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Sample records for chimeric zebrafish embryos

  1. Chimerism in piglets developed from aggregated cloned embryos.

    PubMed

    Huang, Yongye; Li, Zhanjun; Wang, Anfeng; Han, Xiaolei; Song, Yuning; Yuan, Lin; Li, Tianye; Wang, Bing; Lai, Liangxue; Ouyang, Hongsheng; Pang, Daxin

    2016-04-01

    Porcine chimeras are valuable in the study of pluripotency, embryogenesis and development. It would be meaningful to generate chimeric piglets from somatic cell nuclear transfer embryos. In this study, two cell lines expressing the fluorescent markers enhanced green fluorescent protein (EGFP) and tdTomato were used as donor cells to produce reconstructed embryos. Chimeric embryos were generated by aggregating two EGFP-cell derived embryos with two tdTomato-cell derived embryos at the 4-cell stage, and embryo transfer was performed when the aggregated embryos developed into blastocysts. Live porcine chimeras were successfully born and chimerism was observed by their skin color, gene integration, microsatellite loci composition and fluorescent protein expression. The chimeric piglets were largely composed of EGFP-expressing cells, and this phenomenon was possibly due to the hyper-methylation of the promoter of the tdTomato gene. In addition, the expression levels of tumorigenicity-related genes were altered after tdTomato transfection in bladder cancer cells. The results show that chimeric pigs can be produced by aggregating cloned embryos and that the developmental capability of the cloned embryo in the subsequent chimeric development could be affected by the growth characteristics of its donor cell. PMID:27239442

  2. Toxicity induced by emodin on zebrafish embryos.

    PubMed

    He, Qiuxia; Liu, Kechun; Wang, Sifeng; Hou, Hairong; Yuan, Yanqiang; Wang, Ximin

    2012-04-01

    Emodin, a widely available herbal remedy, has a variety of pharmacological actions and valuable clinical applications. The potential effect of emodin on zebrafish (Danio rerio) embryos was evaluated. Zebrafish embryos were incubated with 0.1-2 μg/mL of emodin from 7 hours to 6 days postfertilization (dpf). Emodin, at concentrations of 0.25 μg/mL and above, negatively affected embryo survival and hatching success. Emodin induced a large suite of abnormalities on zebrafish embryos, such as edema, crooked trunk, and abnormal morphogenesis. To elucidate the mechanism of action, the transcript levels of drug-metabolism genes (CYP3A) and a multiple drug-resistance gene (MDR1) were detected by reverse-transcript polymerase chain reaction. Embryos showed increases in mRNA accumulation of CYP3A and MDR1. The above-described results indicated that emodin impaired zebrafish embryo development and some organ morphogenesis, and CYP3A and MDR1 were involved in the process. These findings suggest that emodin was toxic to zebrafish lavae at relatively low concentrations. PMID:21834668

  3. Zebrafish Embryo Model of Bartonella henselae Infection

    PubMed Central

    Lima, Amorce; Cha, Byeong J.; Amin, Jahanshah; Smith, Lisa K.

    2014-01-01

    Abstract Bartonella henselae (Bh) is an emerging zoonotic pathogen that has been associated with a variety of human diseases, including bacillary angiomatosis that is characterized by vasoproliferative tumor-like lesions on the skin of some immunosuppressed individuals. The study of Bh pathogenesis has been limited to in vitro cell culture systems due to the lack of an animal model. Therefore, we wanted to investigate whether the zebrafish embryo could be used to model human infection with Bh. Our data showed that Tg(fli1:egfp)y1 zebrafish embryos supported a sustained Bh infection for 7 days with >10-fold bacterial replication when inoculated in the yolk sac. We showed that Bh recruited phagocytes to the site of infection in the Tg(mpx:GFP)uwm1 embryos. Infected embryos showed evidence of a Bh-induced angiogenic phenotype and an increase in the expression of genes encoding pro-inflammatory factors and pro-angiogenic markers. However, infection of zebrafish embryos with a deletion mutant in the major adhesin (BadA) resulted in little or no bacterial replication and a diminished host response, providing the first evidence that BadA is critical for in vivo infection. Thus, the zebrafish embryo provides the first practical model of Bh infection that will facilitate efforts to identify virulence factors and define molecular mechanisms of Bh pathogenesis. PMID:25026365

  4. Microbead Implantation in the Zebrafish Embryo

    PubMed Central

    Gerlach, Gary F.; Morales, Elvin E.; Wingert, Rebecca A.

    2015-01-01

    The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development. PMID:26274386

  5. Generation and Developmental Characteristics of Porcine Tetraploid Embryos and Tetraploid/diploid Chimeric Embryos

    PubMed Central

    He, Wenteng; Kong, Qingran; Shi, Yongqian; Xie, Bingteng; Jiao, Mingxia; Huang, Tianqing; Guo, Shimeng; Hu, Kui; Liu, Zhonghua

    2013-01-01

    The aim of this study was to optimize electrofusion conditions for generating porcine tetraploid (4n) embryos and produce tetraploid/diploid (4n/2n) chimeric embryos. Different electric field intensities were tested and 2 direct current (DC) pulses of 0.9 kV/cm for 30 μs was selected as the optimum condition for electrofusion of 2-cell embryos to produce 4n embryos. The fusion rate of 2-cell embryos and the development rate to blastocyst of presumably 4n embryos, reached 85.4% and 28.5%, respectively. 68.18% of the fused embryos were found to be 4n as demonstrated by fluorescent in situ hybridization (FISH). Although the number of blastomeres in 4n blastocysts was significantly lower than in 2n blastocysts (P < 0.05), there was no significant difference in developmental rates of blastocysts between 2n and 4n embryos (P > 0.05), suggesting that the blastocyst forming capacity in 4n embryos is similar to those in 2n embryos. Moreover, 4n/2n chimeric embryos were obtained by aggregation of 4n and 2n embryos. We found that the developmental rate and cell number of blastocysts of 4-cell (4n)/4-cell (2n) chimeric embryos were significantly higher than those of 2-cell (4n)/4-cell (2n), 4-cell (4n)/8-cell (2n), 4-cell (4n)/2-cell (2n) chimeric embryos (P < 0.05). Consistent with mouse chimeras, the majority of 4n cells contribute to the trophectoderm (TE), while the 2n cells are mainly present in the inner cell mass (ICM) of porcine 4n/2n chimeric embryos. Our study established a feasible and efficient approach to produce porcine 4n embryos and 4n/2n chimeric embryos. PMID:24120753

  6. Chemical screening with zebrafish embryos.

    PubMed

    Zhong, Hanbing; Lin, Shuo

    2011-01-01

    Functional chemicals are very useful tools for molecular biology studies. Due to its small size, large progeny clutch, and embryonic transparency, zebrafish serves as a superb in vivo animal model for chemical compound screens and characterization. During zebrafish embryogenesis, multiple developmental phenotypes can be easily examined under the microscope, therefore allowing a more comprehensive evaluation for identifying novel functional chemicals than cell-based assays. Ever since the first zebrafish-based chemical screen was conducted in the year 2000, many functional chemicals have been discovered using this strategy. In this chapter, we describe how to perform a typical zebrafish-based chemical screen and discuss the details of the protocol by using the example of the identification and characterization of two new Smo inhibitors with a Gli:GFP transgenic line. PMID:21318908

  7. Survival Study of Zebrafish Embryos Under Gamma Irradiation

    NASA Astrophysics Data System (ADS)

    Mena, Pamela; Allende, Miguel; Morales, José Roberto

    2010-08-01

    Zebrafish embryos have interesting biological properties for the study of human diseases. The present work uses zebrafish embryos in a particular development state, to study biological effects due to gamma radiation, arising from a calibrated 60Co source. Initially, the lethal dose for fish embryos was determined and subsequent irradiations were performed at sub-lethal doses, in order to study more subtle effects.

  8. Toxicity of chlorine to zebrafish embryos

    PubMed Central

    Kent, Michael L.; Buchner, Cari; Barton, Carrie; Tanguay, Robert L.

    2014-01-01

    Surface disinfection of fertilized fish eggs is widely used in aquaculture to reduce extraovum pathogens that may be released from brood fish during spawning, and this is routinely used in zebrafish (Danio rerio) research laboratories. Most laboratories use approximately 25 – 50 ppm unbuffered chlorine solution for 5 – 10 min. Treatment of embryos with chlorine has significant germicidal effects for many Gram-negative bacteria, viruses, and trophozoite stages of protozoa, it has reduced efficacy against cyst or spore stages of protozoa and certain Mycobacterium spp. Therefore, we evaluated the toxicity of unbufferred and buffered chlorine solution to embryos exposed at 6 or 24 hours post-fertilization (hpf) to determine if higher concentrations can be used for treating zebrafish embryos. Most of our experiments entailed using an outbred line (5D), with both mortality and malformations as endpoints. We found that 6 hpf embryos consistently were more resistant than 24 hpf embryos to the toxic effects of chlorine. Chlorine is more toxic and germicidal at lower pHs, and chlorine causes elevated pH. Consistent with this, we found that unbufferred chlorine solutions (pH ca 8–9) were less toxic at corresponding concentrations than solutions buffered to pH 7. Based on our findings here, we recommend treating 6 hpf embryos for 10 min and 24 hpf for 5 min with unbuffered chlorine solution at 100 ppm. One trial indicated that AB fish, a popular outbred line, are more susceptible to toxicity than 5Ds. This suggests that variability between zebrafish lines occurs, and researchers should evaluate each line or strain under their particular laboratory conditions for selection of the optimum chlorine treatment procedure. PMID:24429474

  9. Detection of Smad Signaling in Zebrafish Embryos.

    PubMed

    Liu, Xingfeng; Wang, Qiang; Meng, Anming

    2016-01-01

    Nodal and BMPs play critical roles in germ layer induction and patterning in early zebrafish embryos. Smad2/3 and Smad1/5/8 are intracellular effectors of Nodal and BMPs, respectively. These Smads regulate, in cooperation with other factors, transcription of hundreds of target genes in the nucleus. The activity and stability of Smads are regulated by phosphorylation modifications. To better understand the regulatory network of Smads-mediated signaling and its biological implications, it is necessary to monitor the signaling activity in an in vivo model system. In this chapter, we describe the methods used in zebrafish embryos for dissecting Smads signaling, including TGF-β/Nodal- and BMP-responsive luciferase reporter assays, Western blotting for Smads, co-immunoprecipitation for Smads and their interacting proteins, chromatin-immunoprecipitation for identification of Smad2-binding sites, and immunostaining for detection of active Smad1/5/8. PMID:26520131

  10. Developmental toxicity of cartap on zebrafish embryos.

    PubMed

    Zhou, Shengli; Dong, Qiaoxiang; Li, Shaonan; Guo, Jiangfeng; Wang, Xingxing; Zhu, Guonian

    2009-12-13

    Cartap is a widely used insecticide which belongs to a member of nereistoxin derivatives and acts on nicotinic acetylcholine receptor site. Its effects on aquatic species are of grave concern. To explore the potential developmental toxicity of cartap, zebrafish embryos were continually exposed, from 0.5 to 144h post-fertilization, to a range of concentrations of 25-1000microg/l. Results of the experiment indicated that cartap concentrations of 100microg/l and above negatively affected embryo survival and hatching success. Morphological analysis uncovered a large suite of abnormalities such as less melanin pigmentation, wavy notochord, crooked trunk, fuzzy somites, neurogenesis defects and vasculature defects. The most sensitive organ was proved to be the notochord which displayed defects at concentrations as low as 25microg/l. Both sensitivity towards exposure and localization of the defect were stage specific. To elucidate mechanisms concerning notochord, pigmentation, and hatching defects, enzyme assay, RT Q-PCR, and different exposure strategies were performed. For embryos with hatching failure, chorion was verified not to be digested, while removing cartap from exposure at early pre-hatching stage could significantly increase the hatching success. However, cartap was proved, via vitro assay, to have no effect on proteolytic activity of hatching enzyme. These findings implied that the secretion of hatching enzyme might be blocked. We also revealed that cartap inhibited the activity of melanogenic enzyme tyrosinase and matrix enzyme lysyl oxidase and induced expression of their genes. These suggested that cartap could impaired melanin pigmentation of zebrafish embryos through inhibiting tyrosinase activity, while inhibition of lysyl oxidase activity was responsible for notochord undulation, which subsequently caused somite defect, and at least partially responsible for defects in vasculature and neurogenesis. PMID:19923012

  11. Analysis of Oxidative Stress in Zebrafish Embryos

    PubMed Central

    Mugoni, Vera; Camporeale, Annalisa; Santoro, Massimo M.

    2014-01-01

    High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer. PMID:25046434

  12. Zebrafish embryo development in a microfluidic flow-through system.

    PubMed

    Wielhouwer, Eric M; Ali, Shaukat; Al-Afandi, Abdulrahman; Blom, Marko T; Riekerink, Marinus B Olde; Poelma, Christian; Westerweel, Jerry; Oonk, Johannes; Vrouwe, Elwin X; Buesink, Wilfred; vanMil, Harald G J; Chicken, Jonathan; van't Oever, Ronny; Richardson, Michael K

    2011-05-21

    The zebrafish embryo is a small, cheap, whole-animal model which may replace rodents in some areas of research. Unfortunately, zebrafish embryos are commonly cultured in microtitre plates using cell-culture protocols with static buffer replacement. Such protocols are highly invasive, consume large quantities of reagents and do not readily permit high-quality imaging. Zebrafish and rodent embryos have previously been cultured in static microfluidic drops, and zebrafish embryos have also been raised in a prototype polydimethylsiloxane setup in a Petri dish. Other than this, no animal embryo has ever been shown to undergo embryonic development in a microfluidic flow-through system. We have developed and prototyped a specialized lab-on-a-chip made from bonded layers of borosilicate glass. We find that zebrafish embryos can develop in the chip for 5 days, with continuous buffer flow at pressures of 0.005-0.04 MPa. Phenotypic effects were seen, but these were scored subjectively as 'minor'. Survival rates of 100% could be reached with buffer flows of 2 µL per well per min. High-quality imaging was possible. An acute ethanol exposure test in the chip replicated the same assay performed in microtitre plates. More than 100 embryos could be cultured in an area, excluding infrastructure, smaller than a credit card. We discuss how biochip technology, coupled with zebrafish larvae, could allow biological research to be conducted in massive, parallel experiments, at high speed and low cost. PMID:21491052

  13. A fully automated robotic system for microinjection of zebrafish embryos.

    PubMed

    Wang, Wenhui; Liu, Xinyu; Gelinas, Danielle; Ciruna, Brian; Sun, Yu

    2007-01-01

    As an important embodiment of biomanipulation, injection of foreign materials (e.g., DNA, RNAi, sperm, protein, and drug compounds) into individual cells has significant implications in genetics, transgenics, assisted reproduction, and drug discovery. This paper presents a microrobotic system for fully automated zebrafish embryo injection, which overcomes the problems inherent in manual operation, such as human fatigue and large variations in success rates due to poor reproducibility. Based on computer vision and motion control, the microrobotic system performs injection at a speed of 15 zebrafish embryos (chorion unremoved) per minute, with a survival rate of 98% (n = 350 embryos), a success rate of 99% (n = 350 embryos), and a phenotypic rate of 98.5% (n = 210 embryos). The sample immobilization technique and microrobotic control method are applicable to other biological injection applications such as the injection of mouse oocytes/embryos and Drosophila embryos to enable high-throughput biological and pharmaceutical research. PMID:17848993

  14. A Fully Automated Robotic System for Microinjection of Zebrafish Embryos

    PubMed Central

    Gelinas, Danielle; Ciruna, Brian; Sun, Yu

    2007-01-01

    As an important embodiment of biomanipulation, injection of foreign materials (e.g., DNA, RNAi, sperm, protein, and drug compounds) into individual cells has significant implications in genetics, transgenics, assisted reproduction, and drug discovery. This paper presents a microrobotic system for fully automated zebrafish embryo injection, which overcomes the problems inherent in manual operation, such as human fatigue and large variations in success rates due to poor reproducibility. Based on computer vision and motion control, the microrobotic system performs injection at a speed of 15 zebrafish embryos (chorion unremoved) per minute, with a survival rate of 98% (n = 350 embryos), a success rate of 99% (n = 350 embryos), and a phenotypic rate of 98.5% (n = 210 embryos). The sample immobilization technique and microrobotic control method are applicable to other biological injection applications such as the injection of mouse oocytes/embryos and Drosophila embryos to enable high-throughput biological and pharmaceutical research. PMID:17848993

  15. Phenotype classification of zebrafish embryos by supervised learning.

    PubMed

    Jeanray, Nathalie; Marée, Raphaël; Pruvot, Benoist; Stern, Olivier; Geurts, Pierre; Wehenkel, Louis; Muller, Marc

    2015-01-01

    Zebrafish is increasingly used to assess biological properties of chemical substances and thus is becoming a specific tool for toxicological and pharmacological studies. The effects of chemical substances on embryo survival and development are generally evaluated manually through microscopic observation by an expert and documented by several typical photographs. Here, we present a methodology to automatically classify brightfield images of wildtype zebrafish embryos according to their defects by using an image analysis approach based on supervised machine learning. We show that, compared to manual classification, automatic classification results in 90 to 100% agreement with consensus voting of biological experts in nine out of eleven considered defects in 3 days old zebrafish larvae. Automation of the analysis and classification of zebrafish embryo pictures reduces the workload and time required for the biological expert and increases the reproducibility and objectivity of this classification. PMID:25574849

  16. Phenotype Classification of Zebrafish Embryos by Supervised Learning

    PubMed Central

    Jeanray, Nathalie; Marée, Raphaël; Pruvot, Benoist; Stern, Olivier; Geurts, Pierre; Wehenkel, Louis; Muller, Marc

    2015-01-01

    Zebrafish is increasingly used to assess biological properties of chemical substances and thus is becoming a specific tool for toxicological and pharmacological studies. The effects of chemical substances on embryo survival and development are generally evaluated manually through microscopic observation by an expert and documented by several typical photographs. Here, we present a methodology to automatically classify brightfield images of wildtype zebrafish embryos according to their defects by using an image analysis approach based on supervised machine learning. We show that, compared to manual classification, automatic classification results in 90 to 100% agreement with consensus voting of biological experts in nine out of eleven considered defects in 3 days old zebrafish larvae. Automation of the analysis and classification of zebrafish embryo pictures reduces the workload and time required for the biological expert and increases the reproducibility and objectivity of this classification. PMID:25574849

  17. Neutron induced bystander effect among zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Ng, C. Y. P.; Kong, E. Y.; Kobayashi, A.; Suya, N.; Uchihori, Y.; Cheng, S. H.; Konishi, T.; Yu, K. N.

    2015-12-01

    The present paper reported the first-ever observation of neutron induced bystander effect (NIBE) using zebrafish (Danio rerio) embryos as the in vivo model. The neutron exposure in the present work was provided by the Neutron exposure Accelerator System for Biological Effect Experiments (NASBEE) facility at the National Institute of Radiological Sciences (NIRS), Chiba, Japan. Two different strategies were employed to induce NIBE, namely, through directly partnering and through medium transfer. Both results agreed with a neutron-dose window (20-50 mGy) which could induce NIBE. The lower dose limit corresponded to the threshold amount of neutron-induced damages to trigger significant bystander signals, while the upper limit corresponded to the onset of gamma-ray hormesis which could mitigate the neutron-induced damages and thereby suppress the bystander signals. Failures to observe NIBE in previous studies were due to using neutron doses outside the dose-window. Strategies to enhance the chance of observing NIBE included (1) use of a mono-energetic high-energy (e.g., between 100 keV and 2 MeV) neutron source, and (2) use of a neutron source with a small gamma-ray contamination. It appeared that the NASBEE facility used in the present study fulfilled both conditions, and was thus ideal for triggering NIBE.

  18. Abamectin induces rapid and reversible hypoactivity within early zebrafish embryos.

    PubMed

    Raftery, Tara D; Volz, David C

    2015-01-01

    During early zebrafish embryogenesis, spontaneous tail contractions represent the first sign of locomotion and result from innervation of primary motoneuron axons to target axial muscles. Based on a high-content screen, we previously demonstrated that exposure of zebrafish embryos to abamectin--an avermectin insecticide--from 5-25 hours post-fertilization (hpf) abolished spontaneous activity in the absence of effects on survival and gross morphology. Therefore, the objective of this study was to begin investigating the mechanism of abamectin-induced hypoactivity in zebrafish. Similar to 384-well plates, static exposure of embryos to abamectin from 5-25 hpf in glass beakers resulted in elimination of activity at low micromolar concentrations. However, abamectin did not affect neurite outgrowth from spinal motoneurons and, compared with exposure from 5-25 hpf, embryos were equally susceptible to abamectin-induced hypoactivity when exposures were initiated at 10 and 23 hpf. Moreover, immersion of abamectin-exposed embryos in clean water resulted in complete recovery of spontaneous activity relative to vehicle controls, suggesting that abamectin reversibly activated ligand-gated chloride channels and inhibited neurotransmission. To test this hypothesis, we pretreated embryos to vehicle or non-toxic concentrations of fipronil or endosulfan--two insecticides that antagonize the γ-aminobutyric acid (GABA) receptor--from 5-23 hpf, and then exposed embryos to vehicle or abamectin from 23-25 hpf. Interestingly, activity levels within abamectin-exposed embryos pretreated with either antagonist were similar to embryos exposed to vehicle alone. Using quantitative PCR and phylogenetic analyses, we then confirmed the presence of GABA receptor α1 and β2 subunits at 5, 10, and 23 hpf, and demonstrated that zebrafish GABA receptor subunits are homologous to mammalian GABA receptor subunits. Overall, our data collectively suggest that abamectin induces rapid and reversible

  19. Effects of perfluorinated compounds on development of zebrafish embryos.

    PubMed

    Zheng, Xin-Mei; Liu, Hong-Ling; Shi, Wei; Wei, Si; Giesy, John P; Yu, Hong-Xia

    2011-08-01

    Perfluorinated compounds (PFCs) have been widely used in industrial and consumer products and frequently detected in many environmental media. Potential reproductive effects of perfluorooctanesulfonate (PFOS), perfluorooctanoic acid (PFOA) and perfluorononanoic acid (PFNA) have been reported in mice, rats and water birds. PFOS and PFOA were also confirmed developing toxicants towards zebrafish embryos; however, the reported effect concentrations were contradictory. Polyfluorinated alkylated phosphate ester surfactants (including FC807) are precursor of PFOS and PFOA; however, there is no published information about the effects of FC807 and PFNA on zebrafish embryos. Therefore, this study was conducted to determine the effects of these four PFCs on zebrafish embryos. Normal fertilized zebrafish embryos were selected to be exposed to several concentrations of PFOA, PFNA, PFOS or FC807 in 24-well cell culture plates. A digital camera was used to image morphological anomalies of embryos with a stereomicroscope. Embryos were observed through matching up to 96-h post-fertilization (hpf) and rates of survival and abnormalities recorded. PFCs caused lethality in a concentration-dependent manner with potential toxicity in the order of PFOS > FC807 > PFNA > PFOA based on 72-h LC(50). Forty-eight-hour post-fertilization pericardial edema and 72- or 96-hpf spine crooked malformation were all observed. PFOA, PFNA, PFOS and FC807 all caused structural abnormalities using early stages of development of zebrafish. The PFCs all retarded the development of zebrafish embryos. The toxicity of the PFCs was related to the length of the PFC chain and functional groups. PMID:22828880

  20. Automated Zebrafish Chorion Removal and Single Embryo Placement: Optimizing Throughput of Zebrafish Developmental Toxicity Screens

    PubMed Central

    Mandrell, David; Truong, Lisa; Jephson, Caleb; Sarker, Mushfiqur R.; Moore, Aaron; Lang, Christopher; Simonich, Michael T.; Tanguay, Robert L.

    2012-01-01

    The potential of the developing zebrafish model for toxicology and drug discovery is limited by inefficient approaches to manipulating and chemically exposing zebrafish embryos—namely, manual placement of embryos into 96- or 384-well plates and exposure of embryos while still in the chorion, a barrier of poorly characterized permeability enclosing the developing embryo. We report the automated dechorionation of 1600 embryos at once at 4 h postfertilization (hpf) and placement of the dechorionated embryos into 96-well plates for exposure by 6 hpf. The process removed ≥95% of the embryos from their chorions with 2% embryo mortality by 24 hpf, and 2% of the embryos malformed at 120 hpf. The robotic embryo placement allocated 6-hpf embryos to 94.7% ± 4.2% of the wells in multiple 96-well trials. The rate of embryo mortality was 2.8% (43 of 1536) from robotic handling, the rate of missed wells was 1.2% (18 of 1536), and the frequency of multipicks was <0.1%. Embryo malformations observed at 24 hpf occurred nearly twice as frequently from robotic handling (16 of 864; 1.9%) as from manual pipetting (9 of 864; 1%). There was no statistical difference between the success of performing the embryo placement robotically or manually. PMID:22357610

  1. Evaluation of MWNT toxic effects on daphnia and zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Olasagasti, Maider; Alvarez, Noelia; Vera, Carolina; Rainieri, Sandra

    2009-05-01

    Organisms of daphnia (Daphnia magna) and zebrafish (Danio rerio) embryos were exposed to a range of different concentrations of COOH-functionalized MWCNT suspended in an aqueous solution of Tween 20. Immobilization of daphnia and growth retardation, inhibition and malformation of zebrafish embryos were the endpoints tested after 24 and 48 hours. Immobilization of daphnia could be observed from 3 to 16 ppm and an increasing mortality of zebrafish embryo was detected at all the concentration tested. To identify more subtle toxic effects, we took advantage of the extensive information available on the zebrafish genome and monitored by RT-PCR the expression patterns of different zebrafish genes that could act as toxicity bio-markers. At some of the concentrations tested, changes in the expression profiles of the genes examined were detected. Our results suggest that MWCNT could potentially represent a risk to human health and environment, therefore a wider range of concentrations and further testing of this molecules should be carried out to define possible limitations in their use.

  2. Transferrin-a modulates hepcidin expression in zebrafish embryos

    PubMed Central

    Gibert, Yann; Holzheimer, Jason L.; Lattanzi, Victoria J.; Burnett, Sarah F.; Dooley, Kimberly A.; Wingert, Rebecca A.; Zon, Leonard I.

    2009-01-01

    The iron regulatory hormone hepcidin is transcriptionally up-regulated in response to iron loading, but the mechanisms by which iron levels are sensed are not well understood. Large-scale genetic screens in the zebrafish have resulted in the identification of hypochromic anemia mutants with a range of mutations affecting conserved pathways in iron metabolism and heme synthesis. We hypothesized that transferrin plays a critical role both in iron transport and in regulating hepcidin expression in zebrafish embryos. Here we report the identification and characterization of the zebrafish hypochromic anemia mutant, gavi, which exhibits transferrin deficiency due to mutations in transferrin-a. Morpholino knockdown of transferrin-a in wild-type embryos reproduced the anemia phenotype and decreased somite and terminal gut iron staining, while coinjection of transferrin-a cRNA partially restored these defects. Embryos with transferrin-a or transferrin receptor 2 (TfR2) deficiency exhibited low levels of hepcidin expression, however anemia, in the absence of a defect in the transferrin pathway, failed to impair hepcidin expression. These data indicate that transferrin-a transports iron and that hepcidin expression is regulated by a transferrin-a–dependent pathway in the zebrafish embryo. PMID:19047682

  3. Toxic effects of brominated indoles and phenols on zebrafish embryos.

    PubMed

    Kammann, U; Vobach, M; Wosniok, W

    2006-07-01

    Organobromine compounds in the marine environment have been the focus of growing attention in past years. In contrast to anthropogenic brominated flame retardants, other brominated compounds are produced naturally, e.g., by common polychaete worms and algae. Brominated phenols and indoles assumed to be of biogenic origin have been detected in water and sediment extracts from the German Bight. These substances as well as some of their isomers have been tested with the zebrafish embryo test and were found to cause lethal as well as nonlethal malformations. The zebrafish test was able to detect a log K(OW)-related toxicity for bromophenols, suggesting nonpolar narcosis as a major mode of action. Different effect patterns could be observed for brominated indoles and bromophenols. The comparison of effective concentrations in the zebrafish embryo test with the concentrations determined in water samples suggests the possibility that brominated indoles may affect early life stages of marine fish species in the North Sea. PMID:16418895

  4. Myomaker mediates fusion of fast myocytes in zebrafish embryos

    SciTech Connect

    Landemaine, Aurélie; Rescan, Pierre-Yves; Gabillard, Jean-Charles

    2014-09-05

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

  5. Developmental toxicity assay using high content screening of zebrafish embryos

    PubMed Central

    Lantz-McPeak, Susan; Guo, Xiaoqing; Cuevas, Elvis; Dumas, Melanie; Newport, Glenn D.; Ali, Syed F.; Paule, Merle G.; Kanungo, Jyotshna

    2016-01-01

    Typically, time-consuming standard toxicological assays using the zebrafish (Danio rerio) embryo model evaluate mortality and teratogenicity after exposure during the first 2 days post-fertilization. Here we describe an automated image-based high content screening (HCS) assay to identify the teratogenic/embryotoxic potential of compounds in zebrafish embryos in vivo. Automated image acquisition was performed using a high content microscope system. Further automated analysis of embryo length, as a statistically quantifiable endpoint of toxicity, was performed on images post-acquisition. The biological effects of ethanol, nicotine, ketamine, caffeine, dimethyl sulfoxide and temperature on zebrafish embryos were assessed. This automated developmental toxicity assay, based on a growth-retardation endpoint should be suitable for evaluating the effects of potential teratogens and developmental toxicants in a high throughput manner. This approach can significantly expedite the screening of potential teratogens and developmental toxicants, thereby improving the current risk assessment process by decreasing analysis time and required resources. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. PMID:24871937

  6. Antioxidant Rescue of Selenomethionine-Induced Teratogenesis in Zebrafish Embryos.

    PubMed

    Arnold, M C; Forte, J E; Osterberg, J S; Di Giulio, R T

    2016-02-01

    Selenium (Se) is an essential micronutrient that can be found at toxic concentrations in surface waters contaminated by runoff from agriculture and coal mining. Zebrafish (Danio rerio) embryos were exposed to aqueous Se in the form of selenate, selenite, and l-selenomethionine (SeMet) in an attempt to determine if oxidative stress plays a role in selenium embryo toxicity. Selenate and selenite exposure did not induce embryo deformities (lordosis and craniofacial malformation). l-selenomethionine, however, induced significantly higher deformity rates at 100 µg/L compared with controls. SeMet exposure induced a dose-dependent increase in the catalytic subunit of glutamate-cysteine ligase (gclc) and reached an 11.7-fold increase at 100 µg/L. SeMet exposure also reduced concentrations of TGSH, RGSH, and the TGSH:GSSG ratio. Pretreatment with 100 µM N-acetylcysteine significantly reduced deformities in the zebrafish embryos secondarily treated with 400 µg/L SeMet from approximately 50–10 % as well as rescued all three of the significant glutathione level differences seen with SeMet alone. Selenite exposure induced a 6.6-fold increase in expression of the glutathione-S-transferase pi class 2 (gstp2) gene, which is involved in xenobiotic transformation and possibly oxidative stress. These results suggest that aqueous exposure to SeMet can induce significant embryonic teratogenesis in zebrafish that are at least partially attributed to oxidative stress. PMID:26498942

  7. Nanomaterial Toxicity Screening in Developing Zebrafish Embryos

    EPA Science Inventory

    To assess nanomaterial vertebrate toxicity, a high-content screening assay was created using developing zebrafish, Danio rerio. This included a diverse group of nanomaterials (n=42 total) ranging from metallic (Ag, Au) and metal oxide (CeO2, CuO, TiO2, ZnO) nanoparticles, to non...

  8. Detecting Developmental Neurotoxicants Using Zebrafish Embryos

    EPA Science Inventory

    As part of EPA’s program on the screening and prioritization of chemicals for developmental neurotoxicity, a rapid, cost-effective in vivo vertebrate screen is being developed using an alternative species approach. Zebrafish (Danio rerio), a small freshwater fish with external f...

  9. Leptin expression affects metabolic rate in zebrafish embryos (D. rerio)

    PubMed Central

    Dalman, Mark R.; Liu, Qin; King, Mason D.; Bagatto, Brian; Londraville, Richard L.

    2013-01-01

    We used antisense morpholino oligonucleotide technology to knockdown leptin-(A) gene expression in developing zebrafish embryos and measured its effects on metabolic rate and cardiovascular function. Using two indicators of metabolic rate, oxygen consumption was significantly lower in leptin morphants early in development [<48 hours post-fertilization (hpf)], while acid production was significantly lower in morphants later in development (>48 hpf). Oxygen utilization rates in <48 hpf embryos and acid production in 72 hpf embryos could be rescued to that of wildtype embryos by recombinant leptin coinjected with antisense morpholino. Leptin is established to influence metabolic rate in mammals, and these data suggest leptin signaling also influences metabolic rate in fishes. PMID:23847542

  10. Hormetic effect induced by depleted uranium in zebrafish embryos.

    PubMed

    Ng, C Y P; Cheng, S H; Yu, K N

    2016-06-01

    The present work studied the hormetic effect induced by uranium (U) in embryos of zebrafish (Danio rerio) using apoptosis as the biological endpoint. Hormetic effect is characterized by biphasic dose-response relationships showing a low-dose stimulation and a high-dose inhibition. Embryos were dechorionated at 4h post fertilization (hpf), and were then exposed to 10 or 100μg/l depleted uranium (DU) in uranyl acetate solutions from 5 to 6 hpf. For exposures to 10μg/l DU, the amounts of apoptotic signals in the embryos were significantly increased at 20 hpf but were significantly decreased at 24 hpf, which demonstrated the presence of U-induced hormesis. For exposures to 100μg/l DU, the amounts of apoptotic signals in the embryos were significantly increased at 20, 24 and 30 hpf. Hormetic effect was not shown but its occurrence between 30 and 48 hpf could not be ruled out. In conclusion, hormetic effect could be induced in zebrafish embryos in a concentration- and time-dependent manner. PMID:27060238

  11. Does perfluorooctane sulfonate (PFOS) act as chemosensitizer in zebrafish embryos?

    PubMed

    Keiter, Susanne; Burkhardt-Medicke, Kathleen; Wellner, Peggy; Kais, Britta; Färber, Harald; Skutlarek, Dirk; Engwall, Magnus; Braunbeck, Thomas; Keiter, Steffen H; Luckenbach, Till

    2016-04-01

    Earlier studies have shown that perfluorooctane sulfonate (PFOS) increases the toxicity of other chemicals by enhancing their uptake by cells and tissues. The present study aimed at testing whether the underlying mechanism of enhanced uptake of chemicals by zebrafish (Danio rerio) embryos in the presence of PFOS is by interference of this compound with the cellular efflux transporter Abcb4. Modifications of uptake/clearance and toxicity of two Abcb4 substrates, the fluorescent dye rhodamine B (RhB) and vinblastine, by PFOS were evaluated using 24 and 48h post-fertilization (hpf) embryos. Upon 90min exposure of 24hpf embryos to 1μM RhB and different PFOS concentrations (3-300μM) accumulation of RhB in zebrafish was increased by up to 11.9-fold compared to controls, whereas RhB increases in verapamil treatments were 1.7-fold. Co-administration of PFOS and vinblastine in exposures from 0 to 48hpf resulted in higher vinblastine-caused mortalities in zebrafish embryos indicating increased uptake of this compound. Interference of PFOS with zebrafish Abcb4 activity was further studied using recombinant protein obtained with the baculovirus expression system. PFOS lead to a concentration-dependent decrease of the verapamil-stimulated Abcb4 ATPase activity; at higher PFOS concentrations (250, 500μM), also the basal ATPase activity was lowered indicating PFOS to be an Abcb4 inhibitor. In exposures of 48hpf embryos to a very high RhB concentration (200μM), accumulation of RhB in embryo tissue and adsorption to the chorion were increased in the presence of 50 or 100μM PFOS. In conclusion, the results indicate that PFOS acts as inhibitor of zebrafish Abcb4; however, the exceptionally large PFOS-caused effect amplitude of RhB accumulation in the 1μM RhB experiments and the clear PFOS effects in the experiments with 200μM RhB suggest that an additional mechanism appears to be responsible for the potential of PFOS to enhance uptake of Abcb4 substrates. PMID:26803730

  12. Trimethyltin chloride inhibits neuronal cell differentiation in zebrafish embryo neurodevelopment.

    PubMed

    Kim, Jin; Kim, C-Yoon; Song, Juha; Oh, Hanseul; Kim, Cheol-Hee; Park, Jae-Hak

    2016-01-01

    Trimethyltin chloride (TMT) is a neurotoxicant widely present in the aquatic environment, primarily from effluents of the plastic industry. It is known to cause acute neuronal death in the limbic-cerebellar system, particularly in the hippocampus. However, relatively few studies have estimated the effects of TMT toxicity on neurodevelopment. In this study, we confirmed the dose-dependent effects of TMT on neurodevelopmental stages through analysis of morphological changes and fluorescence assays using HuC-GFP and olig2-dsRed transgenic zebrafish embryos. In addition, we analyzed the expression of genes and proteins related to neurodevelopment. Exposure of embryos to TMT for 4days post fertilization (dpf) elicited a concentration-related decrease in body length and increase in axial malformation. TMT affected the fluorescent CNS structure by decreasing pattern of HuC-GFP and olig2-dsRed transgenic zebrafish. In addition, it significantly modulated the expression patterns of Sonic hedgehog a (Shha), Neurogenin1 (Ngn1), Embryonic lethal abnormal vision like protein 3 (Elavl3), and Glial fibrillary acidic protein (Gfap). The overexpression of Shha and Ngn1, and downregulation of Elavl3 and Gfap, indicate repression of proneural cell differentiation. Our study demonstrates that TMT inhibits specific neurodevelopmental stages in zebrafish embryos and suggests a possible mechanism for the toxicity of TMT in vertebrate neurodevelopment. PMID:26687135

  13. Isolation and Characterization of Single Cells from Zebrafish Embryos.

    PubMed

    Samsa, Leigh Ann; Fleming, Nicole; Magness, Scott; Qian, Li; Liu, Jiandong

    2016-01-01

    The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies. PMID:27022828

  14. Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo.

    PubMed

    Percival, Stefanie M; Parant, John M

    2016-01-01

    Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraB(hi1045) (,) and esco2(hi2865) mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease. PMID:27501381

  15. Uptake and transient expression of chimeric genes in seed-derived embryos.

    PubMed Central

    Töpfer, R; Gronenborn, B; Schell, J; Steinbiss, H H

    1989-01-01

    Uptake of DNA in dry and viable embryos of wheat by imbibition in DNA solution was detected by monitoring the transient expression of chimeric genes. Gene expression vectors used in this study contained a neomycin phosphotransferase (NPT) II reporter gene fused to various promoters. Some of the chimeric "neo" genes were shown to yield reproducibly NPT II activity in germinating embryos. This NPT II activity was increased markedly when the neo genes were carried by a vector capable of autonomous replication. Dimers of wheat dwarf virus, a monopartite gemini virus, were thus shown to be effective in amplifying the transient expressed NPT II activity in embryos of several cereals. These and other observations indicate that the observed transient expression really results from DNA uptake and expression in plant embryo cells and is not due to contaminating microorganisms. PMID:2562504

  16. Whole-mount single molecule FISH method for zebrafish embryo.

    PubMed

    Oka, Yuma; Sato, Thomas N

    2015-01-01

    Noise in gene expression renders cells more adaptable to changing environment by imposing phenotypic and functional heterogeneity on genetically identical individual cells. Hence, quantitative measurement of noise in gene expression is essential for the study of biological processes in cells. Currently, there are two complementary methods for quantitatively measuring noise in gene expression at the single cell level: single molecule FISH (smFISH) and single cell qRT-PCR (or single cell RNA-seq). While smFISH has been developed for culture cells, tissue sections and whole-mount invertebrate organisms, the method has not been reported for whole-mount vertebrate organisms. Here, we report an smFISH method that is suitable for whole-mount zebrafish embryo, a popular vertebrate model organism for the studies of development, physiology and disease. We show the detection of individual transcripts for several cell-type specific and ubiquitously expressed genes at the single cell level in whole-mount zebrafish embryo. We also demonstrate that the method can be adapted to detect two different genes in individual cells simultaneously. The whole-mount smFISH method described in this report is expected to facilitate the study of noise in gene expression and its role in zebrafish, a vertebrate animal model relevant to human biology. PMID:25711926

  17. Transcriptomic response of zebrafish embryos to polyaminoamine (PAMAM) dendrimers.

    PubMed

    Oliveira, Eva; Casado, Marta; Faria, Melissa; Soares, Amadeu M V M; Navas, José María; Barata, Carlos; Piña, Benjamin

    2014-08-01

    The progressive practical applications of engineered nanoparticles results in their ever-increasing release into the environment. Accurate assessment of their environmental and health risks requires the development of methods allowing their monitoring in different environmental compartments and the evaluation of their potential toxicity at different levels of organization. Toxic effects of third-generation (G3) and fourth-generation (G4) poly(amidoamine) dendrimers (ethylenediamine cored, imine-terminated) were assessed on zebrafish embryos during the first two days post-fertilization. Particle characterization by dynamic light scattering showed no tendency to form aggregates in the assay conditions. G3 particles showed somewhat a higher acute toxicity than G4 particles, with LC50 values of 1.8 and 2.3 mg/L, respectively. At sublethal concentrations, both particles affected the zebrafish transcriptome following similar patterns, suggesting a similar mode of action. About 700 transcripts were affected by at least one of the treatments, following a pattern with significant correlations to the effects of bacterial infection in zebrafish embryos. We concluded that the response to G3 and G4 dendrimers was consistent with the activation of the innate immune response, a still unreported potential effect of these particles. These data may contribute to the characterization of hazards of these nanomaterials for both human health and the environment. PMID:24266889

  18. Strategies for analyzing cardiac phenotypes in the zebrafish embryo.

    PubMed

    Houk, A R; Yelon, D

    2016-01-01

    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

  19. Microcystin-LR exposure induces developmental neurotoxicity in zebrafish embryo.

    PubMed

    Wu, Qin; Yan, Wei; Liu, Chunsheng; Li, Li; Yu, Liqin; Zhao, Sujuan; Li, Guangyu

    2016-06-01

    Microcystin-LR (MCLR) is a commonly acting potent hepatotoxin and has been pointed out of potentially causing developmental neurotoxicity, but the exact mechanism is little known. In this study, zebrafish embryos were exposed to 0, 0.8, 1.6 or 3.2 mg/L MCLR for 120 h. MCLR exposure through submersion caused serious hatching delay and body length decrease. The content of MCLR in zebrafish larvae was analyzed and the results demonstrated that MCLR can accumulate in zebrafish larvae. The locomotor speed of zebrafish larvae was decreased. Furthermore, the dopamine and acetylcholine (ACh) content were detected to be significantly decreased in MCLR exposure groups. And the acetylcholinesterase (AChE) activity was significantly increased after exposure to 1.6 and 3.2 mg/L MCLR. The transcription pattern of manf, chrnα7 and ache gene was consistent with the change of the dopamine content, ACh content and AChE activity. Gene expression involved in the development of neurons was also measured. ɑ1-tubulin and shha gene expression were down-regulated, whereas mbp and gap43 gene expression were observed to be significantly up-regulated upon exposure to MCLR. The above results indicated that MCLR-induced developmental toxicity might attribute to the disorder of cholinergic system, dopaminergic signaling, and the development of neurons. PMID:27038211

  20. Low Temperature Mitigates Cardia Bifida in Zebrafish Embryos

    PubMed Central

    Lin, Che-Yi; Huang, Cheng-Chen; Wang, Wen-Der; Hsiao, Chung-Der; Cheng, Ching-Feng; Wu, Yi-Ting; Lu, Yu-Fen; Hwang, Sheng-Ping L.

