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Sample records for zebrafish embryo development

  1. Curcumin affects development of zebrafish embryo.

    PubMed

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

    2007-07-01

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

  2. 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...

  3. EFFECT OF METHYLENE BLUE ON DEVELOPING ZEBRAFISH EMBRYOS Danio rerio

    EPA Science Inventory

    EFFECT OF METHYLENE BLUE ON DEVELOPING ZEBRAFISH EMBRYOS Danio rerioJoan M. Hedge*, Erik Sanders, Kimberly A. Jarema, Deborah Hunter, and Stephanie PadillaIntegrated Systems Toxicology Division, NHEERL, US EPA, Research Triangle Park, NC 27709hedge.joan@epa.govOur laboratory rout...

  4. Imaging retinal progenitor lineages in developing zebrafish embryos.

    PubMed

    Jusuf, Patricia; Harris, William A; Poggi, Lucia

    2013-03-01

    In this protocol, we describe how to make and analyze four dimensional (4D) movies of retinal lineage in the zebrafish embryo in vivo. 4D consists of three spatial dimensions (3D) reconstructed from stacks of confocal planes plus one time dimension. Our imaging is performed on transgenic cells that express fluorescent proteins under the control of cell-specific promoters or on cells that transiently express such reporters in specific retinal cell progenitors. An important aspect of lineage tracing is the ability to follow individual cells as they undergo multiple cell divisions, final migration, and differentiation. This may mean many hours of 4D imaging, requiring that cells be kept healthy and maintained under conditions suitable for normal development. The longest movies we have made are ∼50 h. By analyzing these movies, we can see when a specific cell was born and who its sister was, allowing us to reconstruct its retinal lineages in vivo.

  5. Impact of CdSe/ZnS quantum dots on the development of zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Lei, Yong; Xiao, Qi; Huang, Shan; Xu, Wansu; Zhang, Zhe; He, Zhike; Liu, Yi; Deng, Fengjiao

    2011-12-01

    Due to their unique fluorescent characteristics, quantum dots (QDs) have been successfully applied in the fields of biotechnology and medicine, but there is very limited information regarding their biodistribution and chronic toxicity in vivo. In this article, the biological behavior and toxic effects of mercaptoacetic acid-CdSe/ZnS QDs (MAA-QDs) in developing zebrafish embryos were investigated by in vivo tests. The MAA-QDs were introduced into zebrafish through microinjection at early stage. The results showed that the MAA-QDs at certain concentrations influenced the survival of zebrafish embryos, but treated embryos without developmental defects were also observed. MAA-QDs injected into the cytoplasm at the one-cell stage were allocated to progeny blastoderm cells during proliferation and almost never entered the yolk. The formation of notochord and primordial germ cells with normal morphologies was detected in the treated embryos by whole-mount in situ hybridization. Furthermore, traces of the element cadmium were mainly discovered in the tissue of liver and kidney of 3-month-old-treated zebrafish by quantitative assessment with inductively coupled plasma mass spectrometry. Thus, we hypothesized that low concentration MAA-QDs have chronic toxicities when they were delivered into zebrafish organs.

  6. Dissection and lateral mounting of zebrafish embryos: analysis of spinal cord development.

    PubMed

    Beck, Aaron P; Watt, Roland M; Bonner, Jennifer

    2014-02-28

    The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

  7. Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

    PubMed Central

    Beck, Aaron P.; Watt, Roland M.; Bonner, Jennifer

    2014-01-01

    The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue. PMID:24637734

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

    SciT

    Kwon, Hye-Joo, E-mail: hjkwon@pnu.edu.sa; Biology Department, Princess Nourah University, Riyadh 11671

    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.more » 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. - Highlights: • VDR signaling is involved in embryonic heart development. • Knockdown of vdrb, but not vdra, causes decreased heart rate in zebrafish embryo. • Loss of vdr results in cardiac laterality defects. • Loss of vdra/b alters atrioventricular boundary formation. • Loss of vdra/b causes abnormal cardiac looping.« less

  9. 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...

