model system may provide insights into the development of smoking related disease and could provide a cost-effective, high-throughput platform for the future evaluation of tobacco products.

  1. Developmental Effects and Estrogenicity of Bisphenol A Alternatives in a Zebrafish Embryo Model.

    PubMed

    Mu, Xiyan; Huang, Ying; Li, Xuxing; Lei, Yunlei; Teng, Miaomiao; Li, Xuefeng; Wang, Chengju; Li, Yingren

    2018-03-06

    In order to understand the negative effects of bisphenol A (BPA) alternatives comprehensively, zebrafish embryos were used to assess the lethality, developmental effects, and estrogenic activity of bisphenol analogues. The in silico estrogenic activities of bisphenol analogues were assayed by binding simulation. According to our results, the lethality of bisphenol analogues decreased in order of bisphenol AF (BPAF) > BPA > bisphenol F (BPF) > bisphenol S (BPS). BPAF and BPF induced significant effects on zebrafish embryos, including decreased heart rate, hatching inhibition, and teratogenic effects. The binding potentials of bisphenol analogues toward zebrafish ERs (zfERS) decreased in the following order: BPAF > BPA > BPF > BPS. Among the three subtypes of zfERs, zfERβ2 showed the highest binding activity toward the bisphenols, followed by zfERα and zfERβ1. In vivo estrogenic activity tests showed that BPAF, BPA, and BPF significantly enhanced the protein levels of ERα along with the mRNA levels of esr1, esr2a, esr2b, and vtg1 in zebrafish embryos. Esr2b showed the strongest response to BPAF and BPA exposure among the three esrs. In contrast, BPS did not significantly regulate ER protein level or ER transcription. In conclusion, BPAF showed the highest lethality, developmental effects, and estrogenic activity (both in silico and in vivo) followed by BPA and BPF. BPS showed the weakest toxicity and estrogenic activity. zfERβ2 might act as the main target among the three ER subtypes of zebrafish after exposure to BPAF and BPA.

  2. Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics.

    PubMed

    Brunt, Lucy H; Roddy, Karen A; Rayfield, Emily J; Hammond, Chrissy L

    2016-12-03

    Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

  3. 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.

  4. Embryonic fate map of first pharyngeal arch structures in the sox10: kaede zebrafish transgenic model.

    PubMed

    Dougherty, Max; Kamel, George; Shubinets, Valeriy; Hickey, Graham; Grimaldi, Michael; Liao, Eric C

    2012-09-01

    Cranial neural crest cells follow stereotypic patterns of migration to form craniofacial structures. The zebrafish is a powerful vertebrate genetic model where transgenics with reporter proteins under the transcriptional regulation of lineage-specific promoters can be generated. Numerous studies demonstrate that the zebrafish ethmoid plate is embryologically analogous to the mammalian palate. A fate map correlating embryonic cranial neural crest to defined jaw structures would provide a useful context for the morphogenetic analysis of craniofacial development. To that end, the sox10:kaede transgenic was generated, where sox10 provides lineage restriction to the neural crest. Specific regions of neural crest were labeled at the 10-somite stage by photoconversion of the kaede reporter protein. Lineage analysis was carried out during pharyngeal development in wild-type animals, after miR140 injection, and after estradiol treatment. At the 10-somite stage, cranial neural crest cells anterior of the eye contributed to the median ethmoid plate, whereas cells medial to the eye formed the lateral ethmoid plate and trabeculae and a posterior population formed the mandible. miR-140 overexpression and estradiol inhibition of Hedgehog signaling resulted in cleft development, with failed migration of the anterior cell population to form the median ethmoid plate. The sox10:kaede transgenic line provides a useful tool for neural crest lineage analysis. These studies illustrate the advantages of the zebrafish model for application in morphogenetic studies of vertebrate craniofacial development.

  5. 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.

  6. 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

  7. 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.

  8. Transient Exposure to Ethanol during Zebrafish Embryogenesis Results in Defects in Neuronal Differentiation: An Alternative Model System to Study FASD

    PubMed Central

    Joya, Xavier; Garcia-Algar, Oscar; Vall, Oriol; Pujades, Cristina

    2014-01-01

    Background The exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS). In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines. Methodology/Principal Findings In order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification. Conclusion Our investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s) of ethanol-induced developmental toxicity at very early stages of embryonic development. PMID:25383948

  9. 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.

  10. A Novel Model of Traumatic Brain Injury in Adult Zebrafish Demonstrates Response to Injury and Treatment Comparable with Mammalian Models.

    PubMed

    McCutcheon, Victoria; Park, Eugene; Liu, Elaine; Sobhebidari, Pooya; Tavakkoli, Jahan; Wen, Xiao-Yan; Baker, Andrew J

    2017-04-01

    Traumatic brain injury (TBI) is a leading cause of death and morbidity in industrialized countries with considerable associated health care costs. The cost and time associated with pre-clinical development of TBI therapeutics is lengthy and expensive with a poor track record of successful translation to the clinic. The zebrafish is an emerging model organism in research with unique technical and genomic strengths in the study of disease and development. Its high degree of genetic homology and cell signaling pathways relative to mammalian species and amenability to high and medium throughput assays has potential to accelerate the rate of therapeutic drug identification. Accordingly, we developed a novel closed-head model of TBI in adult zebrafish using a targeted, pulsed, high-intensity focused ultrasound (pHIFU) to induce mechanical injury of the brain. Western blot results indicated altered microtubule and neurofilament expression as well as increased expression of cleaved caspase-3 and beta APP (β-APP; p < 0.05). We used automated behavioral tracking software to evaluate locomotor deficits 24 and 48 h post-injury. Significant behavioral impairment included decreased swim distance and velocity (p < 0.05), as well as heightened anxiety and altered group social dynamics. Responses to injury were pHIFU dose-dependent and modifiable with MK-801, MDL-28170, or temperature modulation. Together, results indicate that the zebrafish exhibits responses to injury and intervention similar to mammalian TBI pathophysiology and suggest the potential for use to rapidly evaluate therapeutic compounds with high efficiency.

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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...

  16. 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.

  17. 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

  18. 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).

  19. MYCN Transgenic Zebrafish Model with the Characterization of Acute Myeloid Leukemia and Altered Hematopoiesis

    PubMed Central

    Shen, Li-Jing; Chen, Fang-Yuan; Zhang, Yong; Cao, Lan-Fang; Kuang, Ying; Zhong, Min; Wang, Ting; Zhong, Hua

    2013-01-01

    Background Amplification of MYCN (N-Myc) oncogene has been reported as a frequent event and a poor prognostic marker in human acute myeloid leukemia (AML). The molecular mechanisms and transcriptional networks by which MYCN exerts its influence in AML are largely unknown. Methodology/Principal Findings We introduced murine MYCN gene into embryonic zebrafish through a heat-shock promoter and established the stable germline Tg(MYCN:HSE:EGFP) zebrafish. N-Myc downstream regulated gene 1 (NDRG1), negatively controlled by MYCN in human and functionally involved in neutrophil maturation, was significantly under-expressed in this model. Using peripheral blood smear detection, histological section and flow cytometric analysis of single cell suspension from kidney and spleen, we found that MYCN overexpression promoted cell proliferation, enhanced the repopulating activity of myeloid cells and the accumulation of immature hematopoietic blast cells. MYCN enhanced primitive hematopoiesis by upregulating scl and lmo2 expression and promoted myelopoiesis by inhibiting gata1 expression and inducing pu.1, mpo expression. Microarray analysis identified that cell cycle, glycolysis/gluconeogenesis, MAPK/Ras, and p53-mediated apoptosis pathways were upregulated. In addition, mismatch repair, transforming and growth factor β (TGFβ) were downregulated in MYCN-overexpressing blood cells (p<0.01). All of these signaling pathways are critical in the proliferation and malignant transformation of blood cells. Conclusion/Significance The above results induced by overexpression of MYCN closely resemble the main aspects of human AML, suggesting that MYCN plays a role in the etiology of AML. MYCN reprograms hematopoietic cell fate by regulating NDRG1 and several lineage-specific hematopoietic transcription factors. Therefore, this MYCN transgenic zebrafish model facilitates dissection of MYCN-mediated signaling in vivo, and enables high-throughput scale screens to identify the potential

  20. Assessment of Toxicological Perturbations and Variants of Pancreatic Islet Development in the Zebrafish Model

    PubMed Central

    Sant, Karilyn E.; Jacobs, Haydee M.; Xu, Jiali; Borofski, Katrina A.; Moss, Larry G.; Moss, Jennifer B.; Timme-Laragy, Alicia R.