    2013-01-01

    The coordinated migration of bilateral cardiomyocytes and the formation of the cardiac cone are essential for heart tube formation. We investigated gene regulatory mechanisms involved in myocardial migration, and regulation of the timing of cardiac cone formation in zebrafish embryos. Through screening of zebrafish treated with ethylnitrosourea, we isolated a mutant with a hypomorphic allele of mil (s1pr2)/edg5, called s1pr2as10 (as10). Mutant embryos with this allele expressed less mil/edg5 mRNA and exhibited cardia bifida prior to 28 hours post-fertilization. Although the bilateral hearts of the mutants gradually fused together, the resulting formation of two atria and one tightly-packed ventricle failed to support normal blood circulation. Interestingly, cardia bifida of s1pr2as10 embryos could be rescued and normal circulation could be restored by incubating the embryos at low temperature (22.5°C). Rescue was also observed in gata5 and bon cardia bifida morphants raised at 22.5°C. The use of DNA microarrays, digital gene expression analyses, loss-of-function, as well as mRNA and protein rescue experiments, revealed that low temperature mitigates cardia bifida by regulating the expression of genes encoding components of the extracellular matrix (fibronectin 1, tenascin-c, tenascin-w). Furthermore, the addition of N-acetyl cysteine (NAC), a reactive oxygen species (ROS) scavenger, significantly decreased the effect of low temperature on mitigating cardia bifida in s1pr2as10 embryos. Our study reveals that temperature coordinates the development of the heart tube and somitogenesis, and that extracellular matrix genes (fibronectin 1, tenascin-c and tenascin-w) are involved. PMID:23922799

  1. Proteomic analysis of zebrafish embryos exposed to simulated-microgravity

    NASA Astrophysics Data System (ADS)

    Hang, Xiaoming; Ma, Wenwen; Wang, Wei; Liu, Cong; Sun, Yeqing

    Microgravity can induce a serial of physiological and pathological changes in human body, such as cardiovascular functional disorder, bone loss, muscular atrophy and impaired immune system function, etc. In this research, we focus on the influence of microgravity to vertebrate embryo development. As a powerful model for studying vertebrate development, zebrafish embryos at 8 hpf (hour past fertilization) and 24 hpf were placed into a NASA developed bioreac-tor (RCCS) to simulate microgravity for 64 and 48 hours, respectively. The same number of control embryos from the same parents were placed in a tissue culture dish at the same temper-ature of 28° C. Each experiment was repeated 3 times and analyzed by two-dimensional (2-D) gel electrophoresis. Image analysis of silver stained 2-D gels revealed that 64 from total 292 protein spots showed quantitative and qualitative variations that were significantly (P<0.05) and reproducibly different between simulate-microgravity treatment and the stationary control samples. 4 protein spots with significant expression alteration (P<0.01) were excised from 2-D gels and analyzed by MALDI-TOF/TOF mass spectra primarily. Of these proteins, 3 down-regulated proteins were identified as bectin 2, centrosomal protein of 135kDa and tropomyosin 4, while the up-regulated protein was identified as creatine kinase muscle B. Other protein spots showed significant expression alteration will be identified successively and the corresponding genes expression will also be measured by Q-PCR method at different development stages. The data presented in this study illustrate that zebrafish embryo can be significantly induced by microgravity on the expression of proteins involved in bone and muscle formation. Key Words: Danio rerio; Simulated-microgravity; Proteomics

  2. Quantum Dot Nanotoxicity Assessment Using the Zebrafish Embryo

    PubMed Central

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

    2009-01-01

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

  3. Three-dimensional printed millifluidic devices for zebrafish embryo tests.

    PubMed

    Zhu, Feng; Skommer, Joanna; Macdonald, Niall P; Friedrich, Timo; Kaslin, Jan; Wlodkowic, Donald

    2015-07-01

    Implementations of Lab-on-a-Chip technologies for in-situ analysis of small model organisms and embryos (both invertebrate and vertebrate) are attracting an increasing interest. A significant hurdle to widespread applications of microfluidic and millifluidic devices for in-situ analysis of small model organisms is the access to expensive clean room facilities and complex microfabrication technologies. Furthermore, these resources require significant investments and engineering know-how. For example, poly(dimethylsiloxane) soft lithography is still largely unattainable to the gross majority of biomedical laboratories willing to pursue development of chip-based platforms. They often turn instead to readily available but inferior classical solutions. We refer to this phenomenon as workshop-to-bench gap of bioengineering science. To tackle the above issues, we examined the capabilities of commercially available Multi-Jet Modelling (MJM) and Stereolithography (SLA) systems for low volume fabrication of optical-grade millifluidic devices designed for culture and biotests performed on millimetre-sized specimens such as zebrafish embryos. The selected 3D printing technologies spanned a range from affordable personal desktop systems to high-end professional printers. The main motivation of our work was to pave the way for off-the-shelf and user-friendly 3D printing methods in order to rapidly and inexpensively build optical-grade millifluidic devices for customized studies on small model organisms. Compared with other rapid prototyping technologies such as soft lithography and infrared laser micromachining in poly(methyl methacrylate), we demonstrate that selected SLA technologies can achieve user-friendly and rapid production of prototypes, superior feature reproduction quality, and comparable levels of optical transparency. A caution need to be, however, exercised as majority of tested SLA and MJM resins were found toxic and caused significant developmental abnormalities

  4. Three-dimensional printed millifluidic devices for zebrafish embryo tests

    PubMed Central

    Zhu, Feng; Skommer, Joanna; Macdonald, Niall P.; Friedrich, Timo; Kaslin, Jan; Wlodkowic, Donald

    2015-01-01

    Implementations of Lab-on-a-Chip technologies for in-situ analysis of small model organisms and embryos (both invertebrate and vertebrate) are attracting an increasing interest. A significant hurdle to widespread applications of microfluidic and millifluidic devices for in-situ analysis of small model organisms is the access to expensive clean room facilities and complex microfabrication technologies. Furthermore, these resources require significant investments and engineering know-how. For example, poly(dimethylsiloxane) soft lithography is still largely unattainable to the gross majority of biomedical laboratories willing to pursue development of chip-based platforms. They often turn instead to readily available but inferior classical solutions. We refer to this phenomenon as workshop-to-bench gap of bioengineering science. To tackle the above issues, we examined the capabilities of commercially available Multi-Jet Modelling (MJM) and Stereolithography (SLA) systems for low volume fabrication of optical-grade millifluidic devices designed for culture and biotests performed on millimetre-sized specimens such as zebrafish embryos. The selected 3D printing technologies spanned a range from affordable personal desktop systems to high-end professional printers. The main motivation of our work was to pave the way for off-the-shelf and user-friendly 3D printing methods in order to rapidly and inexpensively build optical-grade millifluidic devices for customized studies on small model organisms. Compared with other rapid prototyping technologies such as soft lithography and infrared laser micromachining in poly(methyl methacrylate), we demonstrate that selected SLA technologies can achieve user-friendly and rapid production of prototypes, superior feature reproduction quality, and comparable levels of optical transparency. A caution need to be, however, exercised as majority of tested SLA and MJM resins were found toxic and caused significant developmental abnormalities

  5. Embryos of the zebrafish Danio rerio in studies of non-targeted effects of ionizing radiation.

    PubMed

    Choi, V W Y; Yu, K N

    2015-01-01

    The use of embryos of the zebrafish Danio rerio as an in vivo tumor model for studying non-targeted effects of ionizing radiation was reviewed. The zebrafish embryo is an animal model, which enables convenient studies on non-targeted effects of both high-linear-energy-transfer (LET) and low-LET radiation by making use of both broad-beam and microbeam radiation. Zebrafish is also a convenient embryo model for studying radiobiological effects of ionizing radiation on tumors. The embryonic origin of tumors has been gaining ground in the past decades, and efforts to fight cancer from the perspective of developmental biology are underway. Evidence for the involvement of radiation-induced genomic instability (RIGI) and the radiation-induced bystander effect (RIBE) in zebrafish embryos were subsequently given. The results of RIGI were obtained for the irradiation of all two-cell stage cells, as well as 1.5 hpf zebrafish embryos by microbeam protons and broad-beam alpha particles, respectively. In contrast, the RIBE was observed through the radioadaptive response (RAR), which was developed against a subsequent challenging dose that was applied at 10 hpf when <0.2% and <0.3% of the cells of 5 hpf zebrafish embryos were exposed to a priming dose, which was provided by microbeam protons and broad-beam alpha particles, respectively. Finally, a perspective on the field, the need for future studies and the significance of such studies were discussed. PMID:24176822

  6. Proteomic Analysis of Anoxia Tolerance in the Developing Zebrafish Embryo

    PubMed Central

    Mendelsohn, Bryce A.; Malone, James P.; Townsend, R. Reid; Gitlin, Jonathan D.

    2009-01-01

    While some species and tissue types are injured by oxygen deprivation, anoxia tolerant organisms display a protective response that has not been fully elucidated and is well-suited to genomic and proteomic analysis. However, such methodologies have focused on transcriptional responses, prolonged anoxia, or have used cultured cells or isolated tissues. In this study of intact zebrafish embryos, a species capable of >24 h survival in anoxia, we have utilized 2D difference in gel electrophoresis to identify changes in the proteomic profile caused by near-lethal anoxic durations as well as acute anoxia (1 h), a timeframe relevant to ischemic events in human disease when response mechanisms are largely limited to post-transcriptional and post-translational processes. We observed a general stabilization of the proteome in anoxia. Proteins involved in oxidative phosphorylation, antioxidant defense, transcription, and translation changed over this time period. Among the largest proteomic alterations was that of muscle cofilin 2, implicating the regulation of the cytoskeleton and actin assembly in the adaptation to acute anoxia. These studies in an intact embryo highlight proteomic components of an adaptive response to anoxia in a model organism amenable to genetic analysis to permit further mechanistic insight into the phenomenon of anoxia tolerance. PMID:20403745

  7. Ethical acceptability of research on human-animal chimeric embryos: summary of opinions by the Japanese Expert Panel on Bioethics.

    PubMed

    Mizuno, Hiroshi; Akutsu, Hidenori; Kato, Kazuto

    2015-01-01

    Human-animal chimeric embryos are embryos obtained by introducing human cells into a non-human animal embryo. It is envisaged that the application of human-animal chimeric embryos may make possible many useful research projects including producing three-dimensional human organs in animals and verification of the pluripotency of human ES cells or iPS cells in vivo. The use of human-animal chimeric embryos, however, raises several ethical and moral concerns. The most fundamental one is that human-animal chimeric embryos possess the potential to develop into organisms containing human-derived tissue, which may lead to infringing upon the identity of the human species, and thus impairing human dignity. The Japanese Expert Panel on Bioethics in the Cabinet Office carefully considered the scientific significance and ethical acceptability of the issue and released its "Opinions regarding the handling of research using human-animal chimeric embryos". The Panel proposed a framework of case-by-case review, and suggested that the following points must be carefully reviewed from the perspective of ethical acceptability: (a) Types of animal embryos and types of animals receiving embryo transfers, particularly in dealing with non-human primates; (b) Types of human cells and organs intended for production, particularly in dealing with human nerve or germ cells; and (c) Extent of the period required for post-transfer studies. The scientific knowledge that can be gained from transfer into an animal uterus and from the production of an individual must be clarified to avoid unnecessary generation of chimeric animals. The time is ripe for the scientific community and governments to start discussing the ethical issues for establishing a global consensus. PMID:26694481

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  9. Robotic injection of zebrafish embryos for high-throughput screening in disease models.

    PubMed

    Spaink, Herman P; Cui, Chao; Wiweger, Malgorzata I; Jansen, Hans J; Veneman, Wouter J; Marín-Juez, Rubén; de Sonneville, Jan; Ordas, Anita; Torraca, Vincenzo; van der Ent, Wietske; Leenders, William P; Meijer, Annemarie H; Snaar-Jagalska, B Ewa; Dirks, Ron P

    2013-08-15

    The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines. PMID:23769806

  10. Assessment of developmental delay in the zebrafish embryo teratogenicity assay.

    PubMed

    Teixidó, E; Piqué, E; Gómez-Catalán, J; Llobet, J M

    2013-02-01

    In this study we analyzed some aspects of the assessment of developmental delay in the zebrafish embryotoxicity/teratogenicity test and explored the suitability of acetylcholinesterase (AChE) activity as a biochemical marker and as a higher throughput alternative to morphological endpoints such as head-trunk angle, tail length and morphological score. Embryos were exposed from 4 to 52 h post-fertilization (hpf) to a selection of known embryotoxic/teratogen compounds (valproic acid, retinoic acid, caffeine, sodium salicylate, glucose, hydroxyurea, methoxyacetic acid, boric acid and paraoxon-methyl) over a concentration range. They were evaluated for AChE activity, head-trunk angle, tail length and several qualitative parameters integrated in a morphological score. In general, the different patterns of the concentration-response curves allowed distinguishing between chemicals that produced growth retardation (valproic and methoxyacetic acid) and chemicals that produced non-growth-delay related malformations. An acceptable correlation between the morphological score, AChE activity and head-trunk angle as markers of developmental delay was observed, being AChE activity particularly sensitive to detect delay in the absence of malformations. PMID:22898132

  11. Multifaceted toxicity assessment of catalyst composites in transgenic zebrafish embryos.

    PubMed

    Jang, Gun Hyuk; Lee, Keon Yong; Choi, Jaewon; Kim, Sang Hoon; Lee, Kwan Hyi

    2016-09-01

    Recent development in the field of nanomaterials has given rise into the inquiries regarding the toxicological characteristics of the nanomaterials. While many individual nanomaterials have been screened for their toxicological effects, composites that accompany nanomaterials are not common subjects to such screening through toxicological assessment. One of the widely used composites that accompany nanomaterials is catalyst composite used to reduce air pollution, which was selected as a target composite with nanomaterials for the multifaceted toxicological assessment. As existing studies did not possess any significant data regarding such catalyst composites, this study focuses on investigating toxicological characteristics of catalyst composites from various angles in both in-vitro and in-vivo settings. Initial toxicological assessment on catalyst composites was conducted using HUVECs for cell viability assays, and subsequent in-vivo assay regarding their direct influence on living organisms was done. The zebrafish embryo and its transgenic lines were used in the in-vivo assays to obtain multifaceted analytic results. Data obtained from the in-vivo assays include blood vessel formation, mutated heart morphology, and heart functionality change. Our multifaceted toxicological assessment pointed out that chemical composites augmented with nanomaterials can too have toxicological threat as much as individual nanomaterials do and alarms us with their danger. This manuscript provides a multifaceted assessment for composites augmented with nanomaterials, of which their toxicological threats have been overlooked. PMID:27364464

  12. Antisense inhibition of cyclin D1 expression is equivalent to flavopiridol for radiosensitization of zebrafish embryos

    SciTech Connect

    McAleer, Mary Frances; Duffy, Kevin T.; Davidson, William R.; Kari, Gabor; Dicker, Adam P.; Rodeck, Ulrich; Wickstrom, Eric . E-mail: eric@tesla.jci.tju.edu

    2006-10-01

    Purpose: Flavopiridol, a small molecule pan-cyclin inhibitor, has been shown to enhance Radiation response of tumor cells both in vitro and in vivo. The clinical utility of flavopiridol, however, is limited by toxicity, previously attributed to pleiotropic inhibitory effects on several targets affecting multiple signal transduction pathways. Here we used zebrafish embryos to investigate radiosensitizing effects of flavopiridol in normal tissues. Methods and Materials: Zebrafish embryos at the 1- to 4-cell stage were treated with 500 nM flavopiridol or injected with 0.5 pmol antisense hydroxylprolyl-phosphono nucleic acid oligomers to reduce cyclin D1 expression, then subjected to ionizing radiation (IR) or no radiation. Results: Flavopiridol-treated embryos demonstrated a twofold increase in mortality after exposure to 40 Gy by 96 hpf and developed distinct radiation-induced defects in midline development (designated as the 'curly up' phenotype) at higher rates when compared with embryos receiving IR only. Cyclin D1-deficient embryos had virtually identical IR sensitivity profiles when compared with embryos treated with flavopiridol. This was particularly evident for the IR-induced curly up phenotype, which was greatly exacerbated by both flavopriridol and cyclin D1 downregulation. Conclusions: Treatment of zebrafish embryos with flavopiridol enhanced radiation sensitivity of zebrafish embryos to a degree that was very similar to that associated with downregulation of cyclin D1 expression. These results are consistent with the hypothesis that inhibition of cyclin D1 is sufficient to account for the radiosensitizing action of flavopiridol in the zebrafish embryo vertebrate model.

  13. Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae.

    PubMed

    Hagedorn, Elliott J; Cillis, Jennifer L; Curley, Caitlyn R; Patch, Taylor C; Li, Brian; Blaser, Bradley W; Riquelme, Raquel; Zon, Leonard I; Shah, Dhvanit I

    2016-01-01

    Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been  used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development. PMID:27341538

  14. Transient Overexpression of adh8a Increases Allyl Alcohol Toxicity in Zebrafish Embryos

    PubMed Central

    Klüver, Nils; Ortmann, Julia; Paschke, Heidrun; Renner, Patrick; Ritter, Axel P.; Scholz, Stefan

    2014-01-01

    Fish embryos are widely used as an alternative model to study toxicity in vertebrates. Due to their complexity, embryos are believed to more resemble an adult organism than in vitro cellular models. However, concerns have been raised with respect to the embryo's metabolic capacity. We recently identified allyl alcohol, an industrial chemical, to be several orders of magnitude less toxic to zebrafish embryo than to adult zebrafish (embryo LC50 = 478 mg/L vs. fish LC50 = 0.28 mg/L). Reports on mammals have indicated that allyl alcohol requires activation by alcohol dehydrogenases (Adh) to form the highly reactive and toxic metabolite acrolein, which shows similar toxicity in zebrafish embryos and adults. To identify if a limited metabolic capacity of embryos indeed can explain the low allyl alcohol sensitivity of zebrafish embryos, we compared the mRNA expression levels of Adh isoenzymes (adh5, adh8a, adh8b and adhfe1) during embryo development to that in adult fish. The greatest difference between embryo and adult fish was found for adh8a and adh8b expression. Therefore, we hypothesized that these genes might be required for allyl alcohol activation. Microinjection of adh8a, but not adh8b mRNA led to a significant increase of allyl alcohol toxicity in embryos similar to levels reported for adults (LC50 = 0.42 mg/L in adh8a mRNA-injected embryos). Furthermore, GC/MS analysis of adh8a-injected embryos indicated a significant decline of internal allyl alcohol concentrations from 0.23-58 ng/embryo to levels below the limit of detection (< 4.6 µg/L). Injection of neither adh8b nor gfp mRNA had an impact on internal allyl alcohol levels supporting that the increased allyl alcohol toxicity was mediated by an increase in its metabolization. These results underline the necessity to critically consider metabolic activation in the zebrafish embryo. As demonstrated here, mRNA injection is one useful approach to study the role of candidate enzymes involved in

  15. Transient overexpression of adh8a increases allyl alcohol toxicity in zebrafish embryos.

    PubMed

    Klüver, Nils; Ortmann, Julia; Paschke, Heidrun; Renner, Patrick; Ritter, Axel P; Scholz, Stefan

    2014-01-01

    Fish embryos are widely used as an alternative model to study toxicity in vertebrates. Due to their complexity, embryos are believed to more resemble an adult organism than in vitro cellular models. However, concerns have been raised with respect to the embryo's metabolic capacity. We recently identified allyl alcohol, an industrial chemical, to be several orders of magnitude less toxic to zebrafish embryo than to adult zebrafish (embryo LC50 = 478 mg/L vs. fish LC50 = 0.28 mg/L). Reports on mammals have indicated that allyl alcohol requires activation by alcohol dehydrogenases (Adh) to form the highly reactive and toxic metabolite acrolein, which shows similar toxicity in zebrafish embryos and adults. To identify if a limited metabolic capacity of embryos indeed can explain the low allyl alcohol sensitivity of zebrafish embryos, we compared the mRNA expression levels of Adh isoenzymes (adh5, adh8a, adh8b and adhfe1) during embryo development to that in adult fish. The greatest difference between embryo and adult fish was found for adh8a and adh8b expression. Therefore, we hypothesized that these genes might be required for allyl alcohol activation. Microinjection of adh8a, but not adh8b mRNA led to a significant increase of allyl alcohol toxicity in embryos similar to levels reported for adults (LC50 = 0.42 mg/L in adh8a mRNA-injected embryos). Furthermore, GC/MS analysis of adh8a-injected embryos indicated a significant decline of internal allyl alcohol concentrations from 0.23-58 ng/embryo to levels below the limit of detection (< 4.6 µg/L). Injection of neither adh8b nor gfp mRNA had an impact on internal allyl alcohol levels supporting that the increased allyl alcohol toxicity was mediated by an increase in its metabolization. These results underline the necessity to critically consider metabolic activation in the zebrafish embryo. As demonstrated here, mRNA injection is one useful approach to study the role of candidate enzymes involved in

  16. Maternal stress-associated cortisol stimulation may protect embryos from cortisol excess in zebrafish

    PubMed Central

    Faught, Erin; Best, Carol; Vijayan, Mathilakath M.

    2016-01-01

    Abnormal embryo cortisol level causes developmental defects and poor survival in zebrafish (Danio rerio). However, no study has demonstrated that maternal stress leads to higher embryo cortisol content in zebrafish. We tested the hypothesis that maternal stress-associated elevation in cortisol levels increases embryo cortisol content in this asynchronous breeder. Zebrafish mothers were fed cortisol-spiked food for 5 days, to mimic maternal stress, followed by daily breeding for 10 days to monitor temporal embryo cortisol content. Cortisol treatment increased mean embryo yield, but the daily fecundity was variable among the groups. Embryo cortisol content was variable in both groups over a 10-day period. A transient elevation in cortisol levels was observed in the embryos from cortisol-fed mothers only on day 3, but not on subsequent days. We tested whether excess cortisol stimulates 11βHSD2 expression in ovarian follicles as a means to regulate embryo cortisol deposition. Cortisol treatment in vitro increased 11β HSD2 levels sevenfold, and this expression was regulated by actinomycin D and cycloheximide suggesting tight regulation of cortisol levels in the ovarian follicles. We hypothesize that cortisol-induced upregulation of 11βHSD2 activity in the ovarian follicles is a mechanism restricting excess cortisol incorporation into the eggs during maternal stress. PMID:26998341

  17. Transcriptional profiling reveals barcode-like toxicogenomic responses in the zebrafish embryo

    PubMed Central

    Yang, Lixin; Kemadjou, Jules R; Zinsmeister, Christian; Bauer, Matthias; Legradi, Jessica; Müller, Ferenc; Pankratz, Michael; Jäkel, Jens; Strähle, Uwe

    2007-01-01

    Background Early life stages are generally most sensitive to toxic effects. Our knowledge on the action of manmade chemicals on the developing vertebrate embryo is, however, rather limited. We addressed the toxicogenomic response of the zebrafish embryo in a systematic manner by asking whether distinct chemicals would induce specific transcriptional profiles. Results We exposed zebrafish embryos to a range of environmental toxicants and measured the changes in gene-expression profiles by hybridizing cDNA to an oligonucleotide microarray. Several hundred genes responded significantly to at least one of the 11 toxicants tested. We obtained specific expression profiles for each of the chemicals and could predict the identity of the toxicant from the expression profiles with high probability. Changes in gene expression were observed at toxicant concentrations that did not cause morphological effects. The toxicogenomic profiles were highly stage specific and we detected tissue-specific gene responses, underscoring the sensitivity of the assay system. Conclusion Our results show that the genome of the zebrafish embryo responds to toxicant exposure in a highly sensitive and specific manner. Our work provides proof-of-principle for the use of the zebrafish embryo as a toxicogenomic model and highlights its potential for systematic, large-scale analysis of the effects of chemicals on the developing vertebrate embryo. PMID:17961207

  18. Influences of textured substrates on the heart rate of developing zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Chen, Chia-Yun; Chen, Chia-Yuan

    2013-07-01

    Identification of the effects of different textured substrates on zebrafish (Danio rerio) embryos provides insights into the influence of external stimuli on normal cardiovascular functions in the developmental stages of the embryos. This knowledge can be used in numerous genetic studies using zebrafish as an animal model as well as in bioanalytical assays using digital microfluidics. In this study, zebrafish embryos were systematically positioned and in vivo imaged on four types of silicon substrates. These substrates exhibited surface textures and surface wettability that were well modulated by wet chemical etching. The heart rate of the developing embryos significantly increased by 9.1% upon exposure to textured Si substrates with nanostructured surfaces compared with bare Si substrates. Modulation of surface wettability in the tested substrates also responded to the increase in the heart rate of the embryo; however, the effect of surface wettability on heart rate was slight compared with the effect of texture. In-depth experimental and statistical investigations of heart rate under the effects of substrate textures imply a pathway through which the inner mass of the embryo reacts to external stimuli. These findings contribute to zebrafish-related studies and suggest other factors to consider in the design of nanostructure-based microfluidics and other biomedical devices.

  19. Pentachlorophenol exposure causes Warburg-like effects in zebrafish embryos at gastrulation stage

    SciTech Connect

    Xu, Ting; Zhao, Jing; Hu, Ping; Dong, Zhangji; Li, Jingyun; Zhang, Hongchang; Yin, Daqiang; Zhao, Qingshun

    2014-06-01

    Pentachlorophenol (PCP) is a prevalent pollutant in the environment and has been demonstrated to be a serious toxicant to humans and animals. However, little is known regarding the molecular mechanism underlying its toxic effects on vertebrate early development. To explore the impacts and underlying mechanisms of PCP on early development, zebrafish (Danio rerio) embryos were exposed to PCP at concentrations of 0, 20 and 50 μg/L, and microscopic observation and cDNA microarray analysis were subsequently conducted at gastrulation stage. The morphological observations revealed that PCP caused a developmental delay of zebrafish embryos in a concentration-dependent manner. Transcriptomic data showed that 50 μg/L PCP treatment resulted in significant changes in gene expression level, and the genes involved in energy metabolism and cell behavior were identified based on gene functional enrichment analysis. The energy production of embryos was influenced by PCP via the activation of glycolysis along with the inhibition of oxidative phosphorylation (OXPHOS). The results suggested that PCP acts as an inhibitor of OXPHOS at 8 hpf (hours postfertilization). Consistent with the activated glycolysis, the cell cycle activity of PCP-treated embryos was higher than the controls. These characteristics are similar to the Warburg effect, which occurs in human tumors. The microinjection of exogenous ATP confirmed that an additional energy supply could rescue PCP-treated embryos from the developmental delay due to the energy deficit. Taken together, our results demonstrated that PCP causes a Warburg-like effect on zebrafish embryos during gastrulation, and the affected embryos had the phenotype of developmental delay. - Highlights: • We treat zebrafish embryos with PCP at gastrula stage. • PCP acts as an oxidative phosphorylation inhibitor, not an uncoupler, in gastrulation. • Exogenous ATP injection will rescue the development of effected embryos. • The transcriptome of PCP

  20. Nanotherapeutics in angiogenesis: synthesis and in vivo assessment of drug efficacy and biocompatibility in zebrafish embryos

    PubMed Central

    Cheng, Jinping; Gu, Yan-Juan; Wang, Yajun; Cheng, Shuk Han; Wong, Wing-Tak

    2011-01-01

    Background Carbon nanotubes have shown broad potential in biomedical applications, given their unique mechanical, optical, and chemical properties. In this pilot study, carbon nanotubes have been explored as multimodal drug delivery vectors that facilitate antiangiogenic therapy in zebrafish embryos. Methods Three different agents, ie, an antiangiogenic binding site (cyclic arginine-glycin-easpartic acid), an antiangiogenic drug (thalidomide), and a tracking dye (rhodamine), were conjugated onto single-walled carbon nanotubes (SWCNT). The biodistribution, efficacy, and biocompatibility of these triple functionalized SWCNT were tested in mammalian cells and validated in transparent zebrafish embryos. Results Accumulation of SWCNT-associated nanoconjugates in blastoderm cells facilitated drug delivery applications. Mammalian cell xenograft assays demonstrated that these antiangiogenic SWCNT nanoconjugates specifically inhibited ectopic angiogenesis in the engrafted zebrafish embryos. Conclusion This study highlights the potential of using SWCNT for generating efficient nanotherapeutics. PMID:21976976

  1. Semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

    PubMed

    Romano, Shannon N; Gorelick, Daniel A

    2014-01-01

    Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver. PMID:24894681

  2. Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

    PubMed Central

    Romano, Shannon N.; Gorelick, Daniel A.

    2014-01-01

    Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver. PMID:24894681

  3. BMP Signaling Modulates Hepcidin Expression in Zebrafish Embryos Independent of Hemojuvelin

    PubMed Central

    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.

    2011-01-01

    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. PMID:21283739

  4. Construction and Evaluation of a Maize Chimeric Promoter with Activity in Kernel Endosperm and Embryo

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chimeric promoters contain DNA sequences from different promoters. Chimeric promoters are developed to increase the level of recombinant protein expression, precisely control transgene activity, or to escape homology-based gene silencing. Sets of chimeric promoters, each containing different lengt...

  5. Mesoporous silica nanoparticles as a compound delivery system in zebrafish embryos

    PubMed Central

    Sharif, Faiza; Porta, Fabiola; Meijer, Annemarie H; Kros, Alexander; Richardson, Michael K

    2012-01-01

    Silica nanoparticles can be efficiently employed as carriers for therapeutic drugs in vitro. Here, we use zebrafish embryos as a model organism to see whether mesoporous silica nanoparticles (MSNPs) can be incorporated to deliver compounds in vivo. We injected 35–40 nL (10 mg/mL) of custom-synthesized, fluorescently-tagged 200 nm MSNPs into the left flank, behind the yolk sac extension, of 2-day-old zebrafish embryos. We tracked the distribution and translocation of the MSNPs using confocal laser scanning microscopy. Some of the particles remained localized at the injection site, whereas others entered the bloodstream and were carried around the body. Embryo development and survival were not significantly affected by MSNP injection. Acridine orange staining revealed that MSNP injections did not induce significant cell death. We also studied cellular immune responses by means of lysC::DsRED2 transgenic embryos. MSNP-injected embryos showed infiltration of the injection site with neutrophils, similar to controls injected with buffer only. In the same embryos, counterstaining with L-plastin antibody for leukocytes revealed the same amount of cellular infiltration of the injection site in embryos injected with MSNPs or with buffer only. Next, we used MSNPs to deliver two recombinant cytokines (macrophage colony-stimulating factor and receptor for necrosis factor ligand) to zebrafish embryos. These proteins are known to activate cells involved in bone remodeling, and this can be detected with the marker tartrate-resistant acid phosphatase. Coinjection of these proteins loaded onto MSNPs produced a significant increase in the number of tartrate-resistant acid phosphatase-positive cells after 2–3 days of injection. Our results show that MSNPs can be used to deliver bioactive compounds into zebrafish larvae without producing higher mortality or gross evidence of teratogenicity. PMID:22605936

  6. Xenotransplantation of human adipose-derived stem cells in zebrafish embryos.

    PubMed

    Li, Jin; Zeng, Guofang; Qi, Yawei; Tang, Xudong; Zhang, Jingjing; Wu, Zeyong; Liang, Jie; Shi, Lei; Liu, Hongwei; Zhang, Peihua

    2015-01-01

    Zebrafish is a widely used animal model with well-characterized background in developmental biology. The fate of human adipose-derived stem cells (ADSCs) after their xenotransplantation into the developing embryos of zebrafish is unknown. Therefore, human ADSCs were firstly isolated, and then transduced with lentiviral vector system carrying a green fluorescent protein (GFP) reporter gene, and followed by detection of their cell viability and the expression of cell surface antigens. These GFP-expressing human ADSCs were transplanted into the zebrafish embryos at 3.3-4.3 hour post-fertilization (hpf). Green fluorescent signal, the proliferation and differentiation of human ADSCs in recipient embryos were respectively examined using fluorescent microscopy and immunohistochemical staining. The results indicated that human ADSCs did not change their cell viability and the expression levels of cell surface antigens after GFP transduction. Microscopic examination demonstrated that green fluorescent signals of GFP expressed in the transplanted cells were observed in the embryos and larva fish at post-transplantation. The positive staining of Ki-67 revealed the survival and proliferation of human ADSCs in fish larvae after transplantation. The expression of CD105 was observable in the xenotransplanted ADSCs, but CD31 expression was undetectable. Therefore, our results indicate that human ADSCs xenotransplanted in the zebrafish embryos not only can survive and proliferate at across-species circumstance, but also seem to maintain their undifferentiation status in a short term. This xenograft model of zebrafish embryos may provide a promising and useful technical platform for the investigation of biology and physiology of stem cells in vivo. PMID:25849455

  7. Pax7 is required for establishment of the xanthophore lineage in zebrafish embryos

    PubMed Central

    Nord, Hanna; Dennhag, Nils; Muck, Joscha; von Hofsten, Jonas

    2016-01-01

    The pigment pattern of many animal species is a result of the arrangement of different types of pigment-producing chromatophores. The zebrafish has three different types of chromatophores: black melanophores, yellow xanthophores, and shimmering iridophores arranged in a characteristic pattern of golden and blue horizontal stripes. In the zebrafish embryo, chromatophores derive from the neural crest cells. Using pax7a and pax7b zebrafish mutants, we identified a previously unknown requirement for Pax7 in xanthophore lineage formation. The absence of Pax7 results in a severe reduction of xanthophore precursor cells and a complete depletion of differentiated xanthophores in embryos as well as in adult zebrafish. In contrast, the melanophore lineage is increased in pax7a/pax7b double-mutant embryos and larvae, whereas juvenile and adult pax7a/pax7b double-mutant zebrafish display a severe decrease in melanophores and a pigment pattern disorganization indicative of a xanthophore- deficient phenotype. In summary, we propose a novel role for Pax7 in the early specification of chromatophore precursor cells. PMID:27053658

  8. 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. PMID:26838966

  9. The use of microangiography in detecting aberrant vasculature in zebrafish embryos exposed to cadmium.

    PubMed

    Cheng, S H; Chan, P K; Wu, R S

    2001-03-01

    Embryonic vascular patterns in zebrafish (Danio rerio) could be visualised by confocal microscopy coupled with microinjected fluorescent microbeads. This microangiographic technique was adopted here, for the first time, to study the effects of cadmium on cardiovascular development in zebrafish embryos. Zebrafish embryos were incubated in culture medium containing 100 microM cadmium from 5 h post fertilisation (hpf) to 48 hpf. At 48 hpf, embryos were examined for viability and occurrence of malformations. The 100 microM cadmium caused 32.21 +/- 3.65% mortality and 20.33 +/- 4.04% visible malformations in surviving embryos. In the remaining embryos with no visible signs of malformations, further assessments for less obvious abnormalities were performed. Assessments on craniofacial development were made by digital measurements on areas of brains and eyes. Cardiac development was assessed by immunostaining the heart with the antibody MF20 specific for myosin heavy chain. Body lengths of the embryos were also measured. Embryonic development of brains, eyes, hearts and body lengths of visibly healthy embryos in the cadmium treatment group showed no significant difference from the controls. Embryonic vasculature of these visibly healthy embryos was then studied by microinjecting fluorescent microbeads of diameter 0.02 microm into the circulation. All the cadmium treated embryos showed localised vascular defects in the dorsal aortae, segmental and cranial vessels while none of the control embryos showed any aberrant patterns in the networking of the vasculature. Improved image analyses on the anterior regions revealed that cadmium treated embryos had markedly less complex networks of cranial vessels with fewer vessels perfusing the craniofacial regions. The number of branch points in the vascular network was counted. In untreated embryos, there were 135.6 +/- 51 branches in the vasculature in entire body. In the cadmium treated embryos, there were 64.5+/-31 branches. The

  10. Phytohemagglutinin facilitates the aggregation of blastomere pairs from Day 5 donor embryos with Day 4 host embryos for chimeric bovine embryo multiplication.

    PubMed

    Simmet, Kilian; Reichenbach, Myriam; Reichenbach, Horst-Dieter; Wolf, Eckhard

    2015-12-01

    Multiplication of bovine embryos by the production of aggregation chimeras is based on the concept that few blastomeres of a donor embryo form the inner cell mass (ICM) and thus the embryo proper, whereas cells of a host embryo preferentially contribute to the trophectoderm (TE), the progenitor cells of the embryonic part of the placenta. We aggregated two fluorescent blastomeres from enhanced green fluorescent protein (eGFP) transgenic Day 5 morulae with two Day 4 embryos that did not complete their first cleavage until 27 hours after IVF and tested the effect of phytohemagglutinin-L (PHA) on chimeric embryo formation. The resulting blastocysts were characterized by differential staining of cell lineages using the TE-specific factor CDX2 and confocal laser scanning microscopy to facilitate the precise localization of eGFP-positive cells. The proportions of blastocyst development of sandwich aggregates with (n = 99) and without PHA (n = 46) were 85.9% and 54.3% (P < 0.05), respectively. Epifluorescence microscopy showed that the proportion of blastocysts with eGFP-positive cells in the ICM was higher in the PHA group than in the no-PHA group (40% vs. 16%; P < 0.05). Confocal laser scanning microscopy revealed that the total cell numbers of blastocysts from the PHA group of aggregation chimeras (n = 17; 207.8 ± 67.3 [mean ± standard deviation]) were higher (P < 0.05) than those of embryos without ZP and exposed to PHA (n = 30; 159.6 ± 42.2) and of handling control embryos (n = 19; 176.9 ± 53.3). The same was true for ICM cell counts (56.5 ± 22.0 vs. 37.7 ± 14.2 and 38.7 ± 12.4) and TE cell counts (151.2 ± 58.0 vs. 121.9 ± 37.4 and 138.3 ± 53.0), whereas the ICM/total cell number ratio was not different between the groups. Of the 17 chimeric blastocysts analyzed by confocal laser scanning microscopy, nine had eGFP-positive cells (three of them in the ICM, three in the TE, and three in both lineages). When integration in

  11. Divergent teratogenicity of agonists of retinoid X receptors in embryos of zebrafish (Danio rerio).

    PubMed

    Shi, Huahong; Zhu, Pan; Sun, Zhi; Yang, Bo; Zheng, Liang

    2012-07-01

    Zebrafish (Danio rerio) embryos were comparably exposed to seven known agonists of retinoid X receptors (RXRs) including two endogenous compounds (9-cis-retinoic acid and docosahexaenoic acid), four man-made selective ligands (LGD1069, SR11237, fluorobexarotene and CD3254), and a biocide (triphenyltin). The dominant phenotypes of malformation were sharp mouths and small caudal fins in 1 mg/L SR11237-treated group after 5 days exposure. 9-cis-retinoic acid and LGD1069 induced multiple malformations including small eyes, bent notochords, reduced brain, enlarged proctodaems, absence of fins, short tails and edema after 5 days exposure. Fluorobexarotene and CD3254 induced similar phenotypes of malformations after 5 days exposure at low concentration (20 μg/L) to those after the 1st d exposure at high concentrations (50 and 100 μg/L). Triphenlytin induced multiple malformations including deformed eyes, bent notochords, bent tails, and edema in hearts after 5 days exposure at concentrations of 1-10 μg Sn/L. In contrast, no discernible malformations were observed in triphenlytin-treated groups after each separate day exposure. These agonists not only showed different ability of teratogenicity but also induced different phenotypes of malformation in zebrafish embryos. In addition, the sensitive stages of zebrafish embryos were different in response to these agonists. Therefore, our results suggest that the agonists of RXRs had divergent teratogenicity in zebrafish embryos. PMID:22526925

  12. Itaconate and Inflammation, miRs in Zebrafish Embryos, and SHP2 Inhibitor.

    PubMed

    2016-07-21

    Every month the editors of Cell Chemical Biology bring you highlights of the most recent chemical biology literature. Our July 2016 selection includes reports on itaconate as a key metabolite that regulates inflammation, visualization of microRNA in living zebrafish embryos, and the development of a promising SHP2 inhibitor. PMID:27447041

  13. Zebrafish Embryo Disinfection with Povidone-Iodine: Evaluating an Alternative to Chlorine Bleach.

    PubMed

    Chang, Carolyn T; Amack, Jeffrey D; Whipps, Christopher M

    2016-07-01

    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

  14. Optimization of high-throughput nanomaterial developmental toxicity testing in zebrafish embryos

    EPA Science Inventory

    Nanomaterial (NM) developmental toxicities are largely unknown. With an extensive variety of NMs available, high-throughput screening methods may be of value for initial characterization of potential hazard. We optimized a zebrafish embryo test as an in vivo high-throughput assay...

  15. Protective effect of marine algae phlorotannins against AAPH-induced oxidative stress in zebrafish embryo.

    PubMed

    Kang, Min-Cheol; Cha, Seon Heui; Wijesinghe, W A J P; Kang, Sung-Myung; Lee, Seung-Hong; Kim, Eun-A; Song, Choon Bok; Jeon, You-Jin

    2013-06-01

    In this study the protective effect of phlorotannins, including phloroglucinol, eckol, dieckol, eckstolonol and triphloroethol A, isolated from brown algae Ecklonia cava was investigated against AAPH-induced oxidative stress toxicity in zebrafish embryos. Zebrafish embryos were exposed to AAPH and compared with other groups that were co-exposed with phlorotannins until 2-days post-fertilisation. All phlorotannins scavenged intracellular ROS and prevented lipid peroxidation and reduced AAPH-induced cell death in zebrafish embryos. Negative changes in morphological phenomena, such as pericardial oedema, yolk sac oedema, and growth retardation in zebrafish embryos exposed to AAPH were not observed in groups exposed to phlorotannins. These results clearly indicate that phlorotannins possess prominent antioxidant activity against AAPH-mediated toxicity and might be potential therapeutic agents for treating or preventing several diseases implicated with oxidative stress. This study provides a useful tool for examining the protective effect of antioxidants against AAPH-induced oxidative stress in an alternative in vivo model. PMID:23411200

  16. Gambogic acid causes fin developmental defect in zebrafish embryo partially via retinoic acid signaling.

    PubMed

    Jiang, Ling-Ling; Li, Kang; Lin, Qing-Hua; Ren, Jian; He, Zhi-Heng; Li, Huan; Shen, Ning; Wei, Ping; Feng, Feng; He, Ming-Fang

    2016-08-01

    Gambogic acid (GA), the major active ingredient of gamboge, has been approved by the Chinese Food and Drug Administration for clinical trials in cancer patients due to its strong anticancer activity. However, our previous research showed that GA was teratogenic against zebrafish fin development. To explore the teratogenicity and the underlying mechanisms, zebrafish (Danio rerio) embryos were used. The morphological observations revealed that GA caused fin defects in zebrafish embryos in a concentration-dependent manner. The critical exposure time of GA to reveal teratogenicity was before 8 hpf (hours post fertilization). LC/MS/MS analysis revealed that a maximum bioconcentration of GA was occurred at 4 hpf. Q-PCR data showed that GA treatment resulted in significant inactivation of RA signaling which could be partially rescued by the exogenous supply of RA. These results indicate the potential teratogenicity of GA and provide evidence for a caution in its future clinic use. PMID:27288890

  17. A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

    PubMed Central

    Poureetezadi, Shahram Jevin; Donahue, Eric K.; Wingert, Rebecca A.