    2016-01-01

    The pancreatic islets, largely comprised of insulin-producing beta cells, play a critical role in endocrine signaling and glucose homeostasis. Because they have low levels of antioxidant defenses and a high perfusion rate, the endocrine islets may be a highly susceptible target tissue of chemical exposures. However, this endpoint, as well as the integrity of the surrounding exocrine pancreas, is often overlooked in studies of developmental toxicology. Disruption of development by toxicants can alter cell fate and migration, resulting in structural alterations that are difficult to detect in mammalian embryo systems, but that are easily observed in the zebrafish embryo model (Danio rerio). Using endogenously expressed fluorescent protein markers for developing zebrafish beta cells and exocrine pancreas tissue, we documented differences in islet area and incidence rates of islet morphological variants in zebrafish embryos between 48 and 96 h post fertilization (hpf), raised under control conditions commonly used in embryotoxicity assays. We identified critical windows for chemical exposures during which increased incidences of endocrine pancreas abnormalities were observed following exposure to cyclopamine (2–12 hpf), Mono-2-ethylhexyl phthalate (MEHP) (3–48 hpf), and Perfluorooctanesulfonic acid (PFOS) (3–48 hpf). Both islet area and length of the exocrine pancreas were sensitive to oxidative stress from exposure to the oxidant tert-butyl hydroperoxide during a highly proliferative critical window (72 hpf). Finally, pancreatic dysmorphogenesis following developmental exposures is discussed with respect to human disease. PMID:28393070

  1. 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.

  2. 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

  3. 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...

  4. Factor Associated with Neutral Sphingomyelinase Activity Mediates Navigational Capacity of Leukocytes Responding to Wounds and Infection: Live Imaging Studies in Zebrafish Larvae

    PubMed Central

    Boecke, Alexandra; Sieger, Dirk; Neacsu, Cristian Dan; Kashkar, Hamid

    2012-01-01

    Factor associated with neutral sphingomyelinase activity (FAN) is an adaptor protein that specifically binds to the p55 receptor for TNF (TNF-RI). Our previous investigations demonstrated that FAN plays a role in TNF-induced actin reorganization by connecting the plasma membrane with actin cytoskeleton, suggesting that FAN may impact on cellular motility in response to TNF and in the context of immune inflammatory conditions. In this study, we used the translucent zebrafish larvae for in vivo analysis of leukocyte migration after morpholino knockdown of FAN. FAN-deficient zebrafish leukocytes were impaired in their migration toward tail fin wounds, leading to a reduced number of cells reaching the wound. Furthermore, FAN-deficient leukocytes show an impaired response to bacterial infections, suggesting that FAN is generally required for the directed chemotactic response of immune cells independent of the nature of the stimulus. Cell-tracking analysis up to 3 h after injury revealed that the reduced number of leukocytes is not due to a reduction in random motility or speed of movement. Leukocytes from FAN-deficient embryos protrude pseudopodia in all directions instead of having one clear leading edge. Our results suggest that FAN-deficient leukocytes exhibit an impaired navigational capacity, leading to a disrupted chemotactic response. PMID:22802420

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. Zebrafish Embryo Disinfection with Povidone–Iodine: Evaluating an Alternative to Chlorine Bleach

    PubMed Central

    Amack, Jeffrey D.; Whipps, Christopher M.

    2016-01-01

    Abstract Mycobacteriosis is a common bacterial infection in laboratory zebrafish caused by several different species and strains of Mycobacterium, including both rapid and slow growers. One control measure used to prevent mycobacterial spread within and between facilities is surface disinfection of eggs. Recent studies have highlighted the effectiveness of povidone–iodine (PVPI) on preventing propagation of Mycobacterium spp. found in zebrafish colonies. We evaluated the effect of disinfection using 12.5–50 ppm PVPI (unbuffered and buffered) on zebrafish exposed at 6 or 24 h postfertilization (hpf) to determine if this treatment is suitable for use in research zebrafish. Our results show that 6 hpf embryos are less sensitive to treatment as fewer effects on mortality, developmental delay, and deformity were observed. We also found that buffered PVPI treatment results in a greater knockdown of Mycobacterium chelonae and Mycobacterium marinum, as well as results in decreased harmful effects on embryos. Treatments of shorter (2 min vs. 5 min) duration were also more effective at killing mycobacteria in addition to resulting in fewer effects on embryo health. In addition, we compared the efficacy of a rinsing regimen to rinsing and disinfecting. Based on the findings of this study, we recommend disinfecting embryos for 2 min with buffered PVPI at 12.5–25 ppm. PMID:27351620

  11. 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.

  12. 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.

  13. Novel approaches to determine contractile function of the isolated adult zebrafish ventricular cardiac myocyte.

    PubMed

    Dvornikov, Alexey V; Dewan, Sukriti; Alekhina, Olga V; Pickett, F Bryan; de Tombe, Pieter P

    2014-05-01

    The zebrafish (Danio rerio) has been used extensively in cardiovascular biology, but mainly in the study of heart development. The relative ease of its genetic manipulation may indicate the suitability of this species as a cost-effective model system for the study of cardiac contractile biology. However, whether the zebrafish heart is an appropriate model system for investigations pertaining to mammalian cardiac contractile structure-function relationships remains to be resolved. Myocytes were isolated from adult zebrafish hearts by enzymatic digestion, attached to carbon rods, and twitch force and intracellular Ca(2+) were measured. We observed the modulation of twitch force, but not of intracellular Ca(2+), by both extracellular [Ca(2+)] and sarcomere length. In permeabilized cells/myofibrils, we found robust myofilament length-dependent activation. Moreover, modulation of myofilament activation-relaxation and force redevelopment kinetics by varied Ca(2+) activation levels resembled that found previously in mammalian myofilaments. We conclude that the zebrafish is a valid model system for the study of cardiac contractile structure-function relationships.

  14. 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

  15. Zebrafish Model of NF1 for Structure-Function Analysis, Mechanisms of Glial Tumorigenesis, and Chemical Biology

    DTIC Science & Technology

    2014-05-01

    identify modulators of this important disease. 15. SUBJECT TERMS Neurofibromatosis ; learning and memory; glioma; MPNST 16. SECURITY CLASSIFICATION...12-13 Page 3 INTRODUCTION Neurofibromatosis type 1...result indicates that sox10 is highly expressed in high-grade gliomas and MPNSTs in our zebrafish model of type I neurofibromatosis . Figure 4. Sox10

  16. Loss of Type I Collagen Telopeptide Lysyl Hydroxylation Causes Musculoskeletal Abnormalities in a Zebrafish Model of Bruck Syndrome

    PubMed Central

    Gistelinck, Charlotte; Witten, Paul Eckhard; Huysseune, Ann; Symoens, Sofie; Malfait, Fransiska; Larionova, Daria; Simoens, Pascal; Dierick, Manuel; Van Hoorebeke, Luc; De Paepe, Anne; Kwon, Ronald Y; Weis, MaryAnn; Eyre, David R; Willaert, Andy; Coucke, Paul J

    2017-01-01

    Bruck syndrome (BS) is a disorder characterized by joint flexion contractures and skeletal dysplasia that shows strong clinical overlap with the brittle bone disease Osteogenesis Imperfecta (OI). BS is caused by bi-allelic mutations in either the FKBP10 or the PLOD2 gene. PLOD2 encodes the lysyl hydroxylase 2 (LH2) enzyme, which is responsible for the hydroxylation of lysine residues in fibrillar collagen telopeptides. This hydroxylation directs cross-linking of collagen fibrils in the extracellular matrix, which is necessary to provide stability and tensile integrity to the collagen fibrils. To further elucidate the function of LH2 in vertebrate skeletal development, we created a zebrafish model harboring a homozygous plod2 nonsense mutation resulting in reduced telopeptide hydroxylation and cross-linking of bone type I collagen. Adult plod2 mutants present with a shortened body axis and severe skeletal abnormalities with evidence of bone fragility and fractures. The vertebral column of plod2 mutants is short and scoliotic with compressed vertebrae that show excessive bone formation at the vertebral end plates, and increased tissue mineral density in the vertebral centra. The muscle fibers of mutant zebrafish have a reduced diameter near the horizontal myoseptum. The endomysium, a layer of connective tissue ensheathing the individual muscle fibers, is enlarged. Transmission electron microscopy of mutant vertebral bone shows type I collagen fibrils that are less organized with loss of the typical plywood-like structure. In conclusion, plod2 mutant zebrafish show molecular and tissue abnormalities in the musculoskeletal system that are concordant with clinical findings in BS patients. Therefore, the plod2 zebrafish mutant is a promising model for the elucidation of the underlying pathogenetic mechanisms leading to BS and the development of novel therapeutic avenues in this syndrome. PMID:27541483

  17. Loss of Type I Collagen Telopeptide Lysyl Hydroxylation Causes Musculoskeletal Abnormalities in a Zebrafish Model of Bruck Syndrome.

    PubMed

    Gistelinck, Charlotte; Witten, Paul Eckhard; Huysseune, Ann; Symoens, Sofie; Malfait, Fransiska; Larionova, Daria; Simoens, Pascal; Dierick, Manuel; Van Hoorebeke, Luc; De Paepe, Anne; Kwon, Ronald Y; Weis, MaryAnn; Eyre, David R; Willaert, Andy; Coucke, Paul J

    2016-11-01

    Bruck syndrome (BS) is a disorder characterized by joint flexion contractures and skeletal dysplasia that shows strong clinical overlap with the brittle bone disease osteogenesis imperfecta (OI). BS is caused by biallelic mutations in either the FKBP10 or the PLOD2 gene. PLOD2 encodes the lysyl hydroxylase 2 (LH2) enzyme, which is responsible for the hydroxylation of lysine residues in fibrillar collagen telopeptides. This hydroxylation directs crosslinking of collagen fibrils in the extracellular matrix, which is necessary to provide stability and tensile integrity to the collagen fibrils. To further elucidate the function of LH2 in vertebrate skeletal development, we created a zebrafish model harboring a homozygous plod2 nonsense mutation resulting in reduced telopeptide hydroxylation and crosslinking of bone type I collagen. Adult plod2 mutants present with a shortened body axis and severe skeletal abnormalities with evidence of bone fragility and fractures. The vertebral column of plod2 mutants is short and scoliotic with compressed vertebrae that show excessive bone formation at the vertebral end plates, and increased tissue mineral density in the vertebral centra. The muscle fibers of mutant zebrafish have a reduced diameter near the horizontal myoseptum. The endomysium, a layer of connective tissue ensheathing the individual muscle fibers, is enlarged. Transmission electron microscopy of mutant vertebral bone shows type I collagen fibrils that are less organized with loss of the typical plywood-like structure. In conclusion, plod2 mutant zebrafish show molecular and tissue abnormalities in the musculoskeletal system that are concordant with clinical findings in BS patients. Therefore, the plod2 zebrafish mutant is a promising model for the elucidation of the underlying pathogenetic mechanisms leading to BS and the development of novel therapeutic avenues in this syndrome. © 2016 American Society for Bone and Mineral Research. © 2016 American Society for

  18. 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

  19. 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

  20. Zebrafish as a model for monocarboxyl transporter 8-deficiency.

    PubMed

    Vatine, Gad David; Zada, David; Lerer-Goldshtein, Tali; Tovin, Adi; Malkinson, Guy; Yaniv, Karina; Appelbaum, Lior

    2013-01-04

    Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor retardation characterized by neurological impairment and abnormal thyroid hormone (TH) levels. Mutations in the TH transporter, monocarboxylate transporter 8 (MCT8), are associated with AHDS. MCT8 knock-out mice exhibit impaired TH levels; however, they lack neurological defects. Here, the zebrafish mct8 gene and promoter were isolated, and mct8 promoter-driven transgenic lines were used to show that, similar to humans, mct8 is primarily expressed in the nervous and vascular systems. Morpholino-based knockdown and rescue experiments revealed that MCT8 is strictly required for neural development in the brain and spinal cord. This study shows that MCT8 is a crucial regulator during embryonic development and establishes the first vertebrate model for MCT8 deficiency that exhibits a neurological phenotype.

  1. 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

  2. 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.

  3. 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

  4. Looking within the zebrafish to understand the tuberculous granuloma.

    PubMed

    Ramakrishnan, Lalita

    2013-01-01

    Tuberculosis is characterized by the formation of complex immune cell aggregates called granulomas, which for nearly a century have been viewed as critical host-beneficial structures to restrict bacterial growth and spread. A different view has now emerged from real-time visualization of granuloma formation and its consequences in the optically transparent and genetically tractable zebrafish larva. Pathogenic mycobacteria have developed mechanisms to use host granulomas for their expansion and dissemination, at least during the innate phases of infection. Host processes that are intended to be beneficial-death of infected macrophages and their subsequent phagocytosis by macrophages that are newly recruited to the growing granuloma-are harnessed by mycobacteria for their own benefit. Mycobacteria can also render the granuloma a safe-haven in the more advanced stages of infection. An understanding of the host and bacterial pathways involved in tuberculous granuloma formation may suggest new ways to combat mycobacterial infection.

  5. Zebrafish pancreas development.

    PubMed

    Tiso, Natascia; Moro, Enrico; Argenton, Francesco

    2009-11-27

    An accurate understanding of the molecular events governing pancreas development can have an impact on clinical medicine related to diabetes, obesity and pancreatic cancer, diseases with a high impact in public health. Until 1996, the main animal models in which pancreas formation and differentiation could be studied were mouse and, for some instances related to early development, chicken and Xenopus. Zebrafish has penetrated this field very rapidly offering a new model of investigation; by joining functional genomics, genetics and in vivo whole mount visualization, Danio rerio has allowed large scale and fine multidimensional analysis of gene functions during pancreas formation and differentiation.

  6. 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

  7. 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.

  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. 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. Distribution, pharmacokinetics and primary metabolism model of tramadol in zebrafish.

    PubMed

    Zhuo, Huiqin; Jin, Hongwei; Peng, Huifang; Huang, Heqing

    2016-12-01

    The current study aimed to develop a rapid, robust and adequately sensitive method for simultaneous determination of the concentration of tramadol and its active metabolites in zebrafish. The pharmacokinetic and elimination pattern of tramadol and its major phase I metabolites following oral or intramuscular administration in zebrafish tissues was achieved using electrospray ionization‑quadrupole‑time of flight/mass spectrometry (ESI‑Q‑TOF/MS) and gas chromatography/mass spectrometry (GC‑MS). Following administration, the metabolisms were detected in the brain, eyes, muscle and gill tissues within 1 h. Two tramadol metabolites, O‑ and N‑desmethyltramadol, were detected in brain tissue, with N‑desmethyltramadol detected at a higher level. Following GC‑MS detection the curve indicated an initial rapid phase, corresponding to the detection of the tramadol within 1 min, and reached peak value in the brain at 5 min. Faster drug clearance was detected in low‑dose groups, and concentration had dropped around the to initial level (1.11 µg) at 20 min, but was detectable for up to 3 h. However, it took 80 min to fall back to the initial value (1.73 µg) in the high‑dose groups, and tramadol was detectable for up to 4 h. This study developed and validated a simple and high throughput analytical procedure to determine the distribution and pharmacokinetic profiles of tramadol, and its primary metabolites in tissues of zebrafish.