    2014-01-01

    Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications. PMID:25407322

  18. Progress Towards the Development of a Fathead Minnow Embryo Test and Comparison to the Zebrafish Embryo Test for Assessing Acute Fish Toxicity

    EPA Science Inventory

    The Zebrafish Embryo Test (ZFET) for acute fish toxicity is a well developed method nearing adoption as an OECD Test Guideline. Early drafts of the test guideline (TG) envisioned a suite of potential test species to be covered including zebrafish, fathead minnow, Japanese Medaka...

  19. Evaluation of 14 organic solvents and carriers for screening applications in zebrafish embryos and larvae.

    PubMed

    Maes, Jan; Verlooy, Lien; Buenafe, Olivia E; de Witte, Peter A M; Esguerra, Camila V; Crawford, Alexander D

    2012-01-01

    Zebrafish are rapidly growing in popularity as an in vivo model system for chemical genetics, drug discovery, and toxicology, and more recently also for natural product discovery. Experiments involving the pharmacological evaluation of small molecules or natural product extracts in zebrafish bioassays require the effective delivery of these compounds to embryos and larvae. While most samples to be screened are first solubilized in dimethyl sulfoxide (DMSO), which is then diluted in the embryo medium, often this method is not sufficient to prevent the immediate or eventual precipitation of the sample. Certain compounds and extracts are also not highly soluble in DMSO. In such instances the use of carriers and/or other solvents might offer an alternative means to achieve the required sample concentration. Towards this end, we determined the maximum tolerated concentration (MTC) of several commonly used solvents and carriers in zebrafish embryos and larvae at various developmental stages. Solvents evaluated for this study included acetone, acetonitrile, butanone, dimethyl formamide, DMSO, ethanol, glycerol, isopropanol, methanol, polyethylene glycol (PEG-400), propylene glycol, and solketal, and carriers included albumin (BSA) and cyclodextrin (2-hydroxypropyl-beta-cyclodextrin, or HPBCD). This study resulted in the identification of polyethylene glycol (PEG400), propylene glycol, and methanol as solvents that were relatively well-tolerated over a range of developmental stages. In addition, our results showed that acetone was well-tolerated by embryos but not by larvae, and 1% cyclodextrin (HPBCD) was well-tolerated by both embryos and larvae, indicating the utility of this carrier for compound screening in zebrafish. However, given the relatively small differences (2-3 fold) between concentrations that are apparently safe and those that are clearly toxic, further studies - e.g. omics analyses -should be carried out to determine which cellular processes and signalling

  20. [Toxic effects of CdSe/ZnS QDs to zebrafish embryos].

    PubMed

    Chen, Mu-Fei; Huang, Cheng-Zhi; Pu, De-Yong; Zheng, Chao-Yi; Yuan, Kai-Mi; Jin, Xing-Xing; Zhang, Yao-Guang; Jin, Li

    2015-02-01

    The toxic effects of CdSe/ZnS QDs on zebrafish (Danio rerio) embryos at different developmental stages were investigated in this study. The voluntary movement frequency, body length, hatching rate, mortality and malformation rate, SOD activities, MDA contents, mRNA expression of metallothionein (MT) and heat stress protein 70 (Hsp70) were used as indicators. The results showed that the EC50 was 316.994 nmol x L(-1) for zebrafish embryos (72 hpf) when exposed to CdSe/ZnS QDs. After the CdSe/ZnS QDs exposure, the embryos showed a significant increase in mortality and malformation rate, a decrease in hatching rate and body length, an advance in hatching time, and a changing in the spontaneous movement frequency, and many other toxic effects, such as the condensation of embryonic eggs, the formation of pericardial cysts and curvature of the spine. Moreover, it was found that the MDA contents in the embryos in CdSe/ZnS QDs groups were significantly increased, and the SOD activities were changed. In addition, the mRNA expression level of MT and Hsp70 were up-regulated. All the information suggests that exposure of CdSe/ZnS QDs can cause toxic effects on zebrafish embryos, and the effects may be related to the releasing of Cd2+, particle size and oxidative stress. PMID:26031104

  1. Visualizing Compound Distribution during Zebrafish Embryo Development: The Effects of Lipophilicity and DMSO.

    PubMed

    de Koning, Coco; Beekhuijzen, Manon; Tobor-Kapłon, Marysia; de Vries-Buitenweg, Selinda; Schoutsen, Dick; Leeijen, Nico; van de Waart, Beppy; Emmen, Harry

    2015-12-01

    The predictability of the zebrafish embryo model is highly influenced by internal exposure of the embryo/larva. As compound uptake is likely to be influenced by factors such as lipophilicity, solvent use, and chorion presence, this article focuses on investigating their effects on compound distribution within the zebrafish embryo. To visualize compound uptake and distribution, zebrafish embryos were exposed for 96 hr, starting at 4 hr postfertilization, to water-soluble dyes: Schiff's reagent (logP -4.63), Giemsa stain (logP -0.77), Van Gierson stain (logP 1.64), Cresyl fast violet (logP 3.5), Eosine Y (logP 4.8), Sudan III (logP 7.5), and Oil red O (logP 9.81), with and without 1% dimethyl-sulfoxide (DMSO). Three additional compounds were used to analytically determine the uptake and distribution: Acyclovir (logP -1.56), Zidovudine (logP 0.05), and Metoprolol Tartrate Salt (logP 1.8). Examinations were performed every 24 hr. Both methods (visualization and specific analysis) showed that exposure to higher logP values results in higher compound uptake. Specific analysis showed that for lipophilic compounds >90% of compound is taken up by the embryo. For hydrophilic compounds, >90% of compound within the complete egg could not be associated to embryo or chorion and is probably distributed into the perivitelline space. Overall, internal exposure analyses on at least two occasions (i.e., before and after hatching) is crucial for interpretation of zebrafish embryotoxicity data, especially for compounds with extreme logP values. DMSO did not affect exposure when examined with the visualization method, however, this method might be not sensitive enough to draw hard conclusions. PMID:26663754

  2. Cryopreservation of primordial germ cells by rapid cooling of whole zebrafish (Danio rerio) embryos.

    PubMed

    Higaki, Shogo; Mochizuki, Kentaro; Akashi, Yuichiro; Yamaha, Etsuro; Katagiri, Seiji; Takahashi, Yoshiyuki

    2010-04-01

    The feasibility of cryopreservation of zebrafish (Danio rerio) primordial germ cells (PGCs) by rapid cooling (i.e., vitrification) of dechorionated whole embryos at the 14- to 20-somite stage was investigated. Initially, we examined the glass-forming properties and embryo toxicities of six cryoprotectants: methanol (MeOH), ethylene glycol (EG), glycerol (GC), dimethyl sulfoxide (DMSO), propylene glycol (PG) and 1,3-butylene glycol (1,3-BG). According to the results of glass-forming and embryo toxicity tests, pretreatment solution (PS) containing 2 or 3 M cryoprotectant and vitrification solution (VS) containing 5 M cryoprotectant and 0.5 M sucrose were prepared using each cryoprotectant. Dechorionated embryos, the PGCs of which were visualized by injection of green fluorescence protein-nos1 3'UTR mRNA, were cooled rapidly by plunging into liquid nitrogen after serial exposure to PS and VS. All embryos cooled with MeOH, PG and 1,3-BG showed ice formation during cooling, and few embryos had live PGCs after warming. Most embryos cooled with GC did not show ice formation; however, few embryos had live PGCs. All embryos cooled with EG and most embryos cooled with DMSO had live PGCs when the embryos did not show ice formation during cooling. Based on the number of live PGCs in fresh embryos, the maximum survival rates of PGCs recovered from embryos cooled with EG and DMSO were estimated to be about 40 and 20%, respectively. The present study indicates that rapid cooling of dechorionated whole embryos, especially using EG-based solutions, could be utilized as a simple and promising tool for cryopreservation of PGCs. PMID:19996550

  3. No Evidence for AID/MBD4-Coupled DNA Demethylation in Zebrafish Embryos

    PubMed Central

    Kaneto, Reiya; Izawa, Toshiaki; Yokoi, Hayato; Hashimoto, Naohiro; Kikuchi, Yutaka

    2014-01-01

    The mechanisms responsible for active DNA demethylation remain elusive in Metazoa. A previous study that utilized zebrafish embryos provided a potent mechanism for active demethylation in which three proteins, AID, MBD4, and GADD45 are involved. We recently found age-dependent DNA hypomethylation in zebrafish, and it prompted us to examine if AID and MBD4 could be involved in the phenomenon. Unexpectedly, however, we found that most of the findings in the previous study were not reproducible. First, the injection of a methylated DNA fragment into zebrafish eggs did not affect either the methylation of genomic DNA, injected methylated DNA itself, or several loci tested or the expression level of aid, which has been shown to play a role in demethylation. Second, aberrant methylation was not observed at certain CpG islands following the injection of antisense morpholino oligonucleotides against aid and mbd4. Furthermore, we demonstrated that zebrafish MBD4 cDNA lacked a coding region for the methyl-CpG binding domain, which was assumed to be necessary for guidance to target regions. Taken together, we concluded that there is currently no evidence to support the proposed roles of AID and MBD4 in active demethylation in zebrafish embryos. PMID:25536520

  4. High-content screening assay for identification of chemicals impacting spontaneous activity in zebrafish embryos.

    PubMed

    Raftery, Tara D; Isales, Gregory M; Yozzo, Krystle L; Volz, David C

    2014-01-01

    Although cell-based assays exist, rapid and cost-efficient high-content screening (HCS) assays within intact organisms are needed to support prioritization for developmental neurotoxicity testing in rodents. During zebrafish embryogenesis, spontaneous tail contractions occur from late-segmentation (∼19 h postfertilization, hpf) through early pharyngula (∼29 hpf) and represent the first sign of locomotion. Using transgenic zebrafish (fli1:egfp) that stably express eGFP beginning at ∼14 hpf, we have developed and optimized a 384-well-based HCS assay that quantifies spontaneous activity within single zebrafish embryos after exposure to test chemicals in a concentration-response format. Following static exposure of one embryo per well from 5 to 25 hpf, automated image acquisition procedures and custom analysis protocols were used to quantify total body area and spontaneous activity in live embryos. Survival and imaging success rates across control plates ranged from 87.5 to 100% and 93.3-100%, respectively. Using our optimized procedures, we screened 16 chemicals within the US EPA's ToxCast Phase-I library, and found that exposure to abamectin and emamectin benzoate-both potent avermectins-abolished spontaneous activity in the absence of gross malformations. Overall, compared to existing locomotion-based zebrafish assays conducted later in development, this method provides a simpler discovery platform for identifying potential developmental neurotoxicants. PMID:24328182

  5. Aquatic toxicity assessment of single-walled carbon nanotubes using zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Pan, Huichin; Lin, Yu-Jun; Li, Meng-Wei; Chuang, Han-Ni; Chou, Cheng-Chung

    2011-07-01

    Zebrafish embryos selected at the 64-cell stage were exposed to various concentrations of amide functionalized single-walled carbon nanotubes (SWCNTs) ranging from 1 to 10 μg/ml dissolved in 1% Pluronic F-68 (a cell culture grade surfactant), and the development of embryos was examined from 24 to 120 hours post fertilization (hpf). Incubation of embryos in 1% F-68 did not induce overt abnormal phenotype as compared to the wild-type; neither did it cause significant mortality during the exposure period. Generally, there was a slight developmental delay in larvae treated with SWCNTs of 5 μg/ml or above. Only larvae exposed to >= 5 μg/ml SWCNTs showed significantly reduced survival rates. About 50% of the embryos exposed to 5 μg/ml showed abnormal phenotypes at 24 hpf as compared to the control group. As development proceeds to 120 hpf, more embryos displayed defective morphology. A slight hatching delay was observed in embryos exposed to concentrations above 5 μg/ml. There was a general reduction of body axes, including narrowed somite and shortened yolk stalk. In addition, pigmentation in the ventral trunk area was less than that observed in control group. The body lengths of the exposed embryos were decreased significantly at 48 hpf (3.11 mm in control vs. 3.00 mm in SWCNTs-exposed embryos). However, exposure to SWCNTs did not affect the number of somites. Other features that were noticed in the SWCNTs-exposed embryos included edema and shrinkage and blebbling of the epidermal lining. Most of these observed phenotypes persisted from 48 hpf through 120 hpf. Overall, the aforementioned results indicate that soluble amide-functionalized SWCNTs are toxic to zebrafish embryos at a minimum concentration of 5 μg/ml.

  6. Non-induction of radioadaptive response in zebrafish embryos by neutrons.

    PubMed

    Ng, Candy Y P; Kong, Eva Y; Kobayashi, Alisa; Suya, Noriyoshi; Uchihori, Yukio; Cheng, Shuk Han; Konishi, Teruaki; Yu, Kwan Ngok

    2016-06-01

    In vivo neutron-induced radioadaptive response (RAR) was studied using zebrafish (Danio rerio) embryos. The Neutron exposure Accelerator System for Biological Effect Experiments (NASBEE) facility at the National Institute of Radiological Sciences (NIRS), Japan, was employed to provide 2-MeV neutrons. Neutron doses of 0.6, 1, 25, 50 and 100 mGy were chosen as priming doses. An X-ray dose of 2 Gy was chosen as the challenging dose. Zebrafish embryos were dechorionated at 4 h post fertilization (hpf), irradiated with a chosen neutron dose at 5 hpf and the X-ray dose at 10 hpf. The responses of embryos were assessed at 25 hpf through the number of apoptotic signals. None of the neutron doses studied could induce RAR. Non-induction of RAR in embryos having received 0.6- and 1-mGy neutron doses was attributed to neutron-induced hormesis, which maintained the number of damaged cells at below the threshold for RAR induction. On the other hand, non-induction of RAR in embryos having received 25-, 50- and 100-mGy neutron doses was explained by gamma-ray hormesis, which mitigated neutron-induced damages through triggering high-fidelity DNA repair and removal of aberrant cells through apoptosis. Separate experimental results were obtained to verify that high-energy photons could disable RAR. Specifically, 5- or 10-mGy X-rays disabled the RAR induced by a priming dose of 0.88 mGy of alpha particles delivered to 5-hpf zebrafish embryos against a challenging dose of 2 Gy of X-rays delivered to the embryos at 10 hpf. PMID:26850927

  7. Combined effects of depleted uranium and ionising radiation on zebrafish embryos.

    PubMed

    Ng, C Y P; Pereira, S; Cheng, S H; Adam-Guillermin, C; Garnier-Laplace, J; Yu, K N

    2015-11-01

    In the environment, living organisms are exposed to a mixture of stressors, and the combined effects are deemed as multiple stressor effects. In the present work, the authors studied the multiple stressor effect in embryos of the zebrafish (Danio rerio) from simultaneous exposure to alpha particles and depleted uranium (DU) through quantification of apoptotic signals at 24 h post-fertilisation (hpf) revealed by vital dye acridine orange staining. In each set of experiments, dechorionated zebrafish embryos were divided into 4 groups, each having 10 embryos: Group (C) in which the embryos did not receive any further treatment; Group (IU) in which the embryos received an alpha-particle dose of 0.44 mGy at 5 hpf and were then exposed to 100 µg l(-1) of DU from 5 to 6 hpf; Group (I) in which the embryos received an alpha-particle dose of 0.44 mGy at 5 hpf and Group (U) in which the dechorionated embryos were exposed to 100 µg l(-1) of DU from 5 to 6 hpf. The authors confirmed that an alpha-particle dose of 0.44 mGy and a DU exposure for 1 h separately led to hormetic and toxic effects assessed by counting apoptotic signals, respectively, in the zebrafish. Interestingly, the combined exposure led to an effect more toxic than that caused by the DU exposure alone, so effectively DU changed the beneficial effect (hormesis) brought about by alpha-particle irradiation into an apparently toxic effect. This could be explained in terms of the promotion of early death of cells predisposed to spontaneous transformation by the small alpha-particle dose (i.e. hormetic effect) and the postponement of cell death upon DU exposure. PMID:25948823

  8. Non-induction of radioadaptive response in zebrafish embryos by neutrons

    PubMed Central

    Ng, Candy Y.P.; Kong, Eva Y.; Kobayashi, Alisa; Suya, Noriyoshi; Uchihori, Yukio; Cheng, Shuk Han; Konishi, Teruaki; Yu, Kwan Ngok

    2016-01-01

    In vivo neutron-induced radioadaptive response (RAR) was studied using zebrafish (Danio rerio) embryos. The Neutron exposure Accelerator System for Biological Effect Experiments (NASBEE) facility at the National Institute of Radiological Sciences (NIRS), Japan, was employed to provide 2-MeV neutrons. Neutron doses of 0.6, 1, 25, 50 and 100 mGy were chosen as priming doses. An X-ray dose of 2 Gy was chosen as the challenging dose. Zebrafish embryos were dechorionated at 4 h post fertilization (hpf), irradiated with a chosen neutron dose at 5 hpf and the X-ray dose at 10 hpf. The responses of embryos were assessed at 25 hpf through the number of apoptotic signals. None of the neutron doses studied could induce RAR. Non-induction of RAR in embryos having received 0.6- and 1-mGy neutron doses was attributed to neutron-induced hormesis, which maintained the number of damaged cells at below the threshold for RAR induction. On the other hand, non-induction of RAR in embryos having received 25-, 50- and 100-mGy neutron doses was explained by gamma-ray hormesis, which mitigated neutron-induced damages through triggering high-fidelity DNA repair and removal of aberrant cells through apoptosis. Separate experimental results were obtained to verify that high-energy photons could disable RAR. Specifically, 5- or 10-mGy X-rays disabled the RAR induced by a priming dose of 0.88 mGy of alpha particles delivered to 5-hpf zebrafish embryos against a challenging dose of 2 Gy of X-rays delivered to the embryos at 10 hpf. PMID:26850927

  9. Biological Screening of Newly Synthesized BIAN N-Heterocyclic Gold Carbene Complexes in Zebrafish Embryos

    PubMed Central

    Farooq, Muhammad; Abu Taha, Nael; Butorac, Rachel R.; Evans, Daniel Anthony; Elzatahry, Ahmed A.; Elsayed, Elsayed Ahmed; Wadaan, Mohammad A. M.; Al-Deyab, Salem S.; Cowley, Alan H.

    2015-01-01

    N-Heterocyclic carbene (NHC) metal complexes possess diverse biological activities but have yet to be extensively explored as potential chemotherapeutic agents. We have previously reported the synthesis of a new class of NHC metal complexes N-heterocyclic with acetate [IPr(BIAN)AuOAc] and chloride [IPr(BIAN)AuCl] ligands. In the experiments reported herein, the zebrafish embryos were exposed to serial dilutions of each of these complexes for 10–12 h. One hundred percent mortality was observed at concentrations ≥50 µM. At sub-lethal concentrations (10–30 µM), both compounds influenced zebrafish embryonic development. However, quite diverse categories of abnormalities were found in exposed embryos with each compound. Severe brain deformation and notochord degeneration were evident in the case of [IPr(BIAN)AuOAc]. The zebrafish embryos treated with [IPr(BIAN)AuCl] exhibited stunted growth and consequently had smaller body sizes. A depletion of 30%–40% glutathione was detected in the treated embryos, which could account for one of the possible mechanism of neurotoxicity. The fact that these compounds are capable of both affecting the growth and also compromising antioxidant systems by elevating intracellular ROS production implies that they could play an important role as a new breed of therapeutic molecules. PMID:26501273

  10. Imaging Subcellular Structures in the Living Zebrafish Embryo.

    PubMed

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

    2016-01-01

    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. PMID:27078038

  11. Exogenous Nitric Oxide Suppresses in Vivo X-ray-Induced Targeted and Non-Targeted Effects in Zebrafish Embryos

    PubMed Central

    Kong, E.Y.; Yeung, W.K.; Chan, T.K.Y.; Cheng, S.H.; Yu, K.N.

    2016-01-01

    The present paper studied the X-ray-induced targeted effect in irradiated zebrafish embryos (Danio rerio), as well as a non-targeted effect in bystander naïve embryos partnered with irradiated embryos, and examined the influence of exogenous nitric oxide (NO) on these targeted and non-targeted effects. The exogenous NO was generated using an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). The targeted and non-targeted effects, as well as the toxicity of the SNAP, were assessed using the number of apoptotic events in the zebrafish embryos at 24 h post fertilization (hpf) revealed through acridine orange (AO) staining. SNAP with concentrations of 20 and 100 µM were first confirmed to have no significant toxicity on zebrafish embryos. The targeted effect was mitigated in zebrafish embryos if they were pretreated with 100 µM SNAP prior to irradiation with an X-ray dose of 75 mGy but was not alleviated in zebrafish embryos if they were pretreated with 20 µM SNAP. On the other hand, the non-targeted effect was eliminated in the bystander naïve zebrafish embryos if they were pretreated with 20 or 100 µM SNAP prior to partnering with zebrafish embryos having been subjected to irradiation with an X-ray dose of 75 mGy. These findings revealed the importance of NO in the protection against damages induced by ionizing radiations or by radiation-induced bystander signals, and could have important impacts on development of advanced cancer treatment strategies. PMID:27529238

  12. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo.

    PubMed

    Xue, Binghua; Li, Yan; He, Yilong; Wei, Renyue; Sun, Ruizhen; Yin, Zhi; Bou, Gerelchimeg; Liu, Zhonghua

    2016-01-01

    Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs. PMID:26991423

  13. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo

    PubMed Central

    Xue, Binghua; Li, Yan; He, Yilong; Wei, Renyue; Sun, Ruizhen; Yin, Zhi; Bou, Gerelchimeg; Liu, Zhonghua

    2016-01-01

    Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs. PMID:26991423

  14. Cre-mediated site-specific recombination in zebrafish embryos.

    PubMed

    Thummel, Ryan; Burket, Christopher T; Brewer, Jeffrey L; Sarras, Michael P; Li, Li; Perry, Martin; McDermott, Jeffrey P; Sauer, Brian; Hyde, David R; Godwin, Alan R

    2005-08-01

    Cre-mediated site-specific recombination has become an invaluable tool for manipulation of the murine genome. The ability to conditionally activate gene expression or to generate chromosomal alterations with this same tool would greatly enhance zebrafish genetics. This study demonstrates that the HSP70 promoter can be used to inducibly control expression of an enhanced green fluorescent protein (EGFP) -Cre fusion protein. The EGFP-Cre fusion protein is capable of promoting recombination between lox sites in injected plasmids or in stably inherited transgenes as early as 2 hr post-heat shock induction. Finally, the levels of Cre expression achieved in a transgenic fish line carrying the HSP70-EGFP-cre transgene are compatible with viability and both male and female transgenic fish are fertile subsequent to induction of EGFP-Cre expression. Hence, our data suggests that Cre-mediated recombination is a viable means of manipulating gene expression in zebrafish. PMID:15977183

  15. Block the function of nonmuscle myosin II by blebbistatin induces zebrafish embryo cardia bifida.

    PubMed

    Wang, Xueqian; Chong, Mei; Wang, Xin; Wang, Hongkui; Zhang, Jie; Xu, Hui; Zhang, Jingjing; Liu, Dong

    2015-03-01

    Nonmuscle myosin II (NM II) is the name given to the multi-subunit protein product of three genes encoding different nonmuscle myosin heavy chains including NM II-A, NM II-B, and NM II-C. Blebbistatin is a small molecule that has been shown to be a relatively specific inhibitor of NM II. Blocking the function of NM II by blebbistatin induces zebrafish embryo cardia bifida at a dose-dependent manner. In situ hybridization analysis with ventricular marker ventricular myosin heavy chain (vmhc) and atrial marker atrial myosin heavy chain (amhc) showed each of the heart contained both distinct atria and ventricle. However, the cardia bifida embryos had highly variable distance between two separate ventricles. We also provided evidence that time window from 12 to 20 h post fertilization (hpf) is necessary and sufficient for cardia bifida formation caused by blebbistatin treatment. Expression of spinster homolog 2 (spns2) was decreased in blebbistatin-treated embryos, suggesting the cardia bifida phenotype caused by NM II inhibition was relevant to precardiac mesoderm migration defects. Through in situ hybridization analysis, we showed that foxa1 was expressed in endoderm of blebbistatin-treated embryos at 24-hpf stage, suggesting the endoderm formation is normal in cardia bifida embryos caused by blebbistatin treatment. In addition, we demonstrated that blebbistatin treatment resulted in morphology alteration of zebrafish cardiomyocytes in vivo and neonatal mouse cardiomyocytes in vitro. PMID:25403653

  16. Identification and characterization of the RNA-binding protein Rbfox3 in zebrafish embryo.

    PubMed

    Won, Minho; Lee, Siyeo; Choi, Sunkyung; Ro, Hyunju; Kim, Ki-Jung; Kim, Jung-Hwan; Kim, Kyoon Eon; Kim, Kee K

    2016-04-01

    Rbfox3, an RNA-binding fox protein, binds to the antibody to pan-neuronal marker, neuronal nuclei (NeuN). Rbfox3 is expressed in neural tissues across a wide range of species including mammals, birds, and amphibians. However, the molecular identity of Rbfox3 in the zebrafish is largely unknown. In this study, we cloned two zebrafish Rbfox3 genes, Rbfox3a and Rbfox3b. We also cloned the Rbfox3-d31 isoform, which excludes a 93-nucleotide alternative exon within the RNA-recognition motif in both, Rbfox3a and Rbfox3b. Multiple protein sequence alignment revealed that the amino acid sequence for residues 1-20 of the zebrafish Rbfox3, which is the epitope region of NeuN antibody, was different from that of other species. Therefore, NeuN antibody lost its function as a neuronal marker antibody in zebrafish. Reverse transcriptase-polymerase chain reaction showed that both Rbfox3-d31 transcripts were abundant in the early blastula stage, after which they dramatically reduced, suggesting that these isoforms exist mainly as maternal transcripts. In contrast, full-length Rbfox3 transcripts were detected from the 24 h post-fertilization embryo, expression was also maintained at a constant level. Furthermore, full-length Rbfox3-expressing cells were located within the central nervous system during later stages of the zebrafish embryo. Our study provides insight into the molecular structure of zebrafish Rbfox3 as a step towards genetic association studies investigating the developmental role of Rbfox3. PMID:26952657

  17. In vivo dynamics of skeletal muscle Dystrophin in zebrafish embryos revealed by improved FRAP analysis

    PubMed Central

    Bajanca, Fernanda; Gonzalez-Perez, Vinicio; Gillespie, Sean J; Beley, Cyriaque; Garcia, Luis; Theveneau, Eric; Sear, Richard P; Hughes, Simon M

    2015-01-01

    Dystrophin forms an essential link between sarcolemma and cytoskeleton, perturbation of which causes muscular dystrophy. We analysed Dystrophin binding dynamics in vivo for the first time. Within maturing fibres of host zebrafish embryos, our analysis reveals a pool of diffusible Dystrophin and complexes bound at the fibre membrane. Combining modelling, an improved FRAP methodology and direct semi-quantitative analysis of bleaching suggests the existence of two membrane-bound Dystrophin populations with widely differing bound lifetimes: a stable, tightly bound pool, and a dynamic bound pool with high turnover rate that exchanges with the cytoplasmic pool. The three populations were found consistently in human and zebrafish Dystrophins overexpressed in wild-type or dmdta222a/ta222a zebrafish embryos, which lack Dystrophin, and in Gt(dmd-Citrine)ct90a that express endogenously-driven tagged zebrafish Dystrophin. These results lead to a new model for Dystrophin membrane association in developing muscle, and highlight our methodology as a valuable strategy for in vivo analysis of complex protein dynamics. DOI: http://dx.doi.org/10.7554/eLife.06541.001 PMID:26459831

  18. Embryo Microinjection of Selenomethionine Reduces Hatchability and Modifies Oxidant Responsive Gene Expression in Zebrafish

    NASA Astrophysics Data System (ADS)

    Thomas, J. K.; Janz, D. M.

    2016-05-01

    In previous studies we demonstrated that exposure to selenomethionine (SeMet) causes developmental toxicities in zebrafish (Danio rerio). The objectives of this study were to establish a dose-response relationship for developmental toxicities in zebrafish after embryo microinjection of Se (8, 16 or 32 μg/g dry mass of eggs) in the form of SeMet, and to investigate potential underlying mechanism(s) of SeMet-induced developmental toxicities. A dose-dependent increase in frequencies of mortality and total deformities, and reduced hatchability were observed in zebrafish exposed to excess Se via embryo microinjection. The egg Se concentration causing 20% mortality was then used to investigate transcript abundance of proteins involved in antioxidant protection and methylation. Excess Se exposure modified gene expression of oxidant-responsive transcription factors (nuclear factor erythroid 2-related factor nrf2a and nrf2b), and enzymes involved in cellular methylation (methionine adenosyltransferase mat1a and mat2ab) in zebrafish larvae. Notably, excess Se exposure up-regulated transcript abundance of aryl hydrocarbon receptor 2 (ahr2), a signalling pathway involved in the toxicity of dioxin-related compounds. Our findings suggest that oxidative stress or modification of methylation, or a combination of these mechanisms, might be responsible for Se-induced developmental toxicities in fishes.

  19. Embryo Microinjection of Selenomethionine Reduces Hatchability and Modifies Oxidant Responsive Gene Expression in Zebrafish

    PubMed Central

    Thomas, J. K.; Janz, D. M.

    2016-01-01

    In previous studies we demonstrated that exposure to selenomethionine (SeMet) causes developmental toxicities in zebrafish (Danio rerio). The objectives of this study were to establish a dose-response relationship for developmental toxicities in zebrafish after embryo microinjection of Se (8, 16 or 32 μg/g dry mass of eggs) in the form of SeMet, and to investigate potential underlying mechanism(s) of SeMet-induced developmental toxicities. A dose-dependent increase in frequencies of mortality and total deformities, and reduced hatchability were observed in zebrafish exposed to excess Se via embryo microinjection. The egg Se concentration causing 20% mortality was then used to investigate transcript abundance of proteins involved in antioxidant protection and methylation. Excess Se exposure modified gene expression of oxidant-responsive transcription factors (nuclear factor erythroid 2-related factor nrf2a and nrf2b), and enzymes involved in cellular methylation (methionine adenosyltransferase mat1a and mat2ab) in zebrafish larvae. Notably, excess Se exposure up-regulated transcript abundance of aryl hydrocarbon receptor 2 (ahr2), a signalling pathway involved in the toxicity of dioxin-related compounds. Our findings suggest that oxidative stress or modification of methylation, or a combination of these mechanisms, might be responsible for Se-induced developmental toxicities in fishes. PMID:27210033

  20. Novel vector systems optimized for injecting in vitro-synthesized mRNA into zebrafish embryos.

    PubMed

    Ro, Hyunju; Soun, Kyungchull; Kim, Eun-Jung; Rhee, Myungchull

    2004-04-30

    Microinjection of nucleic acids or proteins is a useful way of studying embryonic development. In particular, injection of in vitro-transcribed capped RNA is commonly employed to achieve ectopic or increased expression of genes. Two vector systems, pCS2+ and pT7Ts, have been used for this purpose in zebrafish. However, they were initially optimized for Xenopus embryos not for zebrafish. Here we describe a vector, pcGlobin2, optimized for zebrafish, and its derivative, pcGlobin2-GST. This new vector system offers several advantages. First, pcGlobin 2 contains three critical elements 15' and 3' zebrafish beta-globin UTRs, and a poly(A) tail] for generating stable mRNAs and greatly improving translation efficiency. Second, subcloning and preparation of template DNA is easier because of the larger number of restriction sites. Third, protein-binding assays can be performed directly on the injected embryos with pcGlobin2-GST. Lastly, this vector system can be transfected into animal cells without additional subcloning. PMID:15179057

  1. Toxicological effect of MPA-CdSe QDs exposure on zebrafish embryo and larvae.

    PubMed

    Zhang, Wei; Lin, Kuangfei; Sun, Xue; Dong, Qiaoxiang; Huang, Changjiang; Wang, Huili; Guo, Meijin; Cui, Xinhong

    2012-09-01

    Cadmium selenium (CdSe) quantum dots (QDs) are semiconductor nanocrystals that hold wide range of applications and substantial production volumes. Due to unique composition and nanoscale properties, their potential toxicity to aquatic organisms has increasingly gained a great amount of interest. However, the impact of CdSe QDs exposure on zebrafish embryo and larvae remains almost unknown. Therefore, the lab study was performed to determine the developmental and behavioral toxicities to zebrafish under continuous exposure to low level CdSe QDs (0.05-31.25 mg L(-1)) coated with mercaptopropionic acid (MPA). The results showed MPA-CdSe exposure from embryo to larvae stage affected overall fitness. Our findings for the first time revealed that: (1) The 120 h LC(50) of MPA-CdSe for zebrafish was 1.98 mg L(-1); (2) embryos exposed to MPA-CdSe resulted in malformations incidence and lower hatch rate; (3) abnormal vascular of FLI-1 transgenic zebrafish larvae appeared after exposure to MPA-CdSe including vascular junction, bifurcation, crossing and particle appearance; (4) larvae behavior assessment showed during MPA-CdSe exposure a rapid transition from light-to-dark elicited a similar, brief burst and a higher basal swimming rate; (5) MPA-CdSe induced embryos cell apoptosis in the head and tail region. Results of the observations provide a basic understanding of MPA-CdSe toxicity to aquatic organisms and suggest the need for additional research to identify the toxicological mechanism. PMID:22595531

  2. Abcb4 acts as multixenobiotic transporter and active barrier against chemical uptake in zebrafish (Danio rerio) embryos

    PubMed Central

    2013-01-01

    Background In mammals, ABCB1 constitutes a cellular “first line of defense” against a wide array of chemicals and drugs conferring cellular multidrug or multixenobiotic resistance (MDR/MXR). We tested the hypothesis that an ABCB1 ortholog serves as protection for the sensitive developmental processes in zebrafish embryos against adverse compounds dissolved in the water. Results Indication for ABCB1-type efflux counteracting the accumulation of chemicals in zebrafish embryos comes from experiments with fluorescent and toxic transporter substrates and inhibitors. With inhibitors present, levels of fluorescent dyes in embryo tissue and sensitivity of embryos to toxic substrates were generally elevated. We verified two predicted sequences from zebrafish, previously annotated as abcb1, by cloning; our synteny analyses, however, identified them as abcb4 and abcb5, respectively. The abcb1 gene is absent in the zebrafish genome and we explored whether instead Abcb4 and/or Abcb5 show toxicant defense properties. Quantitative real-time polymerase chain reaction (qPCR) analyses showed the presence of transcripts of both genes throughout the first 48 hours of zebrafish development. Similar to transporter inhibitors, morpholino knock-down of Abcb4 increased accumulation of fluorescent substrates in embryo tissue and sensitivity of embryos toward toxic compounds. In contrast, morpholino knock-down of Abcb5 did not exert this effect. ATPase assays with recombinant protein obtained with the baculovirus expression system confirmed that dye and toxic compounds act as substrates of zebrafish Abcb4 and inhibitors block its function. The compounds tested comprised model substrates of human ABCB1, namely the fluorescent dyes rhodamine B and calcein-am and the toxic compounds vinblastine, vincristine and doxorubicin; cyclosporin A, PSC833, MK571 and verapamil were applied as inhibitors. Additionally, tests were performed with ecotoxicologically relevant compounds: phenanthrene (a

  3. Adaptive response to ionising radiation induced by cadmium in zebrafish embryos.

    PubMed

    Choi, V W Y; Ng, C Y P; Kong, M K Y; Cheng, S H; Yu, K N

    2013-03-01

    An adaptive response is a biological response where the exposure of cells or animals to a low priming exposure induces mechanisms that protect the cells or animals against the detrimental effects of a subsequent larger challenging exposure. In realistic environmental situations, living organisms can be exposed to a mixture of stressors, and the resultant effects due to such exposures are referred to as multiple stressor effects. In the present work we demonstrated, via quantification of apoptosis in the embryos, that embryos of the zebrafish (Danio rerio) subjected to a priming exposure provided by one environmental stressor (cadmium in micromolar concentrations) could undergo an adaptive response against a subsequent challenging exposure provided by another environmental stressor (alpha particles). We concluded that zebrafish embryos treated with 1 to 10 μM Cd at 5 h postfertilisation (hpf) for both 1 and 5 h could undergo an adaptive response against subsequent ~4.4 mGy alpha-particle irradiation at 10 hpf, which could be interpreted as an antagonistic multiple stressor effect between Cd and ionising radiation. The zebrafish has become a popular vertebrate model for studying the in vivo response to ionising radiation. As such, our results suggested that multiple stressor effects should be carefully considered for human radiation risk assessment since the risk may be perturbed by another environmental stressor such as a heavy metal. PMID:23296313

  4. Identification of phenolic compounds in red wine extract samples and zebrafish embryos by HPLC-ESI-LTQ-Orbitrap-MS.

    PubMed

    Vallverdú-Queralt, Anna; Boix, Nuria; Piqué, Ester; Gómez-Catalan, Jesús; Medina-Remon, Alexander; Sasot, Gemma; Mercader-Martí, Mercè; Llobet, Juan M; Lamuela-Raventos, Rosa M

    2015-08-15

    The zebrafish embryo is a highly interesting biological model with applications in different scientific fields, such as biomedicine, pharmacology and toxicology. In this study, we used liquid chromatography/electrospray ionisation-linear ion trap quadrupole-Orbitrap-mass spectrometry (HPLC/ESI-LTQ-Orbitrap-MS) to identify the polyphenol compounds in a red wine extract and zebrafish embryos. Phenolic compounds and anthocyanin metabolites were determined in zebrafish embryos previously exposed to the red wine extract. Compounds were identified by injection in a high-resolution system (LTQ-Orbitrap) using accurate mass measurements in MS, MS(2) and MS(3) modes. To our knowledge, this research constitutes the first comprehensive identification of phenolic compounds in zebrafish by HPLC coupled to high-resolution mass spectrometry. PMID:25794733

  5. In vivo imaging of zebrafish from embryo to adult stage with optical projection tomography

    NASA Astrophysics Data System (ADS)

    Bassi, Andrea; Fieramonti, Luca; D'Andrea, Cosimo; Valentini, Gianluca; Cubeddu, Rinaldo; De Silvestri, Sandro; Cerullo, Giulio; Foglia, Efrem; Cotelli, Franco

    2013-02-01

    Optical Projection Tomography (OPT) is a three dimensional imaging technique that is particularly suitable for studying millimeter sized biological samples and organisms. Similarly to x-ray computed tomography, OPT is based on the acquisition of a sequence of images taken through the sample at many angles (projections). Assuming the linearity of the optical absorption process, the projections are combined to reconstruct the 3-D volume of the sample, typically using a filtered back-projection algorithm. OPT has been applied to in-vivo imaging of zebrafish (Danio rerio). The instrument and the protocol for in vivo imaging of zebrafish embryos and juvenile specimens are described. Light scattering remains a challenge for in vivo OPT, especially when samples at the upper size limit, like zebrafish at the adult stage, are under study. We describe Time-Gated Optical Projection Tomography (TGOPT), a technique able to reconstruct adult zebrafish internal structures by counteracting the scattering effects through a fast time-gate. The time gating mechanism is based on non-linear optical upconversion of an infrared ultrashort laser pulse and allows the detection of quasi-ballistic photons within a 100 fs temporal gate. This results in a strong improvement in contrast and resolution with respect to conventional OPT. Artifacts in the reconstructed images are reduced as well. We show that TGOPT is suited for imaging the skeletal system and nervous structures of adult zebrafish.