  11. 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.

  12. 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.

  13. Molecular and functional characterization of the scavenger receptor CD36 in zebrafish and common carp.

    PubMed

    Fink, Inge R; Benard, Erica L; Hermsen, Trudi; Meijer, Annemarie H; Forlenza, Maria; Wiegertjes, Geert F

    2015-02-01

    CD36 is a scavenger receptor which has been studied closely in mammals where it is expressed by many different cell types and plays a role in highly diverse processes, both homeostatic and pathologic. It is among other things important in the innate immune system, in angiogenesis, and in clearance of apoptotic cells, and it is also involved in lipid metabolism and atherosclerosis. Recently, in the cephalochordate amphioxus a primitive CD36 family member was described, which was present before the divergence of CD36 from other scavenger receptor B family members, SCARB1 and SCARB2. Not much is known on the Cd36 molecule in teleost fish. We therefore studied Cd36 in both zebrafish and common carp, two closely related cyprinid fish species. Whereas a single cd36 gene is present in zebrafish, carp has two cd36 genes, and all show conserved synteny compared to mammalian CD36. The gene expression of carp cd36 is high in brain, ovary and testis but absent in immune organs. Although in mammals CD36 expression in erythrocytes, monocytes and macrophages is high, gene expression studies in leukocyte subtypes of adult carp and zebrafish larvae, including thrombocytes and macrophages provided no indication for any substantial expression of cd36 in immune cell types. Surprisingly, analysis of the cd36 promoter region does show the presence of several binding sites for transcription factors known to regulate immune responses. Overexpression of carp cd36 locates the receptor on the cell surface of mammalian cell lines consistent with the predicted topology of cyprinid Cd36 with a large extracellular domain, two transmembrane domains, and short cytoplasmic tails at both ends. Gene expression of cd36 is down-regulated during infection of zebrafish with Mycobacterium marinum, whereas knockdown of cd36 in zebrafish larvae led to higher bacterial burden upon such infection. We discuss the putative role for Cd36 in immune responses of fish in the context of other members of the

  14. 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.

  15. 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.

  16. 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

  17. Fusaric acid induces a notochord malformation in zebrafish via copper chelation.

    PubMed

    Yin, Emily S; Rakhmankulova, Malika; Kucera, Kaury; de Sena Filho, Jose Guedes; Portero, Carolina E; Narváez-Trujillo, Alexandra; Holley, Scott A; Strobel, Scott A

    2015-08-01

    Over a thousand extracts were tested for phenotypic effects in developing zebrafish embryos to identify bioactive molecules produced by endophytic fungi. One extract isolated from Fusarium sp., a widely distributed fungal genus found in soil and often associated with plants, induced an undulated notochord in developing zebrafish embryos. The active compound was isolated and identified as fusaric acid. Previous literature has shown this phenotype to be associated with copper chelation from the active site of lysyl oxidase, but the ability of fusaric acid to bind copper ions has not been well described. Isothermal titration calorimetry revealed that fusaric acid is a modest copper chelator with a binding constant of 4.4 × 10(5) M(-1). These results shed light on the toxicity of fusaric acid and the potential teratogenic effects of consuming plants infected with Fusarium sp.

  18. 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.

  19. 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

  20. 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.

  1. 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

  2. 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

  3. 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.

  4. 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

  5. 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.

  6. NAD+ Biosynthesis Ameliorates a Zebrafish Model of Muscular Dystrophy

    PubMed Central

    Goody, Michelle F.; Kelly, Meghan W.; Reynolds, Christine J.; Khalil, Andre; Crawford, Bryan D.; Henry, Clarissa A.

    2012-01-01

    Muscular dystrophies are common, currently incurable diseases. A subset of dystrophies result from genetic disruptions in complexes that attach muscle fibers to their surrounding extracellular matrix microenvironment. Cell-matrix adhesions are exquisite sensors of physiological conditions and mediate responses that allow cells to adapt to changing conditions. Thus, one approach towards finding targets for future therapeutic applications is to identify cell adhesion pathways that mediate these dynamic, adaptive responses in vivo. We find that nicotinamide riboside kinase 2b-mediated NAD+ biosynthesis, which functions as a small molecule agonist of muscle fiber-extracellular matrix adhesion, corrects dystrophic phenotypes in zebrafish lacking either a primary component of the dystrophin-glycoprotein complex or integrin alpha7. Exogenous NAD+ or a vitamin precursor to NAD+ reduces muscle fiber degeneration and results in significantly faster escape responses in dystrophic embryos. Overexpression of paxillin, a cell adhesion protein downstream of NAD+ in this novel cell adhesion pathway, reduces muscle degeneration in zebrafish with intact integrin receptors but does not improve motility. Activation of this pathway significantly increases organization of laminin, a major component of the extracellular matrix basement membrane. Our results indicate that the primary protective effects of NAD+ result from changes to the basement membrane, as a wild-type basement membrane is sufficient to increase resilience of dystrophic muscle fibers to damage. The surprising result that NAD+ supplementation ameliorates dystrophy in dystrophin-glycoprotein complex– or integrin alpha7–deficient zebrafish suggests the existence of an additional laminin receptor complex that anchors muscle fibers to the basement membrane. We find that integrin alpha6 participates in this pathway, but either integrin alpha7 or the dystrophin-glycoprotein complex is required in conjunction with integrin

  7. 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.

  8. Zebrafish cardiac muscle thick filaments: isolation technique and three-dimensional structure.

    PubMed

    González-Solá, Maryví; Al-Khayat, Hind A; Behra, Martine; Kensler, Robert W

    2014-04-15

    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. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  9. 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.

  10. Live imaging reveals a conserved role of fatty acid β-oxidation in early lymphatic development in zebrafish.

    PubMed

    Zecchin, Annalisa; Wong, Brian W; Tembuyser, Bieke; Souffreau, Joris; Van Nuffelen, An; Wyns, Sabine; Vinckier, Stefan; Carmeliet, Peter; Dewerchin, Mieke

    2018-06-18

    During embryonic development, lymphatic endothelial cells (LECs) differentiate from venous endothelial cells (VECs), a process that is tightly regulated by several genetic signals. While the aquatic zebrafish model is regularly used for studying lymphangiogenesis and offers the unique advantage of time-lapse video-imaging of lymphatic development, some aspects of lymphatic development in this model differ from those in the mouse. It therefore remained to be determined whether fatty acid β-oxidation (FAO), which we showed to regulate lymphatic formation in the mouse, also co-determines lymphatic development in this aquatic model. Here, we took advantage of the power of the zebrafish embryo model to visualize the earliest steps of lymphatic development through time-lapse video-imaging. By targeting zebrafish isoforms of carnitine palmitoyltransferase 1a (cpt1a), a rate controlling enzyme of FAO, with multiple morpholinos, we demonstrate that reducing CPT1A levels and FAO flux during zebrafish development impairs lymphangiogenic secondary sprouting, the initiation of lymphatic development in the zebrafish trunk, and the formation of the first lymphatic structures. These findings not only show evolutionary conservation of the importance of FAO for lymphatic development, but also suggest a role for FAO in co-regulating the process of VEC-to-LEC differentiation in zebrafish in vivo. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Botulinum Toxin Induces Muscle Paralysis and Inhibits Bone Regeneration in Zebrafish

    PubMed Central

    Recidoro, Anthony M.; Roof, Amanda C.; Schmitt, Michael; Worton, Leah E.; Petrie, Timothy; Strand, Nicholas; Ausk, Brandon J.; Srinivasan, Sundar; Moon, Randall T.; Gardiner, Edith M.; Kaminsky, Werner; Bain, Steven D.; Allan, Christopher H.; Gross, Ted S.; Kwon, Ronald Y.