  6. Toxic effects of perfluorononanoic acid on the development of Zebrafish (Danio rerio) embryos.

    PubMed

    Liu, Hui; Sheng, Nan; Zhang, Wei; Dai, Jiayin

    2015-06-01

    Perfluorononanoic acid (PFNA) is a nine-carbon perfluoroalkyl acid widely used in industrial and domestic products. It is a persistent organic pollutant found in the environment as well as in the tissues of humans and wildlife. There is a concern that this chemical might be a developmental toxicant and teratogen in various ecosystems. In the present study, the toxic effects of PFNA were evaluated in zebrafish (Danio rerio) embryos. One hour post-fertilization embryos were treated with 0, 25, 50, 100, 200, 300, 350, and 400 μmol/L PFNA for 96 hr in 6-well plates. Developmental phenotypes and hatching rates were observed and recorded. Nineteen genes related to oxidative stress and lipid metabolism were examined using Quantitative RT-PCR and confirmed by whole mount in situ hybridization (WISH). Results showed that PFNA delayed the development of zebrafish embryos, reduced the hatching rate, and caused ventricular edema and malformation of the spine. In addition, the amount of reactive oxygen species in the embryo bodies increased significantly after exposure to PFNA compared with that of the control group. The Quantitative RT-PCR and WISH experiments demonstrated that mRNA expression of the lfabp and ucp2 genes increased significantly while that of sod1 and mt-nd1 decreased significantly after PFNA exposure. The mRNA expression levels of gpx1 and mt-atp6 decreased significantly in the high concentration group. However, the mRNA expression levels of both ppara and pparg did not show any significant variation after exposure. These findings suggest that PFNA affected the development of zebrafish embryos at relatively low concentrations. PMID:26040728

  7. Bioaccumulation and molecular effects of sediment-bound metals in zebrafish embryos.

    PubMed

    Redelstein, R; Zielke, H; Spira, D; Feiler, U; Erdinger, L; Zimmer, H; Wiseman, S; Hecker, M; Giesy, J P; Seiler, T-B; Hollert, H

    2015-11-01

    Predicting the bioavailability and effects of metals in sediments is of major concern in context with sediment risk assessment. This study aimed to investigate the bioavailability and molecular effects of metals spiked into riverine sediments to zebrafish (Danio rerio) embryos. Embryos were exposed to a natural and an artificial sediment spiked with cadmium (Cd), copper (Cu), nickel (Ni) and zinc (Zn) individually or as a mixture at concentrations ranging from 150 to 3000 mg/kg dry weight (dw) over 48 h, and uptake of metals was determined. Furthermore, transcript abundances of the metallothioneins MT1 and MT2, the metal-responsive element-binding transcription factor (MTF) and the genes sod1, hsp70 and hsp90α1 were measured as indicators of metal-induced or general cellular stress. D. rerio embryos accumulated metals from sediments at concentrations up to 100 times greater than those spiked to the sediment with the greatest bioaccumulation factor (BAF) for Cu from artificial sediment (275.4 ± 41.9 (SD)). Embryos accumulated greater concentrations of all metals from artificial than from natural sediment, and accumulation was greater when embryos were exposed to individual metals than when they were exposed to the mixture. Exposure of embryos to Zn or the mixture exhibited up to 30-fold greater transcript abundances of MT1, MT2 and hsp70 compared to controls which is related to significant uptake of Zn from the sediment. Further changes in transcript abundances could not be related to a significant uptake of metals from sediments. These studies reveal that metals from spiked sediments are bioavailable to D. rerio embryos directly exposed to sediments and that the induction of specific genes can be used as biomarkers for the exposure of early life stages of zebrafish to metal-contaminated sediments. PMID:26354112

  8. A representative retinoid X receptor antagonist UVI3003 induced teratogenesis in zebrafish embryos.

    PubMed

    Zheng, Liang; Xu, Ting; Li, Daoji; Zhou, Junliang

    2015-03-01

    Retinoid X receptor (RXR) interfering activity has been detected in different water resources. To study RXR disruptor-induced toxicological effects on vertebrates, embryos of zebrafish (Danio rerio) were exposed to a representative RXR antagonist UVI3003. Results showed that the teratogenic index (LC50 /EC50 ) of UVI3003 was as high as 5.4. UVI3003 induced multiple malformations of embryos, including deformed fins, reduced brains, small jaws, bent tails and edema in hearts, the degree of which became more severe with increasing exposure concentration. Although no significant difference was observed in the hatching rates between the exposure group and control, the whole body length was significantly reduced by 6.5% and 8.9% when exposed to 200 and 300 µg l(-1) of UVI3003, respectively. The heart rate also significantly decreased by 8.8-50.2% during exposure. Further experiments revealed that the pharyngula stage was the most sensitive development phase in terms of embryo response to UVI3003. The results demonstrated severe teratogenicity of RXR antagonist in zebrafish embryos and provided important data for ecotoxicological evaluation of RXR antagonists. PMID:25186191

  9. Effects of acoustic levitation on the development of zebrafish, Danio rerio, embryos

    PubMed Central

    Sundvik, Maria; Nieminen, Heikki J.; Salmi, Ari; Panula, Pertti; Hæggström, Edward

    2015-01-01

    Acoustic levitation provides potential to characterize and manipulate material such as solid particles and fluid in a wall-less environment. While attempts to levitate small animals have been made, the biological effects of such levitation have been scarcely documented. Here, our goal was to explore if zebrafish embryos can be levitated (peak pressures at the pressure node and anti-node: 135 dB and 144 dB, respectively) with no effects on early development. We levitated the embryos (n = 94) at 2–14 hours post fertilization (hpf) for 1000 (n = 47) or 2000 seconds (n = 47). We compared the size and number of trunk neuromasts and otoliths in sonicated samples to controls (n = 94), and found no statistically significant differences (p > 0.05). While mortality rate was lower in the control group (22.3%) compared to that in the 1000 s (34.0%) and 2000 s (42.6%) levitation groups, the differences were statistically insignificant (p > 0.05). The results suggest that acoustic levitation for less than 2000 sec does not interfere with the development of zebrafish embryos, but may affect mortality rate. Acoustic levitation could potentially be used as a non-contacting wall-less platform for characterizing and manipulating vertebrae embryos without causing major adverse effects to their development. PMID:26337364

  10. Tjp3/zo-3 is critical for epidermal barrier function in zebrafish embryos.

    PubMed

    Kiener, Tanja K; Selptsova-Friedrich, Inna; Hunziker, Walter

    2008-04-01

    TJP3/ZO-3 is a scaffolding protein that tethers tight junction integral membrane proteins to the actin cytoskeleton and links the conserved Crumbs polarity complex to tight junctions. The physiological function of TJP3/ZO-3 is not known and mice lacking TJP3/ZO-3 show no apparent phenotype. Here we show that Tjp3/Zo-3 is a component of tight junctions present in the enveloping cell layer of zebrafish embryos. Silencing tjp3/zo-3 using morpholinos leads to edema, loss of blood circulation and tail fin malformations in the embryos. The ultrastructure of tight junctions of the enveloping cell layer is disrupted, without affecting the asymmetric distribution of plasma membrane proteins. Morphants show a loss of the epidermal barrier, as assessed by an increased permeability of the enveloping cell layer to low molecular weight tracers and a higher sensitivity of the embryos to osmotic stress. Subjecting wild-type embryos to osmotic stress mimicks the morphant phenotype, consistent with the phenotype being a direct consequence of failed osmoregulation. Thus, Tjp3/Zo-3 is critical for barrier function of the enveloping cell layer and osmoregulation in early stages of zebrafish development. PMID:18275946

  11. Effects of acoustic levitation on the development of zebrafish, Danio rerio, embryos.

    PubMed

    Sundvik, Maria; Nieminen, Heikki J; Salmi, Ari; Panula, Pertti; Hæggström, Edward

    2015-01-01

    Acoustic levitation provides potential to characterize and manipulate material such as solid particles and fluid in a wall-less environment. While attempts to levitate small animals have been made, the biological effects of such levitation have been scarcely documented. Here, our goal was to explore if zebrafish embryos can be levitated (peak pressures at the pressure node and anti-node: 135 dB and 144 dB, respectively) with no effects on early development. We levitated the embryos (n = 94) at 2-14 hours post fertilization (hpf) for 1000 (n = 47) or 2000 seconds (n = 47). We compared the size and number of trunk neuromasts and otoliths in sonicated samples to controls (n = 94), and found no statistically significant differences (p > 0.05). While mortality rate was lower in the control group (22.3%) compared to that in the 1000 s (34.0%) and 2000 s (42.6%) levitation groups, the differences were statistically insignificant (p > 0.05). The results suggest that acoustic levitation for less than 2000 sec does not interfere with the development of zebrafish embryos, but may affect mortality rate. Acoustic levitation could potentially be used as a non-contacting wall-less platform for characterizing and manipulating vertebrae embryos without causing major adverse effects to their development. PMID:26337364

  12. Endocrine, teratogenic and neurotoxic effects of cyanobacteria detected by cellular in vitro and zebrafish embryos assays.

    PubMed

    Jonas, Adam; Scholz, Stefan; Fetter, Eva; Sychrova, Eliska; Novakova, Katerina; Ortmann, Julia; Benisek, Martin; Adamovsky, Ondrej; Giesy, John P; Hilscherova, Klara

    2015-02-01

    Cyanobacteria contain various types of bioactive compounds, which could cause adverse effects on organisms. They are released into surface waters during cyanobacterial blooms, but there is little information on their potential relevance for effects in vivo. In this study presence of bioactive compounds was characterized in cyanobacteria Microcystis aeruginosa (Chroococcales), Planktothrix agardhii (Oscillatoriales) and Aphanizomenon gracile (Nostocales) with selected in vitro assays. The in vivo relevance of detected bioactivities was analysed using transgenic zebrafish embryos tg(cyp19a1b-GFP). Teratogenic potency was assessed by analysis of developmental disorders and effects on functions of the neuromuscular system by video tracking of locomotion. Estrogenicity in vitro corresponded to 0.95-54.6 ng estradiol equivalent(g dry weight (dw))(-1). In zebrafish embryos, estrogenic effects could not be detected potentially because they were masked by high toxicity. There was no detectable (anti)androgenic/glucocorticoid activity in any sample. Retinoid-like activity was determined at 1-1.3 μg all-trans-retinoic acid equivalent(g dw)(-1). Corresponding to the retinoid-like activity A. gracile extract also caused teratogenic effects in zebrafish embryos. Furthermore, exposure to biomass extracts at 0.3 gd wL(-1) caused increase of body length in embryos. There were minor effects on locomotion caused by 0.3 gd wL(-1)M. aeruginosa and P. agardhii extracts. The traditionally measured cyanotoxins microcystins did not seem to play significant role in observed effects. This indicates importance of other cyanobacterial compounds at least towards some species or their developmental phases. More attention should be paid to activity of retinoids, estrogens and other bioactive substances in phytoplankton using in vitro and in vivo bioassays. PMID:25170595

  13. Combined effects of alpha particles and depleted uranium on Zebrafish (Danio rerio) embryos

    PubMed Central

    Ng, Candy Y.P.; Pereira, Sandrine; Cheng, Shuk Han; Adam-Guillermin, Christelle; Garnier-Laplace, Jacqueline; Yu, Kwan Ngok

    2016-01-01

    The combined effects of low-dose or high-dose alpha particles and depleted uranium (DU) in Zebrafish (Danio rerio) embryos were studied. Three schemes were examined—(i) [ILUL]: 0.44 mGy alpha-particle dose + 10 µg/l DU exposure, (ii) [IHUH]: 4.4 mGy alpha-particle dose + 100 µg/l DU exposure and (iii) [IHUL]: 4.4 mGy alpha-particle dose + 10 µg/l DU exposure—in which Zebrafish embryos were irradiated with alpha particles at 5 h post fertilization (hpf) and/or exposed to uranium at 5–6 hpf. The results were also compared with our previous work, which studied the effects of [ILUH]: 0.44 mGy alpha-particle dose + 100 µg/l DU exposure. When the Zebrafish embryos developed to 24 hpf, the apoptotic signals in the entire embryos, used as the biological endpoint for this study, were quantified. Our results showed that [ILUL] and [IHUL] led to antagonistic effects, whereas [IHUH] led to an additive effect. The effect found for the previously studied case of [ILUH] was difficult to define because it was synergistic with reference to the 100 µg/l DU exposure, but it was antagonistic with reference to the 0.44 mGy alpha-particle dose. All the findings regarding the four different schemes showed that the combined effects critically depended on the dose response to each individual stressor. We also qualitatively explained these findings in terms of promotion of early death of cells predisposed to spontaneous transformation by alpha particles, interacting with the delay in cell death resulting from various concentrations of DU exposure. PMID:26937024

  14. Nanodiamond for biolabelling and toxicity evaluation in the zebrafish embryo in vivo.

    PubMed

    Lin, Y-C; Wu, K-T; Lin, Z-R; Perevedentseva, E; Karmenyan, A; Lin, M-D; Cheng, C-L

    2016-08-01

    Nanodiamond (ND) has been proposed for various biomedical applications, including bioimaging, biosensing and drug delivery, owing to its physical-chemical properties and biocompatibility. Particularly, ND has been demonstrated as fluorescence- and Raman-detectable labels in many cellular models. Different surface functionalization methods have been developed, varying the ND's surface properties and rendering the possibility to attach biomolecules to provide interaction with biological targets. For this, toxicity is of major concern in animal models. Aside from cellular models, a cost-effective animal test will greatly facilitate the development of applications. In this study, we use the rapid, sensitive and reproducible zebrafish embryo model for in vivo nanotoxicity test. We optimize the conditions for using this animal model and analyze the zebrafish embryonic development in the presence of ND. ND is observed in the embryo in vivo using laser confocal fluorescence microscopy and fluorescence lifetime imaging. Using the zebrafish model for a safety evaluation of ND-based nanolabel is discussed. PMID:27093912

  15. Developmental toxicity and alteration of gene expression in zebrafish embryos exposed to PFOS

    SciTech Connect

    Shi Xiongjie; Du Yongbing; Lam, Paul K.S.; Wu, Rudolf S.S.; Zhou Bingsheng

    2008-07-01

    Perfluorooctanesulfonate (PFOS) is a persistent organic pollutant, the potential toxicity of which is causing great concern. In the present study, we employed zebrafish embryos to investigate the developmental toxicity of this compound. Four-hour post-fertilization (hpf) zebrafish embryos were exposed to 0.1, 0.5, 1, 3 and 5 mg/L PFOS. Hatching was delayed and hatching rates as well as larval survivorship were significantly reduced after the embryos were exposed to 1, 3 and 5 mg/L PFOS until 132 hpf. The fry displayed gross developmental malformations, including epiboly deformities, hypopigmentation, yolk sac edema, tail and heart malformations and spinal curvature upon exposure to PFOS concentrations of 1 mg/L or greater. Growth (body length) was significantly reduced in the 3 and 5 mg/L PFOS-treated groups. To test whether developmental malformation was mediated via apoptosis, flow cytometry analysis of DNA content, acridine orange staining and TUNEL assay was used. These techniques indicated that more apoptotic cells were present in the PFOS-treated embryos than in the control embryos. Certain genes related to cell apoptosis, p53 and Bax, were both significantly up-regulated upon exposure to all the concentrations tested. In addition, we investigated the effects of PFOS on marker genes related to early thyroid development (hhex and pax8) and genes regulating the balance of androgens and estrogens (cyp19a and cyp19b). For thyroid development, the expression of hhex was significantly up-regulated at all concentrations tested, whereas pax8 expression was significantly up-regulated only upon exposure to lower concentrations of PFOS (0.1, 0.5, 1 mg/L). The expression of cyp19a and of cyp19b was significantly down-regulated at all exposure concentrations. The overall results indicated that zebrafish embryos constitute a reliable model for testing the developmental toxicity of PFOS, and the gene expression patterns in the embryos were able to reveal some potential

  16. DNA Damage Assessment in Zebrafish Embryos Exposed to Monceren(®) 250 SC Fungicide Using the Alkaline Comet Assay.

    PubMed

    Ku-Centurión, Marco; González-Marín, Berenyce; Calderón-Ezquerro, María C; Martínez-Valenzuela, María C; Maldonado, Ernesto; Calderón-Segura, María E

    2016-10-01

    Monceren 250 SC is a commercial fungicide with the active ingredient 1-(4-chlorobenzyl)-1-(cyclopentyl)-3-phenylurea, also known as pencycuron. This compound inhibits the growth of fungi as Rhizoctonia solani that invades potato, rice, and cotton or as Pellicularia spp, which contaminates lettuce and tomato crops. In this study, we assessed genotoxicity or DNA damage by the alkaline comet assay in zebrafish blastula-stage embryos exposed to 250 to 1250 μg/mL of the Monceren fungicide or to Bleomycin (0.25 μg/mL) used as a positive control. Additionally, survival and spontaneous movement were monitored in embryos after exposure to different concentrations of fungicide. DNA damage was evaluated using three genotoxicity parameters of the alkaline comet assay: tail length, tail moment, and tail intensity. We found that Monceren 250 SC fungicide induces DNA damage, as shown by significant increases in the three genotoxicity parameters in zebrafish embryos compared with control embryos nonexposed to Monceren. Tail intensity was the more accurate parameter to evaluate genotoxicity levels in zebrafish embryos. At 48 h after exposure to the fungicide, the survival rate of larvae-embryos was reduced to 40-45%. This study shows that the Monceren 250 SC fungicide exerts genotoxic effects in zebrafish during early stages of development. PMID:27557408

  17. Reconstruction and representation of caudal vasculature of zebrafish embryo from confocal scanning laser fluorescence microscopic images.

    PubMed

    Feng, Jun; Cheng, Shuk Han; Chan, Po K; Ip, Horace H S

    2005-12-01

    Three-dimensional (3D) reconstruction from a series of sections is an important technique in medical imaging, particularly for visualization of blood vessels from angiography. Here, we present a framework for automatic segmentation and registration of different kind of blood vessels from 2-day-old zebrafish embryos. Series of optical sections were acquired from confocal microscopy with the blood vessels labeled by fluorescent microbeads (0.02 microm) injected into blood stream of 2-day-old zebrafish embryos. Blood vessels were extracted and their morphological parameters, including length and diameter, were calculated. At the same time, individual blood vessels were registered automatically. Vasculature was represented by attributed vessel represent graph (AVRG), which contained morphological data and connectivity of every blood vessel. Using AVRG to represent a vasculature made the comparison between vasculatures of different embryos more easy. Visualization, as well as quantification, of reconstructed 3D model of AVRG was presented in an interactive interface. The framework was implemented by Visual C++ as Windows-based program. PMID:16263106

  18. Toxicity, uptake kinetics and behavior assessment in zebrafish embryos following exposure to perfluorooctanesulphonicacid (PFOS)

    PubMed Central

    Huang, Haihua; Huang, Changjiang; Wang, Lijun; Ye, Xiaowei; Bai, Chenglian; Simonich, Michael T.; Tanguay, Robert L.; Dong, Qiaoxiang

    2014-01-01

    Perfluorooctanesulphonicacid (PFOS), a persistent organic contaminant, has been widely detected in the environment, wildlife and humans, but few studies have assessed its effect on aquatic organisms. The present study evaluated the effect of PFOS on zebrafish embryos. Zebrafish embryos exhibited bent spine and developmental toxicity after exposure to various PFOS concentrations (0.01-16.0 μM) from 6 to 120 hour post-fertilization (hpf). The LC50 at 120 hpf was 4.39 μM and the EC50 at 120 hpf was 2.23 μM. PFOS induced apoptosis at 24 hpf was consistently located in the brain, eye, and tail region of embryos. PFOS elevated the basal rate of swimming after 4 days of exposure, and larvae exposed to PFOS (0.5-8.0μM) for only 1 h at 6 dpf swam faster with increasing PFOS concentration. Larvae exposed to 16.0 μM PFOS for 24 h periods from 1 to 121 hpf showed the highest incidence of malformations in the 97-121 hpf window. Continuous exposure to PFOS from 1 to 121 hpf resulted in a steady accumulation with no evidence of elimination. Our results further the understanding of the health risks of PFOS to aquatic organisms and identify additional research needed on PFOS toxicology. PMID:20171748

  19. Toxicity of sediment cores from Yangtze River estuary to zebrafish (Danio rerio) embryos.

    PubMed

    Wang, Peipei; Zhang, Lili; Liu, Li; Chen, Ling; Gao, Hongwen; Wu, Lingling

    2015-11-01

    Toxicity evaluation is an important segment in sediment quality monitoring in order to protect aquatic organisms and human health. The purpose of this study is to assess the toxicity of sediments from three sediment cores in Yangtze River Estuary, China, using the zebrafish (Danio rerio) embryo tests. Fertilized zebrafish eggs were exposed to both whole sediments and sediment organic extracts prepared from collected sediments, in order to provide a comprehensive and realistic insight into the bioavailable toxicity potential of the sediments. As end points, development parameters (mortality, hatching rate, and abnormality) in the developing embryos were recorded during the 96-h exposure. The results showed that some samples increased mortality, inhibited the hatching of embryos, and induced morphological abnormalities. The embryonic toxicities presented serrated changes and irregular distribution with depth, which may be related to hydrodynamic effect and unstable environmental input. However, lethal and sub-lethal effects were more significant at the sub-surface sediments (10∼40 cm), which indicated that the pollution is more serious in recent decades. PMID:25163567

  20. Real-time prediction of cell division timing in developing zebrafish embryo.

    PubMed

    Kozawa, Satoshi; Akanuma, Takashi; Sato, Tetsuo; Sato, Yasuomi D; Ikeda, Kazushi; Sato, Thomas N

    2016-01-01

    Combination of live-imaging and live-manipulation of developing embryos in vivo provides a useful tool to study developmental processes. Identification and selection of target cells for an in vivo live-manipulation are generally performed by experience- and knowledge-based decision-making of the observer. Computer-assisted live-prediction method would be an additional approach to facilitate the identification and selection of the appropriate target cells. Herein we report such a method using developing zebrafish embryos. We choose V2 neural progenitor cells in developing zebrafish embryo as their successive shape changes can be visualized in real-time in vivo. We developed a relatively simple mathematical method of describing cellular geometry of V2 cells to predict cell division-timing based on their successively changing shapes in vivo. Using quantitatively measured 4D live-imaging data, features of V2 cell-shape at each time point prior to division were extracted and a statistical model capturing the successive changes of the V2 cell-shape was developed. By applying sequential Bayesian inference method to the model, we successfully predicted division-timing of randomly selected individual V2 cells while the cell behavior was being live-imaged. This system could assist pre-selecting target cells desirable for real-time manipulation-thus, presenting a new opportunity for in vivo experimental systems. PMID:27597656

  1. Vitamin D receptor signaling is required for heart development in zebrafish embryo.

    PubMed

    Kwon, Hye-Joo

    2016-02-12

    Vitamin D has been found to be associated with cardiovascular diseases. However, the role of vitamin D in heart development during embryonic period is largely unknown. Vitamin D induces its genomic effects through its nuclear receptor, the vitamin D receptor (VDR). The present study investigated the role of VDR on heart development by antisense-mediated knockdown approaches in zebrafish model system. In zebrafish embryos, two distinct VDR genes (vdra and vdrb) have been identified. Knockdown of vdra has little effect on heart development, whereas disrupting vdrb gene causes various cardiac phenotypes, characterized by pericardial edema, slower heart rate and laterality defects. Depletion of both vdra and vdrb (vdra/b) produce additive, but not synergistic effects. To determine whether atrioventricular (AV) cardiomyocytes are properly organized in these embryos, the expression of bmp4, which marks the developing AV boundary at 48 h post-fertilization, was examined. Notably, vdra/b-deficient embryos display ectopic expression of bmp4 towards the ventricle or throughout atrial and ventricular chambers. Taken together, these results suggest that VDR signaling plays an essential role in heart development. PMID:26797277

  2. Embryotoxicity and genotoxicity evaluation of sediments from Yangtze River estuary using zebrafish (Danio rerio) embryos.

    PubMed

    Li, Qian; Chen, Ling; Liu, Li; Wu, Lingling

    2016-03-01

    Sediments function both as a sink and a source of pollutants in aquatic ecosystems and may impose serious effects on benthic organisms and human health. As one of the largest estuaries in the world, the Yangtze River estuary suffers from abundant wastewater from the coastal cities. In this study, the zebrafish (Danio rerio) embryos were employed in the fish embryo test and a comet assay to evaluate the embryotoxicity and genotoxicity of the sediments from the Yangtze River estuary, respectively. Results showed that the sediments from the Yangtze River estuary significantly increased mortality, induced development abnormalities, and reduced hatching rate and heart rate of zebrafish embryos after 96 h of exposure. Significant genotoxicity was observed in the samples relative to the controls. Relatively low-level embryotoxicity and genotoxicity of sediments were found in the Yangtze River compared with other river systems. Toxic responses were also discussed in relation to the analyzed organic contaminants in sediments. More attention should be paid to non-priority pollutant monitoring in the Yangtze River estuary. PMID:26545894

  3. Selective disruption of vascular endothelium of zebrafish embryos by ultrafast laser microsurgical treatment

    PubMed Central

    Woo, Suk-Yi; Moon, Heh-Young; Kim, Tag Gyum; Lee, Heung Soon; Sidhu, Mehra S.; Kim, Changho; Jeon, Jae-Phil; Jeoung, Sae Chae

    2015-01-01

    In this work, we demonstrate that ultrafast laser irradiation could selectively disrupt vascular endothelium of zebrafish embryos in vivo. Ultrafast lasers minimize the collateral damage in the vicinity of the laser focus and eventually reduce coagulation in the tissues. We have also found that the threshold fluence for lesion formation of the vascular endothelium strongly depends on the developmental stage of the embryos. The threshold laser fluence required to induce apparent lesions in the vascular structure for Somite 14, 20 and 25 stages is about 5 J/cm2 ~7 J/cm2, which is much lower than that for the later development stages of Prim 16 and Prim 20 of 30 J/cm2 ~50 J/cm2. The proposed method for treating the vascular cord of zebrafish embryos in the early stage of development has potential as a selective and effective method to induce a fatal lesion in the vascular endothelium without damaging the developed blood vessels. PMID:26713187

  4. Acoustic radiation force impulse (ARFI) imaging of zebrafish embryo by high-frequency coded excitation sequence.

    PubMed

    Park, Jinhyoung; Lee, Jungwoo; Lau, Sien Ting; Lee, Changyang; Huang, Ying; Lien, Ching-Ling; Kirk Shung, K

    2012-04-01

    Acoustic radiation force impulse (ARFI) imaging has been developed as a non-invasive method for quantitative illustration of tissue stiffness or displacement. Conventional ARFI imaging (2-10 MHz) has been implemented in commercial scanners for illustrating elastic properties of several organs. The image resolution, however, is too coarse to study mechanical properties of micro-sized objects such as cells. This article thus presents a high-frequency coded excitation ARFI technique, with the ultimate goal of displaying elastic characteristics of cellular structures. Tissue mimicking phantoms and zebrafish embryos are imaged with a 100-MHz lithium niobate (LiNbO₃) transducer, by cross-correlating tracked RF echoes with the reference. The phantom results show that the contrast of ARFI image (14 dB) with coded excitation is better than that of the conventional ARFI image (9 dB). The depths of penetration are 2.6 and 2.2 mm, respectively. The stiffness data of the zebrafish demonstrate that the envelope is harder than the embryo region. The temporal displacement change at the embryo and the chorion is as large as 36 and 3.6 μm. Consequently, this high-frequency ARFI approach may serve as a remote palpation imaging tool that reveals viscoelastic properties of small biological samples. PMID:22101757

  5. Assessment of biocompatibility of 3D printed photopolymers using zebrafish embryo toxicity assays.

    PubMed

    Macdonald, N P; Zhu, F; Hall, C J; Reboud, J; Crosier, P S; Patton, E E; Wlodkowic, D; Cooper, J M

    2016-01-21

    3D printing has emerged as a rapid and cost-efficient manufacturing technique to enable the fabrication of bespoke, complex prototypes. If the technology is to have a significant impact in biomedical applications, such as drug discovery and molecular diagnostics, the devices produced must be biologically compatible to enable their use with established reference assays and protocols. In this work we demonstrate that we can adapt the Fish Embryo Test (FET) as a new method to quantify the toxicity of 3D printed microfluidic devices. We assessed the biocompatibility of four commercially available 3D printing polymers (VisiJetCrystal EX200, Watershed 11122XC, Fototec SLA 7150 Clear and ABSplus P-430), through the observation of key developmental markers in the developing zebrafish embryos. Results show all of the photopolymers to be highly toxic to the embryos, resulting in fatality, although we do demonstrate that post-printing treatment of Fototec 7150 makes it suitable for zebrafish culture within the FET. PMID:26646354

  6. Real-time prediction of cell division timing in developing zebrafish embryo

    PubMed Central

    Kozawa, Satoshi; Akanuma, Takashi; Sato, Tetsuo; Sato, Yasuomi D.; Ikeda, Kazushi; Sato, Thomas N.

    2016-01-01

    Combination of live-imaging and live-manipulation of developing embryos in vivo provides a useful tool to study developmental processes. Identification and selection of target cells for an in vivo live-manipulation are generally performed by experience- and knowledge-based decision-making of the observer. Computer-assisted live-prediction method would be an additional approach to facilitate the identification and selection of the appropriate target cells. Herein we report such a method using developing zebrafish embryos. We choose V2 neural progenitor cells in developing zebrafish embryo as their successive shape changes can be visualized in real-time in vivo. We developed a relatively simple mathematical method of describing cellular geometry of V2 cells to predict cell division-timing based on their successively changing shapes in vivo. Using quantitatively measured 4D live-imaging data, features of V2 cell-shape at each time point prior to division were extracted and a statistical model capturing the successive changes of the V2 cell-shape was developed. By applying sequential Bayesian inference method to the model, we successfully predicted division-timing of randomly selected individual V2 cells while the cell behavior was being live-imaged. This system could assist pre-selecting target cells desirable for real-time manipulation–thus, presenting a new opportunity for in vivo experimental systems. PMID:27597656

  7. Nucleolar protein 4-like has a complex expression pattern in zebrafish embryos.

    PubMed

    Borah, Supriya; Barrodia, Praveen; Swain, Rajeeb K

    2016-01-01

    The nucleolar protein 4-like (NOL4L) gene is present on chromosome 20 (20q11.21) in humans. Parts of this gene have been shown to fuse with RUNX1 and PAX5 in acute myeloid leukemia and acute lymphoblastic leukemia, respectively. The normal function of NOL4L in humans and other organisms is not well understood. The expression patterns and functions of NOL4L homologs during vertebrate development have not been reported. We sought to address these questions by studying the expression pattern of zebrafish nol4l during embryogenesis. Our data show that Znol4l mRNA is expressed in multiple organs in zebrafish embryos. The sites of expression include parts of the brain, spinal cord, pronephros, hematopoietic cells and gut. PMID:26934290

  8. Zebrafish embryos as a screen for DNA methylation modifications after compound exposure.

    PubMed

    Bouwmeester, Manon C; Ruiter, Sander; Lommelaars, Tobias; Sippel, Josefine; Hodemaekers, Hennie M; van den Brandhof, Evert-Jan; Pennings, Jeroen L A; Kamstra, Jorke H; Jelinek, Jaroslav; Issa, Jean-Pierre J; Legler, Juliette; van der Ven, Leo T M

    2016-01-15

    Modified epigenetic programming early in life is proposed to underlie the development of an adverse adult phenotype, known as the Developmental Origins of Health and Disease (DOHaD) concept. Several environmental contaminants have been implicated as modifying factors of the developing epigenome. This underlines the need to investigate this newly recognized toxicological risk and systematically screen for the epigenome modifying potential of compounds. In this study, we examined the applicability of the zebrafish embryo as a screening model for DNA methylation modifications. Embryos were exposed from 0 to 72 h post fertilization (hpf) to bisphenol-A (BPA), diethylstilbestrol, 17α-ethynylestradiol, nickel, cadmium, tributyltin, arsenite, perfluoroctanoic acid, valproic acid, flusilazole, 5-azacytidine (5AC) in subtoxic concentrations. Both global and site-specific methylation was examined. Global methylation was only affected by 5AC. Genome wide locus-specific analysis was performed for BPA exposed embryos using Digital Restriction Enzyme Analysis of Methylation (DREAM), which showed minimal wide scale effects on the genome, whereas potential informative markers were not confirmed by pyrosequencing. Site-specific methylation was examined in the promoter regions of three selected genes vasa, vtg1 and cyp19a2, of which vasa (ddx4) was the most responsive. This analysis distinguished estrogenic compounds from metals by direction and sensitivity of the effect compared to embryotoxicity. In conclusion, the zebrafish embryo is a potential screening tool to examine DNA methylation modifications after xenobiotic exposure. The next step is to examine the adult phenotype of exposed embryos and to analyze molecular mechanisms that potentially link epigenetic effects and altered phenotypes, to support the DOHaD hypothesis. PMID:26712470

  9. Acute ZnO nanoparticles exposure induces developmental toxicity, oxidative stress and DNA damage in embryo-larval zebrafish.

    PubMed

    Zhao, Xuesong; Wang, Shutao; Wu, Yuan; You, Hong; Lv, Lina

    2013-07-15

    Nano-scale zinc oxide (nano-ZnO) is widely used in various industrial and commercial applications. However, the available toxicological information was inadequate to assess the potential ecological risk of nano-ZnO to aquatic organisms and the publics. In this study, the developmental toxicity, oxidative stress and DNA damage of nano-ZnO embryos were investigated in the embryo-larval zebrafish, the toxicity of Zn(2+) releasing from nano-ZnO were also investigated to ascertain the relationship between the nano-ZnO and corresponding Zn(2+). Zebrafish embryos were exposed to 1, 5, 10, 20, 50, and 100mg/L nano-ZnO and 0.59, 2.15, 3.63, 4.07, 5.31, and 6.04 mg/L Zn(2+) for 144 h post-fertilisation (hpf), respectively. Up to 144 hpf, activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and malondialdehyde (MDA) contents, the genes related to oxidative damage, reactive oxygen species (ROS) generation and DNA damage in zebrafish embryos were measured. The nano-ZnO was found to exert a dose-dependent toxicity to zebrafish embryos and larvae, reducing the hatching rate and inducing malformation and the acute toxicity to zebrafish embryos was greater than that of the Zn(2+) solution. The generation of ROS was significantly increased at 50 and 100mg/L nano-ZnO. DNA damage of zebrafish embryo was evaluated by single-cell gel electrophoresis and was enhanced with increasing nano-ZnO concentration. Moreover, the transcriptional expression of mitochondrial inner membrane genes related to ROS production, such as Bcl-2, in response to oxidative damage, such as Nqo1, and related to antioxidant response element such as Gstp2 were significantly down-regulated in the nano-ZnO treatment groups. However, the nano-ZnO up-regulated the transcriptional expression of Ucp2-related to ROS production. In conclusion, nano-ZnO induces developmental toxicity, oxidative stress and DNA damage on zebrafish embryos and the dissolved Zn(2+) only partially

  10. Automated detection and quantification of single RNAs at cellular resolution in zebrafish embryos.

    PubMed

    Stapel, L Carine; Lombardot, Benoit; Broaddus, Coleman; Kainmueller, Dagmar; Jug, Florian; Myers, Eugene W; Vastenhouw, Nadine L

    2016-02-01

    Analysis of differential gene expression is crucial for the study of cell fate and behavior during embryonic development. However, automated methods for the sensitive detection and quantification of RNAs at cellular resolution in embryos are lacking. With the advent of single-molecule fluorescence in situ hybridization (smFISH), gene expression can be analyzed at single-molecule resolution. However, the limited availability of protocols for smFISH in embryos and the lack of efficient image analysis pipelines have hampered quantification at the (sub)cellular level in complex samples such as tissues and embryos. Here, we present a protocol for smFISH on zebrafish embryo sections in combination with an image analysis pipeline for automated transcript detection and cell segmentation. We use this strategy to quantify gene expression differences between different cell types and identify differences in subcellular transcript localization between genes. The combination of our smFISH protocol and custom-made, freely available, analysis pipeline will enable researchers to fully exploit the benefits of quantitative transcript analysis at cellular and subcellular resolution in tissues and embryos. PMID:26700682

  11. Toxicity and cardiac effects of carbaryl in early developing zebrafish (Danio rerio) embryos

    SciTech Connect

    Lin, C.C.; Hui, Michelle N.Y.; Cheng, S.H. E-mail: bhcheng@cityu.edu.hk

    2007-07-15

    Carbaryl, an acetylcholinesterase inhibitor, is known to be moderately toxic to adult zebrafish and has been reported to cause heart malformations and irregular heartbeat in medaka. We performed experiments to study the toxicity of carbaryl, specifically its effects on the heart, in early developing zebrafish embryos. LC50 and EC50 values for carbaryl at 28 h post-fertilization were 44.66 {mu}g/ml and 7.52 {mu}g/ml, respectively, and 10 {mu}g/ml carbaryl was used in subsequent experiments. After confirming acetylcholinesterase inhibition by carbaryl using an enzymatic method, we observed red blood cell accumulation, delayed hatching and pericardial edema, but not heart malformation as described in some previous reports. Our chronic exposure data also demonstrated carbaryl-induced bradycardia, which is a common effect of acetylcholinesterase inhibitors due to the accumulation of acetylcholine, in embryos from 1 day post-fertilization (dpf) to 5 dpf. The distance between the sinus venosus, the point where blood enters the atrium, and the bulbus arteriosus, the point where blood leaves the ventricle, indicated normal looping of the heart tube. Immunostaining of myosin heavy chains with the ventricle-specific antibody MF20 and the atrium-specific antibody S46 showed normal development of heart chambers. At the same time, acute exposure resulted in carbaryl-induced bradycardia. Heart rate dropped significantly after a 10-min exposure to 100 {mu}g/ml carbaryl but recovered when carbaryl was removed. The novel observation of carbaryl-induced bradycardia in 1- and 2-dpf embryos suggested that carbaryl affected cardiac function possibly through an alternative mechanism other than acetylcholinesterase inhibition such as inhibition of calcium ion channels, since acetylcholine receptors in zebrafish are not functional until 3 dpf. However, the exact nature of this mechanism is currently unknown, and thus further studies are required.

  12. Transcriptomic Changes in Zebrafish Embryos and Larvae Following Benzo[a]pyrene Exposure

    PubMed Central

    Fang, Xiefan; Corrales, Jone; Thornton, Cammi; Clerk, Tracy; Scheffler, Brian E.; Willett, Kristine L.

    2015-01-01

    Benzo[a]pyrene (BaP) is an environmentally relevant carcinogenic and endocrine disrupting compound that causes immediate, long-term, and multigenerational health deficits in mammals and fish. Previously, we found that BaP alters DNA methylation patterns in developing zebrafish, which may affect gene expression. Herein, we performed a genome-wide transcriptional analysis and discovered differential gene expression and splicing in developing zebrafish. Adult zebrafish were exposed to control or 42.0 ± 1.9 µg/l BaP for 7 days. Eggs were collected and raised in control conditions or continuously exposed to BaP until 3.3 and 96 h post–fertilization (hpf). RNA sequencing (RNA-Seq) was conducted on zebrafish embryos and larvae. Data were analyzed to identify differentially expressed (DE) genes (changed at the gene or transcript variant level) and genes with differential exon usage (DEU; changed at the exon level). At 3.3 hpf, BaP exposure resulted in 8 DE genes and 51 DEU genes. At 96 hpf, BaP exposure altered expression in 1153 DE genes and 159 DEU genes. Functional ontology analysis by Ingenuity Pathway Analysis revealed that many disease pathways, including organismal death, growth failure, abnormal morphology of embryonic tissue, congenital heart disease, and adverse neuritogenesis, were significantly enriched for the DE and DEU genes, providing novel insights on the mechanisms of action of BaP-induced developmental toxicities. Collectively, we discovered substantial transcriptomic changes at the gene, transcript variant, and exon levels in developing zebrafish after early life BaP waterborne exposure, and these changes may lead to long-term adverse physiological consequences. PMID:26001963

  13. Zebrafish (Danio rerio) embryo as a platform for the identification of novel angiogenesis inhibitors of retinal vascular diseases.

    PubMed

    Rezzola, Sara; Paganini, Giuseppe; Semeraro, Francesco; Presta, Marco; Tobia, Chiara

    2016-07-01

    Pathological angiogenesis of the retina is a main cause of blindness. Therapeutic approaches targeting vascular endothelial growth factor, a main angiogenesis inducer in retinal vascular diseases, show significant limitations. Thus, experimental models of retinal neovascularization remain crucial for investigating novel anti-angiogenic strategies and bringing them to patients. Recent observations have shown that eye neovascularization in zebrafish (Danio rerio) embryo may represent a novel target for the identification of angiogenesis inhibitors. This review highlights the use of zebrafish embryo as an innovative model system for the screening of anti-angiogenic molecules to be employed for the treatment of angiogenesis-dependent eye diseases. PMID:27085972

  14. Short-term chilled storage of zebrafish (Danio rerio) embryos in cryoprotectant as an alternative to cryopreservation.

    PubMed

    Desai, Kunjan; Spikings, Emma; Zhang, Tiantian

    2015-02-01

    As zebrafish embryos have never been cryopreserved, we developed a protocol to store zebrafish embryos (50% epiboly-5.3 hour post fertilization) for up to 18 h at 0°C. Initial experiments to optimize the cryoprotectant (CPA) solution demonstrated improved embryo hatching rate following chilling at 0°C for 18 h with 1 M MeOH+0.1 M sucrose (56 ± 5%) compared with other combinations of methanol (0.2-0.5 M) and sucrose (0.05-0.1 M). This combination of CPAs that protects against chilling injury was further tested to assess its impact on sox gene and protein expression. Significant decreases in sox3 gene expression were observed in hatched embryos that had been chilled for 18 h in 1 M MeOH+0.1 sucrose compared with non-chilled controls, however the expression of both sox2 and sox3 proteins was unaffected. Significant decreases in sox2 protein expression were, however, observed in embryos that had been chilled without CPAs and these embryos also had lower hatching rates than those chilled with the optimal CPA solution. We, therefore, conclude that the CPA combination of 1 M MeOH+0.1 M sucrose facilitates chilled storage of early stage (50% epiboly) zebrafish embryos for up to 18 h without compromising transcriptional response. PMID:25545702

  15. Random Walk of Single Gold Nanoparticles in Zebrafish Embryos Leading to Stochastic Toxic Effects on Embryonic Developments

    PubMed Central

    Browning, Lauren M.; Lee, Kerry J.; Huang, Tao; Nallathamby, Prakash D.; Lowman, Jill E.; Xu, Xiao-Hong Nancy

    2010-01-01

    We have synthesized and characterized stable (non-aggregation, non-photobleaching and non-blinking), nearly monodisperse and highly-purified Au nanoparticles, and used them to probe transport of cleavage-stage zebrafish embryos and to study their effects on embryonic development in real time. We found that single Au nanoparticles (11.6 ± 0.9 nm in diameter) passively diffused into chorionic space of the embryos via their chorionic-pore-canals and continued their random-walk through chorionic space and into inner mass of embryos. Diffusion coefficients of single nanoparticles vary dramatically (2.8×10-11 to 1.3×10-8 cm2/s) as nanoparticles diffuse through various parts of embryos, suggesting highly diverse transport barriers and viscosity gradients of embryos. The amount of Au nanoparticles accumulated in embryos increase with its concentration. Interestingly, their effects on embryonic development are not proportionally related to the concentration. Majority of embryos (74% on average) incubated chronically with 0.025-1.2 nM Au nanoparticles for 120 h developed to normal zebrafish, with some (24%) being dead and few (2%) deformed. We developed a new approach to image and characterize individual Au nanoparticles embedded in tissues using histology sample preparation methods and LSRP spectra of single nanoparticles. We found that Au nanoparticles in various parts of normally developed and deformed zebrafish, suggesting that random-walk of nanoparticles in embryos during their development might have led to stochastic effects on embryonic development. These results show that Au nanoparticles are much more biocompatible (less toxic) to the embryos than Ag nanoparticles that we reported previously, suggesting that they are better suited as biocompatible probes for imaging embryos in vivo. The results provide powerful evidences that biocompatibility and toxicity of nanoparticles highly depend on their chemical properties, and the embryos can serve as effective in

  16. Muscular contractions in the zebrafish embryo are necessary to reveal thiuram-induced notochord distortions

    SciTech Connect

    Teraoka, Hiroki . E-mail: hteraoka@rakuno.ac.jp; Urakawa, Satsuki; Nanba, Satomi; Nagai, Yuhki; Wu Dong; Imagawa, Tomohiro; Tanguay, Robert L.; Svoboda, Kurt; Handley-Goldstone, Heather M.; Stegeman, John J.; Hiraga, Takeo

    2006-04-01

    Dithiocarbamates form a large group of chemicals that have numerous uses in agriculture and medicine. It has been reported that dithiocarbamates, including thiuram (tetramethylthiuram disulfide), cause wavy distortions of the notochord in zebrafish and other fish embryos. In the present study, we investigated the mechanism underlying the toxicity of thiuram in zebrafish embryos. When embryos were exposed to thiuram (2-1000 nM: 0.48-240 {mu}g/L) from 3 h post fertilization (hpf) (30% epiboly) until 24 hpf (Prim-5), all embryos develop wavy notochords, disorganized somites, and have shortened yolk sac extensions. The thiuram response was specific and did not cause growth retardation or mortality at 24 hpf. The thiuram-dependent responses showed the same concentration dependence with a waterborne EC{sub 5} values of approximately 7 nM. Morphometric measurements revealed that thiuram does not affect the rate of notochord lengthening. However, the rate of overall body lengthening was significantly reduced in thiuram-exposed animals. Other dithiocarbamates, such as ziram, caused similar malformations to thiuram. While expression of genes involved in somitogenesis was not affected, the levels of notochord-specific transcripts were altered after the onset of malformations. Distortion of the notochord started precisely at 18 hpf, which is concomitant with onset of spontaneous rhythmic trunk contractions. Abolishment of spontaneous contractions using tricaine, {alpha}-bungarotoxin, and a paralytic mutant sofa potato, resulted in normal notochord morphology in the presence of thiuram. These results indicate that muscle activity is necessary to reveal the underlying functional deficit and suggest that the developmental target of dithiocarbamates impairs trunk plasticity through an unknown mechanism.

  17. Developing a Novel Embryo-Larval Zebrafish Xenograft Assay to Prioritize Human Glioblastoma Therapeutics.

    PubMed

    Wehmas, Leah Christine; Tanguay, Robert L; Punnoose, Alex; Greenwood, Juliet A

    2016-08-01

    Glioblastoma is an aggressive brain cancer requiring improved treatments. Existing methods of drug discovery and development require years before new therapeutics become available to patients. Zebrafish xenograft models hold promise for prioritizing drug development. We have developed an embryo-larval zebrafish xenograft assay in which cancer cells are implanted in a brain microenvironment to discover and prioritize compounds that impact glioblastoma proliferation, migration, and invasion. We illustrate the utility of our assay by evaluating the well-studied, phosphatidylinositide 3-kinase inhibitor LY294002 and zinc oxide nanoparticles (ZnO NPs), which demonstrate selective cancer cytotoxicity in cell culture, but the in vivo effectiveness has not been established. Exposures of 3.125-6.25 μM LY294002 significantly decreased proliferation up to 34% with concentration-dependent trends. Exposure to 6.25 μM LY294002 significantly inhibited migration/invasion by ∼27% within the glioblastoma cell mass (0-80 μm) and by ∼32% in the next distance region (81-160 μm). Unexpectedly, ZnO enhanced glioblastoma proliferation by ∼19% and migration/invasion by ∼35% at the periphery of the cell mass (161+ μm); however, dissolution of these NPs make it difficult to discern whether this was a nano or ionic effect. These results demonstrate that we have a short, relevant, and sensitive zebrafish-based assay to aid glioblastoma therapeutic development. PMID:27158859

  18. Developmental enhancers revealed by extensive DNA methylome maps of zebrafish early embryos

    PubMed Central

    Lee, Hyung Joo; Lowdon, Rebecca F; Maricque, Brett; Zhang, Bo; Stevens, Michael; Li, Daofeng; Johnson, Stephen L; Wang, Ting

    2015-01-01

    DNA methylation undergoes dynamic changes during development and cell differentiation. Recent genome-wide studies discovered that tissue-specific differentially methylated regions (DMRs) often overlap tissue-specific distal cis-regulatory elements. However, developmental DNA methylation dynamics of the majority of the genomic CpGs outside gene promoters and CpG islands has not been extensively characterized. Here we generate and compare comprehensive DNA methylome maps of zebrafish developing embryos. From these maps we identify thousands of developmental stage-specific DMRs (dsDMR) across zebrafish developmental stages. The dsDMRs contain evolutionarily conserved sequences, are associated with developmental genes, and are marked with active enhancer histone post-translational modifications. Their methylation pattern correlates much stronger than promoter methylation with expression of putative target genes. When tested in vivo using a transgenic zebrafish assay, 20 out of 20 selected candidate dsDMRs exhibit functional enhancer activities. Our data suggest that developmental enhancers are a major target of DNA methylation changes during embryogenesis. PMID:25697895

  19. Interactions of Hydroxyapatite with Proteins and Its Toxicological Effect to Zebrafish Embryos Development

    PubMed Central

    Xu, Zhen; Zhang, Ya-Lei; Song, Cao; Wu, Ling-Ling; Gao, Hong-Wen

    2012-01-01

    The increased application of nanomaterials has raised the level of public concern regarding possible toxicities caused by exposure to nanostructures. The interactions of nanosized hydroxyapatite (HA) with cytochrome c and hemoglobin were investigated by zeta-potential, UV-vis, fluorescence and circular dichroism. The experimental results indicated that the interactions were formed via charge attraction and hydrogen bond and obeyed Langmuir adsorption isotherm. The two functional proteins bridged between HA particles to aggregate into the coralloid form, where change of the secondary structure of proteins occurred. From effects of nanosized HA, SiO2 and TiO2 particles on the zebrafish embryos development, they were adsorbed on the membrane surface confirmed by the electronic scanning microscopy. Nano-HA aggregated into the biggest particles around the membrane protein and then caused a little toxicity to development of zebrafish embryos. The SiO2 particles were distributed throughout the outer surface and caused jam of membrane passage, delay of the hatching time and axial malformation. Maybe owing to the oxygen free radical activity, TiO2 caused some serious deformity characters in the cardiovascular system. PMID:22509249

  20. Interactions of hydroxyapatite with proteins and its toxicological effect to zebrafish embryos development.

    PubMed

    Xu, Zhen; Zhang, Ya-Lei; Song, Cao; Wu, Ling-Ling; Gao, Hong-Wen

    2012-01-01

    The increased application of nanomaterials has raised the level of public concern regarding possible toxicities caused by exposure to nanostructures. The interactions of nanosized hydroxyapatite (HA) with cytochrome c and hemoglobin were investigated by zeta-potential, UV-vis, fluorescence and circular dichroism. The experimental results indicated that the interactions were formed via charge attraction and hydrogen bond and obeyed Langmuir adsorption isotherm. The two functional proteins bridged between HA particles to aggregate into the coralloid form, where change of the secondary structure of proteins occurred. From effects of nanosized HA, SiO(2) and TiO(2) particles on the zebrafish embryos development, they were adsorbed on the membrane surface confirmed by the electronic scanning microscopy. Nano-HA aggregated into the biggest particles around the membrane protein and then caused a little toxicity to development of zebrafish embryos. The SiO(2) particles were distributed throughout the outer surface and caused jam of membrane passage, delay of the hatching time and axial malformation. Maybe owing to the oxygen free radical activity, TiO(2) caused some serious deformity characters in the cardiovascular system. PMID:22509249

  1. Intrinsic Expression of a Multiexon Type 3 Deiodinase Gene Controls Zebrafish Embryo Size

    PubMed Central

    Guo, Cuicui; Chen, Xia; Song, Huaidong; Maynard, Michelle A.; Zhou, Yi; Lobanov, Alexei V.; Gladyshev, Vadim N.; Ganis, Jared J.; Wiley, David; Jugo, Rebecca H.; Lee, Nicholas Y.; Castroneves, Luciana A.; Zon, Leonard I.; Scanlan, Thomas S.; Feldman, Henry A.

    2014-01-01

    Thyroid hormone is a master regulator of differentiation and growth, and its action is terminated by the enzymatic removal of an inner-ring iodine catalyzed by the selenoenzyme type 3 deiodinase (dio3). Our studies of the zebrafish reveal that the dio3 gene is duplicated in this species and that embryonic deiodination is an important determinant of embryo size. Although both dio3 paralogs encode enzymatically active proteins with high affinity for thyroid hormones, their anatomic patterns of expression are markedly divergent and only embryos with knockdown of dio3b, a biallelically expressed selenoenzyme expressed in the developing central nervous system, manifest severe thyroid hormone-dependent growth restriction at 72 hours post fertilization. This indicates that the embryonic deficiency of dio3, once considered only a placental enzyme, causes microsomia independently of placental physiology and raises the intriguing possibility that fetal abnormalities in human deiodination may present as intrauterine growth retardation. By mapping the gene structures and enzymatic properties of all four zebrafish deiodinases, we also identify dio3b as the first multiexon dio3 gene, containing a large intron separating its open reading frame from its selenocysteine insertion sequence (SECIS) element. PMID:25004091

  2. Comparative analysis of goitrogenic effects of phenylthiourea and methimazole in zebrafish embryos.

    PubMed

    Fetter, Eva; Baldauf, Lisa; Da Fonte, Dillon F; Ortmann, Julia; Scholz, Stefan

    2015-11-01

    Craniofacial malformations, reduced locomotion and induction of genes encoding for enzymes involved in thyroid hormone synthesis were assessed using methimazole and N-phenylthiourea in zebrafish embryos. Gene expression, the most sensitive endpoint (EC50_MMI=372-765μM, EC50_PTU=7.6-8.6μM), was analysed in wild-type and in a transgenic strain, tg(tg:mCherry), expressing mCherry fluorescence protein under the control of the thyroglobulin gene. Reduction of locomotion and craniofacial malformations were observed at one or two orders of magnitude above concentrations affecting gene expression, respectively. Both effects could be linked to the malformations caused by reduced thyroxin levels. Our results show that due to the presence of the autoregulatory loop of the hypothalamus-pituitary-thyroid axis, various molecular initiating events of thyroid disruption are amenable for the zebrafish embryo. We propose the tg(tg:mCherry) bioassay as a sensitive tool in medium scale screening of goitrogens, given the minimal effort for sample preparation and analysis of gene expression. PMID:25962731

  3. Enantioselective separation and zebrafish embryo toxicity of insecticide beta-cypermethrin.

    PubMed

    Xu, Chao; Tu, Wenqing; Lou, Chun; Hong, Yingying; Zhao, Meirong

    2010-01-01

    Enantioselectivity of chiral pollutants is receiving growing concern due to the difference in toxicology and environment fate between enantiomers. In this study, enantiomers of insecticide beta-cypermethrin (beta-CP) were separated on selected chiral column by HPLC, and the toxicity of enantiomers was evaluated using the zebrafish embryo-larval assays. The enantiomers of beta-CP were baseline separated on Chiralcel OD and Chiralpak AD columns and detected by circular dichroism (CD) at 236 nm. Better separation could be achieved at lower temperature (e.g., 20 degrees C) and with lower levels of polar modifiers. Pure enantiomers were obtained on Chiralcel OD. The CD spectra of enantiomers were recorded. By comparing the elution order with a previous similar study, the absolute configuration of beta-CP enantiomers was determined. The individual enantiomers were used in zebrafish embryo test, and the results showed that beta-CP enantioselectively induced yolk sac edema, pericardial edema and crooked body. The IR-cis-alphaS and 1R-trans-alphaS enantiomers showed strong developmental toxicities at concentration of 0.1 mg/L, while the 1S-cis-alphaR and 1S-trans-alphaR induced no malformations at higher concentration (e.g., 0.3 mg/L). The results suggest that the enantioselective toxicological effects of beta-CP should be considered when evaluating its ecotoxicological effects. PMID:20608511

  4. Evaluation of cytotoxicity and genotoxicity of insecticide carbaryl to flounder gill cells and its teratogenicity to zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Pandey, Manish Raj; Guo, Huarong

    2015-04-01

    In this study, we determined the cytotoxicity and genotoxicity of carbamate insecticide carbaryl to flounder gill (FG) cells and its teratogenicity to zebrafish embryos. The cytotoxicity of carbaryl to FG cells was determined with methods including MTT and neutral red uptaking (NRU), lactate dehydrogenase (LDH) releasing and Hoechst 33342 and propidium idodide (PI) double staining. Moderate cytotoxicity in a concentration-dependent manner was observed. The 24 h-IC50 value of 53.48 ± 1.21, 59.13 ± 1.19 and 46.21 ± 1.24 mg L-1 carbaryl was obtained through MTT, NRU and LDH assays, respectively. Double fluorescence staining demonstrated that carbaryl induced the death of FG cells mainly through necrosis. There was no significant genotoxicity found in the FG cells exposed to the highest testing concentration of carbaryl (20 mg L-1, P > 0.05) as was demonstrated by Comet assay. Zebrafish embryos exposed to carbaryl at concentrations ≥10 mg L-1 displayed moderate toxic effects on the survival, spontaneous movement, hatching, heart rates of the embryos and their development, which were evidenced by yolk and pericardial sac edemas, body length reduction and tail flexure in time- and concentration-dependent manners at specific stages. The 24 h-, 48 h- and 96 h-LC50 values of carbaryl to zebrafish embryos were 41.80 ± 1.10, 17.80 ± 1.04 and 14.46 ± 1.05 mg L-1, respectively. These results suggested that carbaryl is moderately toxic to FG cells cultured in vitro and zebrafish embryos, and the FG cells were similar to zebrafish embryos in their sensitivity to carbaryl as 24 h-IC50 and LC50 indicated.

  5. Thymosin beta4 regulates cardiac valve formation via endothelial-mesenchymal transformation in zebrafish embryos.

    PubMed

    Shin, Sun-Hye; Lee, Sangkyu; Bae, Jong-Sup; Jee, Jun-Goo; Cha, Hee-Jae; Lee, You Mie

    2014-04-01

    Thymosin beta4 (TB4) has multiple functions in cellular response in processes as diverse as embryonic organ development and the pathogeneses of disease, especially those associated with cardiac coronary vessels. However, the specific roles played by TB4 during heart valve development in vertebrates are largely unknown. Here, we identified a novel function of TB4 in endothelialmesenchymal transformation (EMT) in cardiac valve endocardial cushions in zebrafish. The expressions of thymosin family members in developing zebrafish embryos were determined by whole mount in situ hybridization. Of the thymosin family members only zTB4 was expressed in the developing heart region. Cardiac valve development at 48 h post fertilization was defected in zebrafish TB4 (zTB4) morpholino-injected embryos (morphants). In zTB4 morphants, abnormal linear heart tube development was observed. The expressions of bone morphogenetic protein (BMP) 4, notch1b, and hyaluronic acid synthase (HAS) 2 genes were also markedly reduced in atrio-ventricular canal (AVC). Endocardial cells in the AVC region were stained with anti-Zn5 antibody reactive against Dm-grasp (an EMT marker) to observe EMT in developing cardiac valves in zTB4 morphants. EMT marker expression in valve endothelial cells was confirmed after transfection with TB4 siRNA in the presence of transforming growth factor β (TGFβ) by RT-PCR and immunofluorescent assay. Zn5-positive endocardial AVC cells were not observed in zTB4 morphants, and knockdown of TB4 suppressed TGF-β-induced EMT in ovine valve endothelial cells. Taken together, our results demonstrate that TB4 plays a pivotal role in cardiac valve formation by increasing EMT.1. PMID:24732964

  6. The Chromosomal Passenger Protein Birc5b Organizes Microfilaments and Germ Plasm in the Zebrafish Embryo

    PubMed Central

    Nair, Sreelaja; Marlow, Florence; Abrams, Elliott; Kapp, Lee; Mullins, Mary C.; Pelegri, Francisco

    2013-01-01

    Microtubule-microfilament interactions are important for cytokinesis and subcellular localization of proteins and mRNAs. In the early zebrafish embryo, astral microtubule-microfilament interactions also facilitate a stereotypic segregation pattern of germ plasm ribonucleoparticles (GP RNPs), which is critical for their eventual selective inheritance by germ cells. The precise mechanisms and molecular mediators for both cytoskeletal interactions and GP RNPs segregation are the focus of intense research. Here, we report the molecular identification of a zebrafish maternal-effect mutation motley as Birc5b, a homolog of the mammalian Chromosomal Passenger Complex (CPC) component Survivin. The meiosis and mitosis defects in motley/birc5b mutant embryos are consistent with failed CPC function, and additional defects in astral microtubule remodeling contribute to failures in the initiation of cytokinesis furrow ingression. Unexpectedly, the motley/birc5b mutation also disrupts cortical microfilaments and GP RNP aggregation during early cell divisions. Birc5b localizes to the tips of astral microtubules along with polymerizing cortical F-actin and the GP RNPs. Mutant Birc5b co-localizes with cortical F-actin and GP RNPs, but fails to associate with astral microtubule tips, leading to disorganized microfilaments and GP RNP aggregation defects. Thus, maternal Birc5b localizes to astral microtubule tips and associates with cortical F-actin and GP RNPs, potentially linking the two cytoskeletons to mediate microtubule-microfilament reorganization and GP RNP aggregation during early embryonic cell cycles in zebrafish. In addition to the known mitotic function of CPC components, our analyses reveal a non-canonical role for an evolutionarily conserved CPC protein in microfilament reorganization and germ plasm aggregation. PMID:23637620

  7. Cortisol Regulates Acid Secretion of H+-ATPase-rich Ionocytes in Zebrafish (Danio rerio) Embryos

    PubMed Central

    Lin, Chia-Hao; Shih, Tin-Han; Liu, Sian-Tai; Hsu, Hao-Hsuan; Hwang, Pung-Pung

    2015-01-01

    Systemic acid-base regulation is vital for physiological processes in vertebrates. Freshwater (FW) fish live in an inconstant environment, and thus frequently face ambient acid stress. FW fish have to efficiently modulate their acid secretion processes for body fluid acid-base homeostasis during ambient acid challenge; hormonal control plays an important role in such physiological regulation. The hormone cortisol was previously proposed to be associated with acid base regulation in FW fish; however, the underlying mechanism has not been fully described. In the present study, mRNA expression of acid-secreting related transporters and cyp11b (encoding an enzyme involved in cortisol synthesis) in zebrafish embryos was stimulated by treatment with acidic FW (AFW, pH 4.0) for 3 d. Exogenous cortisol treatment (20 mg/L, 3 d) resulted in upregulated expression of transporters related to acid secretion and increased acid secretion function at the organism level in zebrafish embryos. Moreover, cortisol treatment also significantly increased the acid secretion capacity of H+-ATPase-rich cells (HRCs) at the cellular level. In loss-of-function experiments, microinjection of glucocorticoid receptor (GR) morpholino (MO) suppressed the expression of acid-secreting related transporters, and decreased acid secretion function at both the organism and cellular levels; on the other hand, mineralocorticoid receptor (MR) MO did not induce any effects. Such evidence supports the hypothesized role of cortisol in fish acid-base regulation, and provides new insights into the roles of cortisol; cortisol-GR signaling stimulates zebrafish acid secretion function through transcriptional/translational regulation of the transporters and upregulation of acid secretion capacity in each acid-secreting ionocyte. PMID:26635615

  8. Retinoic acid receptor subtype-specific transcriptotypes in the early zebrafish embryo.

    PubMed

    Samarut, Eric; Gaudin, Cyril; Hughes, Sandrine; Gillet, Benjamin; de Bernard, Simon; Jouve, Pierre-Emmanuel; Buffat, Laurent; Allot, Alexis; Lecompte, Odile; Berekelya, Liubov; Rochette-Egly, Cécile; Laudet, Vincent

    2014-02-01

    Retinoic acid (RA) controls many aspects of embryonic development by binding to specific receptors (retinoic acid receptors [RARs]) that regulate complex transcriptional networks. Three different RAR subtypes are present in vertebrates and play both common and specific roles in transducing RA signaling. Specific activities of each receptor subtype can be correlated with its exclusive expression pattern, whereas shared activities between different subtypes are generally assimilated to functional redundancy. However, the question remains whether some subtype-specific activity still exists in regions or organs coexpressing multiple RAR subtypes. We tackled this issue at the transcriptional level using early zebrafish embryo as a model. Using morpholino knockdown, we specifically invalidated the zebrafish endogenous RAR subtypes in an in vivo context. After building up a list of RA-responsive genes in the zebrafish gastrula through a whole-transcriptome analysis, we compared this panel of genes with those that still respond to RA in embryos lacking one or another RAR subtype. Our work reveals that RAR subtypes do not have fully redundant functions at the transcriptional level but can transduce RA signal in a subtype-specific fashion. As a result, we define RAR subtype-specific transcriptotypes that correspond to repertoires of genes activated by different RAR subtypes. Finally, we found genes of the RA pathway (cyp26a1, raraa) the regulation of which by RA is highly robust and can even resist the knockdown of all RARs. This suggests that RA-responsive genes are differentially sensitive to alterations in the RA pathway and, in particular, cyp26a1 and raraa are under a high pressure to maintain signaling integrity. PMID:24422634

  9. Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos

    PubMed Central

    2012-01-01

    Background Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish) and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38) in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff), contains three copies of oestrogen response elements (3ERE) that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS) elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein). Results The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1–2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17ß-oestradiol (E2), the synthetic oestrogen 17α- ethinyloestradiol (EE2), and the relatively weak environmental oestrogen nonylphenol (NP), and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10 ng/L, E2; 100 ng/L, after 72 h exposures). For the NP exposures, GFP expression was observed at 10 μg NP/L after 72 h (100 μg NP/L was toxic to the fish). We also demonstrate that

  10. High-content screening in zebrafish embryos identifies butafenacil as a potent inducer of anemia.

    PubMed

    Leet, Jessica K; Lindberg, Casey D; Bassett, Luke A; Isales, Gregory M; Yozzo, Krystle L; Raftery, Tara D; Volz, David C

    2014-01-01

    Using transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's) zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05-50 µM) using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO)--an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors--flumioxazin--resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1) screening chemicals for cardiovascular toxicity and (2) prioritizing chemicals for future hypothesis-driven and mechanism

  11. High-Content Screening in Zebrafish Embryos Identifies Butafenacil as a Potent Inducer of Anemia

    PubMed Central

    Leet, Jessica K.; Lindberg, Casey D.; Bassett, Luke A.; Isales, Gregory M.; Yozzo, Krystle L.; Raftery, Tara D.; Volz, David C.

    2014-01-01

    Using transgenic zebrafish (fli1:egfp) that stably express enhanced green fluorescent protein (eGFP) within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS) assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's) zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05–50 µM) using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO) – an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors – flumioxazin – resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1) screening chemicals for cardiovascular toxicity and (2) prioritizing chemicals for future hypothesis

  12. Efficacy of UV-C photolysis of bisphenol A on transcriptome alterations of genes in zebrafish embryos.

    PubMed

    Saeed, Asma; Hashmi, Imran; Zare, Ava; Mehrabani-Zeinabad, Mitra; Achari, Gopal; Habibi, Hamid R

    2016-09-18

    The purpose of this study was to investigate the efficacy of UV-C direct photolysis of bisphenol A (BPA) as a remediation method of BPA contamination. We used zebrafish embryos as a model organism to test the toxicity and residual biological activity by measuring cytochrome P4501A1 (CYP1A), aromatase B (Aro B) and heat shock proteins (HSP-70) transcript levels. The mRNA levels of CYP1A gene increased about two fold while exposure of zebrafish embryos at 72 hpf resulted in significant induction (P = 0.048) of Aro B at 100 µg/L of BPA. Exposure of zebrafish embryos at 72 hpf to increasing concentrations of BPA resulted in significant induction (P = 0.0031) of HSP-70 transcript level. UV treatment of BPA resulted in a significant reduction in toxicity by reducing mortality of zebrafish embryos. The results suggest that UV-C direct photolysis may be an effective method for remediation of BPA contamination. Further studies will be necessary for better understanding of the identity and relative activity of the UV degradation by-products. PMID:27314163

  13. Individual and joint toxic effects of cadmium sulfate and α-naphthoflavone on the development of zebrafish embryo.

    PubMed

    Yin, Jian; Yang, Jian-ming; Zhang, Feng; Miao, Peng; Lin, Ying; Chen, Ming-li

    2014-09-01

    This paper aims to evaluate the individual and joint toxicities of cadmium sulfate (CdSO4) and α-naphthoflavone (ANF) in zebrafish embryos. As a result, CdSO4 caused both lethal and sub-lethal effects, such as 24 h post-fertilization (hpf) death and 72 hpf delayed hatching. However, ANF only caused sub-lethal effects, including 48 hpf cardiac edema and 72 hpf delayed hatching. Taking 24 hpf death and 48 hpf cardiac edema as endpoints, the toxicities of CdSO4 and ANF were significantly enhanced by each other. Consistently, both CdSO4 and ANF caused significant oxidative stress, including decreases in the reduced glutathione (GSH) level, inhibition of superoxide dismutase (SOD) activity, as well as increases in malondialdehyde (MDA) content in zebrafish embryos, but these mixtures produced much more significant alterations on the biomarkers. Co-treatment of CdSO4 and ANF significantly down-regulated the mRNA level of multidrug resistance-associated protein (mrp) 1 and cytochrome P450 (cyp) 1a, which constituted the protective mechanisms for zebrafish embryos to chemical toxins. In conclusion, co-treatment of CdSO4 and ANF exhibited a much more severe damage in zebrafish embryos than individual treatment. Meanwhile, production of oxidative stress and altered expression of mrp1 and cyp1a could be important components of such joint toxicity. PMID:25183031

  14. Total synthesis of Herbarin A and B, determination of their antioxidant properties and toxicity in zebrafish embryo model.

    PubMed

    Heimberger, Julia; Cade, Hannah C; Padgett, Jihan; Sittaramane, Vinoth; Shaikh, Abid

    2015-03-15

    Herbarin A and B were isolated from the fungal strains of Cladosporium herbarum found in marine sponges Aplysina aerophoba and Callyspongia aerizusa. Total synthesis of Herbarin A and B was achieved by carrying out a multi-step synthesis approach, and the antioxidant properties were evaluated using FRAP assay. Toxicity of these compounds was determined using a zebrafish embryo model. PMID:25690788

  15. Whole-Mount Immunohistochemistry for Anti-F59 in Zebrafish Embryos (1-5 Days Post Fertilization (dpf)).

    PubMed

    Doganli, Canan; Bukata, Lucas; Lykke-Hartmann, Karin

    2016-01-01

    Immunohistochemistry (IHC) is a powerful method to determine localization of tissue components by the interaction of target antigens with labeled antibodies. Here we describe an IHC protocol for localizing the myosin heavy chain of zebrafish embryos at 1-2 and 3-5 days post fertilization (dpf). PMID:26695047

  16. Individual and joint toxic effects of cadmium sulfate and α-naphthoflavone on the development of zebrafish embryo*

    PubMed Central

    Yin, Jian; Yang, Jian-ming; Zhang, Feng; Miao, Peng; Lin, Ying; Chen, Ming-li

    2014-01-01

    This paper aims to evaluate the individual and joint toxicities of cadmium sulfate (CdSO4) and α-naphthoflavone (ANF) in zebrafish embryos. As a result, CdSO4 caused both lethal and sub-lethal effects, such as 24 h post-fertilization (hpf) death and 72 hpf delayed hatching. However, ANF only caused sub-lethal effects, including 48 hpf cardiac edema and 72 hpf delayed hatching. Taking 24 hpf death and 48 hpf cardiac edema as endpoints, the toxicities of CdSO4 and ANF were significantly enhanced by each other. Consistently, both CdSO4 and ANF caused significant oxidative stress, including decreases in the reduced glutathione (GSH) level, inhibition of superoxide dismutase (SOD) activity, as well as increases in malondialdehyde (MDA) content in zebrafish embryos, but these mixtures produced much more significant alterations on the biomarkers. Co-treatment of CdSO4 and ANF significantly down-regulated the mRNA level of multidrug resistance-associated protein (mrp) 1 and cytochrome P450 (cyp) 1a, which constituted the protective mechanisms for zebrafish embryos to chemical toxins. In conclusion, co-treatment of CdSO4 and ANF exhibited a much more severe damage in zebrafish embryos than individual treatment. Meanwhile, production of oxidative stress and altered expression of mrp1 and cyp1a could be important components of such joint toxicity. PMID:25183031

  17. Acetylcholinesterase in zebrafish embryos as a tool to identify neurotoxic effects in sediments.

    PubMed

    Kais, Britta; Stengel, Daniel; Batel, Annika; Braunbeck, Thomas

    2015-11-01

    In order to clarify the suitability of zebrafish (Danio rerio) embryos for the detection of neurotoxic compounds, the acetylcholinesterase assay was adapted and validated with a series of priority pollutants listed as relevant for the European water policy (Aroclor 1254, 2,3-benzofuran, bisphenol A, chlorpyrifos, paraoxon-methyl, quinoline, and methyl mercury chloride) as well as acetonic extracts from three sediments of known contamination. The acute toxicities of the model substances and the sediment extracts were determined by means of the fish embryo test as specified in OECD TG 236, and concentrations as low as the effective concentration at 10% inhibition (EC10) were used as the highest test concentration in the acetylcholinesterase test in order to avoid nonspecific systemic effects mimicking neurotoxicity. Among the model compounds, only the known acetylcholinesterase inhibitors paraoxon-methyl and chlorpyrifos produced a strong inhibition to about 20 and 33%, respectively, of the negative controls. For the sediment extracts, a reduction of acetylcholinesterase activity to about 60% could only be shown for the Vering Canal sediment extracts; this could be correlated to high contents of acetylcholinesterase-inhibiting polycyclic aromatic hydrocarbons (PAHs) as identified by chemical analyses. Co-incubation of the Vering Canal sediment extracts with chlorpyrifos at EC10 concentrations each did not significantly increase the inhibitory effect of chlorpyrifos, indicating that the mode of action of acetylcholinesterase inhibition by the sediment-borne PAHs is different to that of the typical acetylcholinesterase blocker chlorpyrifos. Overall, the study documents that zebrafish embryos represent a suitable model not only to reveal acetylcholinesterase inhibition, but also to investigate various modes of neurotoxic action. PMID:25567057

  18. The toxicity of silver nanoparticles to zebrafish embryos increases through sewage treatment processes.

    PubMed

    Muth-Köhne, Elke; Sonnack, Laura; Schlich, Karsten; Hischen, Florian; Baumgartner, Werner; Hund-Rinke, Kerstin; Schäfers, Christoph; Fenske, Martina

    2013-10-01

    Silver nanoparticles (AgNPs) are widely believed to be retained in the sewage sludge during sewage treatment. The AgNPs and their derivatives, however, re-enter the environment with the sludge and via the effluent. AgNP were shown to occur in surface water, while evidence of a potential toxicity of AgNPs in aquatic organisms is growing. This study aims to examine the toxicity of AgNPs to the embryos of the aquatic vertebrate model zebrafish (Danio rerio) before and after sewage treatment plants (STPs) processes. Embryos were treated with AgNP (particle size: >90 % <20 nm) and AgNO3 in ISO water for 48 h and consequently displayed effects such as delayed development, tail malformations and edema. For AgNP, the embryos were smaller than the controls with conspicuously smaller yolk sacs. The corresponding EC50 values of 48 hours post fertilization (hpf) were determined as 73 μg/l for AgNO3 and 1.1 mg/l for AgNP. Whole-mount immunostainings of primary and secondary motor neurons also revealed secondary neurotoxic effects. A TEM analysis confirmed uptake of the AgNPs, and the distribution within the embryo suggested absorption across the skin. Embryos were also exposed (for 48 h) to effluents of AgNP-spiked model STP with AgNP influent concentrations of 4 and 16 mg/l. These embryos exhibited the same malformations than for AgNO3 and AgNPs, but the embryo toxicity of the sewage treatment effluent was higher (EC50 = 142 μg/l; 48 hpf). On the other hand, control STP effluent spiked with AgNPs afterwards was less toxic (EC50 = 2.9 mg/l; 48 hpf) than AgNPs in ISO water. This observation of an increased fish embryo toxicity of STP effluents with increasing AgNP influent concentrations identifies the accumulation of AgNP in the STP as a potential source of effluent toxicity. PMID:23975539

  19. Assessment of Jatropha curcas L. biodiesel seed cake toxicity using the zebrafish (Danio rerio) embryo toxicity (ZFET) test.

    PubMed

    Hallare, Arnold V; Ruiz, Paulo Lorenzo S; Cariño, J C Earl D

    2014-05-01

    Consequent to the growing demand for alternative sources of energy, the seeds from Jatropha curcas remain to be the favorite for biodiesel production. However, a significant volume of the residual organic mass (seed cake) is produced during the extraction process, which raises concerns on safe waste disposal. In the present study, we assessed the toxicity of J. curcas seed cake using the zebrafish (Danio rerio) embryotoxicity test. Within 1-h post-fertilization (hpf), the fertilized eggs were exposed to five mass concentrations of J. curcas seed cake and were followed through 24, 48, and 72 hpf. Toxicity was evaluated based on lethal endpoints induced on zebrafish embryos namely egg coagulation, non-formation of somites, and non-detachment of tail. The lowest concentration tested, 1 g/L, was not able to elicit toxicity on embryos whereas 100 % mortality (based also on lethal endpoints) was recorded at the highest concentration at 2.15 g/L. The computed LC50 for the J. curcas seed cake was 1.61 g/L. No further increase in mortality was observed in the succeeding time points (48 and 72 hpf) indicating that J. curcas seed cake exerted acute toxicity on zebrafish embryos. Sublethal endpoints (yolk sac and pericardial edema) were noted at 72 hpf in zebrafish embryos exposed to higher concentrations. The observed lethal endpoints induced on zebrafish embryos were discussed in relation to the active principles, notably, phorbol esters that have remained in the seed cake even after extraction. PMID:24464135

  20. Toxicity assessment and bioaccumulation in zebrafish embryos exposed to carbon nanotubes suspended in Pluronic® F-108.

    PubMed

    Wang, Ruhung; N Meredith, Alicea; Lee, Michael; Deutsch, Dakota; Miadzvedskaya, Lizaveta; Braun, Elizabeth; Pantano, Paul; Harper, Stacey; Draper, Rockford

    2016-08-01

    Carbon nanotubes (CNTs) are often suspended in Pluronic® surfactants by sonication, which may confound toxicity studies because sonication of surfactants can create degradation products that are toxic to mammalian cells. Here, we present a toxicity assessment of Pluronic® F-108 with and without suspended CNTs using embryonic zebrafish as an in vivo model. Pluronic® sonolytic degradation products were toxic to zebrafish embryos just as they were to mammalian cells. When the toxic Pluronic® fragments were removed, there was little effect of pristine multi-walled CNTs (pMWNTs), carboxylated MWNTs (cMWNTs) or pristine single-walled carbon nanotubes (pSWNTs) on embryo viability and development, even at high concentrations. A gel electrophoretic method coupled with Raman imaging was developed to measure the bioaccumulation of CNTs by zebrafish embryos, and dose-dependent uptake of CNTs was observed. These data indicate that embryos accumulate pMWNTs, cMWNTs and pSWNTs yet there is very little embryo toxicity. PMID:26559437

  1. Guarding Embryo Development of Zebrafish by Shell Engineering: A Strategy to Shield Life from Ozone Depletion

    PubMed Central

    Wang, Ben; Liu, Peng; Tang, Yanyan; Pan, Haihua; Xu, Xurong; Tang, Ruikang

    2010-01-01

    Background The reduced concentration of stratospheric ozone results in an increased flux of biologically damaging mid-ultraviolet radiation (UVB, 280 to 320 nm) reaching earth surfaces. Environmentally relevant levels of UVB negatively impact various natural populations of marine organisms, which is ascribed to suppressed embryonic development by increased radiation. Methodology/Principal Findings Inspired by strategies in the living systems generated by evolution, we induce an extra UVB-adsorbed coat on the chorion (eggshell surrounding embryo) of zebrafish, during the blastula period. Short and long UV exposure experiments show that the artificial mineral-shell reduces the UV radiation effectively and the enclosed embryos become more robust. In contrast, the uncoated embryos cannot survive under the enhanced UVB condition. Conclusions We suggest that an engineered shell of functional materials onto biological units can be developed as a strategy to shield lives to counteract negative changes of global environment, or to provide extra protection for the living units in biological research. PMID:20376356

  2. Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos.

    PubMed

    Giger, Florence A; Dumortier, Julien G; David, Nicolas B

    2016-01-01

    Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo. PMID:27168357

  3. Indole Alkaloids from Fischerella Inhibit Vertebrate Development in the Zebrafish (Danio rerio) Embryo Model

    PubMed Central

    Walton, Katherine; Gantar, Miroslav; Gibbs, Patrick D. L.; Schmale, Michael C.; Berry, John P.

    2014-01-01

    Cyanobacteria are recognized producers of toxic or otherwise bioactive metabolite associated, in particular, with so-called “harmful algal blooms” (HABs) and eutrophication of freshwater systems. In the present study, two apparently teratogenic indole alkaloids from a freshwater strain of the widespread cyanobacterial genus, Fischerella (Stigonemataceae), were isolated by bioassay-guided fractionation, specifically using the zebrafish (Danio rerio) embryo, as a model of vertebrate development. The two alkaloids include the previously known 12-epi-hapalindole H isonitrile (1), and a new nitrile-containing variant, 12-epi-ambiguine B nitrile (2). Although both compounds were toxic to developing embryos, the former compound was shown to be relatively more potent, and to correlate best with the observed embryo toxicity. Related indole alkaloids from Fischerella, and other genera in the Stigonemataceae, have been widely reported as antimicrobial compounds, specifically in association with apparent allelopathy. However, this is the first report of their vertebrate toxicity, and the observed teratogenicity of these alkaloids supports a possible contribution to the toxicity of this widespread cyanobacterial family, particularly in relation to freshwater HABs and eutrophication. PMID:25533520

  4. Biosurfactant templated quantum sized fluorescent gold nanoclusters for in vivo bioimaging in zebrafish embryos.

    PubMed

    S, Chandirasekar; C, Chandrasekaran; T, Muthukumarasamyvel; G, Sudhandiran; N, Rajendiran

    2016-07-01

    We report the biosurfactant (sodium cholate) templated bright bluish-green emitting gold nanoclusters (AuNCs) by green chemical approach. Optical properties of the AuNCs were studied using UV-vis and luminescence spectroscopy. Lifetime of the fluorescent AuNCs was measured using time correlated single photon counting technique (TCSPC). High-resolution transmission electron microscopy (HR-TEM) and dynamic light scattering (DLS) were used to measure the sizes of the clusters. In-vivo toxicity and bioimaging studies of sodium cholate (NaC) templated AuNCs were carried out at different developmental stages of zebrafish embryos. The survival rate, hatching rate, heart rate, malformation and apoptotic gene expression experiments shows no significant toxicity in developing embryos up to 100μL/mL of AuNCs concentration and the AuNCs stained embryos exhibited green fluorescence with high intensity over the period from 4 to 96hpf (hours post fertilization) which shows that AuNCs were stable in living organisms. PMID:27037785

  5. Parental exposure to natural mixtures of POPs reduced embryo production and altered gene transcription in zebrafish embryos.

    PubMed

    Lyche, Jan L; Grześ, Irena M; Karlsson, Camilla; Nourizadeh-Lillabadi, Rasoul; Berg, Vidar; Kristoffersen, Anja B; Skåre, Janneche U; Alestrøm, Peter; Ropstad, Erik

    2013-01-15

    Determination of toxicity of complex mixtures has been proposed to be one of the most important challenges for modern toxicology. In this study we performed genome wide transcriptome profiling to assess potential toxicant induced changes in gene regulation in zebrafish embryos following parental exposure to two natural mixtures of persistent organic pollutants (POPs). The mixtures used were extracted from burbot (Lota lota) liver originating from two lakes (Lake Mjøsa and Lake Losna) belonging to the same freshwater system in Norway. The dominating groups of contaminants were polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane metabolites (DDTs). Because both mixtures used in the present study induced similar effects, it is likely that the same toxicants are involved. The Mjøsa mixture contains high levels of PBDEs while this group of pollutants is low in the Losna mixture. However, both mixtures contain substantial concentrations of PCB and DDT suggesting these contaminants as the predominant contributors to the toxicity observed. The observed effects included phenotypic traits, like embryo production and survival, and gene transcription changes corresponding with disease and biological functions such as cancer, reproductive system disease, cardiovascular disease, lipid and protein metabolism, small molecule biochemistry and cell cycle. The changes in gene transcription included genes regulated by HNF4A, insulin, LH, FSH and NF-κB which are known to be central regulators of endocrine signaling, metabolism, metabolic homeostasis, immune functions, cancer development and reproduction. The results suggest that relative low concentrations of the natural mixtures of POPs used in the present study might pose a threat to wild freshwater fish living in the lakes from which the POPs mixtures originated. PMID:23063069

  6. Comparison of static immersion and intravenous injection systems for exposure of zebrafish embryos to the natural pathogen Edwardsiella tarda

    PubMed Central

    2011-01-01

    Background The zebrafish embryo is an important in vivo model to study the host innate immune response towards microbial infection. In most zebrafish infectious disease models, infection is achieved by micro-injection of bacteria into the embryo. Alternatively, Edwardsiella tarda, a natural fish pathogen, has been used to treat embryos by static immersion. In this study we used transcriptome profiling and quantitative RT-PCR to analyze the immune response induced by E. tarda immersion and injection. Results Mortality rates after static immersion of embryos in E. tarda suspension varied between 25-75%, while intravenous injection of bacteria resulted in 100% mortality. Quantitative RT-PCR analysis on the level of single embryos showed that expression of the proinflammatory marker genes il1b and mmp9 was induced only in some embryos that were exposed to E. tarda in the immersion system, whereas intravenous injection of E. tarda led to il1b and mmp9 induction in all embryos. In addition, microarray expression profiles of embryos subjected to immersion or injection showed little overlap. E. tarda-injected embryos displayed strong induction of inflammatory and defense genes and of regulatory genes of the immune response. E. tarda-immersed embryos showed transient induction of the cytochrome P450 gene cyp1a. This gene was also induced after immersion in Escherichia coli and Pseudomonas aeruginosa suspensions, but, in contrast, was not induced upon intravenous E. tarda injection. One of the rare common responses in the immersion and injection systems was induction of irg1l, a homolog of a murine immunoresponsive gene of unknown function. Conclusions Based on the differences in mortality rates between experiments and gene expression profiles of individual embryos we conclude that zebrafish embryos cannot be reproducibly infected by exposure to E. tarda in the immersion system. Induction of il1b and mmp9 was consistently observed in embryos that had been systemically

  7. A Caged Ret Kinase Inhibitor and its Effect on Motoneuron Development in Zebrafish Embryos

    PubMed Central

    Bliman, David; Nilsson, Jesper R.; Kettunen, Petronella; Andréasson, Joakim; Grøtli, Morten

    2015-01-01

    Proto-oncogene tyrosine-protein kinase receptor RET is implicated in the development and maintenance of neurons of the central and peripheral nervous systems. Attaching activity-compromising photocleavable groups (caging) to inhibitors could allow for external spatiotemporally controlled inhibition using light, potentially providing novel information on how these kinase receptors are involved in cellular processes. Here, caged RET inhibitors were obtained from 3-substituted pyrazolopyrimidine-based compounds by attaching photolabile groups to the exocyclic amino function. The most promising compound displayed excellent inhibitory effect in cell-free, as well as live-cell assays upon decaging. Furthermore, inhibition could be efficiently activated with light in vivo in zebrafish embryos and was shown to effect motoneuron development. PMID:26300345

  8. Mixture toxicity of water contaminants-effect analysis using the zebrafish embryo assay (Danio rerio).

    PubMed

    Schmidt, Susanne; Busch, Wibke; Altenburger, Rolf; Küster, Eberhard

    2016-06-01

    Three water contaminants were selected to be tested in the zebrafish embryo toxicity test (DarT) in order to investigate the sensitivity of the zebrafish embryo toxicity test with respect to mixture effect detection. The concentration-response curves for the observed effects lethality and hypo-pigmentation were calculated after an exposure of the embryos for 96 h with a fungicide (carbendazim), a plasticizer or propellent precursor (2,4-DNT: 2,4- dinitrotoluene) and an aromatic compound (AαC: 2-amino-9H-pyrido[2,3-b]indol), respectively. Follow-up mixture tests were based on the calculated LC50 or EC50 of the single compounds and combined effects were predicted according to the mixture concepts of concentration addition (CA) and independent action (IA). The order of toxicity for the single substances was carbendazim (LC50 = 1.25 μM) < AαC (LC50 = 8.16 μM) < 2,4-DNT (LC50 = 177.05 μM). For AαC and 2,4 DNT hypo-pigmentation was observed in addition (AαC EC50 = 1.81 μM; 2,4-DNT EC50 = 8.81 μM). Two binary and one ternary mixture were studied on lethality and one on hypo-pigmentation: 2,4-DNT/AαC (LC50 = 119.21 μM, EC50 = 5.37 μM), carbendazim/AαC (LC50 = 4.49 μM) and AαC/Carbendazim/2,4 DNT (LC50 = 108.62 μM). Results showed that the effects were in agreement with the CA model when substances were tested in mixtures. Therefore, in a reasonable worst case scenario substance combination effects in fish embryos were at maximum only prone to overestimation when using CA as the mixture concept. PMID:27011319

  9. Modulation by Cocaine of Dopamine Receptors through miRNA-133b in Zebrafish Embryos

    PubMed Central

    Barreto-Valer, Katherine; López-Bellido, Roger; Macho Sánchez-Simón, Fátima; Rodríguez, Raquel E.