    2016-01-01

    Intramuscular administration of Botulinum toxin (BTx) has been associated with impaired osteogenesis in diverse conditions of bone formation (e.g., development, growth, and healing), yet the mechanisms of neuromuscular-bone crosstalk underlying these deficits have yet to be identified. Motivated by the emerging utility of zebrafish (Danio rerio) as a rapid, genetically tractable, and optically transparent model for human pathologies (as well as the potential to interrogate neuromuscular-mediated bone disorders in a simple model that bridges in vitro and more complex in vivo model systems), in this study we developed a model of BTx-induced muscle paralysis in adult zebrafish, and examined its effects on intramembranous ossification during tail fin regeneration. BTx administration induced rapid muscle paralysis in adult zebrafish in a manner that was dose-dependent, transient, and focal, mirroring the paralytic phenotype observed in animal and human studies. During fin regeneration, BTx impaired continued bone ray outgrowth, morphology, and patterning, indicating defects in early osteogenesis. Further, BTx significantly decreased mineralizing activity and crystalline mineral accumulation, suggesting delayed late-stage osteoblast differentiation and/or altered secondary bone apposition. Bone ray transection proximal to the amputation site focally inhibited bone outgrowth in the affected ray, implicating intra- and/or inter-ray nerves in this process. Taken together, these studies demonstrate the potential to interrogate pathological features of BTx-induced osteoanabolic dysfunction in the regenerating zebrafish fin, define the technological toolbox for detecting bone growth and mineralization deficits in this process, and suggest that pathways mediating neuromuscular regulation of osteogenesis may be conserved beyond established mammalian models of bone anabolic disorders. PMID:24806738

  12. Imaging of viral neuroinvasion in the zebrafish reveals that Sindbis and chikungunya viruses favour different entry routes

    PubMed Central

    Passoni, Gabriella; Langevin, Christelle; Palha, Nuno; Mounce, Bryan C.; Briolat, Valérie; Affaticati, Pierre; De Job, Elodie; Joly, Jean-Stéphane; Vignuzzi, Marco; Saleh, Maria-Carla; Herbomel, Philippe; Boudinot, Pierre

    2017-01-01

    ABSTRACT Alphaviruses, such as chikungunya virus (CHIKV) and Sindbis virus (SINV), are vector-borne pathogens that cause acute illnesses in humans and are sometimes associated with neuropathies, especially in infants and elderly patients. Little is known about their mechanism of entry into the central nervous system (CNS), even for SINV, which has been used extensively as a model for viral encephalopathies. We previously established a CHIKV infection model in the optically transparent zebrafish larva; here we describe a new SINV infection model in this host. We imaged in vivo the onset and progression of the infection caused by intravenous SINV inoculation. Similar to that described for CHIKV, infection in the periphery was detected early and was transient, whereas CNS infection started at later time points and was persistent or progressive. We then tested the possible mechanisms of neuroinvasion by CHIKV and SINV. Neither virus relied on macrophage-mediated transport to access the CNS. CHIKV, but not SINV, always infects endothelial cells of the brain vasculature. By contrast, axonal transport was much more efficient with SINV than CHIKV, both from the periphery to the CNS and between neural tissues. Thus, the preferred mechanisms of neuroinvasion by these two related viruses are distinct, providing a powerful imaging-friendly system to compare mechanisms and prevention methods of encephalopathies. PMID:28483796

  13. 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

  14. 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.

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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.

  20. Pro-NP™ protect against TiO2 nanoparticle-induced phototoxicity in zebrafish model: exploring potential application for skin care.

    PubMed

    Kim, Min-Sik; Stees, Melinda; Karuturi, Bala Vamsi K; Vijayaraghavalu, Sivakumar; Peterson, Richard E; Madsen, Gary L; Labhasetwar, Vinod

    2017-06-01

    Titanium dioxide nanoparticles (TiO 2 NPs) are used in sunscreen products to protect the skin from the sun's ultraviolet rays. However, following exposure to sunlight, the photocatalytic activity of TiO 2 NPs can produce an excess of reactive oxygen species (ROS), causing skin cell damage, triggering an inflammatory response. In zebrafish model, we evaluated how well Pro-NP™ (biodegradable NPs containing superoxide dismutase and catalase) could protect them from TiO 2 NP-induced photo-oxidative stress. We hypothesized that the antioxidant properties of Pro-NP™ would protect zebrafish embryos from the phototoxic effects of TiO 2 NPs, improving overall survival and growth. Dechorionated embryos were treated with TiO 2 NPs alone or co-treated with Pro-NP™, and then exposed to simulated sunlight. Pro-NP™ by itself caused no toxicity; however, for embryos exposed to 100 μg/ml TiO 2 NPs, zebrafish survival was reduced to ∼40% and at 500 μg/ml to ∼10%. In contrast, at 100 μg/ml TiO 2 NP, co-treatment with Pro-NP™ increased zebrafish survival in a dose-dependent manner. Co-treatment also improved percent of embryos hatching and resulted in normal growth of zebrafish. On the other hand, embryos treated with TiO 2 NPs alone developed deformities, had reduced pigmentation, and showed severely truncated growth. Pro-NP™ afforded a greater level of protection against TiO 2 NP-induced phototoxicity than other antioxidants (vitamin E or N-acetylcysteine) commonly used in topical skin care formulations. We conclude that Pro-NP™ exert significant protective effects against TiO 2 NP-induced phototoxicity and could be developed as a safe, effective skin care product, used alone or in combination with sunscreen products to protect the skin from sun's UV radiation.

  1. 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.

  2. Whole-Volume Clustering of Time Series Data from Zebrafish Brain Calcium Images via Mixture Modeling.

    PubMed

    Nguyen, Hien D; Ullmann, Jeremy F P; McLachlan, Geoffrey J; Voleti, Venkatakaushik; Li, Wenze; Hillman, Elizabeth M C; Reutens, David C; Janke, Andrew L

    2018-02-01

    Calcium is a ubiquitous messenger in neural signaling events. An increasing number of techniques are enabling visualization of neurological activity in animal models via luminescent proteins that bind to calcium ions. These techniques generate large volumes of spatially correlated time series. A model-based functional data analysis methodology via Gaussian mixtures is suggested for the clustering of data from such visualizations is proposed. The methodology is theoretically justified and a computationally efficient approach to estimation is suggested. An example analysis of a zebrafish imaging experiment is presented.

  3. Neoplasia and Neoplasm Associated Lesions in Laboratory Colonies of Zebrafish Emphasizing Key Influences of Diet and Aquaculture System Design

    PubMed Central

    Spitsbergen, Jan M.; Buhler, Donald R.; Peterson, Tracy S.

    2014-01-01

    During the past decade the zebrafish has emerged as a leading model for mechanistic cancer research due to its sophisticated genetic and genomic resources, its tractability for tissue targeting of transgene expression, its efficiency for forward genetic approaches to cancer model development, and its cost-effectiveness for enhancer and suppressor screens once a cancer model is established. However, in contrast to other laboratory animal species widely used as cancer models, much basic cancer biology information is lacking in zebrafish. As yet data are not published regarding dietary influences on neoplasm incidences in zebrafish. Little information is available regarding spontaneous tumor incidences or histologic types in wild-type (wt) lines of zebrafish. So far a comprehensive database documenting the full spectrum of neoplasia in various organ systems and tissues in not available for zebrafish as it is for other intensely studied laboratory animal species. This manuscript confirms that as in other species diet and husbandry can profoundly influence tumor incidences and histologic spectra in zebrafish. We show that in many laboratory colonies wt lines of zebrafish exhibit elevated neoplasm incidences and neoplasm associated lesions such as heptocyte megalocytosis. We present experimental evidence showing that certain diet and water management regimens can result in high incidences of neoplasia and neoplasm associated lesions. We document the wide array of benign and malignant neoplasms affecting nearly every organ, tissue and cell type in zebrafish, in some cases as a spontaneous aging change, and in other cases due to carcinogen treatment or genetic manipulation. PMID:23382343

  4. Embryonic Zebrafish Model - A Well-Established Method for Rapidly Assessing the Toxicity of Homeopathic Drugs

    PubMed Central

    Gupta, Himanshu R; Patil, Yogesh; Singh, Dipty

    2016-01-01

    in this study. The embryonic zebrafish model is recommended as a well-established method for rapidly assessing the toxicity of homeopathic drugs. PMID:28127503

  5. 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.