    2012-01-01

    The use of cocaine during pregnancy can affect the mother and indirectly might alter the development of the embryo/foetus. Accordingly, in the present work our aim was to study in vivo (in zebrafish embryos) the effects of cocaine on the expression of dopamine receptors and on miR-133b. These embryos were exposed to cocaine hydrochloride (HCl) at 5 hours post-fertilization (hpf) and were then collected at 8, 16, 24, 48 and 72 hpf to study the expression of dopamine receptors, drd1, drd2a, drd2b and drd3, by quantitative real time PCR (qPCR) and in situ hybridization (ISH, only at 24 hpf). Our results indicate that cocaine alters the expression of the genes studied, depending on the stage of the developing embryo and the type of dopamine receptor. We found that cocaine reduced the expression of miR-133b at 24 and 48 hpf in the central nervous system (CNS) and at the periphery by qPCR and also that the spatial distribution of miR-133b was mainly seen in somites, a finding that suggests the involvement of miR-133b in the development of the skeletal muscle. In contrast, at the level of the CNS miR-133b had a weak and moderate expression at 24 and 48 hpf. We also analysed the interaction of miR-133b with the Pitx3 and Pitx3 target genes drd2a and drd2b, tyrosine hydroxylase (th) and dopamine transporter (dat) by microinjection of the Pitx3-3'UTR sequence. Microinjection of Pitx3-3'UTR affected the expression of pitx3, drd2a, drd2b, th and dat. In conclusion, in the present work we describe a possible mechanism to account for cocaine activity by controlling miR-133b transcription in zebrafish. Via miR-133b cocaine would modulate the expression of pitx3 and subsequently of dopamine receptors, dat and th. These results indicate that miRNAs can play an important role during embryogenesis and in drug addiction. PMID:23285158

  10. Toxicological implications of microcystins for zebrafish embryos in the presence of other environmental pollutants.

    PubMed

    Pavagadhi, Shruti; Gong, Zhiyuan; Balasubramanian, Rajasekhar

    2013-07-01

    Microcystins (MCs) interact with environmental contaminants as well as various other congeners of the MC family in the natural environment and with antioxidants in the exposed organisms. These interactions are likely to modify the toxicological behavior of MCs at the cellular level. The present study was conducted to determine the toxicological response of extracellular MCs in aquatic systems under environmentally relevant conditions. Microcystin-leucine-arginine (MCLR) and microcystin-arginine-arginine (MCRR) were introduced at different concentrations in a single-component (MCLR or MCRR) or dual-component (MCLR and MCRR) system to zebrafish embryos in the presence of inorganic elements (Hg, As, Pb, and Cd) and nutrient species (NO3 (-) , PO4 (3-) , and Cl(-1) ). Hatchability, heart rate, and mortality of zerbrafish embryos were monitored together with changes in the activity of glutathione-S-transferase (GST) to evaluate their response on exposure to MCLR and MCRR. There was a significant reduction in all these parameters at higher doses of MCLR and MCRR (>100 ng/mL), implying bioaccumulation of these MCs in embryos and adverse effects on early development stages of the fish. It was further observed that PO4 (3-) and Cl(-) enhanced the toxic effects of MCLR and MCRR while NO3 (-) attenuated their toxic effects. In contrast, all 4 toxic elements together increased the toxicity of MCLR and MCRR to embryos compared with their single-component counterparts. Thus, the toxic effects of MCs depend not only on their relative environmental concentrations, but also on those of other environmental pollutants and the levels of antioxidants in exposed organisms. PMID:23440872

  11. Hypoxia-induced retinal neovascularization in zebrafish embryos: a potential model of retinopathy of prematurity.

    PubMed

    Wu, Yu-Ching; Chang, Chao-Yuan; Kao, Alex; Hsi, Brian; Lee, Shwu-Huey; Chen, Yau-Hung; Wang, I-Jong

    2015-01-01

    Retinopathy of prematurity, formerly known as a retrolental fibroplasia, is a leading cause of infantile blindness worldwide. Retinopathy of prematurity is caused by the failure of central retinal vessels to reach the retinal periphery, creating a nonperfused peripheral retina, resulting in retinal hypoxia, neovascularization, vitreous hemorrhage, vitreoretinal fibrosis, and loss of vision. We established a potential retinopathy of prematurity model by using a green fluorescent vascular endothelium zebrafish transgenic line treated with cobalt chloride (a hypoxia-inducing agent), followed by GS4012 (a vascular endothelial growth factor inducer) at 24 hours postfertilization, and observed that the number of vascular branches and sprouts significantly increased in the central retinal vascular trunks 2-4 days after treatment. We created an angiography method by using tetramethylrhodamine dextran, which exhibited severe vascular leakage through the vessel wall into the surrounding retinal tissues. The quantification of mRNA extracted from the heads of the larvae by using real-time quantitative polymerase chain reaction revealed a twofold increase in vegfaa and vegfr2 expression compared with the control group, indicating increased vascular endothelial growth factor signaling in the hypoxic condition. In addition, we demonstrated that the hypoxic insult could be effectively rescued by several antivascular endothelial growth factor agents such as SU5416, bevacizumab, and ranibizumab. In conclusion, we provide a simple, highly reproducible, and clinically relevant retinopathy of prematurity model based on zebrafish embryos; this model may serve as a useful platform for clarifying the mechanisms of human retinopathy of prematurity and its progression. PMID:25978439

  12. Exploring the Effects of Different Types of Surfactants on Zebrafish Embryos and Larvae

    PubMed Central

    Wang, Yanan; Zhang, Yuan; Li, Xu; Sun, Mingzhu; Wei, Zhuo; Wang, Yu; Gao, Aiai; Chen, Dongyan; Zhao, Xin; Feng, Xizeng

    2015-01-01

    Currently, surfactants are widely distributed in the environment. As organic pollutants, their toxicities have drawn extensive attention. In this study, the effects of anionic [sodium dodecyl sulphate (SDS) ], cationic [dodecyl dimethyl benzyl ammonium chloride (1227)] and non-ionic [fatty alcohol polyoxyethylene ether (AEO) ] surfactants on zebrafish larval behaviour were evaluated. Five behavioural parameters were recorded using a larval rest/wake assay, including rest total, number of rest bouts, rest bouts length, total activity and waking activity. The results revealed that 1227 and AEO at 1 μg/mL were toxic to larval locomotor activity and that SDS had no significant effects. Moreover, we tested the toxicities of the three surfactants in developing zebrafish embryos. AEO exposure resulted in smaller head size, smaller eye size and shorter body length relative to SDS and 1227. All three surfactants incurred concentration-dependent responses. Furthermore, in situ hybridisation indicated that smaller head size may be associated with a decreased expression of krox20. The altered expression of ntl demonstrated that the developmental retardation stemmed from inhibited cell migration and growth. These findings provide references for ecotoxicological assessments of different types of surfactants, and play a warning role in the application of surfactants. PMID:26053337

  13. Transcriptomic analysis in the developing zebrafish embryo after compound exposure: Individual gene expression and pathway regulation

    SciTech Connect

    Hermsen, Sanne A.B.; Pronk, Tessa E.; Brandhof, Evert-Jan van den; Ven, Leo T.M. van der; Piersma, Aldert H.

    2013-10-01

    The zebrafish embryotoxicity test is a promising alternative assay for developmental toxicity. Classically, morphological assessment of the embryos is applied to evaluate the effects of compound exposure. However, by applying differential gene expression analysis the sensitivity and predictability of the test may be increased. For defining gene expression signatures of developmental toxicity, we explored the possibility of using gene expression signatures of compound exposures based on commonly expressed individual genes as well as based on regulated gene pathways. Four developmental toxic compounds were tested in concentration-response design, caffeine, carbamazepine, retinoic acid and valproic acid, and two non-embryotoxic compounds, D-mannitol and saccharin, were included. With transcriptomic analyses we were able to identify commonly expressed genes, which were mostly development related, after exposure to the embryotoxicants. We also identified gene pathways regulated by the embryotoxicants, suggestive of their modes of action. Furthermore, whereas pathways may be regulated by all compounds, individual gene expression within these pathways can differ for each compound. Overall, the present study suggests that the use of individual gene expression signatures as well as pathway regulation may be useful starting points for defining gene biomarkers for predicting embryotoxicity. - Highlights: • The zebrafish embryotoxicity test in combination with transcriptomics was used. • We explored two approaches of defining gene biomarkers for developmental toxicity. • Four compounds in concentration-response design were tested. • We identified commonly expressed individual genes as well as regulated gene pathways. • Both approaches seem suitable starting points for defining gene biomarkers.

  14. Tyrosine glycosylation of Rho by Yersinia toxin impairs blastomere cell behaviour in zebrafish embryos

    PubMed Central

    Jank, Thomas; Eckerle, Stephanie; Steinemann, Marcus; Trillhaase, Christoph; Schimpl, Marianne; Wiese, Sebastian; van Aalten, Daan M. F.; Driever, Wolfgang; Aktories, Klaus

    2015-01-01

    Yersinia species cause zoonotic infections, including enterocolitis and plague. Here we studied Yersinia ruckeri antifeeding prophage 18 (Afp18), the toxin component of the phage tail-derived protein translocation system Afp, which causes enteric redmouth disease in salmonid fish species. Here we show that microinjection of the glycosyltransferase domain Afp18G into zebrafish embryos blocks cytokinesis, actin-dependent motility and cell blebbing, eventually abrogating gastrulation. In zebrafish ZF4 cells, Afp18G depolymerizes actin stress fibres by mono-O-GlcNAcylation of RhoA at tyrosine-34; thereby Afp18G inhibits RhoA activation by guanine nucleotide exchange factors, and blocks RhoA, but not Rac and Cdc42 downstream signalling. The crystal structure of tyrosine-GlcNAcylated RhoA reveals an open conformation of the effector loop distinct from recently described structures of GDP- or GTP-bound RhoA. Unravelling of the molecular mechanism of the toxin component Afp18 as glycosyltransferase opens new perspectives in studies of phage tail-derived protein translocation systems, which are preserved from archaea to human pathogenic prokaryotes. PMID:26190758

  15. Ruthenium-Caged Antisense Morpholinos for Regulating Gene Expression in Zebrafish Embryos

    PubMed Central

    Griepenburg, J.C.; Rapp, T.L.; Carroll, P.J.; Eberwine, J.; Dmochowski, I.J.

    2015-01-01

    Photochemical approaches afford high spatiotemporal control over molecular structure and function, for broad applications in materials and biological science. Here, we present the first example of a visible light responsive ruthenium-based photolinker, Ru(bipyridine)2(3-ethynylpyridine)2 (RuBEP), which was reacted stoichiometrically with a 25mer DNA or morpholino (MO) oligonucleotide functionalized with 3′ and 5′ terminal azides, via Cu(I)-mediated [3+2] Huisgen cycloaddition reactions. RuBEP-caged circular morpholinos (Ru-MOs) targeting two early developmental zebrafish genes, chordin and notail, were synthesized and tested in vivo. One-cell-stage zebrafish embryos microinjected with Ru-MO and incubated in the dark for 24 h developed normally, consistent with caging, whereas irradiation at 450 nm dissociated one 3-ethynylpyridine ligand (ϕ = 0.33) and uncaged the MO to achieve gene knockdown. As demonstrated, Ru photolinkers provide a versatile method for controlling structure and function of biopolymers. PMID:26023327

  16. Hypoxia-Induced Retinal Neovascularization in Zebrafish Embryos: A Potential Model of Retinopathy of Prematurity

    PubMed Central

    Kao, Alex; Hsi, Brian; Lee, Shwu-Huey; Chen, Yau-Hung; Wang, I-Jong

    2015-01-01

    Retinopathy of prematurity, formerly known as a retrolental fibroplasia, is a leading cause of infantile blindness worldwide. Retinopathy of prematurity is caused by the failure of central retinal vessels to reach the retinal periphery, creating a nonperfused peripheral retina, resulting in retinal hypoxia, neovascularization, vitreous hemorrhage, vitreoretinal fibrosis, and loss of vision. We established a potential retinopathy of prematurity model by using a green fluorescent vascular endothelium zebrafish transgenic line treated with cobalt chloride (a hypoxia-inducing agent), followed by GS4012 (a vascular endothelial growth factor inducer) at 24 hours postfertilization, and observed that the number of vascular branches and sprouts significantly increased in the central retinal vascular trunks 2–4 days after treatment. We created an angiography method by using tetramethylrhodamine dextran, which exhibited severe vascular leakage through the vessel wall into the surrounding retinal tissues. The quantification of mRNA extracted from the heads of the larvae by using real-time quantitative polymerase chain reaction revealed a twofold increase in vegfaa and vegfr2 expression compared with the control group, indicating increased vascular endothelial growth factor signaling in the hypoxic condition. In addition, we demonstrated that the hypoxic insult could be effectively rescued by several antivascular endothelial growth factor agents such as SU5416, bevacizumab, and ranibizumab. In conclusion, we provide a simple, highly reproducible, and clinically relevant retinopathy of prematurity model based on zebrafish embryos; this model may serve as a useful platform for clarifying the mechanisms of human retinopathy of prematurity and its progression. PMID:25978439

  17. Exploring the Effects of Different Types of Surfactants on Zebrafish Embryos and Larvae.

    PubMed

    Wang, Yanan; Zhang, Yuan; Li, Xu; Sun, Mingzhu; Wei, Zhuo; Wang, Yu; Gao, Aiai; Chen, Dongyan; Zhao, Xin; Feng, Xizeng

    2015-01-01

    Currently, surfactants are widely distributed in the environment. As organic pollutants, their toxicities have drawn extensive attention. In this study, the effects of anionic [sodium dodecyl sulphate (SDS)], cationic [dodecyl dimethyl benzyl ammonium chloride (1227)] and non-ionic [fatty alcohol polyoxyethylene ether (AEO)] surfactants on zebrafish larval behaviour were evaluated. Five behavioural parameters were recorded using a larval rest/wake assay, including rest total, number of rest bouts, rest bouts length, total activity and waking activity. The results revealed that 1227 and AEO at 1 μg/mL were toxic to larval locomotor activity and that SDS had no significant effects. Moreover, we tested the toxicities of the three surfactants in developing zebrafish embryos. AEO exposure resulted in smaller head size, smaller eye size and shorter body length relative to SDS and 1227. All three surfactants incurred concentration-dependent responses. Furthermore, in situ hybridisation indicated that smaller head size may be associated with a decreased expression of krox20. The altered expression of ntl demonstrated that the developmental retardation stemmed from inhibited cell migration and growth. These findings provide references for ecotoxicological assessments of different types of surfactants, and play a warning role in the application of surfactants. PMID:26053337

  18. Tyrosine glycosylation of Rho by Yersinia toxin impairs blastomere cell behaviour in zebrafish embryos.

    PubMed

    Jank, Thomas; Eckerle, Stephanie; Steinemann, Marcus; Trillhaase, Christoph; Schimpl, Marianne; Wiese, Sebastian; van Aalten, Daan M F; Driever, Wolfgang; Aktories, Klaus

    2015-01-01

    Yersinia species cause zoonotic infections, including enterocolitis and plague. Here we studied Yersinia ruckeri antifeeding prophage 18 (Afp18), the toxin component of the phage tail-derived protein translocation system Afp, which causes enteric redmouth disease in salmonid fish species. Here we show that microinjection of the glycosyltransferase domain Afp18(G) into zebrafish embryos blocks cytokinesis, actin-dependent motility and cell blebbing, eventually abrogating gastrulation. In zebrafish ZF4 cells, Afp18(G) depolymerizes actin stress fibres by mono-O-GlcNAcylation of RhoA at tyrosine-34; thereby Afp18(G) inhibits RhoA activation by guanine nucleotide exchange factors, and blocks RhoA, but not Rac and Cdc42 downstream signalling. The crystal structure of tyrosine-GlcNAcylated RhoA reveals an open conformation of the effector loop distinct from recently described structures of GDP- or GTP-bound RhoA. Unravelling of the molecular mechanism of the toxin component Afp18 as glycosyltransferase opens new perspectives in studies of phage tail-derived protein translocation systems, which are preserved from archaea to human pathogenic prokaryotes. PMID:26190758

  19. Rereplication in emi1-Deficient Zebrafish Embryos Occurs through a Cdh1-Mediated Pathway

    PubMed Central

    Robu, Mara E.; Zhang, Yong; Rhodes, Jennifer

    2012-01-01

    Disruption of early mitotic inhibitor 1 (Emi1) interferes with normal cell cycle progression and results in early embryonic lethality in vertebrates. During S and G2 phases the ubiquitin ligase complex APC/C is inhibited by Emi1 protein, thereby enabling the accumulation of Cyclins A and B so they can regulate replication and promote the transition from G2 phase to mitosis, respectively. Depletion of Emi1 prevents mitotic entry and causes rereplication and an increase in cell size. In this study, we show that the developmental and cell cycle defects caused by inactivation of zebrafish emi1 are due to inappropriate activation of APC/C through its cofactor Cdh1. Inhibiting/slowing progression into S-phase by depleting Cdt1, an essential replication licensing factor, partially rescued emi1 deficiency-induced rereplication and the increased cell size. The cell size effect was enhanced by co-depletion of cell survival regulator p53. These data suggest that the increased size of emi1-deficient cells is either directly or indirectly caused by the rereplication defects. Moreover, enforced expression of Cyclin A partially ablated the rereplicating population in emi1-deficient zebrafish embryos, consistent with the role of Cyclin A in origin licensing. Forced expression of Cyclin B partially restored the G1 population, in agreement with the established role of Cyclin B in mitotic progression and exit. However, expression of Cyclin B also partially inhibited rereplication in emi1-deficient embryos, suggesting a role for Cyclin B in regulating replication in this cellular context. As Cyclin A and B are substrates for APC/C-Cdh1 - mediated degradation, and Cdt1 is under control of Cyclin A, these data indicate that emi1 deficiency-induced defects in vivo are due to the dysregulation of an APC/C-Cdh1 molecular axis. PMID:23082190

  20. Effects of copper oxide nanoparticles on developing zebrafish embryos and larvae.

    PubMed

    Sun, Yan; Zhang, Gong; He, Zizi; Wang, Yajie; Cui, Jianlin; Li, Yuhao

    2016-01-01

    Copper oxide nanoparticles (CuO NPs) are used for a variety of purposes in a wide range of commercially available products. Some CuO NPs probably end up in the aquatic systems, thus raising concerns about aqueous exposure toxicity, and the impact of CuO NPs on liver development and neuronal differentiation remains unclear. In this study, particles were characterized using Fourier transform infrared spectra, scanning electron microscopy, and transmission electron microscopy. Zebrafish embryos were continuously exposed to CuO NPs from 4 hours postfertilization at concentrations of 50, 25, 12.5, 6.25, or 1 mg/L. The expression of gstp1 and cyp1a was examined by quantitative reverse transcription polymerase chain reaction. The expression of tumor necrosis factor alpha and superoxide dismutase 1 was examined by quantitative reverse transcription polymerase chain reaction and Western blotting. Liver development and retinal neurodifferentiation were analyzed by whole-mount in situ hybridization, hematoxylin-eosin staining, and immunohistochemistry, and a behavioral test was performed to track the movement of larvae. We show that exposure of CuO NPs at low doses has little effect on embryonic development. However, exposure to CuO NPs at concentrations of 12.5 mg/L or higher leads to abnormal phenotypes and induces an inflammatory response in a dose-dependent pattern. Moreover, exposure to CuO NPs at high doses results in an underdeveloped liver and a delay in retinal neurodifferentiation accompanied by reduced locomotor ability. Our data demonstrate that short-term exposure to CuO NPs at high doses shows hepatotoxicity and neurotoxicity in zebrafish embryos and larvae. PMID:27022258

  1. Carbendazim exposure induces developmental, biochemical and behavioural disturbance in zebrafish embryos.

    PubMed

    Andrade, Thayres S; Henriques, Jorge F; Almeida, Ana Rita; Machado, Ana Luísa; Koba, Olga; Giang, Pham Thai; Soares, Amadeu M V M; Domingues, Inês

    2016-01-01

    Carbendazim is a widely used broad spectrum benzimidazole fungicide; however, its effects to non-target aquatic organisms are poorly studied. The aim of this study was to investigate the toxic effects of carbendazim to zebrafish early life stages at several levels of biological organization, including developmental, biochemical and behavioural levels. The embryo assay was done following the OECD guideline 236 and using a concentration range between 1.1 and 1.8mg/L. Lethal and developmental endpoints such as hatching, edemas, malformations, heart beat rate, body growth and delays were assessed in a 96h exposure. A sub-teratogenic range (from 0.16 to 500μg/L) was then used to assess effects at biochemical and behavioural levels. Biochemical markers included cholinesterase (ChE), glutathione-S-transferase (GST), lactate dehydrogenase (LDH) and catalase (CAT) and were assessed at 96h. The locomotor behaviour was assessed using an automated video tracking system at 120h. Carbendazim (96h-LC50 of 1.75mg/L) elicited several developmental anomalies in zebrafish embryos with EC50 values ranging from 0.85 to 1.6mg/L. ChE, GST and LDH activities were increased at concentrations equal or above 4μg/L. The locomotor assay showed to be extremely sensitive, detecting effects in time that larvae spent swimming at concentrations of 0.16μg/L and thus, being several orders of magnitude more sensitive that developmental parameters or lethality. These are ecological relevant concentrations and highlight the potential of behavioural endpoints as early warning signs for environmental stress. Further studies should focus on understanding how the behavioural disturbances measured in these types of studies translate into fitness impairment at the adult stage. PMID:26653011

  2. Effects of copper oxide nanoparticles on developing zebrafish embryos and larvae

    PubMed Central

    Sun, Yan; Zhang, Gong; He, Zizi; Wang, Yajie; Cui, Jianlin; Li, Yuhao

    2016-01-01

    Copper oxide nanoparticles (CuO NPs) are used for a variety of purposes in a wide range of commercially available products. Some CuO NPs probably end up in the aquatic systems, thus raising concerns about aqueous exposure toxicity, and the impact of CuO NPs on liver development and neuronal differentiation remains unclear. In this study, particles were characterized using Fourier transform infrared spectra, scanning electron microscopy, and transmission electron microscopy. Zebrafish embryos were continuously exposed to CuO NPs from 4 hours postfertilization at concentrations of 50, 25, 12.5, 6.25, or 1 mg/L. The expression of gstp1 and cyp1a was examined by quantitative reverse transcription polymerase chain reaction. The expression of tumor necrosis factor alpha and superoxide dismutase 1 was examined by quantitative reverse transcription polymerase chain reaction and Western blotting. Liver development and retinal neurodifferentiation were analyzed by whole-mount in situ hybridization, hematoxylin–eosin staining, and immunohistochemistry, and a behavioral test was performed to track the movement of larvae. We show that exposure of CuO NPs at low doses has little effect on embryonic development. However, exposure to CuO NPs at concentrations of 12.5 mg/L or higher leads to abnormal phenotypes and induces an inflammatory response in a dose-dependent pattern. Moreover, exposure to CuO NPs at high doses results in an underdeveloped liver and a delay in retinal neurodifferentiation accompanied by reduced locomotor ability. Our data demonstrate that short-term exposure to CuO NPs at high doses shows hepatotoxicity and neurotoxicity in zebrafish embryos and larvae. PMID:27022258

  3. Functional expressions of adenosine triphosphate-binding cassette transporters during the development of zebrafish embryos and their effects on the detoxification of cadmium chloride and β-naphthoflavone.

    PubMed

    Yin, Huancai; Bai, Pengli; Miao, Peng; Chen, Mingli; Hu, Jun; Deng, Xudong; Yin, Jian

    2016-07-01

    Adenosine triphosphate-binding cassette (ABC) transporters, including ABCB, ABCC and ABCG families represent general biological defenses against environmental toxicants in varieties of marine and freshwater organisms, but their physiological functions at differential developmental stages of zebrafish embryos remain undefined. In this work, functional expressions of typical ABC transporters including P-glycoprotein (Pgp), multiresistance associated protein 1 (Mrp1) and Mrp2 were studied in zebrafish embryos at 4, 24, 48 and 72 h post-fertilization (hpf). As a result, both the gene expressions and activities of Pgp and Mrps increased with the development of embryos. Correspondingly, 4-72 hpf embryos exhibited an increased tolerance to the toxicity caused by cadmium chloride (CdCl2 ) and β-naphthoflavone (BNF) with time. Such a correlation was assumed caused by the involvement of ABC transporters in the detoxification of chemicals. In addition, the assumption was supported by the fact that model efflux inhibitors of Pgp and Mrps such as reversine 205 and MK571 significantly inhibited the efflux of toxicants and increased the toxicity of Cd and BNF in zebrafish embryos. Moreover, exposure to CdCl2 and BNF induced the gene expressions of Pgp and Mrp1 in 72 hpf embryos. Thus, functional expressions of Pgp and Mrps increased with the development of zebrafish embryos, which could cause an increasing tolerance of zebrafish embryos to CdCl2 and BNF. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26387481

  4. Toxicity of cylindrospermopsin, and other apparent metabolites from Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum, to the zebrafish (Danio rerio) embryo

    PubMed Central

    Berry, John P.; Gibbs, Patrick D.L.; Schmale, Michael C.; Saker, Martin L.

    2012-01-01

    Cyanobacteria produce a diverse array of toxic or otherwise bioactive compounds that pose growing threats to human and environmental health. We utilized the zebrafish (Danio rerio) embryo, as a model of vertebrate development, to investigate the inhibition of development pathways (i.e. developmental toxicity) by the cyanobacterial toxin, cylindrospermopsin (CYN), as well as extracts from various isolates of Cylindrospermopsis raciborskii and Aphanizomenon ovalisporum. CYN was toxic only when injected directly into embryos, but not by direct immersion at doses up to 50 μg/ml. Despite the dose dependency of toxicity observed following injection of CYN, no consistent patterns of developmental defects were observed, suggesting that toxic effects of CYN may not target specific developmental pathways. In contrast, direct immersion of embryos in all of the extracts resulted in both increased mortality and reproducible, consistent, developmental dysfunctions. Interestingly, there was no correlation of developmental toxicity observed for these extracts with the presence of CYN or with previously reported toxicity for these strains. These results suggest that CYN is lethal to zebrafish embryos, but apparently inhibits no specific developmental pathways, whereas other apparent metabolites from C. raciborskii and A. ovalisporum seem to reproducibly inhibit development in the zebrafish model. Continued investigation of these apparent, unknown metabolites is needed. PMID:19087885

  5. Rapid high performance liquid chromatography-high resolution mass spectrometry methodology for multiple prenol lipids analysis in zebrafish embryos.

    PubMed

    Martano, Chiara; Mugoni, Vera; Dal Bello, Federica; Santoro, Massimo M; Medana, Claudio

    2015-09-18

    The analysis of lipid molecules in living organism is an important step in deciphering metabolic pathways. Recently, the zebrafish has been adopted as a valuable animal model system to perform in vivo metabolomics studies, however limited methodologies and protocols are currently available to investigate zebrafish lipidome and even fewer to analyze specific classes of lipids. Here we present an HPLC-HRMS based method to rapidly measure multiple prenol lipid molecules from zebrafish tissues. In particular, we have optimized our method for concurrent detection of ubiquinones (Coenzyme Q6, Coenzyme Q9, Coenzyme Q10), cholesterol, vitamin E (α-tocopherol), vitamin K1 and vitamin K2. The purpose of this study was to compare different ionization modes, mobile phases and stationary phases in order to optimize lipid molecules separation. After HPLC-HRMS parameters selection, several extraction conditions from zebrafish embryos were evaluated. We assessed our methodology by quantitation of analytical recovery on zebrafish extracts from wild-type or zebrafish mutants (barolo) affected by impaired biosynthesis of ubiquinones. PMID:26283533

  6. Early exclusion of hand1-deficient cells from distinct regions of the left ventricular myocardium in chimeric mouse embryos.

    PubMed

    Riley, P R; Gertsenstein, M; Dawson, K; Cross, J C

    2000-11-01

    The basic helix-loop-helix transcription factor gene Hand1 has been implicated in development of the heart. However, the early lethality of Hand1-null mutant mouse embryos has precluded a precise understanding of its function. In this study, we have generated Hand1 homozygous mutant ES cells and performed in vitro differentiation experiments and chimeric analysis to study the role of Hand1 function during cardiac development. Hand1-null ES cells were able to differentiate into beating cardiomyocytes in vitro that expressed cardiac myosin and several cardiac-specific transcripts including Nkx2-5, alpha-cardiac actin, and the myofilament genes myosin light chain 2a and 2v. In chimeras derived from Hand1-null ES cells and ROSA26 embryos, mutant cells were underrepresented in the left caudal region of the linear heart tube at E8.0. By E9.5, after cardiac looping, mutant cells were underrepresented in the anterior region of the outer curvature of the left ventricular myocardium, but did contribute to other parts of the left ventricle and to other cardiac chambers. These results imply that Hand1 is not essential for differentiation of ventricular cardiomyocytes. Hand1-null cells were also underrepresented in several other regions of later embryos, including the rhombencephalic neural tube that was associated with a deficiency of mutant cells in the neural crest cell-derived cardiac outflow tract and first branchial arch. In summary, Hand1 has cell-autonomous functions during cardiac morphogenesis in both mesodermal and neural crest derivatives. PMID:11076684

  7. Quantitative toxicoproteomic analysis of zebrafish embryos exposed to a retinoid X receptor antagonist UVI3003.

    PubMed

    Zheng, Liang; Yu, Jianlan; Shi, Huahong; Xia, Liang; Xin, Qi; Zhang, Qiang; Zhao, Heng; Luo, Ji; Jin, Wenhai; Li, Daoji; Zhou, Junliang

    2015-09-01

    Retinoid X receptor (RXR) antagonists, including some environmental endocrine disruptors, have a teratogenic effect on vertebrate embryos. To investigate the toxicological mechanism on the protein expression level, a quantitative proteomic study was conducted to analyze the proteome alterations of zebrafish (Danio rerio) embryos exposed to gradient concentrations of a representative RXR antagonist UVI3003. Using isobaric Tags for Relative and Absolute Quantitation (iTRAQ) labeling coupled nano high-performance liquid chromatography-tandem mass spectrometry (nano HPLC-MS/MS), in total 6592 proteins were identified, among which 195 proteins were found to be differentially expressed by more than a two-fold change in exposed groups compared with the control. Gene ontology analysis showed that these differential proteins were mostly involved in anatomical structure development, biosynthetic process, ion binding and oxidoreductase activity. Moreover, the biological pathways of translation, lipoprotein metabolism, cell survival and gluconeogenesis were intensively inhibited after exposure. Some significantly downregulated proteins such as apolipoprotein A-I and vitellogenin and upregulated proteins such as calcium activated nucleotidase 1b, glutathione S-transferase and glucose 6-dehydrogenases showed a strong dose-dependent response. The results provided new insight into the molecular details of RXR antagonist-induced teratogenicity and added novel information of pathways and potential biomarkers for evaluation of RXR interfering activity. PMID:25581642

  8. River waters induced neurotoxicity in an embryo-larval zebrafish model.

    PubMed

    García-Cambero, Jesús Pablo; Catalá, Myriam; Valcárcel, Yolanda

    2012-10-01

    Some investigations have revealed an increased release of psychoactive drugs into the aquatic environments near big cities. However, despite the alert generated by the presence of such neurotoxic compounds, there is a lack of studies evaluating the impact on living organisms. One solution consists in the development of bioassays able to address specific risks, such as neurotoxicity, but on the other hand suitable to assess complex matrices like river samples. The objective of this work was to assess surface water toxicity by means of a zebrafish embryo-larval combined toxicity assay, which is based on a variety of toxicological endpoints, especially those related to neurodevelopment. For such a purpose, we selected the Tagus River in which a previous monitoring study revealed the presence of psychoactive drugs. Results showed that most of the toxicological endpoints evaluated remained unaltered in the exposed embryos, except for the tail length that was larger in the exposed larvae, and the locomotor activity in the 6-day larvae, which was decreased in four groups of exposure (n=5 sampling points). In the absence of systemic toxicity, changes in larval locomotion are indicative of neurotoxicity. This result suggests that the Tagus River can convey neurotoxic compounds at levels that may represent an early and specific threat over the aquatic species of vertebrates, what can have dramatic consequences under the ecological point of view. PMID:22906717

  9. Formation of copper complexes in landfill leachate and their toxicity to zebrafish embryos

    SciTech Connect

    Fraser, J.K.; Butler, C.A.; Timperley, M.H.; Evans, C.W.

    2000-05-01

    Toxic metal organic complexes have not been found in natural waters, although some organic acids form bioavailable lipophilic and metabolite-type metal complexes. Landfill leachates usually contain organic acids and in the urban environment these leachates, when mixed with storm waters containing Cu, could be a source of toxic Cu organic complexes in streams and estuaries. The authors investigated the formation of Cu complexes in the leachate from an active urban landfill and found that some of the complexes formed were toxic to zebrafish embryos. High and low nominal molecular weight (NMWT) fractions; >5,000 Da and <700 Da, of leachate both formed Cu complexes with almost identical Cu complexing characteristics but the toxicity was due solely to the low NMWT complexes formed in the <700 Da fraction. Chemical equilibrium modeling with MINTEQA2 and H and Cu complex conditional association constants and ligand concentrations obtained from pH and Cu titrations with a Cu ion-selective electrode and van den Berg-Ruzic analyses of the titration data was used to calculate the copper speciation in the embryo test solutions. This calculated speciation, which was confirmed by measurements of Cu{sup 2+} in the test solutions, enabled the toxicity due to the free Cu ion and to the Cu complexes to be distinguished.

  10. Cortisol-treated zebrafish embryos develop into pro-inflammatory adults with aberrant immune gene regulation.

    PubMed

    Hartig, Ellen I; Zhu, Shusen; King, Benjamin L; Coffman, James A

    2016-01-01

    Chronic early-life stress increases adult susceptibility to numerous health problems linked to chronic inflammation. One way that this may occur is via glucocorticoid-induced developmental programming. To gain insight into such programming we treated zebrafish embryos with cortisol and examined the effects on both larvae and adults. Treated larvae had elevated whole-body cortisol and glucocorticoid signaling, and upregulated genes associated with defense response and immune system processes. In adulthood the treated fish maintained elevated basal cortisol levels in the absence of exogenous cortisol, and constitutively mis-expressed genes involved in defense response and its regulation. Adults derived from cortisol-treated embryos displayed defective tailfin regeneration, heightened basal expression of pro-inflammatory genes, and failure to appropriately regulate those genes following injury or immunological challenge. These results support the hypothesis that chronically elevated glucocorticoid signaling early in life directs development of a pro-inflammatory adult phenotype, at the expense of immunoregulation and somatic regenerative capacity. PMID:27444789

  11. The combined toxicological effects of titanium dioxide nanoparticles and bisphenol A on zebrafish embryos

    PubMed Central

    2014-01-01

    Environmental pollutants co-exist and exhibit interaction effects that are different from those associated with a single pollutant. As one of the more commonly manufactured nanomaterials, titanium dioxide nanoparticles (TiO2-NPs) are most likely to bind to other contaminants in water. In this paper, we aimed to study the combined toxicological effects of TiO2-NPs and bisphenol A (BPA) on organism. First, in vitro adsorption experiments were conducted to determine the adsorptive interaction between TiO2-NPs and BPA. Second, zebrafish embryo toxicity tests were performed to monitor for changes in the toxicological effects associated with the two chemicals. The study results demonstrated that adsorptive interactions exist between the two chemicals and increased toxicity effects which included an advanced toxicological effect time, decreased survival, increased morphological abnormalities, and delayed embryo hatching. Also, we suggest that the mode of combined action has a synergistic effect. Based on this, we postulate that concomitant exposure to TiO2-NPs and BPA increased BPA bioavailability and uptake into cells and organisms. Further studies are required to understand the mechanisms of interactions of this mixture. PMID:25177222

  12. Steroid androgen 17α-methyltestosterone induces malformations and biochemical alterations in zebrafish embryos.

    PubMed

    Rivero-Wendt, Carla Letícia Gediel; Oliveira, Rhaul; Monteiro, Marta Sofia; Domingues, Inês; Soares, Amadeu Mortágua Velho Maia; Grisolia, Cesar Koppe

    2016-06-01

    The synthetic androgen 17α-methyltestosterone is widely used in fish aquaculture for sex reversion of female individuals. Little is known about the amount of MT residues reaching the aquatic environment and further impacts in non-target organisms, including fish early-life stages. Thus, in this work, zebrafish embryos were exposed to two forms of 17α-methyltestosterone: the pure compound (MT) and a formulation commonly used in Brazil (cMT). For MT, a 96h-LC50 of 10.09mg/l was calculated. MT also affected embryo development inducing tail malformations, edemas, abnormal development of the head, and hatching delay. At biochemical level MT inhibited vitellogenin (VTG) and inhibited cholinesterase and lactate dehydrogenase. cMT elicited similar patterns of toxicity as the pure compound (MT). Effects reported in this study suggest a potential environmental risk of MT, especially since the VTG effects occurred at environmental relevant concentrations (0.004mg/l). PMID:27137108

  13. Leptospira interrogans stably infects zebrafish embryos, altering phagocyte behavior and homing to specific tissues.

    PubMed

    Davis, J Muse; Haake, David A; Ramakrishnan, Lalita

    2009-01-01

    Leptospirosis is an extremely widespread zoonotic infection with outcomes ranging from subclinical infection to fatal Weil's syndrome. Despite the global impact of the disease, key aspects of its pathogenesis remain unclear. To examine in detail the earliest steps in the host response to leptospires, we used fluorescently labelled Leptospira interrogans serovar Copenhageni to infect 30 hour post fertilization zebrafish embryos by either the caudal vein or hindbrain ventricle. These embryos have functional innate immunity but have not yet developed an adaptive immune system. Furthermore, they are optically transparent, allowing direct visualization of host-pathogen interactions from the moment of infection. We observed rapid uptake of leptospires by phagocytes, followed by persistent, intracellular infection over the first 48 hours. Phagocytosis of leptospires occasionally resulted in formation of large cellular vesicles consistent with apoptotic bodies. By 24 hours, clusters of infected phagocytes were accumulating lateral to the dorsal artery, presumably in early hematopoietic tissue. Our observations suggest that phagocytosis may be a key defense mechanism in the early stages of leptospirosis, and that phagocytic cells play roles in immunopathogenesis and likely in the dissemination of leptospires to specific target tissues. PMID:19547748

  14. Effect of titanium dioxide nanoparticles on zebrafish embryos and developing retina

    PubMed Central

    Wang, Ya-Jie; He, Zi-Zi; Fang, Yang-Wu; Xu, Yang; Chen, Ya-Nan; Wang, Guan-Qun; Yang, Yong-Qiang; Yang, Zhuo; Li, Yu-Hao

    2014-01-01

    AIM To investigate the impact of titanium dioxide nanoparticles (TiO2 NPs) on embryonic development and retinal neurogenesis. METHODS The agglomeration and sedimentation of TiO2 NPs solutions at different dilutions were observed, and the ultraviolet-visible spectra of their supernatants were measured. Zebrafish embryos were experimentally exposed to TiO2 NPs until 72h postfertilization (hpf). The retinal neurogenesis and distribution of the microglia were analyzed by immunohistochemistry and whole mount in situ hybridization. RESULTS The 1 mg/L was determined to be an appropriate exposure dose. Embryos exposed to TiO2 NPs had a normal phenotype. The neurogenesis was initiated on time, and ganglion cells, cones and rods were well differentiated at 72 hpf. The expression of fms mRNA and the 4C4 antibody, which were specific to microglia in the central nervous system (CNS), closely resembled their endogenous profile. CONCLUSION These data demonstrate that short-term exposure to TiO2 NPs at a low dose does not lead to delayed embryonic development or retinal neurotoxicity. PMID:25540739

  15. Cortisol-treated zebrafish embryos develop into pro-inflammatory adults with aberrant immune gene regulation

    PubMed Central

    Hartig, Ellen I.; Zhu, Shusen; King, Benjamin L.

    2016-01-01

    ABSTRACT Chronic early-life stress increases adult susceptibility to numerous health problems linked to chronic inflammation. One way that this may occur is via glucocorticoid-induced developmental programming. To gain insight into such programming we treated zebrafish embryos with cortisol and examined the effects on both larvae and adults. Treated larvae had elevated whole-body cortisol and glucocorticoid signaling, and upregulated genes associated with defense response and immune system processes. In adulthood the treated fish maintained elevated basal cortisol levels in the absence of exogenous cortisol, and constitutively mis-expressed genes involved in defense response and its regulation. Adults derived from cortisol-treated embryos displayed defective tailfin regeneration, heightened basal expression of pro-inflammatory genes, and failure to appropriately regulate those genes following injury or immunological challenge. These results support the hypothesis that chronically elevated glucocorticoid signaling early in life directs development of a pro-inflammatory adult phenotype, at the expense of immunoregulation and somatic regenerative capacity. PMID:27444789

  16. The combined toxicological effects of titanium dioxide nanoparticles and bisphenol A on zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Yan, Jun; Lin, Bencheng; Hu, Chuanlu; Zhang, Huashan; Lin, Zhiqing; Xi, Zhuge

    2014-08-01

    Environmental pollutants co-exist and exhibit interaction effects that are different from those associated with a single pollutant. As one of the more commonly manufactured nanomaterials, titanium dioxide nanoparticles (TiO2-NPs) are most likely to bind to other contaminants in water. In this paper, we aimed to study the combined toxicological effects of TiO2-NPs and bisphenol A (BPA) on organism. First, in vitro adsorption experiments were conducted to determine the adsorptive interaction between TiO2-NPs and BPA. Second, zebrafish embryo toxicity tests were performed to monitor for changes in the toxicological effects associated with the two chemicals. The study results demonstrated that adsorptive interactions exist between the two chemicals and increased toxicity effects which included an advanced toxicological effect time, decreased survival, increased morphological abnormalities, and delayed embryo hatching. Also, we suggest that the mode of combined action has a synergistic effect. Based on this, we postulate that concomitant exposure to TiO2-NPs and BPA increased BPA bioavailability and uptake into cells and organisms. Further studies are required to understand the mechanisms of interactions of this mixture.

  17. Transcriptional Regulation During Zygotic Genome Activation in Zebrafish and Other Anamniote Embryos.

    PubMed

    Wragg, J; Müller, F

    2016-01-01

    Embryo development commences with the fusion of two terminally differentiated haploid gametes into the totipotent fertilized egg, which through a series of major cellular and molecular transitions generate a pluripotent cell mass. The activation of the zygotic genome occurs during the so-called maternal to zygotic transition and prepares the embryo for zygotic takeover from maternal factors, in the control of the development of cellular lineages during differentiation. Recent advances in next generation sequencing technologies have allowed the dissection of the genomic and epigenomic processes mediating this transition. These processes include reorganization of the chromatin structure to a transcriptionally permissive state, changes in composition and function of structural and regulatory DNA-binding proteins, and changeover of the transcriptome as it is overhauled from that deposited by the mother in the oocyte to a zygotically transcribed complement. Zygotic genome activation in zebrafish occurs 10 cell cycles after fertilization and provides an ideal experimental platform for elucidating the temporal sequence and dynamics of establishment of a transcriptionally active chromatin state and helps in identifying the determinants of transcription activation at polymerase II transcribed gene promoters. The relatively large number of pluripotent cells generated by the fast cell divisions before zygotic transcription provides sufficient biomass for next generation sequencing technology approaches to establish the temporal dynamics of events and suggest causative relationship between them. However, genomic and genetic technologies need to be improved further to capture the earliest events in development, where cell number is a limiting factor. These technologies need to be complemented with precise, inducible genetic interference studies using the latest genome editing tools to reveal the function of candidate determinants and to confirm the predictions made by classic

  18. The effects of copper pyrithione, an antifouling agent, on developing zebrafish embryos.

    PubMed

    Almond, Kelly M; Trombetta, Louis D

    2016-03-01

    A substitute for the organotins has been the use of metal pyrithiones, principally zinc and copper (CuPT) as antifouling agents. Zebrafish, Danio rerio, embryos were exposed after fertilization to increasing concentrations of CuPT (2, 4, 8, 12, 16, 32 and 64 μg/L) for 24 h. Morphological abnormalities at 30, 96 and 120 hours post fertilization (hpf) were recorded. Abnormalities at concentrations of 12 μg/L and higher were observed. Notochords became severely twisted as concentrations increased. These distortions of the notochord originated in the tail at the lower concentrations and proceeded rostrally with increasing dose. Edema was observed in the cardiac and yolk sac regions at the 12 and 16 μg/L CuPT concentrations. Light microscopy showed disorganization of muscle fibers, disruption and distortion of the transverse myoseptum and vacuolization of the myocyte. Hatching was measured every 12 h for 5 days following the 24 h exposure. Hatching decreased in a dose dependent manner. At 120 hpf, 47 % of the 64 μg/L CuPT treated embryos hatched. Inductively coupled plasma atomic absorbance spectrophotometry (ICPAAS) revealed copper bioaccumulation in whole embryo tissue and was significantly elevated in 32 and 64 μg/L CuPT treatment groups as compared to controls. Lipid peroxidation end products were significantly increased in animals exposed to 32 and 64 μg/L of CuPT. These data demonstrate that oxidative stress may play a role in the toxicity. The abnormalities and deformities observed in fish larvae would significantly decrease survival in polluted aqua-systems and question the use of this product as an antifouling agent. PMID:26686506

  19. Exploring the zebrafish embryo as an alternative model for the evaluation of liver toxicity by histopathology and expression profiling.

    PubMed

    Driessen, Marja; Kienhuis, Anne S; Pennings, Jeroen L A; Pronk, Tessa E; van de Brandhof, Evert-Jan; Roodbergen, Marianne; Spaink, Herman P; van de Water, Bob; van der Ven, Leo T M

    2013-05-01

    The whole zebrafish embryo model (ZFE) has proven its applicability in developmental toxicity testing. Since functional hepatocytes are already present from 36 h post fertilization onwards, whole ZFE have been proposed as an attractive alternative to mammalian in vivo models in hepatotoxicity testing. The goal of the present study is to further underpin the applicability of whole ZFE for hepatotoxicity testing by combining histopathology and next-generation sequencing-based gene expression profiling. To this aim, whole ZFE and adult zebrafish were exposed to a set of hepatotoxic reference compounds. Histopathology revealed compound and life-stage-specific effects indicative of toxic injury in livers of whole ZFE and adult zebrafish. Next-generation sequencing (NGS) was used to compare transcript profiles in pooled individual RNA samples of whole ZFE and livers of adult zebrafish. This revealed that hepatotoxicity-associated expression can be detected beyond the overall transcription noise in the whole embryo. In situ hybridization verified liver specificity of selected highly expressed markers in whole ZFE. Finally, cyclosporine A (CsA) was used as an illustrative case to support applicability of ZFE in hepatotoxicity testing by comparing CsA-induced gene expression between ZFE, in vivo mouse liver and HepaRG cells on the levels of single genes, pathways and transcription factors. While there was no clear overlap on single gene level between the whole ZFE and in vivo mouse liver, strong similarities were observed between whole ZFE and in vivo mouse liver in regulated pathways related to hepatotoxicity, as well as in relevant overrepresented transcription factors. In conclusion, both the use of NGS of pooled RNA extracts analysis combined with histopathology and traditional microarray in single case showed the potential to detect liver-related genes and processes within the transcriptome of a whole zebrafish embryo. This supports the applicability of the whole ZFE

  20. Effects of cyanobacterial lipopolysaccharides from microcystis on glutathione-based detoxification pathways in the zebrafish (Danio rerio) embryo.

    PubMed

    Jaja-Chimedza, Asha; Gantar, Miroslav; Mayer, Gregory D; Gibbs, Patrick D L; Berry, John P

    2012-06-01

    Cyanobacteria ("blue-green algae") are recognized producers of a diverse array of toxic secondary metabolites. Of these, the lipopolysaccharides (LPS), produced by all cyanobacteria, remain to be well investigated. In the current study, we specifically employed the zebrafish (Danio rerio) embryo to investigate the effects of LPS from geographically diverse strains of the widespread cyanobacterial genus, Microcystis, on several detoxifying enzymes/pathways, including glutathione-S-transferase (GST), glutathione peroxidase (GPx)/glutathione reductase (GR), superoxide dismutase (SOD), and catalase (CAT), and compared observed effects to those of heterotrophic bacterial (i.e., E. coli) LPS. In agreement with previous studies, cyanobacterial LPS significantly reduced GST in embryos exposed to LPS in all treatments. In contrast, GPx moderately increased in embryos exposed to LPS, with no effect on reciprocal GR activity. Interestingly, total glutathione levels were elevated in embryos exposed to Microcystis LPS, but the relative levels of reduced and oxidized glutathione (i.e., GSH/GSSG) were, likewise, elevated suggesting that oxidative stress is not involved in the observed effects as typical of heterotrophic bacterial LPS in mammalian systems. In further support of this, no effect was observed with respect to CAT or SOD activity. These findings demonstrate that Microcystis LPS affects glutathione-based detoxification pathways in the zebrafish embryo, and more generally, that this model is well suited for investigating the apparent toxicophore of cyanobacterial LPS, including possible differences in structure-activity relationships between heterotrophic and cyanobacterial LPS, and teleost fish versus mammalian systems. PMID:22822454

  1. Pretilachlor has the potential to induce endocrine disruption, oxidative stress, apoptosis and immunotoxicity during zebrafish embryo development.

    PubMed

    Jiang, Jinhua; Chen, Yanhong; Yu, Ruixian; Zhao, Xueping; Wang, Qiang; Cai, Leiming

    2016-03-01

    The objectives of the present study were to investigate the toxic effects of pretilachlor on zebrafish during its embryo development. The results demonstrated that the transcription of genes involved in the hypothalamic-pituitary-gonadal/thyroid (HPG/HPT) axis was increased after exposure to 50, 100, 200μg/L pretilachlor for 96h, the aromatase activity, vitellogenin (VTG) and thyroid hormones T3 and T4 levels in zebrafish were also up-regulated simultaneously. Pretilachlor exposure induced a noticeable increase in ROS level, increased the transcription and level of antioxidant proteins (e.g., CAT, SOD and GPX). Moreover, the up-regulation of P53, Mdm2, Bbc3 expression and Caspase3 and Caspase9 activities in the apoptosis pathway suggested pretilachlor might trigger cell apoptosis in zebrafish. In addition, the transcription of CXCL-C1C, IL-1β and IL-8 related to the innate immunity was down-regulated after pretilachlor exposure. These data suggested that pretilachlor could simultaneously induce endocrine disruption, apoptosis, oxidative stress and immunotoxicity during zebrafish embryo development. PMID:26851375

  2. Transcriptional profiles of glutathione-S-Transferase isoforms, Cyp, and AOE genes in atrazine-exposed zebrafish embryos.

    PubMed

    Glisic, Branka; Hrubik, Jelena; Fa, Svetlana; Dopudj, Nela; Kovacevic, Radmila; Andric, Nebojsa

    2016-02-01

    Glutathione-S-transferase (GST) superfamily consists of multiple members involved in xenobiotic metabolism. Expressional pattern of the GST isoforms in adult fish has been used as a biomarker of exposure to environmental chemicals. However, GST transcriptional responses vary across organs, thus requiring a cross-tissue examination of multiple mRNAs for GST profiling in an animal after chemical exposure. Zebrafish embryos express all GST isoforms as adult fish and could therefore represent an alternative model for identification of biomarkers of exposure. To evaluate such a possibility, we studied a set of cytosolic and microsomal GST isoform-specific expression profiles in the zebrafish embryos after exposure to atrazine, a widely used herbicide. Expression of the GST isoforms was compared with that of CYP genes involved in the phase I of xenobiotic metabolism and antioxidant enzyme (AOE) genes. Using quantitative real-time PCR, we showed dynamic changes in the expressional pattern of twenty GST isoforms, cyp1a, cyp3a65, ahr2, and four AOEs in early development of zebrafish. Acute (48 and 72 h) exposure of 24 h-old embryos to atrazine, from environmentally relevant (0.005 mg/L) to high (40 mg/L) concentrations, caused a variety of transient, albeit minor changes (<2.5-fold) in the GST isoforms, ahr2 and AOE genes response. However, expression of cyp1a and cyp3a65 mRNA was markedly and consistently induced by high doses of atrazine (5 and 40 mg/L). In summary, an analysis of the response of multiple systems in the zebrafish embryos provided a comprehensive understanding of atrazine toxicity and its potential impact on biological processes. PMID:25158112

  3. Development of a pheasant interspecies primordial germ cell transfer to chicken embryo: effect of donor cell sex on chimeric semen production.

    PubMed

    Kang, S J; Choi, J W; Park, K J; Lee, Y M; Kim, T M; Sohn, S H; Lim, J M; Han, J Y

    2009-09-01

    This study was conducted to evaluate whether the sex of donor primordial germ cells (PGCs) influences production of chimeric semen from recipient hatchlings produced by interspecies transfer between pheasant (Phasianus colchicus) and chicken (Gallus gallus). Pheasant PGCs were retrieved from 7-d-old embryos and subsequently transferred into circulatory blood of 2.5-d-old (Stage 17) embryos. The sex of embryos was discerned 3 to 6 days after laying, and in preliminary study, overall rate of embryo survival after sexing was 74.6% with male-to-female ratio of 0.49 to 0.51. In Experiment 1, magnetic-activated cell sorting (MACS) using QCR1 antibody was effective for enriching the population of male and female PGCs in gonadal cells (9.2- to 12.5-fold and 10.8- to 19.5-fold increase, respectively). In Experiment 2, an increase in the number of hatchlings producing chimeric semen was detected after the homosexual transfer of male-to-male compared with that after the heterosexual transfer of female-to-male (68% to 88%). Significant increase was found in the frequency of chimeric semen production (0.96 to 1.68 times); production of pheasant progenies by artificial insemination using chimeric semen was also increased in the homosexual transfer (0 to 3 cases). In conclusion, the homosexual PGC transfer of male-to-male yielded better rate of generating pheasant progenies after test cross-reproduction than that of the heterosexual transfer of female-to-male, which could improve the efficiency of interspecies germ cell transfer system. PMID:19515408

  4. The impact of endocrine-disrupting chemicals on oxidative stress and innate immune response in zebrafish embryos.

    PubMed

    Xu, Hai; Yang, Ming; Qiu, Wenhui; Pan, Chenyuan; Wu, Minghong

    2013-08-01

    Bisphenol A (BPA) and nonylphenol (NP) are well known endocrine-disrupting chemicals (EDCs) ubiquitous in the aquatic environment and are an ecotoxicological risk for the health of aquatic organisms. Limited attention has been given to the immunotoxicity of these chemicals. The present study revealed a concentration-dependent increase of reactive oxygen species content and an induced expression of redox-sensitive transcription factors in zebrafish embryos after exposure to various concentrations of BPA, NP, and BPA/NP mixture for 4 h to 168 h postfertilization. Transcription of genes related to the immune response, including IFNγ, IL1β, IL10, Mx, TNFα, CC-chemokine, and CXCL-clc, were significantly up-regulated on exposure to EDCs. A significant induction of concentrations of proinflammatory mediator, nitric oxide, accompanied by an increase in the activity of nitric oxide synthase (NOS) and an upregulation of inducible NOS gene expression, was detected in zebrafish embryos on exposures to EDCs. To elucidate the potential mechanisms by which BPA and NP activate the innate immune response, expression profiles of genes related to the Toll-like receptors (TLRs) signaling pathway were examined. Expressions of TLR3, TRIF, MyD88, SARM, IRAK4, and TRAF6 were altered on exposure to EDCs. The authors' results demonstrate that exposure to BPA and NP significantly affects the expression of genes related to immune response in zebrafish embryos following oxidative stress. PMID:23606268

  5. Transcriptomic Analysis of Purified Embryonic Neural Stem Cells from Zebrafish Embryos Reveals Signaling Pathways Involved in Glycine-Dependent Neurogenesis

    PubMed Central

    Samarut, Eric; Bekri, Abdelhamid; Drapeau, Pierre

    2016-01-01

    How is the initial set of neurons correctly established during the development of the vertebrate central nervous system? In the embryo, glycine and GABA are depolarizing due the immature chloride gradient, which is only reversed to become hyperpolarizing later in post-natal development. We previously showed that glycine regulates neurogenesis via paracrine signaling that promotes calcium transients in neural stem cells (NSCs) and their differentiation into interneurons within the spinal cord of the zebrafish embryo. However, the subjacent molecular mechanisms are not yet understood. Our previous work suggests that early neuronal progenitors were not differentiating correctly in the developing spinal cord. As a result, we aimed at identifying the downstream molecular mechanisms involved specifically in NSCs during glycine-dependent embryonic neurogenesis. Using a gfap:GFP transgenic line, we successfully purified NSCs by fluorescence-activated cell sorting from whole zebrafish embryos and in embryos in which the glycine receptor was knocked down. The strength of this approach is that it focused on the NSC population while tackling the biological issue in an in vivo context in whole zebrafish embryos. After sequencing the transcriptome by RNA-sequencing, we analyzed the genes whose expression was changed upon disruption of glycine signaling and we confirmed the differential expression by independent RTqPCR assay. While over a thousand genes showed altered expression levels, through pathway analysis we identified 14 top candidate genes belonging to five different canonical signaling pathways (signaling by calcium, TGF-beta, sonic hedgehog, Wnt, and p53-related apoptosis) that are likely to mediate the promotion of neurogenesis by glycine. PMID:27065799

  6. The multiple stressor effect in zebrafish embryos from simultaneous exposure to ionising radiation and cadmium.

    PubMed

    Ng, C Y P; Choi, V W Y; Lam, A C L; Cheng, S H; Yu, K N

    2013-03-01

    Living organisms are exposed to a mixture of environmental stressors, and the resultant effects are referred to as multiple stressor effects. In the present work, we studied the multiple stressor effect in embryos of the zebrafish (Danio rerio) from simultaneous exposure to ionising radiation (alpha particles) and cadmium through quantification of apoptotic signals at 24 h postfertilisation (hpf) revealed by vital dye acridine orange staining. For each set of experiments, 32-40 dechorionated embryos were deployed, which were divided into four groups each having 8-10 embryos. The four groups of embryos were referred to as (1) the control group (C), which received no further treatments after dechorionation; (2) the Cd-dosed and irradiated group (CdIr), which was exposed to 100 μM Cd from 5 to 24 hpf, and also received about 4.4 mGy from alpha particles at 5 hpf; (3) the irradiated group (Ir), which received about 4.4 mGy from alpha particles at 5 hpf; and (4) the Cd-dosed group (Cd), which was exposed to 100 μM Cd from 5 to 24 hpf. In general, the CdIr, Ir and Cd groups had more apoptotic signals than the C group. Within the 12 sets of experimental results, two showed significant synergistic effects, one showed a weakly synergistic effect and nine showed additive effects. The multiple stressor effect of 100 μM Cd with ~4.4 mGy alpha-particle radiation resulted in an additive or synergistic effect, but no antagonistic effect. The failure to identify significant synergistic effects for some sets of data, and thus their subsequent classification as additive effects, might be a result of the relatively small magnitude of the synergistic effects. The results showed that the radiation risk could be perturbed by another environmental stressor such as a heavy metal, and as such a realistic human radiation risk assessment should in general take into account the multiple stressor effects. PMID:23296360

  7. Differences in toxicity of anionic and cationic PAMAM and PPI dendrimers in zebrafish embryos and cancer cell lines.

    PubMed

    Bodewein, Lambert; Schmelter, Frank; Di Fiore, Stefano; Hollert, Henner; Fischer, Rainer; Fenske, Martina

    2016-08-15

    Dendrimers are an emerging class of polymeric nanoparticles with beneficial biomedical applications like early diagnostics, in vitro gene transfection or controlled drug delivery. However, the potential toxic impact of exposure on human health or the environment is often inadequately defined. Thus, polyamidoamine (PAMAM) dendrimers of generations G3.0, 3.5, 4.0, 4.5 and 5.0 and polypropylenimine (PPI) dendrimers G3.0, 4.0 and 5.0 were tested in zebrafish embryos for 96h and human cancer cell lines for 24h, to assess and compare developmental in vivo toxicity with cytotoxicity. The zebrafish embryo toxicity of cationic PAMAM and PPI dendrimers increased over time, with EC50 values ranging from 0.16 to just below 1.7μM at 24 and 48hpf. The predominant effects were mortality, plus reduced heartbeat and blood circulation for PPI dendrimers. Apoptosis in the embryos increased in line with the general toxicity concentration-dependently. Hatch and dechorionation of the embryos increased the toxicity, suggesting a protective role of the chorion. Lower generation dendrimers were more toxic in the embryos whereas the toxicity in the HepG2 and DU145 cell lines increased with increasing generation of cationic PAMAMs and PPI dendrimers. HepG2 were less sensitive than DU145 cells, with IC50 values≥402μM (PAMAMs) and ≤240μM (PPIs) for HepG2 and ≤13.24μM (PAMAMs) and ≤12.84μM (PPIs) for DU145. Neither in fish embryos nor cells toxicity thresholds were determinable for anionic PAMAM G3.5 and G4.5. The study demonstrated that the cytotoxicity underestimated the in-vivo toxicity of the dendrimers in the fish embryos. PMID:27288734

  8. Dynamic analysis of angiogenesis in transgenic zebrafish embryos using a 3D multilayer chip-based technology

    NASA Astrophysics Data System (ADS)

    Akagi, Jin; Zhu, Feng; Hall, Chris J.; Khoshmanesh, Khashayar; Kalantar-Zadeh, Kourosh; Mitchell, Arnan; Crosier, Kathryn E.; Crosier, Philip S.; Wlodkowic, Donald

    2013-03-01

    Transgenic zebrafish (Danio rerio) models of human diseases have recently emerged as innovative experimental systems in drug discovery and molecular pathology. None of the currently available technologies, however, allow for automated immobilization and treatment of large numbers of spatially encoded transgenic embryos during real-time developmental analysis. This work describes the proof-of-concept design and validation of an integrated 3D microfluidic chip-based system fabricated directly in the poly(methyl methacrylate) transparent thermoplastic using infrared laser micromachining. At its core, the device utilizes an array of 3D micro-mechanical traps to actively capture and immobilize single embryos using a low-pressure suction. It also features built-in piezoelectric microdiaphragm pumps, embryo trapping suction manifold, drug delivery manifold and optically transparent indium tin oxide (ITO) heating element to provide optimal temperature during embryo development. Furthermore, we present design of the proof-of-concept off-chip electronic interface equipped with robotic servo actuator driven stage, innovative servomotor-actuated pinch valves and miniaturized fluorescent USB microscope. Our results show that the innovative device has 100% embryo trapping efficiency while supporting normal embryo development for up to 72 hours in a confined microfluidic environment. We also present data that this microfluidic system can be readily applied to kinetic analysis of a panel of investigational anti-angiogenic agents in transgenic zebrafish Tg(fli1a:EGFP) line. The optical transparency and embryo immobilization allow for convenient visualization of developing vasculature patterns in response to drug treatment without the need for specimen re-positioning. The integrated electronic interfaces bring the Lab-on-a-Chip systems a step closer to realization of complete analytical automation.

  9. Nanosilver-coated socks and their toxicity to zebrafish (Danio rerio) embryos.

    PubMed

    Gao, Jiejun; Sepúlveda, Maria S; Klinkhamer, Christopher; Wei, Alexander; Gao, Yu; Mahapatra, Cecon T

    2015-01-01

    Silver nanoparticles (AgNPs) are being incorporated and are known to be released from various consumer products such as textiles. However, no data are available on the toxicity of AgNPs released from any of these commercial products. In this study, we quantified total silver released from socks into wash water by inductively coupled plasma mass spectrometry (ICP-MS) and determined the presence of AgNPs using transmission electron microscopy (TEM). We then exposed zebrafish (Danio rerio) embryos for 72 h to either this leachate ("sock-AgNP") or to the centrifugate ("spun-AgNP") free of AgNPs and compared their toxicity to that of ionic silver (Ag(+)). Our data suggest that AgNPs do get released into the wash water, and centrifugation eliminated AgNPs but did not decrease total silver concentrations, indicating that most of the silver in the sock-AgNP solution was in the ionic form. All embryos died during the first 24 h when exposed to undiluted sock-AgNP and spun-AgNP solutions resulting in significantly lower LC50 values (0.14 and 0.26 mg L(-1)) compared to AgNO3 (0.80 mg L(-1)). Similarly, at 72 hpf, both sock-derived solutions were more potent at affecting hatching and inducing abnormal development. These results suggest that both sock-AgNP and spun-AgNP solutions were more toxic than AgNO3. Previous studies have consistently shown the opposite, i.e., AgNPs are about 10 times less toxic that Ag(+). All together our results show that the high toxicity induced by the leachate of these socks is likely not caused by AgNPs or Ag(+). More studies are needed to evaluate the toxicity of the myriad of AgNP-coated commercial products that are now estimated to be close to 500. PMID:25303653

  10. Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos.

    PubMed

    Rothschild, Sarah C; Francescatto, Ludmila; Tombes, Robert M

    2016-01-01

    The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr. Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol. This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer's Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data. PMID:26967668

  11. Transcription regulation of the vegf gene by the BMP/Smad pathway in the angioblast of zebrafish embryos

    SciTech Connect

    He Chen; Chen Xiaozhuo . E-mail: chenx@ohiou.edu

    2005-04-01

    Vascular endothelial growth factor (VEGF) is a mitogen that is critically involved in vasculogenesis, angiogenesis, and hematopoiesis. However, what and how transcription factors participate in the regulation of vegf gene expression are not fully understood. Here we report the cloning and sequencing of the zebrafish vegf promoter which revealed that the promoter contains a number of bone morphogenetic protein (BMP)-activated Smad binding elements (SBE), implicating Smad1 and Smad5 in the regulation of BMP-induced expression of vegf. Electrophoretic mobility shift assays of adding recombinant Smad proteins to the SBE-containing DNA oligonucleotides that represent portions of zebrafish vegf promoter resulted in mobility shift of the oligonucleotides. These changes demonstrate potential interactions between Smad1/5 and the vegf promoter. Reporter activity assays using the wild-type or SBE-deleted vegf promoters to drive the luciferase reporter gene expression revealed that Smad1 stimulated while Smad5 repressed the vegf promoter activity in zebrafish embryos. These data indicate that the BMP/Smad signaling pathway is involved in the regulation of zebrafish vegf transcription. In addition, we demonstrate that transgenic expression of human BMP4 in zebrafish embryos induced an expansion of the posterior intermediate cell mass (ICM, also commonly called blood island), a population of cells containing endothelial and hematopoietic precursors. In the expanded ICM, vegf and VEGF receptor 2 (flk-1) were ectopically co-expressed, suggesting that an autocrine/paracrine regulation of vegf expression may exist and contribute to the BMP-induced hemangiogenic cell proliferation.

  12. Pomalidomide is nonteratogenic in chicken and zebrafish embryos and nonneurotoxic in vitro

    PubMed Central

    Mahony, Chris; Erskine, Lynda; Niven, Jennifer; Greig, Nigel H.; Figg, William Douglas; Vargesson, Neil

    2013-01-01

    Thalidomide and its analog, Lenalidomide, are in current use clinically for treatment of multiple myeloma, complications of leprosy and cancers. An additional analog, Pomalidomide, has recently been licensed for treatment of multiple myeloma, and is purported to be clinically more potent than either Thalidomide or Lenalidomide. Using a combination of zebrafish and chicken embryos together with in vitro assays we have determined the relative anti-inflammatory activity of each compound. We demonstrate that in vivo embryonic assays Pomalidomide is a significantly more potent anti-inflammatory agent than either Thalidomide or Lenalidomide. We tested the effect of Pomalidomide and Lenalidomide on angiogenesis, teratogenesis, and neurite outgrowth, known detrimental effects of Thalidomide. We found that Pomalidomide, displays a high degree of cell specificity, and has no detectable teratogenic, antiangiogenic or neurotoxic effects at potent anti-inflammatory concentrations. This is in marked contrast to Thalidomide and Lenalidomide, which had detrimental effects on blood vessels, nerves, and embryonic development at anti-inflammatory concentrations. This work has implications for Pomalidomide as a treatment for conditions Thalidomide and Lenalidomide treat currently. PMID:23858438

  13. Organization and function of microfilaments during late epiboly in zebrafish embryos.

    PubMed

    Cheng, Jackie C; Miller, Andrew L; Webb, Sarah E

    2004-10-01

    We report that, during epiboly in zebrafish, three F-actin--based structures appear only after the blastoderm migrates past the embryonic equator. They are composed of two ring-like F-actin structures that form at the deep cell and enveloping layer margins of the blastoderm and a punctate actin band that develops in the external yolk syncytial layer. Treatment with cytochalasin B or the calcium chelator dibromo-BAPTA results in the disruption of all three of these actin-based structures, leading to the slowing or immediate arrest of epiboly, respectively, followed by a failure of yolk cell occlusion and the eventual lysis of the embryo through the vegetal pole region. We suggest, therefore, that these structures function in the occlusion of the vegetal portion of the yolk cell during the latter stages of epiboly. Possible roles for these new structures, their modulation by Ca2+, as well as the functions of other previously described F-actin--based structures observed throughout epiboly, are discussed. PMID:15366008

  14. The developmental effects of pentachlorophenol on zebrafish embryos during segmentation: A systematic view

    NASA Astrophysics Data System (ADS)

    Xu, Ting; Zhao, Jing; Xu, Zhifa; Pan, Ruijie; Yin, Daqiang

    2016-05-01

    Pentachlorophenol (PCP) is a typical toxicant and prevailing pollutant whose toxicity has been broadly investigated. However, previous studies did not specifically investigate the underlying mechanisms of its developmental toxicity. Here, we chose zebrafish embryos as the model, exposed them to 2 different concentrations of PCP, and sequenced their entire transcriptomes at 10 and 24 hours post-fertilization (hpf). The sequencing analysis revealed that high concentrations of PCP elicited systematic responses at both time points. By combining the enrichment terms with single genes, the results were further analyzed using three categories: metabolism, transporters, and organogenesis. Hyperactive glycolysis was the most outstanding feature of the transcriptome at 10 hpf. The entire system seemed to be hypoxic, although hypoxia-inducible factor-1α (HIF1α) may have been suppressed by the upregulation of prolyl hydroxylase domain enzymes (PHDs). At 24 hpf, PCP primarily affected somitogenesis and lens formation probably resulting from the disruption of embryonic body plan at earlier stages. The proposed underlying toxicological mechanism of PCP was based on the crosstalk between each clue. Our study attempted to describe the developmental toxicity of environmental pollutants from a systematic view. Meanwhile, some features of gene expression profiling could serve as markers of human health or ecological risk.

  15. 3D Imaging of Transition Metals in the Zebrafish Embryo by X-ray Fluorescence Microtomography

    PubMed Central

    Bourassa, Daisy; Gleber, Sophie-Charlotte; Vogt, Stefan; Yi, Hong; Will, Fabian; Richter, Heiko; Shin, Chong Hyun; Fahrni, Christoph J.

    2014-01-01

    Synchrotron X-ray fluorescence (SXRF) microtomography has emerged as a powerful technique for the 3D visualization of the elemental distribution in biological samples. The mechanical stability, both of the instrument and the specimen, is paramount when acquiring tomographic projection series. By combining the progressive lowering of temperature method (PLT) with femtosecond laser sectioning, we were able to embed, excise, and preserve a zebrafish embryo at 24 hours post fertilization in an X-ray compatible, transparent resin for tomographic elemental imaging. Based on a data set comprised of 60 projections, acquired with a step size of 2 μm during 100 hours of beam time, we reconstructed the 3D distribution of zinc, iron, and copper using the iterative maximum likelihood expectation maximization (MLEM) reconstruction algorithm. The volumetric elemental maps, which entail over 124 million individual voxels for each transition metal, revealed distinct elemental distributions that could be correlated with characteristic anatomical features at this stage of embryonic development. PMID:24992831

  16. The developmental effects of pentachlorophenol on zebrafish embryos during segmentation: A systematic view.

    PubMed

    Xu, Ting; Zhao, Jing; Xu, Zhifa; Pan, Ruijie; Yin, Daqiang

    2016-01-01

    Pentachlorophenol (PCP) is a typical toxicant and prevailing pollutant whose toxicity has been broadly investigated. However, previous studies did not specifically investigate the underlying mechanisms of its developmental toxicity. Here, we chose zebrafish embryos as the model, exposed them to 2 different concentrations of PCP, and sequenced their entire transcriptomes at 10 and 24 hours post-fertilization (hpf). The sequencing analysis revealed that high concentrations of PCP elicited systematic responses at both time points. By combining the enrichment terms with single genes, the results were further analyzed using three categories: metabolism, transporters, and organogenesis. Hyperactive glycolysis was the most outstanding feature of the transcriptome at 10 hpf. The entire system seemed to be hypoxic, although hypoxia-inducible factor-1α (HIF1α) may have been suppressed by the upregulation of prolyl hydroxylase domain enzymes (PHDs). At 24 hpf, PCP primarily affected somitogenesis and lens formation probably resulting from the disruption of embryonic body plan at earlier stages. The proposed underlying toxicological mechanism of PCP was based on the crosstalk between each clue. Our study attempted to describe the developmental toxicity of environmental pollutants from a systematic view. Meanwhile, some features of gene expression profiling could serve as markers of human health or ecological risk. PMID:27181905

  17. The developmental effects of pentachlorophenol on zebrafish embryos during segmentation: A systematic view

    PubMed Central

    Xu, Ting; Zhao, Jing; Xu, Zhifa; Pan, Ruijie; Yin, Daqiang

    2016-01-01

    Pentachlorophenol (PCP) is a typical toxicant and prevailing pollutant whose toxicity has been broadly investigated. However, previous studies did not specifically investigate the underlying mechanisms of its developmental toxicity. Here, we chose zebrafish embryos as the model, exposed them to 2 different concentrations of PCP, and sequenced their entire transcriptomes at 10 and 24 hours post-fertilization (hpf). The sequencing analysis revealed that high concentrations of PCP elicited systematic responses at both time points. By combining the enrichment terms with single genes, the results were further analyzed using three categories: metabolism, transporters, and organogenesis. Hyperactive glycolysis was the most outstanding feature of the transcriptome at 10 hpf. The entire system seemed to be hypoxic, although hypoxia-inducible factor-1α (HIF1α) may have been suppressed by the upregulation of prolyl hydroxylase domain enzymes (PHDs). At 24 hpf, PCP primarily affected somitogenesis and lens formation probably resulting from the disruption of embryonic body plan at earlier stages. The proposed underlying toxicological mechanism of PCP was based on the crosstalk between each clue. Our study attempted to describe the developmental toxicity of environmental pollutants from a systematic view. Meanwhile, some features of gene expression profiling could serve as markers of human health or ecological risk. PMID:27181905

  18. Comparative toxicity of lead (Pb2+), copper (Cu2+), and mixtures of lead and copper to zebrafish embryos on a microfluidic chip

    PubMed Central

    Li, Yinbao; Yang, Xiujuan; Zhang, Beibei; Pan, Jianbin; Li, Xinchun; Yang, Fan; Sun, Duanping

    2015-01-01

    Investigations were conducted to determine acute effects of Pb2+ and Cu2+ presented individually and collectively on zebrafish embryos. Aquatic safety testing requires a cheap, fast, and highly efficient platform for real-time evaluation of single and mixture of metal toxicity. In this study, we have developed a microfluidic system for phenotype-based evaluation of toxic effects of Pb2+ and Cu2+ using zebrafish (Danio rerio) embryos. The microfluidic chip is composed of a disc-shaped concentration gradient generator and 24 culture chambers, which can generate one blank solution, seven mixture concentrations, and eight single concentrations for each metal solution, thus enabling the assessment of zebrafish embryos. To test the accuracy of this new chip platform, we have examined the toxicity and teratogenicity of Pb2+ and Cu2+ on embryos. The individual and combined impact of Pb2+ and Cu2+ on zebrafish embryonic development was quantitatively assessed by recording a series of physiological indicators, such as spontaneous motion at 22 hours post fertilization (hpf), mortality at 24 hpf, heartbeat and body length at 96 hpf, etc. It was found that Pb2+ or Cu2+ could induce deformity and cardiovascular toxicity in zebrafish embryos and the mixture could induce more severe toxicity. This chip is a multiplexed testing apparatus that allows for the examination of toxicity and teratogenicity for substances and it also can be used as a potentially cost-effective and rapid aquatic safety assessment tool. PMID:25825620

  19. Teratogenicity of Ochratoxin A and the Degradation Product, Ochratoxin α, in the Zebrafish (Danio rerio) Embryo Model of Vertebrate Development

    PubMed Central

    Haq, Mehreen; Gonzalez, Nelson; Mintz, Keenan; Jaja-Chimedza, Asha; De Jesus, Christopher Lawrence; Lydon, Christina; Welch, Aaron Z.; Berry, John P.

    2016-01-01

    Ochratoxins, and particularly ochratoxin A (OTA), are toxic fungal-derived contaminants of food and other agricultural products. Growing evidence supports the degradation of OTA by chemical, enzymatic and/or microbial means as a potential approach to remove this mycotoxin from food products. In particular, hydrolysis of OTA to ochratoxin α (OTα) and phenylalanine is the presumptive product of degradation in most cases. In the current study, we employed the zebrafish (Danio rerio) embryo, as a model of vertebrate development to evaluate, the teratogenicity of OTA and OTα. These studies show that OTA is potently active in the zebrafish embryo toxicity assay (ZETA), and that toxicity is both concentration- and time-dependent with discernible and quantifiable developmental toxicity observed at nanomolar concentrations. On the other hand, OTα had no significant effect on embryo development at all concentrations tested supporting a decreased toxicity of this degradation product. Taken together, these results suggest that ZETA is a useful, and highly sensitive, tool for evaluating OTA toxicity, as well as its degradation products, toward development of effective detoxification strategies. Specifically, the results obtained with ZETA, in the present study, further demonstrate the toxicity of OTA, and support its degradation via hydrolysis to OTα as an effective means of detoxification. PMID:26861395

  20. Lipid Abundance in Zebrafish Embryos Is Regulated by Complementary Actions of the Endocannabinoid System and Retinoic Acid Pathway.

    PubMed

    Fraher, Daniel; Ellis, Megan K; Morrison, Shona; McGee, Sean L; Ward, Alister C; Walder, Ken; Gibert, Yann

    2015-10-01

    The endocannabinoid system (ECS) and retinoic acid (RA) signaling have been associated with influencing lipid metabolism. We hypothesized that modulation of these pathways could modify lipid abundance in developing vertebrates and that these pathways could have a combinatorial effect on lipid levels. Zebrafish embryos were exposed to chemical treatments altering the activity of the ECS and RA pathway. Embryos were stained with the neutral lipid dye Oil-Red-O (ORO) and underwent whole-mount in situ hybridization (WISH). Mouse 3T3-L1 fibroblasts were differentiated under exposure to RA-modulating chemicals and subsequently stained with ORO and analyzed for gene expression by qRT-PCR. ECS activation and RA exposure increased lipid abundance and the expression of lipoprotein lipase. In addition, RA treatment increased expression of CCAAT/enhancer-binding protein alpha. Both ECS receptors and RA receptor subtypes were separately involved in modulating lipid abundance. Finally, increased ECS or RA activity ameliorated the reduced lipid abundance caused by peroxisome proliferator-activated receptor gamma (PPARγ) inhibition. Therefore, the ECS and RA pathway influence lipid abundance in zebrafish embryos and have an additive effect when treated simultaneously. Furthermore, we demonstrated that these pathways act downstream or independently of PPARγ to influence lipid levels. Our study shows for the first time that the RA and ECS pathways have additive function in lipid abundance during vertebrate development. PMID:26181105

  1. Teratogenicity of Ochratoxin A and the Degradation Product, Ochratoxin α, in the Zebrafish (Danio rerio) Embryo Model of Vertebrate Development.

    PubMed

    Haq, Mehreen; Gonzalez, Nelson; Mintz, Keenan; Jaja-Chimedza, Asha; De Jesus, Christopher Lawrence; Lydon, Christina; Welch, Aaron; Berry, John P

    2016-02-01

    Ochratoxins, and particularly ochratoxin A (OTA), are toxic fungal-derived contaminants of food and other agricultural products. Growing evidence supports the degradation of OTA by chemical, enzymatic and/or microbial means as a potential approach to remove this mycotoxin from food products. In particular, hydrolysis of OTA to ochratoxin α (OTα) and phenylalanine is the presumptive product of degradation in most cases. In the current study, we employed the zebrafish (Danio rerio) embryo, as a model of vertebrate development to evaluate, the teratogenicity of OTA and OTα. These studies show that OTA is potently active in the zebrafish embryo toxicity assay (ZETA), and that toxicity is both concentration- and time-dependent with discernible and quantifiable developmental toxicity observed at nanomolar concentrations. On the other hand, OTα had no significant effect on embryo development at all concentrations tested supporting a decreased toxicity of this degradation product. Taken together, these results suggest that ZETA is a useful, and highly sensitive, tool for evaluating OTA toxicity, as well as its degradation products, toward development of effective detoxification strategies. Specifically, the results obtained with ZETA, in the present study, further demonstrate the toxicity of OTA, and support its degradation via hydrolysis to OTα as an effective means of detoxification. PMID:26861395

  2. Body Mass Parameters, Lipid Profiles and Protein Contents of Zebrafish Embryos and Effects of 2,4-Dinitrophenol Exposure

    PubMed Central

    Hachicho, Nancy; Reithel, Sarah; Miltner, Anja; Heipieper, Hermann J.; Küster, Eberhard; Luckenbach, Till

    2015-01-01

    Morphology and physiology of fish embryos undergo dramatic changes during their development until the onset of feeding, supplied only by endogenous yolk reserves. For obtaining an insight how these restructuring processes are reflected by body mass related parameters, dry weights (dw), contents of the elements carbon and nitrogen and lipid and protein levels were quantified in different stages within the first four days of embryo development of the zebrafish (Danio rerio). The data show age dependent changes in tissue composition. Dry weights decreased significantly from 79μgdw/egg at 0hours post fertilization (hpf) to 61 μgdw/egg after 96 hpf. The amounts of total carbon fluctuated between 460 mg g-1 and 540 mg g-1 dw, nitrogen was at about 100 mg g-1 dw and total fatty acids were between 48–73 mg g-1 dw. In contrast to these parameters that remained relatively constant, the protein content, which was 240 mg g-1 at 0 hpf, showed an overall increase of about 40%. Comparisons of intact eggs and dechorionated embryos at stages prior to hatching (24, 30, 48 hpf) showed that the differences seen for dry weight and for carbon and nitrogen contents became smaller at more advanced stages, consistent with transition of material from the chorion to embryo tissue. Further, we determined the effect of 2,4-dinitrophenol at a subacutely toxic concentration (14 μM, LC10) as a model chemical challenge on the examined body mass related parameters. The compound caused significant decreases in phospholipid and glycolipid fatty acid contents along with a decrease in the phospholipid fatty acid unsaturation index. No major changes were observed for the other examined parameters. Lipidomic studies as performed here may thus be useful for determining subacute effects of lipophilic organic compounds on lipid metabolism and on cellular membranes of zebrafish embryos. PMID:26292096

  3. The zebrafish embryo as a tool for screening and characterizing pleurocidin host-defense peptides as anti-cancer agents

    PubMed Central

    Morash, Michael G.; Douglas, Susan E.; Robotham, Anna; Ridley, Christina M.; Gallant, Jeffrey W.; Soanes, Kelly H.