  6. 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

  7. Sqstm1 knock-down causes a locomotor phenotype ameliorated by rapamycin in a zebrafish model of ALS/FTLD.

    PubMed

    Lattante, Serena; de Calbiac, Hortense; Le Ber, Isabelle; Brice, Alexis; Ciura, Sorana; Kabashi, Edor

    2015-03-15

    Mutations in SQSTM1, encoding for the protein SQSTM1/p62, have been recently reported in 1-3.5% of patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration (ALS/FTLD). Inclusions positive for SQSTM1/p62 have been detected in patients with neurodegenerative disorders, including ALS/FTLD. In order to investigate the pathogenic mechanisms induced by SQSTM1 mutations in ALS/FTLD, we developed a zebrafish model. Knock-down of the sqstm1 zebrafish ortholog, as well as impairment of its splicing, led to a specific phenotype, consisting of behavioral and axonal anomalies. Here, we report swimming deficits associated with shorter motor neuronal axons that could be rescued by the overexpression of wild-type human SQSTM1. Interestingly, no rescue of the loss-of-function phenotype was observed when overexpressing human SQSTM1 constructs carrying ALS/FTLD-related mutations. Consistent with its role in autophagy regulation, we found increased mTOR levels upon knock-down of sqstm1. Furthermore, treatment of zebrafish embryos with rapamycin, a known inhibitor of the mTOR pathway, yielded an amelioration of the locomotor phenotype in the sqstm1 knock-down model. Our results suggest that loss-of-function of SQSTM1 causes phenotypic features characterized by locomotor deficits and motor neuron axonal defects that are associated with a misregulation of autophagic processes. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  8. 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

  9. Regulation of zebrafish sleep and arousal states: current and prospective approaches

    PubMed Central

    Chiu, Cindy N.; Prober, David A.

    2013-01-01

    Every day, we shift among various states of sleep and arousal to meet the many demands of our bodies and environment. A central puzzle in neurobiology is how the brain controls these behavioral states, which are essential to an animal's well-being and survival. Mammalian models have predominated sleep and arousal research, although in the past decade, invertebrate models have made significant contributions to our understanding of the genetic underpinnings of behavioral states. More recently, the zebrafish has emerged as a promising model system for sleep and arousal research. Here we review experimental evidence that the zebrafish, a diurnal vertebrate, exhibits fundamental behavioral and neurochemical characteristics of mammalian sleep and arousal. We also propose how specific advantages of the zebrafish can be harnessed to advance the field. These include tractable genetics to identify and manipulate molecular and cellular regulators of behavioral states, optical transparency to facilitate in vivo observation of neural structure and function, and amenability to high-throughput drug screens to discover novel therapies for neurological disorders. PMID:23576957

  10. Reversal learning and resurgence of operant behavior in zebrafish (Danio rerio).

    PubMed

    Kuroda, Toshikazu; Mizutani, Yuto; Cançado, Carlos R X; Podlesnik, Christopher A

    2017-09-01

    Zebrafish are used extensively as vertebrate animal models in biomedical research for having such features as a fully sequenced genome and transparent embryo. Yet, operant-conditioning studies with this species are scarce. The present study investigated reversal learning and resurgence of operant behavior in zebrafish. A target response (approaching a sensor) was reinforced in Phase 1. In Phase 2, the target response was extinguished while reinforcing an alternative response (approaching a different sensor). In Phase 3, extinction was in effect for the target and alternative responses. Reversal learning was demonstrated when responding tracked contingency changes between Phases 1 and 2. Moreover, resurgence occurred in 10 of 13 fish in Phase 3: Target response rates increased transiently and exceeded rates of an unreinforced control response. The present study provides the first evidence with zebrafish supporting reversal learning between discrete operant responses and a laboratory model of relapse. These findings open the possibility to assessing genetic influences of operant behavior generally and in models of relapse (e.g., resurgence, renewal, reinstatement). Copyright © 2017 Elsevier B.V. All rights reserved.

  11. l-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome

    PubMed Central

    Xu, Baoshan; Sowa, Nenja; Cardenas, Maria E.; Gerton, Jennifer L.

    2015-01-01

    Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (α-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite α-KIC can partially rescue development in zebrafish models for CdLS. PMID:25378554

  12. L-leucine partially rescues translational and developmental defects associated with zebrafish models of Cornelia de Lange syndrome.

    PubMed

    Xu, Baoshan; Sowa, Nenja; Cardenas, Maria E; Gerton, Jennifer L

    2015-03-15

    Cohesinopathies are human genetic disorders that include Cornelia de Lange syndrome (CdLS) and Roberts syndrome (RBS) and are characterized by defects in limb and craniofacial development as well as mental retardation. The developmental phenotypes of CdLS and other cohesinopathies suggest that mutations in the structure and regulation of the cohesin complex during embryogenesis interfere with gene regulation. In a previous project, we showed that RBS was associated with highly fragmented nucleoli and defects in both ribosome biogenesis and protein translation. l-leucine stimulation of the mTOR pathway partially rescued translation in human RBS cells and development in zebrafish models of RBS. In this study, we investigate protein translation in zebrafish models of CdLS. Our results show that phosphorylation of RPS6 as well as 4E-binding protein 1 (4EBP1) was reduced in nipbla/b, rad21 and smc3-morphant embryos, a pattern indicating reduced translation. Moreover, protein biosynthesis and rRNA production were decreased in the cohesin morphant embryo cells. l-leucine partly rescued protein synthesis and rRNA production in the cohesin morphants and partially restored phosphorylation of RPS6 and 4EBP1. Concomitantly, l-leucine treatment partially improved cohesinopathy embryo development including the formation of craniofacial cartilage. Interestingly, we observed that alpha-ketoisocaproate (α-KIC), which is a keto derivative of leucine, also partially rescued the development of rad21 and nipbla/b morphants by boosting mTOR-dependent translation. In summary, our results suggest that cohesinopathies are caused in part by defective protein synthesis, and stimulation of the mTOR pathway through l-leucine or its metabolite α-KIC can partially rescue development in zebrafish models for CdLS. © The Author 2014. Published by Oxford University Press.

  13. Leadership emergence in a data-driven model of zebrafish shoals with speed modulation

    NASA Astrophysics Data System (ADS)

    Zienkiewicz, A.; Barton, D. A. W.; Porfiri, M.; Di Bernardo, M.

    2015-11-01

    Models of collective animal motion can greatly aid in the design and interpretation of behavioural experiments that seek to unravel, isolate, and manipulate the determinants of leader-follower relationships. Here, we develop an initial model of zebrafish social behaviour, which accounts for both speed and angular velocity regulatory interactions among conspecifics. Using this model, we analyse the macroscopic observables of small shoals influenced by an "informed" agent, showing that leaders which actively modulate their speed with respect to their neighbours can entrain and stabilise collective dynamics of the naïve shoal. Supplementary material in the form of two mp4 files available from the Journal web page at http://dx.doi.org/10.1140/epjst/e2015-50093-5

  14. 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.

  15. 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.