    2011-01-01

    SUMMARY The emergence of multidrug-resistant cancers and the lack of targeted therapies for many cancers underscore an unmet need for new therapeutics with novel modes of action towards cancer cells. Host-defense peptides often exhibit selective cytotoxicity towards cancer cells and show potential as anti-cancer therapeutics. Here, we screen 26 naturally occurring variants of the peptide pleurocidin for cytotoxic and anti-cancer activities, and investigate the underlying mechanism of action. Cytotoxicities were assessed in vitro using cell-based assays and in vivo using zebrafish embryos. Morphological changes were assessed by both transmission and scanning electron microscopy, and functional assays were performed on zebrafish embryos to investigate the mechanism of cell death. A total of 14 peptides were virtually inactive against HL60 human leukemia cells, whereas 12 caused >50% death at ≤32 μg/ml. Morphological changes characteristic of oncosis were evident by electron microscopy after only 1 minute of treatment with 32 μg/ml of variant NRC-03. Only two peptides were hemolytic. Four peptides showed no toxicity towards zebrafish embryos at the highest concentration tested (25 μM; ∼64 μg/ml) and one peptide was highly toxic, killing 4-hour-post-fertilization (hpf) embryos immediately after exposure to 1 μM peptide. Four other peptides killed embryos after 24 hours of exposure at 1 μM. Most peptides caused mortality at one or more developmental stages only after continuous exposure (24 hours) with higher lethal doses (≥5 μM). Pleurocidin NRC-03 bound to embryos and induced the release of superoxide, caused an increase in the number of TUNEL-positive nuclei, and caused membrane damage and the loss of embryonic epithelial integrity, marked by the exclusion of cells from the outer epithelium and the appearance of F-actin within the circumferential cells of the repair site. Our results indicate that specific pleurocidin variants are attractive cancer

  4. Enantioselectivity in Developmental Toxicity of rac-metalaxyl and R-metalaxyl in Zebrafish (Danio rerio) Embryo.

    PubMed

    Zhang, Yinjun; Zhang, Yi; Chen, An; Zhang, Wei; Chen, Hao; Zhang, Quan

    2016-06-01

    Enantioselectivity of chiral pesticides in environmental safety has attracted more and more attention. In this study, we evaluated the enantioselective toxicity of rac-metalaxyl and R-metalaxyl to zebrafish (Danio rerio) embryos through various malformations including pericardial edema, yolk sac edema, crooked body, and short tails. The results showed that there were significant differences in toxicity to zebrafish embryos caused by rac-metalaxyl and R-metalaxyl, and the LC50 s at 96 h are 416.41 (353.91, 499.29) mg · L(-1) and 320.650 (279.80, 363.46) mg · L(-1) , respectively. In order to explore the possible mechanism of the development defects, the genes involved in the hypothalamic-pituitary-gonadal axis (vtg1, vtg2, cyp17, cyp19a, cyp19b) and hypothalamic-pituitary-thyroid axis (dio1, dio2, nis, tg, tpo) were quantified by quantitative real-time polymerase chain reaction (qRT-PCR). The results revealed that there were no significant differences in the expression of vtg1, vtg2, cyp17, cyp19a, and cyp19b after exposure to rac-metalaxyl. However, the expression of vtg1, cyp19a, and cyp19b decreased significantly after exposure to R-metalaxyl. And likewise, rac-metalaxyl only caused the upregulation of dio2, while R-metalaxyl suppressed the expression of dio1 and tpo and induced the expression of dio2 and nis. The change of gene expression may cause the enantioselectivity in developmental toxicity in zebrafish embryo. The data provided here will be helpful for us to comprehensively understand the potential ecological risks of the currently used chiral fungicides. Chirality 28:489-494, 2016. © 2016 Wiley Periodicals, Inc. PMID:27103609

  5. Melatonin rescues zebrafish embryos from the parkinsonian phenotype restoring the parkin/PINK1/DJ-1/MUL1 network.

    PubMed

    Díaz-Casado, María E; Lima, Elena; García, José A; Doerrier, Carolina; Aranda, Paula; Sayed, Ramy Ka; Guerra-Librero, Ana; Escames, Germaine; López, Luis C; Acuña-Castroviejo, Darío

    2016-08-01

    Multiple studies reporting mitochondrial impairment in Parkinson's disease (PD) involve knockout or knockdown models to inhibit the expression of mitochondrial-related genes, including parkin, PINK1, and DJ-1 ones. Melatonin has significant neuroprotective properties, which have been related to its ability to boost mitochondrial bioenergetics. The meaning and molecular targets of melatonin in PD are yet unclear. Zebrafish are an outstanding model of PD because they are vertebrates, their dopaminergic system is comparable to the nigrostriatal system of humans, and their brains express the same genes as mammals. The exposure of 24 hpf zebrafish embryos to MPTP leads to a significant inhibition of the mitochondrial complex I and the induction of sncga gene, responsible for enhancing γ-synuclein accumulation, which is related to mitochondrial dysfunction. Moreover, MPTP inhibited the parkin/PINK1/DJ-1 expression, impeding the normal function of the parkin/PINK1/DJ-1/MUL1 network to remove the damaged mitochondria. This situation remains over time, and removing MPTP from the treatment did not stop the neurodegenerative process. On the contrary, mitochondria become worse during the next 2 days without MPTP, and the embryos developed a severe motor impairment that cannot be rescued because the mitochondrial-related gene expression remained inhibited. Melatonin, added together with MPTP or added once MPTP was removed, prevented and recovered, respectively, the parkinsonian phenotype once it was established, restoring gene expression and normal function of the parkin/PINK1/DJ-1/MUL1 loop and also the normal motor activity of the embryos. The results show, for the first time, that melatonin restores brain function in zebrafish suffering with Parkinson-like disease. PMID:27064726

  6. Adsorption characteristics of nano-TiO2 onto zebrafish embryos and its impacts on egg hatching.

    PubMed

    Shih, Yu-Jen; Su, Chia-Chi; Chen, Chiu-Wen; Dong, Cheng-Di; Liu, Wen-Sheng; Huang, C P

    2016-07-01

    The characteristics of nanoparticles (NPs) uptake may fundamentally alter physicochemical effects of engineered NPs on aquatic organisms, thereby yielding different ecotoxicology assessment results. The adsorption behavior of nano-TiO2 (P-25) on zebrafish embryos in Holtfreter's medium (pH 7.2, I ∼ 7.2 × 10(-2) M) and the presence of sodium alginate (100 mg/L) as dispersant was investigated. Zebrafish embryos (total 100) were exposed to nano-TiO2 at different concentrations (e.g., 0, 10, 20, 60, 120 mg/L) in batch-mode assay. The adsorption capacity of nano-TiO2 on fish eggs was determined by measuring the Ti concentration on the egg surface using ICP-OES analysis. Results showed that the adsorption capacity increased rapidly in the first hour, and then declined to reach equilibrium in 8 h. The adsorption characteristics was visualized as a three-step process of rapid initial layer formation, followed by break-up of aggregates and finally rearrangement of floc structures; the maximum adsorption capacity was the sum of an inner rigid layers of aggregates of 0.81-0.84 μg-TiO2/#-egg and an outer softly flocculated layers of 1.01 μg-TiO2/#-egg. The Gibbs free energy was 543.29-551.26 and 100.75 kJ/mol, respectively, for the inner-layer and the outer-layer aggregates. Adsorption capacity at 0.5-1.0 μg-TiO2/#-egg promoted egg hatching; but hatching was inhibited at higher adsorption capacity. Results clearly showed that the configuration of TiO2 aggregates could impact the hatching efficiency of zebrafish embryos. PMID:27043376

  7. Is UV radiation changing the toxicity of compounds to zebrafish embryos?

    PubMed

    Almeida, Ana Rita; Andrade, Thayres S; Burkina, Viktoriia; Fedorova, Ganna; Loureiro, Susana; Soares, Amadeu M V M; Domingues, Inês

    2015-12-01

    At ecosystems level, environmental parameters such as temperature, pH, dissolved oxygen concentration and intensity of UV radiation (UVR) have an important role on the efficiency of organisms' physiological and behavioral performances and consequently on the capacity of response to contaminants. Insignificant alterations of these parameters may compromise this response. In addition, these parameters can additionally alter chemical compounds by inducing their degradation, producing thereafter other metabolites. Understanding the combined effects of chemicals and environmental parameters is absolutely necessary for an adequate prediction of risk in aquatic environments. According to this scenario, this work aims at studying the combined toxicity of UVR and three xenobiotics: the biocide triclosan (TCS), the metal chromium (as potassium dichromate, PD) and the fungicide prochloraz (PCZ). To achieve this goal zebrafish (Danio rerio) embryos (3h post fertilization (hpf)) were exposed to several concentrations of each chemical combined with different UV intensities; mortality and eggs were recorded every 24h for the all test duration (96 h). Results showed different response patterns depending on the toxicant, stress levels and duration of exposure. The combination of UVR and TCS indicated a dose ratio deviation where synergism was observed when UVR was the dominant stressor (day 2). The combination of UVR and PD presented a dose level dependency at day 3 indicating antagonism at low stress levels, changing with time where at day 4, a dose ratio deviation showed statistically that synergism occurred at higher PD concentrations. Finally, UVR combined with PCZ indicated a dose ratio at day 3 and dose level deviation at day 4 of exposure, suggesting a synergistic response when PCZ is the dominant stressor in the combination. The obtained results in this study highlighted the importance of taking into account the possible interaction of stressors and time of exposure to

  8. Light-Addressable Measurement of in Vivo Tissue Oxygenation in an Unanesthetized Zebrafish Embryo via Phase-Based Phosphorescence Lifetime Detection

    PubMed Central

    Huang, Shih-Hao; Yu, Chu-Hung; Chien, Yi-Lung

    2015-01-01

    We have developed a digital light modulation system that utilizes a modified commercial projector equipped with a laser diode as a light source for quantitative measurements of in vivo tissue oxygenation in an unanesthetized zebrafish embryo via phase-based phosphorescence lifetime detection. The oxygen-sensitive phosphorescent probe (Oxyphor G4) was first inoculated into the bloodstream of 48 h post-fertilization (48 hpf) zebrafish embryos via the circulation valley to rapidly disperse probes throughout the embryo. The unanesthetized zebrafish embryo was introduced into the microfluidic device and immobilized on its lateral side by using a pneumatically actuated membrane. By controlling the illumination pattern on the digital micromirror device in the projector, the modulated excitation light can be spatially projected to illuminate arbitrarily-shaped regions of tissue of interest for in vivo oxygen measurements. We have successfully measured in vivo oxygen changes in the cardiac region and cardinal vein of a 48 hpf zebrafish embryo that experience hypoxia and subsequent normoxic conditions. Our proposed platform provides the potential for the real-time investigation of oxygen distribution in tissue microvasculature that relates to physiological stimulation and diseases in a developing organism. PMID:25856326

  9. Development of inter-family nuclear transplant embryos by transplanting the nuclei from the loach blastulae into the non-enucleated zebrafish eggs

    NASA Astrophysics Data System (ADS)

    Li, Li; Zhang, Shicui; Yuan, Jinduo; Li, Hongyan

    2003-03-01

    The developmental fate of the pronuclei in recombined embryos obtained by transplanting the donor nuclei into the non-enucleated eggs remains controversial in the case of fish. In the present study, the nuclei from the loach blastulae were transplanted into non-enucleated zebrafish eggs, the resulting 9 inter-family nuclear transplant embryos developed to larval stages. Although the development timing of the nuclear transplants resembled that of zebrafish, chromosome examination revealed that most of the recombined embryos were diploids with karyotype characteristic of loach, which was also proved by RAPD analysis. Moreover, 3 out of the 9 larval fish formed barb rudiments specific to loach. It was therefore concluded that the nuclear transplant larval fish were inter-family nucleo-cytoplasmic hybrids; and that only the donor nuclei were involved in the development of the nuclear transplant embryos, while the pronuclei in the non-enucleated eggs were likely automatically eliminated during the development.

  10. Low-dose exposure of silica nanoparticles induces cardiac dysfunction via neutrophil-mediated inflammation and cardiac contraction in zebrafish embryos.

    PubMed

    Duan, Junchao; Yu, Yang; Li, Yang; Li, Yanbo; Liu, Hongcui; Jing, Li; Yang, Man; Wang, Ji; Li, Chunqi; Sun, Zhiwei

    2016-06-01

    The toxicity mechanism of nanoparticles on vertebrate cardiovascular system is still unclear, especially on the low-level exposure. This study was to explore the toxic effect and mechanisms of low-dose exposure of silica nanoparticles (SiNPs) on cardiac function in zebrafish embryos via the intravenous microinjection. The dosage of SiNPs was based on the no observed adverse effect level (NOAEL) of malformation assessment in zebrafish embryos. The mainly cardiac toxicity phenotypes induced by SiNPs were pericardial edema and bradycardia but had no effect on atrioventricular block. Using o-Dianisidine for erythrocyte staining, the cardiac output of zebrafish embryos was decreased in a dose-dependent manner. Microarray analysis and bioinformatics analysis were performed to screen the differential expression genes and possible pathway involved in cardiac function. SiNPs induced whole-embryo oxidative stress and neutrophil-mediated cardiac inflammation in Tg(mpo:GFP) zebrafish. Inflammatory cells were observed in atrium of SiNPs-treated zebrafish heart by histopathological examination. In addition, the expression of TNNT2 protein, a cardiac contraction marker in heart tissue had been down-regulated compared to control group using immunohistochemistry. Confirmed by qRT-PCR and western blot assays, results showed that SiNPs inhibited the calcium signaling pathway and cardiac muscle contraction via the down-regulated of related genes, such as ATPase-related genes (atp2a1l, atp1b2b, atp1a3b), calcium channel-related genes (cacna1ab, cacna1da) and the regulatory gene tnnc1a for cardiac troponin C. Moreover, the protein level of TNNT2 was decreased in a dose-dependent manner. For the first time, our results demonstrated that SiNPs induced cardiac dysfunction via the neutrophil-mediated cardiac inflammation and cardiac contraction in zebrafish embryos. PMID:26551753

  11. [A comparison of the knockout efficiencies of two codon-optimized Cas9 coding sequences in zebrafish embryos].

    PubMed

    Fenghua, Zhang; Houpeng, Wang; Siyu, Huang; Feng, Xiong; Zuoyan, Zhu; Yonghua, Sun

    2016-02-01

    Recent years have witnessed the rapid development of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas9)system. In order to realize gene knockout with high efficiency and specificity in zebrafish, several labs have synthesized distinct Cas9 cDNA sequences which were cloned into different vectors. In this study, we chose two commonly used zebrafish-codon-optimized Cas9 coding sequences (zCas9_bz, zCas9_wc) from two different labs, and utilized them to knockout seven genes in zebrafish embryos, including the exogenous egfp and six endogenous genes (chd, hbegfa, th, eef1a1b, tyr and tcf7l1a). We compared the knockout efficiencies resulting from the two zCas9 coding sequences, by direct sequencing of PCR products, colony sequencing and phenotypic analysis. The results showed that the knockout efficiency of zCas9_wc was higher than that of zCas9_bz in all conditions. PMID:26907778

  12. The cellular and molecular progression of mitochondrial dysfunction induced by 2,4-dinitrophenol in developing zebrafish embryos

    PubMed Central

    Bestman, Jennifer E.; Stackley, Krista D.; Rahn, Jennifer J.; Williamson, Tucker J.; Chan, Sherine S. L.

    2015-01-01

    The etiology of mitochondrial disease is poorly understood. Furthermore, treatment options are limited, and diagnostic methods often lack the sensitivity to detect disease in its early stages. Disrupted oxidative phosphorylation (OXPHOS) that inhibits ATP production is a common phenotype of mitochondrial disorders that can be induced in zebrafish by exposure to 2,4-dinitrophenol (DNP), a FDA-banned weight-loss agent and EPA-regulated environmental toxicant, traditionally used in research labs as an uncoupler of OXPHOS. Despite the DNP-induced OXPHOS inhibition we observed using in vivo respirometry, the development of the DNP-treated and control zebrafish were largely similar during the first half of embryogenesis. During this period, DNP-treated embryos induced gene expression of mitochondrial and nuclear genes that stimulated the production of new mitochondria and increased glycolysis to yield normal levels of ATP. DNP-treated embryos were incapable of sustaining this mitochondrial biogenic response past mid-embryogenesis, as shown by significantly lowered ATP production and ATP levels, decreased gene expression, and the onset of developmental defects. Examining neural tissues commonly affected by mitochondrial disease, we found that DNP exposure also inhibited motor neuron axon arbor outgrowth and the proper formation of the retina. We observed and quantified the molecular and physiological progression of mitochondrial dysfunction during development with this new model of OXPHOS dysfunction, which has great potential for use in diagnostics and therapies for mitochondrial disease. PMID:25771346

  13. The Nicotine-Evoked Locomotor Response: A Behavioral Paradigm for Toxicity Screening in Zebrafish (Danio rerio) Embryos and Eleutheroembryos Exposed to Methylmercury

    PubMed Central

    Mora-Zamorano, Francisco X.; Svoboda, Kurt R.; Carvan, Michael J.

    2016-01-01

    This study is an adaptation of the nicotine-evoked locomotor response (NLR) assay, which was originally utilized for phenotype-based neurotoxicity screening in zebrafish embryos. Zebrafish embryos do not exhibit spontaneous swimming until roughly 4 days post-fertilization (dpf), however, a robust swimming response can be induced as early as 36 hours post-fertilization (hpf) by means of acute nicotine exposure (30–240μM). Here, the NLR was tested as a tool for early detection of locomotor phenotypes in 36, 48 and 72 hpf mutant zebrafish embryos of the non-touch-responsive maco strain; this assay successfully discriminated mutant embryos from their non-mutant siblings. Then, methylmercury (MeHg) was used as a proof-of-concept neurotoxicant to test the effectiveness of the NLR assay as a screening tool in toxicology. The locomotor effects of MeHg were evaluated in 6 dpf wild type eleutheroembryos exposed to waterborne MeHg (0, 0.01, 0.03 and 0.1μM). Afterwards, the NLR assay was tested in 48 hpf embryos subjected to the same MeHg exposure regimes. Embryos exposed to 0.01 and 0.03μM of MeHg exhibited significant increases in locomotion in both scenarios. These findings suggest that similar locomotor phenotypes observed in free swimming fish can be detected as early as 48 hpf, when locomotion is induced with nicotine. PMID:27123921

  14. Effect of fluorescent whitening agent on the transcription of cell damage-related genes in zebrafish embryos.

    PubMed

    Jung, Hyun; Seok, Seung-Hyeok; Han, Ju-Hee; Abdelkader, Tamer Said; Kim, Tae-Hyoun; Chang, Seo-Na; Ko, Ae-Sun; Choi, Seung-Kyu; Lee, Cho-Rong; Seo, Ji-Eun; Byun, Soo-Hyun; Kim, Jung-A; Park, Jae-Hak

    2012-09-01

    7-Diethylamino-4-methylcoumarin (DEMC) is a fluorescent whitening agent (FWAs). There have been some studies on DEMC's protective effects against biological activity but there are few papers about the in vivo toxicity of DEMC. In this study, we used wild-type zebrafish embryos 3 days post fertilization (dpf). Test solutions with DEMC concentrations were negative control (without vehicle), 0 (with vehicle, 0.01% v/v ethanol), 0.25, 0.5, 0.75, 1.0, 1.25, 1.5 and 2 ppm. Embryos and larvae were counted for survival rate and hatching rate. Heart rates were also counted at 2.5 and 3.0 dpf. At 3.0 dpf, quantitative RT-PCR was performed with some samples (0, 0.25, 0.75 and 1.25 ppm) to determine the toxic effect to DEMC by detecting the expression levels of toxic-responsive genes. We used 11 genes, which included oxidative stress-related genes [sod(Mn), sod(Cu,Zn) and hsp70], mitochondrial metabolism-related genes (coxI, pyc, cyt and cyclinG1) and apoptosis-related genes (c-jun, bcl2, bax and p53). High-concentration DEMC-treated groups showed significant different survival rate, hatching rate and heart rate compared with low-concentration DEMC-treated groups. The LC50 of this chemical, 0.959 ppm, was calculated. We also confirmed that some genes in the DEMC exposure groups showed significantly up-regulations in expression levels compared with control groups. We concluded that the fluorescence agent, DEMC, has possible developmental toxicities and alteration effect of gene expression, which are related to oxidative stress, mitochondrial metabolism and apoptosis in zebrafish embryos. PMID:21538407

  15. Impacts of oxidative stress on acetylcholinesterase transcription, and activity in embryos of zebrafish (Danio rerio) following Chlorpyrifos exposure.

    PubMed

    Rodríguez-Fuentes, Gabriela; Rubio-Escalante, Fernando J; Noreña-Barroso, Elsa; Escalante-Herrera, Karla S; Schlenk, Daniel

    2015-01-01

    Organophosphate pesticides cause irreversible inhibition of AChE which leads to neuronal overstimulation and death. Thus, dogma indicates that the target of OP pesticides is AChE, but many authors postulate that these compounds also disturb cellular redox processes, and change the activities of antioxidant enzymes. Interestingly, it has also been reported that oxidative stress plays also a role in the regulation and activity of AChE. The aims of this study were to determine the effects of the antioxidant, vitamin C (VC), the oxidant, t-butyl hydroperoxide (tBOOH) and the organophosphate Chlorpyrifos (CPF), on AChE gene transcription and activity in zebrafish embryos after 72h exposure. In addition, oxidative stress was evaluated by measuring antioxidant enzymes activities and transcription, and quantification of total glutathione. Apical effects on the development of zebrafish embryos were also measured. With the exception of AChE inhibition and enhanced gene expression, limited effects of CPF on oxidative stress and apical endpoints were found at this developmental stage. Addition of VC had little effect on oxidative stress or AChE, but increased pericardial area and heartbeat rate through an unknown mechanism. TBOOH diminished AChE gene expression and activity, and caused oxidative stress when administered alone. However, in combination with CPF, only reductions in AChE activity were observed with no significant changes in oxidative stress suggesting the adverse apical endpoints in the embryos may have been due to AChE inhibition by CPF rather than oxidative stress. These results give additional evidence to support the role of prooxidants in AChE activity and expression. PMID:25937383

  16. Toxicity to embryo and adult zebrafish of copper complexes with two malonic acids as models for dissolved organic matter

    SciTech Connect

    Palmer, F.B.; Evans, C.W.; Butler, C.A.; Timperley, M.H.

    1998-08-01

    The toxicity to embryo and adult zebrafish (Danio rerio) of Cu complexes with two substituted malonic acids, benzyl- and n-hexadecyl-, chosen as models for low-molecular-weight natural dissolved organic matter, were investigated. Toxicity test solutions at pH 6.5 {+-} 0.1 with the required Cu ion-specific electrode. In the absence of malonic acids, concentrations of Cu{sup 2+} up to 1.13 {mu}mol/L increased the embryo hatching time from approx. 2 d in control solutions (no Cu or malonic acid) and solutions containing malonic acids without Cu to approx. 8 d. The Cu-benzylmalonic acid complex in the presence of inorganic Cu species did not delay hatching beyond that attributable to Cu{sup 2+}. In contrast, 0.60 {mu}mol/L Cu-n-hexadecylmalonic complexes delayed hatching by 5.5 d in excess of that attributable to 1.13 {mu}mol/L Cu{sup 2+}, assuming that the hatching delays caused by the different Cu species were additive, possibly because of Cu entry into the embryo as the lipophilic Cu-n-hexadecylmalonic complex. None of the Cu-malonic acid complexes was acutely toxic to adult zebrafish at concentrations up to 1.4 {mu}mol/L, possibly because Cu was removed from the Cu-malonic acid complexes by stronger chelating groups at the gill surface. Substituted malonic acids with similar proton and Cu association constants can be readily prepared with a variety of simple substituents, radiolabeled if required. Their results show that these acids could be useful ligands for investigating intracellular transport and metabolism of metal-organic complexes.

  17. Study of Pluripotency Markers in Zebrafish Embryos and Transient Embryonic Stem Cell Cultures

    PubMed Central

    Robles, Vanesa; Martí, Mercé

    2011-01-01

    Abstract Targeted genomic manipulation using embryonic stem (ES) cells has not yet been achieved in zebrafish, although methods for zebrafish ES cell culture has been described in literature. The knowledge of pluripotency markers in this species is almost nonexistent and this is a very limiting factor in the definition of the ideal culture conditions for ES cells. Here, we studied the expression of several genes associated with pluripotency in zebrafish embryonic cells versus differentiated cells and the expression of some of these genes is recorded throughout embryonic development. Some of the commonly accepted pluripotency markers are also tested in embryonic cells, transient embryonic cell cultures, and differentiated cells. Our results support the hypothesis that stage-specific embryonic antigen 1 (SSEA1) is a marker that precedes the expression of pluripotency genes in a zebrafish embryonic cell colony, in the same way that SOX2 precedes nestin expression in those colonies that have already started differentiation toward neurons. We consider this study a step forward in the knowledge of zebrafish pluripotency markers and, therefore, an important tool for the monitoring of zebrafish embryonic cell cultures. PMID:21563922

  18. A high-throughput lab-on-a-chip interface for zebrafish embryo tests in drug discovery and ecotoxicology

    NASA Astrophysics Data System (ADS)

    Zhu, Feng; Akagi, Jin; Hall, Chris J.; Crosier, Kathryn E.; Crosier, Philip S.; Delaage, Pierre; Wlodkowic, Donald

    2013-12-01

    Drug discovery screenings performed on zebrafish embryos mirror with a high level of accuracy. The tests usually performed on mammalian animal models, and the fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, conventional methods utilising 96-well microtiter plates and manual dispensing of fish embryos are very time-consuming. They rely on laborious and iterative manual pipetting that is a main source of analytical errors and low throughput. In this work, we present development of a miniaturised and high-throughput Lab-on-a-Chip (LOC) platform for automation of FET assays. The 3D high-density LOC array was fabricated in poly-methyl methacrylate (PMMA) transparent thermoplastic using infrared laser micromachining while the off-chip interfaces were fabricated using additive manufacturing processes (FDM and SLA). The system's design facilitates rapid loading and immobilization of a large number of embryos in predefined clusters of traps during continuous microperfusion of drugs/toxins. It has been conceptually designed to seamlessly interface with both upright and inverted fluorescent imaging systems and also to directly interface with conventional microtiter plate readers that accept 96-well plates. We also present proof-of-concept interfacing with a high-speed imaging cytometer Plate RUNNER HD® capable of multispectral image acquisition with resolution of up to 8192 x 8192 pixels and depth of field of about 40 μm. Furthermore, we developed a miniaturized and self-contained analytical device interfaced with a miniaturized USB microscope. This system modification is capable of performing rapid imaging of multiple embryos at a low resolution for drug toxicity analysis.

  19. Role of the cyclooxygenase 2-thromboxane pathway in 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced decrease in mesencephalic vein blood flow in the zebrafish embryo

    SciTech Connect

    Teraoka, Hiroki Kubota, Akira; Dong, Wu; Kawai, Yusuke; Yamazaki, Koji; Mori, Chisato; Harada, Yoshiteru; Peterson, Richard E.; Hiraga, Takeo

    2009-01-01

    Previously, we reported that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) evoked developmental toxicity required activation of aryl hydrocarbon receptor type 2 (AHR2), using zebrafish embryos. However, the downstream molecular targets of AHR2 activation are largely unknown and are the focus of the present investigation. TCDD induces cyclooxygenase 2 (COX2), a rate-limiting enzyme for prostaglandin synthesis in certain cells. In the present study, we investigated the role of the COX2-thromboxane pathway in causing a specific endpoint of TCDD developmental toxicity in the zebrafish embryo, namely, a decrease in regional blood flow in the dorsal midbrain. It was found that the TCDD-induced reduction in mesencephalic vein blood flow was markedly inhibited by selective COX2 inhibitors, NS-398 and SC-236, and by a general COX inhibitor, indomethacin, but not by a selective COX1 inhibitor, SC-560. Gene knock-down of COX2 by two different types of morpholino antisense oligonucleotides, but not by their negative homologs, also protected the zebrafish embryos from mesencephalic vein circulation failure caused by TCDD. This inhibitory effect of TCDD on regional blood flow in the dorsal midbrain was also blocked by selective antagonists of the thromboxane receptor (TP). Treatment of control zebrafish embryos with a TP agonist also caused a reduction in mesencephalic vein blood flow and it too was blocked by a TP antagonist, without any effect on trunk circulation. Finally, gene knock-down of thromboxane A synthase 1 (TBXS) with morpholinos but not by the morpholinos' negative homologs provided significant protection against TCDD-induced mesencephalic circulation failure. Taken together, these results point to a role of the prostanoid synthesis pathway via COX2-TBXS-TP in the local circulation failure induced by TCDD in the dorsal midbrain of the zebrafish embryo.

  20. Acute and sub-lethal exposure to copper oxide nanoparticles causes oxidative stress and teratogenicity in zebrafish embryos.

    PubMed

    Ganesan, Santhanamari; Anaimalai Thirumurthi, Naveenkumar; Raghunath, Azhwar; Vijayakumar, Savitha; Perumal, Ekambaram

    2016-04-01

    Nano-copper oxides are a versatile inorganic material. As a result of their versatility, the immense applications and usage end up in the environment causing a concern for the lifespan of various beings. The ambiguities surround globally on the toxic effects of copper oxide nanoparticles (CuO-NPs). Hence, the present study endeavored to study the sub-lethal acute exposure effects on the developing zebrafish embryos. The 48 hpf LC50 value was about 64 ppm. Therefore, we have chosen the sub-lethal dose of 40 and 60 ppm for the study. Accumulation of CuO-NPs was evidenced from the SEM-EDS and AAS analyzes. The alterations in the AChE and Na(+)/K(+)-ATPase activities disrupted the development process. An increment in the levels of oxidants with a concomitant decrease in the antioxidant enzymes confirmed the induction of oxidative stress. Oxidative stress triggered apoptosis in the exposed embryos. Developmental anomalies were observed with CuO-NPs exposure in addition to oxidative stress in the developing embryos. Decreased heart rate and hatching delay hindered the normal developmental processes. Our work has offered valuable data on the connection between oxidative stress and teratogenicity leading to lethality caused by CuO-NPs. A further molecular mechanism unraveling the uncharted connection between oxidative stress and teratogenicity will aid in the safe use of CuO-NPs. PMID:26493272

  1. High-throughput screening of zebrafish embryos using automated heart detection and imaging.

    PubMed

    Spomer, Waldemar; Pfriem, Alexander; Alshut, Rüdiger; Just, Steffen; Pylatiuk, Christian

    2012-12-01

    Over the past decade, the zebrafish has become a key model organism in genetic screenings and drug discovery. A number of genes have been identified to affect the development of the shape and functioning of the heart, leading to zebrafish mutants with heart defects. The development of semiautomated microscopy systems has allowed for the investigation of drugs that reverse a disease phenotype on a larger scale. However, there is a lack of automated feature detection, and commercially available computer-aided microscopes are expensive. Screening of the zebrafish heart for drug discovery typically includes the identification of heart parameters, such as the frequency or fractional shortening. Until now, screening processes have been characterized by manual handling of the larvae and manual microscopy. Here, an intelligent robotic microscope is presented, which automatically identifies the orientation of a zebrafish in a micro well. A predefined region of interest, such as the heart, is detected automatically, and a video with higher magnification is recorded. Screening of a 96-well plate takes 35 to 55 min, depending on the length of the videos. Of the zebrafish hearts, 75% are recorded accurately without any user interaction. A description of the system, including the graphical user interface, is given. PMID:23053930

  2. A relational-tubular (ReTu) deformable model for vasculature quantification of zebrafish embryo from microangiography image series.

    PubMed

    Feng, Jun; Ip, Horace H S; Cheng, Shuk H; Chan, Po K

    2004-09-01

    Embryonic cardiovascular system plays a vital role in embryonic development of human and animal. In this work, we introduce a novel deformable model, which we called Relational-tubular (ReTu) deformable model for segmenting and quantifying the embryonic vasculature of zebrafish embryo from microangiography image series. Particularly, to incorporate additional constraints on the spatial relationships among vessel branches, we introduce a new energy term called relation energy into the model energy function. This energy item acts as a repulsion force between neighboring vessels during the deformation to encourage them to move towards their respective volume data. Using the ReTu deformable model, the deformation process is an iterative two-stage procedure: vascular axis deformation and vascular surface deformation. The efficiency and robustness of this approach are demonstrated by experiments which show that satisfactory quantifications of the vasculature can be obtained after 3-4 iterations. PMID:15294311

  3. Rapid targeting of plasmid DNA to zebrafish embryo nuclei by the nuclear localization signal of SV40 T antigen.

    PubMed

    Collas, P; Aleström, P

    1997-03-01

    Binding SV40 T antigen nuclear localization signals (NLSs) to plasmid DNA promotes transgene expression following injection of DNA-NLS complexes into the cytoplasm of zebrafish eggs. We now demonstrate that NLS peptides mediate import of DNA from the cytoplasm into embryo nuclei, under conditions in which naked DNA is not imported. Plasmid DNA was localized by polymerase chain reaction (PCR) in isolated nuclei, and relative amounts were quantified by densitometry. Binding DNA to NLSs, but not to nuclear-import-deficient peptides, promoted rapid targeting of DNA-NLS complexes to nuclei, and transport across the nuclear envelope. Import of DNA-NLS complexes was competed by co-injected albumin-NLS conjugates. NLS, but not reverse NLS, was detected on blots of nuclei probed with 32P-labeled DNA. The results suggest that NLS-mediated DNA transfer into nuclei may constitute a valuable tool for several gene transfer applications. PMID:9116870

  4. Developmental toxicity and neurotoxicity of two matrine-type alkaloids, matrine and sophocarpine, in zebrafish (Danio rerio) embryos/larvae.

    PubMed

    Lu, Zhao-Guang; Li, Ming-Hui; Wang, Jun-Song; Wei, Dan-Dan; Liu, Qing-Wang; Kong, Ling-Yi

    2014-08-01

    Matrine and sophocarpine are two major matrine-type alkaloids included in the traditional Chinese medicine (TCM) Kushen (the root of Sophora flavescens Ait.). They have been widely used clinically in China, however with few reports concerning their potential toxicities. This study investigated the developmental toxicity and neurotoxicity of matrine and sophocarpine on zebrafish embryos/larvae from 0 to 96/120h post fertilization (hpf). Both drugs displayed teratogenic and lethal effects with the EC50 and LC50 values at 145 and 240mg/L for matrine and 87.1 and 166mg/L for sophocarpine, respectively. Exposure of matrine and sophocarpine significantly altered spontaneous movement and inhibited swimming performance at concentrations below those causing lethality and malformations, indicating a neurotoxic potential of both drugs. The results are in agreement with most mammalian studies and clinical observations. PMID:24911943

  5. Chromatin states of developmentally-regulated genes revealed by DNA and histone methylation patterns in zebrafish embryos.

    PubMed

    Lindeman, Leif C; Winata, Cecilia L; Aanes, Hvard; Mathavan, Sinnakaruppan; Alestrom, Peter; Collas, Philippe

    2010-01-01

    Embryo development proceeds from a cascade of gene activation and repression events controlled by epigenetic modifications of DNA and histones. Little is known about epigenetic states in the developing zebrafish, despite its importance as a model organism. We report here DNA methylation and histone modification profiles of promoters of developmentally-regulated genes (pou5f1, sox2, sox3, klf4, nnr, otx1b, nes, vasa), as well as tert and bactin2, in zebrafish embryos at the mid-late blastula transition, shortly after embryonic genome activation. We identify four classes of promoters based on the following profiles: (i) those enriched in marks of active genes (H3K9ac, H4ac, H3K4me3) without transcriptionally repressing H3K9me3 or H3K27me3; (ii) those enriched in H3K9ac, H4ac and H3K27me3, without H3K9me3; one such gene was klf4, shown by in situ hybridization to be mosaically expressed, likely accounting for the detection of both activating and repressive marks on its promoter; (iii) those enriched in H3K4me3 and H3K27me3 without acetylation; and (iv) those enriched in all histone modifications examined. Culture of embryo-derived cells under differentiation conditions leads to H3K9 and H4 deacetylation and H3K9 and H3K27 trimethylation on genes that are inactivated, yielding an epigenetic profile similar to those of fibroblasts or muscle. All promoters however retain H3K4me3, indicating an uncoupling of H3K4me3 occupancy and gene expression. All non-CpG island developmentally-regulated promoters are DNA unmethylated in embryos, but hypermethylated in fibroblasts. Our results suggest that differentially expressed embryonic genes are regulated by various patterns of histone modifications on unmethylated DNA, which create a developmentally permissive chromatin state. PMID:20336603

  6. Effects of 4-methylbenzylidene camphor (4-MBC) on neuronal and muscular development in zebrafish (Danio rerio) embryos.

    PubMed

    Li, Vincent Wai Tsun; Tsui, Mei Po Mirabelle; Chen, Xueping; Hui, Michelle Nga Yu; Jin, Ling; Lam, Raymond H W; Yu, Richard Man Kit; Murphy, Margaret B; Cheng, Jinping; Lam, Paul Kwan Sing; Cheng, Shuk Han

    2016-05-01

    The negative effects of overexposure to ultraviolet (UV) radiation in humans, including sunburn and light-induced cellular injury, are of increasing public concern. 4-Methylbenzylidene camphor (4-MBC), an organic chemical UV filter, is an active ingredient in sunscreen products. To date, little information is available about its neurotoxicity during early vertebrate development. Zebrafish embryos were exposed to various concentrations of 4-MBC in embryo medium for 3 days. In this study, a high concentration of 4-MBC, which is not being expected at the current environmental concentrations in the environment, was used for the purpose of phenotypic screening. Embryos exposed to 15 μM of 4-MBC displayed abnormal axial curvature and exhibited impaired motility. Exposure effects were found to be greatest during the segmentation period, when somite formation and innervation occur. Immunostaining of the muscle and axon markers F59, znp1, and zn5 revealed that 4-MBC exposure leads to a disorganized pattern of slow muscle fibers and axon pathfinding errors during the innervation of both primary and secondary motor neurons. Our results also showed reduction in AChE activity upon 4-MBC exposure both in vivo in the embryos (15 μM) and in vitro in mammalian Neuro-2A cells (0.1 μM), providing a possible mechanism for 4-MBC-induced muscular and neuronal defects. Taken together, our results have shown that 4-MBC is a teratogen and influences muscular and neuronal development, which may result in developmental defects. PMID:26888529

  7. No correlation between multilamellar bodies in the inner ear and further organs of mutant (backstroke, bks) and wildtype zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Anken, R.; Ibsch, M.; Kniesel, U.; Rahmann, H.

    2004-01-01

    The origin of the proteinacious matrix of the inner ear stones (otoliths) of vertebrates has not yet been clarified. Using the backstroke mutant (bks) of the zebrafish Danio rerio, which is characterized by a complete lack of otoliths, we searched for possibly missing or aberrant structural components within the macular epithelia of the inner ears of embryos on the ultrastructural level. Numerous multilamellar bodies (MLBs) were found. The MLBs were, however, not restricted to the inner ears of mutants but were also found in wildtype individuals and in further organs such as brain and liver. MLBs have hitherto never been described from the inner ear of fish and are generally estimated to be rare structures. Their occurrence in fish liver can, however, be induced by using particular chemical substances, which seem to effect adaptive compensatory processes on the cellular level. Such a chemical treatment also affects the ultrastructure of further organelles. Since the occurrence of MLBs in the liver of zebrafish was not accompanied by an alteration of the morphology of other organelles, their occurrence seems not to be due to environmental stress. The findings indicate that the MLBs cannot be correlated with bks-inherent features as well as with missing otolith development/growth. Since the occurrence of MLBs was independent from the developmental stage of a specimen and its overall tissue preservation, it can moreover be excluded that these MLBs merely represent fixation artifacts. Their presence more likely indicates cellular remodelling processes of hitherto unknown significance.

  8. Deficiency in the mRNA export mediator Gle1 impairs Schwann cell development in the zebrafish embryo.

    PubMed

    Seytanoglu, A; Alsomali, N I; Valori, C F; McGown, A; Kim, H R; Ning, K; Ramesh, T; Sharrack, B; Wood, J D; Azzouz, M

    2016-05-13

    GLE1 mutations cause lethal congenital contracture syndrome 1 (LCCS1), a severe autosomal recessive fetal motor neuron disease, and more recently have been associated with amyotrophic lateral sclerosis (ALS). The gene encodes a highly conserved protein with an essential role in mRNA export. The mechanism linking Gle1 function to motor neuron degeneration in humans has not been elucidated, but increasing evidence implicates abnormal RNA processing as a key event in the pathogenesis of several motor neuron diseases. Homozygous gle1(-/-) mutant zebrafish display various aspects of LCCS, showing severe developmental abnormalities including motor neuron arborization defects and embryonic lethality. A previous gene expression study on spinal cord from LCCS fetuses indicated that oligodendrocyte dysfunction may be an important factor in LCCS. We therefore set out to investigate the development of myelinating glia in gle1(-/-) mutant zebrafish embryos. While expression of myelin basic protein (mbp) in hindbrain oligodendrocytes appeared relatively normal, our studies revealed a prominent defect in Schwann cell precursor proliferation and differentiation in the posterior lateral line nerve. Other genes mutated in LCCS have important roles in Schwann cell development, thereby suggesting that Schwann cell deficits may be a common factor in LCCS pathogenesis. These findings illustrate the potential importance of glial cells such as myelinating Schwann cells in motor neuron diseases linked to RNA processing defects. PMID:26921650

  9. Cartilage and bone malformations in the head of zebrafish (Danio rerio) embryos following exposure to disulfiram and acetic acid hydrazide

    SciTech Connect

    Strecker, Ruben; Weigt, Stefan; Braunbeck, Thomas

    2013-04-15

    In order to investigate teratogenic effects, especially on cartilage and bone formation, zebrafish embryos were exposed for 144 h to the dithiocarbamate pesticide disulfiram (20–320 μg/L) and acetic acid hydrazide (0.375–12 g/L), a degradation product of isoniazid. After fixation and full-mount staining, disulfiram could be shown to induce strong cartilage malformations after exposure to ≥ 80 μg/L, whereas acetic acid hydrazide caused cartilage alterations only from 1.5 g/L. Undulating notochords occurred after exposure to disulfiram even at the lowest test concentration of 20 μg/L, whereas at the two lowest concentrations of acetic acid hydrazide (0.375 and 0.75 g/L) mainly fractures of the notochord were observed. Concentrations of acetic acid hydrazide ≥ 1.5 g/L resulted in undulated notochords similar to disulfiram. Cartilages and ossifications of the cranium, including the cleithrum, were individually analyzed assessing the severity of malformation and the degree of ossification in a semi-quantitative approach. Cartilages of the neurocranium such as the ethmoid plate proved to be more stable than cartilages of the pharyngeal skeleton such as Meckel's cartilage. Hence, ossification proved significantly more susceptible than cartilage. The alterations induced in the notochord as well as in the cranium might well be of ecological relevance, since notochord malformation is likely to result in impaired swimming and cranial malformation might compromise regular food uptake. - Highlights: ► Disulfiram and acetic acid hydrazide as notochord, cartilage and bone teratogens ► Zebrafish embryos to model effects on single cartilages and bones in the head ► LC50 calculation and head length measurements after six days post-fertilization ► Lethality, head length and teratogenic effects are dose-dependent. ► Cartilages of the neurocranium are the most stable elements in the head.

  10. Teratogenicity, genotoxicity and oxidative stress in zebrafish embryos (Danio rerio) co-exposed to arsenic and atrazine.

    PubMed

    Adeyemi, Joseph A; da Cunha Martins-Junior, Airton; Barbosa, Fernando

    2015-01-01

    Arsenic and atrazine are common environmental contaminants probably due to their extensive use as pesticides on agricultural farmlands. In this study, zebrafish embryos were exposed to 0.8mM arsenic, 0.1mM atrazine or mixture of both for 96h, and various indices which are indicative of teratogenicity (egg coagulation, growth retardation, edema formation, hatching success, scoliosis), genotoxicity (DNA tail moments) and oxidative stress (lipid peroxidation and reduced glutathione (GSH) levels, catalase and glutathione peroxidase activities) were determined. The negative control were exposed to 0.5% DMSO while the positive control group were exposed to 4mg/L 3,4 dichloroaniline. Egg coagulation was highest in the positive control (85%), followed by the group that was exposed to mixture of arsenic and atrazine (30%) and least in the arsenic-exposed group (20%). The incidences of edema (59%) and growth retardation (35.2%) were more frequent in the group that was exposed to contaminant mixture and least in atrazine-exposed group where incidences of both edema and growth retardation were 15%. The incidence of scoliosis ranged between 20% in arsenic-exposed group and 10% in atrazine-exposed group. Hatching success was generally high in all the groups ranging between 95% in atrazine-exposed group and 88% in the group that was exposed to mixture of arsenic and atrazine. There was no evidence of teratogenic effect in the negative control group. DNA tail moments and lipid peroxidation levels increased significantly while GSH levels and catalase activity decreased significantly in contaminant-exposed groups, especially the mixture compared to the negative control. There was no significant change in GPx activity in the exposed groups compared to the negative control. The results of this study demonstrate that both arsenic and atrazine are potentially teratogenic and genotoxic, and can cause oxidative stress in zebrafish embryos, and these effects are potentiated by toxic