  16. Screening for angiogenic inhibitors in zebrafish to evaluate a predictive model for developmental vascular toxicity.

    PubMed

    Tal, Tamara; Kilty, Claire; Smith, Andrew; LaLone, Carlie; Kennedy, Brendán; Tennant, Alan; McCollum, Catherine W; Bondesson, Maria; Knudsen, Thomas; Padilla, Stephanie; Kleinstreuer, Nicole

    2017-06-01

    Chemically-induced vascular toxicity during embryonic development may cause a wide range of adverse effects. To identify putative vascular disrupting chemicals (pVDCs), a predictive pVDC signature was constructed from 124 U.S. EPA ToxCast high-throughput screening (HTS) assays and used to rank 1060 chemicals for their potential to disrupt vascular development. Thirty-seven compounds were selected for targeted testing in transgenic Tg(kdrl:EGFP) and Tg(fli1:EGFP) zebrafish embryos to identify chemicals that impair developmental angiogenesis. We hypothesized that zebrafish angiogenesis toxicity data would correlate with human cell-based and cell-free in vitro HTS ToxCast data. Univariate statistical associations used to filter HTS data based on correlations with zebrafish angiogenic inhibition in vivo revealed 132 total significant associations, 33 of which were already captured in the pVDC signature, and 689 non-significant assay associations. Correlated assays were enriched in cytokine and extracellular matrix pathways. Taken together, the findings indicate the utility of zebrafish assays to evaluate an HTS-based predictive toxicity signature and also provide an experimental basis for expansion of the pVDC signature with novel HTS assays. Published by Elsevier Inc.

  17. 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

  18. 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

  19. 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

  20. Strengths and Limitations of Model Systems for the Study of Urinary Tract Infections and Related Pathologies

    PubMed Central

    Barber, Amelia E.; Norton, J. Paul; Wiles, Travis J.

    2016-01-01

    SUMMARY Urinary tract infections (UTIs) are some of the most common bacterial infections worldwide and are a source of substantial morbidity among otherwise healthy women. UTIs can be caused by a variety of microbes, but the predominant etiologic agent of these infections is uropathogenic Escherichia coli (UPEC). An especially troubling feature of UPEC-associated UTIs is their high rate of recurrence. This problem is compounded by the drastic increase in the global incidence of antibiotic-resistant UPEC strains over the past 15 years. The need for more-effective treatments for UTIs is driving research aimed at bettering our understanding of the virulence mechanisms and host-pathogen interactions that occur during the course of these infections. Surrogate models of human infection, including cell culture systems and the use of murine, porcine, avian, teleost (zebrafish), and nematode hosts, are being employed to define host and bacterial factors that modulate the pathogenesis of UTIs. These model systems are revealing how UPEC strains can avoid or overcome host defenses and acquire scarce nutrients while also providing insight into the virulence mechanisms used by UPEC within compromised individuals, such as catheterized patients. Here, we summarize our current understanding of UTI pathogenesis while also giving an overview of the model systems used to study the initiation, persistence, and recurrence of UTIs and life-threatening sequelae like urosepsis. Although we focus on UPEC, the experimental systems described here can also provide valuable insight into the disease processes associated with other bacterial pathogens both within the urinary tract and elsewhere within the host. PMID:26935136

  1. Learning and memory in zebrafish (Danio rerio).

    PubMed

    Gerlai, R

    2016-01-01

    Learning and memory are defining features of our own species inherently important to our daily lives and to who we are. Without our memories we cease to exist as a person. Without our ability to learn individuals and collectively our society would cease to function. Diseases of the mind still remain incurable. The interest in understanding of the mechanisms of learning and memory is thus well founded. Given the complexity of such mechanisms, concerted efforts have been made to study them under controlled laboratory conditions, ie, with laboratory model organisms. The zebrafish, although new in this field, is one such model organism. The rapidly developing forward- and reverse genetic methods designed for the zebrafish and the increasing use of pharmacological tools along with numerous neurobiology techniques make this species perhaps the best model for the analysis of the mechanisms of complex central nervous system characteristics. The fact that it is an evolutionarily ancient and simpler vertebrate, but at the same time it possesses numerous conserved features across multiple levels of biological organization makes this species an excellent tool for the analysis of the mechanisms of learning and memory. The bottleneck lies in our understanding of its cognitive and mnemonic features, the topic of this chapter. The current paper builds on a chapter published in the previous edition and continues to focus on associative learning, but now it extends the discussion to other forms of learning and to recent discoveries on memory-related features and findings obtained both in adults and larval zebrafish. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Zebrafish: a model animal for analyzing the impact of environmental pollutants on muscle and brain mitochondrial bioenergetics.

    PubMed

    Bourdineaud, Jean-Paul; Rossignol, R; Brèthes, D

    2013-01-01

    Mercury, anthropogenic release of uranium (U), and nanoparticles constitute hazardous environmental pollutants able to accumulate along the aquatic food chain with severe risk for animal and human health. The impact of such pollutants on living organisms has been up to now approached by classical toxicology in which huge doses of toxic compounds, environmentally irrelevant, are displayed through routes that never occur in the lifespan of organisms (for instance injecting a bolus of mercury to an animal although the main route is through prey and fish eating). We wanted to address the effect of such pollutants on the muscle and brain mitochondrial bioenergetics under realistic conditions, at unprecedented low doses, using an aquatic model animal, the zebrafish Danio rerio. We developed an original method to measure brain mitochondrial respiration: a single brain was put in 1.5 mL conical tube containing a respiratory buffer. Brains were gently homogenized by 13 strokes with a conical plastic pestle, and the homogenates were immediately used for respiration measurements. Skinned muscle fibers were prepared by saponin permeabilization. Zebrafish were contaminated with food containing 13 μg of methylmercury (MeHg)/g, an environmentally relevant dose. In permeabilized muscle fibers, we observed a strong inhibition of both state 3 mitochondrial respiration and cytochrome c oxidase activity after 49 days of MeHg exposure. We measured a dramatic decrease in the rate of ATP release by skinned muscle fibers. Contrarily to muscles, brain mitochondrial respiration was not modified by MeHg exposure although brain accumulated twice as much MeHg than muscles. When zebrafish were exposed to 30 μg/L of waterborne U, the basal mitochondrial respiratory control ratio was decreased in muscles after 28 days of exposure. This was due to an increase of the inner mitochondrial membrane permeability. The impact of a daily ration of food containing gold nanoparticles of two sizes (12 and

  3. 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.

  4. 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

  5. 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.

  6. 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…

  7. 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

  8. In vitro antagonistic activity and the protective effect of probiotic Bacillus licheniformis Dahb1 in zebrafish challenged with GFP tagged Vibrio parahaemolyticus Dahv2.

    PubMed

    Girija, Vairavan; Malaikozhundan, Balasubramanian; Vaseeharan, Baskaralingam; Vijayakumar, Sekar; Gobi, Narayanan; Del Valle Herrera, Marian; Chen, Jiann-Chu; Santhanam, Perumal

    2018-01-01

    In vitro antagonistic activity and the protective effect of probiotic Bacillus licheniformis Dahb1 in zebrafish (Danio rerio) challenged with GFP tagged Vibrio parahaemolyticus Dahv2 was studied. The cell free extract of probiotic B. licheniformis Dahb1 at 100 μg mL -1 showed growth inhibition of V. parahaemolyticus Dahv2 in vitro. B. licheniformis Dahb1 also inhibited the biofilm growth of GFP tagged V. parahaemolyticus Dahv2 at 100 μg mL -1 in vitro. The growth and survival of zebrafish was tested using probiotic B. licheniformis Dahb1. Weight (1.28 g) of zebrafish that received the cell free extract was much higher than in control (1.04 g). The mortality of zebrafish infected with GFP tagged V. parahaemolyticus Dahv2 at 10 7 Cfu mL -1 (Group IV) was 100%, whereas a complete survival of zebrafish that received the cell free extract of B. licheniformis Dahb1 at 10 7 Cfu mL -1 (Group VII) was observed after 30 days. The number of GFP tagged V. parahaemolyticus Dahv2 colonies in the intestine and gills significantly reduced after treatment with the cell free extract of B. licheniformis Dahb1. Furthermore, a significant decrease in the fluorescent colonies of GFP tagged V. parahaemolyticus Dahv2 was observed after treatment with the cell free extract of B. licheniformis Dahb1 under confocal laser scanning microscopy (CLSM). In conclusion, the cell free extract of B. licheniformis Dahb1 could prevent Vibrio infection by enhancing the growth and survival of zebrafish. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. 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.

  10. Insights from Molecular Dynamics Simulations: Structural Basis for the V567D Mutation-Induced Instability of Zebrafish Alpha-Dystroglycan and Comparison with the Murine Model

    PubMed Central

    Pirolli, Davide; Sciandra, Francesca; Bozzi, Manuela; Giardina, Bruno; Brancaccio, Andrea; De Rosa, Maria Cristina

    2014-01-01

    A missense amino acid mutation of valine to aspartic acid in 567 position of alpha-dystroglycan (DG), identified in dag1-mutated zebrafish, results in a reduced transcription and a complete absence of the protein. Lacking experimental structural data for zebrafish DG domains, the detailed mechanism for the observed mutation-induced destabilization of the DG complex and membrane damage, remained unclear. With the aim to contribute to a better clarification of the structure-function relationships featuring the DG complex, three-dimensional structural models of wild-type and mutant (V567D) C-terminal domain of alpha-DG from zebrafish were constructed by a template-based modelling approach. We then ran extensive molecular dynamics (MD) simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability. A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish. Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein. A biochemical analysis of the murine alpha-DG mutant I591D confirmed a pronounced instability of the protein. Taken together, the computational and biochemical analysis suggest that the V567D/I591D mutation, belonging to the G beta-strand, plays a key role in inducing a destabilization of the alpha-DG C-terminal Ig-like domain that could possibly affect and propagate to the entire DG complex. The structural features herein identified may be of crucial help to understand the molecular basis of primary dystroglycanopathies. PMID:25078606

  11. Insights from molecular dynamics simulations: structural basis for the V567D mutation-induced instability of zebrafish alpha-dystroglycan and comparison with the murine model.

    PubMed

    Pirolli, Davide; Sciandra, Francesca; Bozzi, Manuela; Giardina, Bruno; Brancaccio, Andrea; De Rosa, Maria Cristina

    2014-01-01

    A missense amino acid mutation of valine to aspartic acid in 567 position of alpha-dystroglycan (DG), identified in dag1-mutated zebrafish, results in a reduced transcription and a complete absence of the protein. Lacking experimental structural data for zebrafish DG domains, the detailed mechanism for the observed mutation-induced destabilization of the DG complex and membrane damage, remained unclear. With the aim to contribute to a better clarification of the structure-function relationships featuring the DG complex, three-dimensional structural models of wild-type and mutant (V567D) C-terminal domain of alpha-DG from zebrafish were constructed by a template-based modelling approach. We then ran extensive molecular dynamics (MD) simulations to reveal the structural and dynamic properties of the C-terminal domain and to evaluate the effect of the single mutation on alpha-DG stability. A comparative study has been also carried out on our previously generated model of murine alpha-DG C-terminal domain including the I591D mutation, which is topologically equivalent to the V567D mutation found in zebrafish. Trajectories from MD simulations were analyzed in detail, revealing extensive structural disorder involving multiple beta-strands in the mutated variant of the zebrafish protein whereas local effects have been detected in the murine protein. A biochemical analysis of the murine alpha-DG mutant I591D confirmed a pronounced instability of the protein. Taken together, the computational and biochemical analysis suggest that the V567D/I591D mutation, belonging to the G beta-strand, plays a key role in inducing a destabilization of the alpha-DG C-terminal Ig-like domain that could possibly affect and propagate to the entire DG complex. The structural features herein identified may be of crucial help to understand the molecular basis of primary dystroglycanopathies.

  12. 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

  13. Zebrafish: swimming towards a role for fanconi genes in DNA repair.

    PubMed

    Scata, Kimberly A; El-Deiry, Wafik S

    2004-06-01

    The zebrafish, Danio rerio, has become a favorite model organism for geneticists and developmental biologists. Recently cancer biologists have turned to this tiny fish to help them unravel the mysteries of conserved pathways such as the Fanconi Anemia (FA) pathway. Although a relatively rare disease, the genes involved in FA are part of a large network of DNA damage response/repair genes. Liu and colleagues have recapitulated some of the clinical manifestations of human FA by knocking down the zebrafish FANC-D2 gene thereby providing a new model for probing the underlying causes of these phenotypes.

  14. 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.

  15. 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

  16. 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.

  17. Patient-derived xenograft in zebrafish embryos: a new platform for translational research in neuroendocrine tumors.

    PubMed

    Gaudenzi, Germano; Albertelli, Manuela; Dicitore, Alessandra; Würth, Roberto; Gatto, Federico; Barbieri, Federica; Cotelli, Franco; Florio, Tullio; Ferone, Diego; Persani, Luca; Vitale, Giovanni

    2017-08-01

    Preclinical research on neuroendocrine tumors usually involves immortalized cell lines and few animal models. In the present study we described an in vivo model based on patient-derived xenografts of neuroendocrine tumor cells in zebrafish (Danio rerio) embryos, allowing a rapid analysis of the angiogenic and invasive potential. Patient-derived neuroendocrine tumor cells were transplanted in 48 hours post-fertilization Tg(fli1a:EGFP) y1 zebrafish embryos that express enhanced green fluorescent protein in the entire vasculature. Neuroendocrine tumor cells, stained with CM-Dil, were injected into the subperidermal (perivitelline) space, close to the developing subintestinal venous plexus. A proper control group, represented by zebrafish injected with only D-PBS, was included in this study. Angiogenic and invasive potentials of each patient-derived xenograft were evaluated by both epifluorescence and confocal microscopes. Six out of eight neuroendocrine tumor samples were successfully transplanted in zebrafish embryos. Although the implanted tumor mass had a limited size (about 100 cells for embryos), patient-derived xenografts showed pro-angiogenic (5 cases) and invasive (6 cases) behaviors within 48 hours post injection. Patient-derived xenograft in zebrafish embryos appears to be a reliable in vivo preclinical model for neuroendocrine tumors, tumors with often limited cell availability. The rapidity of this procedure makes our model a promising platform to perform preclinical drug screening and opens a new scenario for personalized treatment in patients with neuroendocrine tumors.

  18. 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

  19. Characterization of the fusion core in zebrafish endogenous retroviral envelope protein

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

    Shi, Jian; State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, Hubei 430071; Zhang, Huaidong

    2015-05-08

    Zebrafish endogenous retrovirus (ZFERV) is the unique endogenous retrovirus in zebrafish, as yet, containing intact open reading frames of its envelope protein gene in zebrafish genome. Similarly, several envelope proteins of endogenous retroviruses in human and other mammalian animal genomes (such as syncytin-1 and 2 in human, syncytin-A and B in mouse) were identified and shown to be functional in induction of cell–cell fusion involved in placental development. ZFERV envelope protein (Env) gene appears to be also functional in vivo because it is expressible. After sequence alignment, we found ZFERV Env shares similar structural profiles with syncytin and other type Imore » viral envelopes, especially in the regions of N- and C-terminal heptad repeats (NHR and CHR) which were crucial for membrane fusion. We expressed the regions of N + C protein in the ZFERV Env (residues 459–567, including predicted NHR and CHR) to characterize the fusion core structure. We found N + C protein could form a stable coiled-coil trimer that consists of three helical NHR regions forming a central trimeric core, and three helical CHR regions packing into the grooves on the surface of the central core. The structural characterization of the fusion core revealed the possible mechanism of fusion mediated by ZFERV Env. These results gave comprehensive explanation of how the ancient virus infects the zebrafish and integrates into the genome million years ago, and showed a rational clue for discovery of physiological significance (e.g., medicate cell–cell fusion). - Highlights: • ZFERV Env shares similar structural profiles with syncytin and other type I viral envelopes. • The fusion core of ZFERV Env forms stable coiled-coil trimer including three NHRs and three CHRs. • The structural mechanism of viral entry mediated by ZFERV Env is disclosed. • The results are helpful for further discovery of physiological function of ZFERV Env in zebrafish.« less

  20. 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