  10. Muscle development is disrupted in zebrafish embryos deficient for Fibronectin

    PubMed Central

    Snow, Chelsi J.; Peterson, Matthew T.; Khalil, Andre; Henry, Clarissa A.

    2008-01-01

    After somitogenesis, skeletal muscle precursors elongate into muscle fibers that anchor to the somite boundary, which becomes the myotome boundary. Fibronectin (Fn) is a major component of the extracellular matrix in both boundaries. Although Fn is required for somitogenesis, effects of Fn disruption on subsequent muscle development are unknown. We show fn knockdown disrupts myogenesis. Muscle morphogenesis is more disrupted in fn morphants than in a mutant where initial somite boundaries did not form, aei/deltaD. We quantified this disruption using the 2D Wavelet-Transform Modulus Maxima method, which uses the variation of intensity in an image with respect to the direction considered to characterize the structure in a cell lattice. We show that fibers in fn morphants are less organized than in aei/deltaD mutant embryos. Fast- and slow-twitch muscle lengths are also more frequently uncoupled. These data suggest fn may function to regulate fiber organization and limit fast-twitch muscle fiber length. PMID:18729220

  11. 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

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

    PubMed

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

    2015-09-04

    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.

  13. The effects of cobalt on the development, oxidative stress, and apoptosis in zebrafish embryos.

    PubMed

    Cai, Guiquan; Zhu, Junfeng; Shen, Chao; Cui, Yimin; Du, Jiulin; Chen, Xiaodong

    2012-12-01

    Metal-on-metal hip arthroplasty has been performed with increasing frequency throughout the world, particularly in younger and more active patients, including women of childbearing age. The potential toxicity of cobalt exposure on fetus is concerned since cobalt ions generated by metal-on-metal bearings can traverse the placenta and be detected in fetal blood and amniotic fluid. This study examined the effects of cobalt exposure on early embryonic development and the mechanisms underlying its toxicity. Zebrafish embryos were exposed to a range of cobalt concentrations (0-100 mg/L) between 1 and 144 h postfertilization. The survival and early development of embryos were not significantly affected by cobalt at concentrations <100 μg/L. However, embryos exposed to higher concentrations (>100 μg/L) displayed reduced survival rates and abnormal development, including delayed hatching, aberrant morphology, retarded growth, and bradycardia. Furthermore, this study examined oxidative stress and apoptosis in embryos exposed to cobalt at concentrations of 0-500 μg/L. Lipid peroxidation levels were increased in cobalt-treated embryos at concentrations of 100 and 500 μg/L. The mRNA levels of catalase, superoxide dismutase 2, p53, caspase-3, and caspase-9 genes were upregulated in a dose-dependent manner. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays also revealed abnormal apoptotic signals in the brain, trunk, and tail when treated with 500 μg/L cobalt. These data suggest that oxidative stress and apoptosis are associated with cobalt toxicity in zebrafish embryos.

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

    SciT

    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 inmore » 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.« less

  15. 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.

  16. 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

  17. 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

  18. Acute exposure to tris (2-butoxyethyl) phosphate (TBOEP) affects growth and development of embryo-larval zebrafish.

    PubMed

    Liu, Yiran; Wu, Ding; Xu, Qinglong; Yu, Liqin; Liu, Chunsheng; Wang, Jianghua

    2017-10-01

    Tris (2-butoxyethyl) phosphate (TBOEP), is used as a flame retardant worldwide. It is an additive in materials and can be easily discharged into the surrounding environment. There is evidence linking TBOEP exposure to abnormal development and growth in zebrafish embryos/larvae. Here, using zebrafish embryo as a model, we investigated toxicological effects on developing zebrafish (Danio rerio) caused by TBOEP at concentrations of 0, 20, 200, 1000, 2000μg/L starting from 2h post-fertilization (hpf). Our findings revealed that TBOEP exposure caused developmental toxicity, such as malformation, growth delay and decreased heart rate in zebrafish larvae. Correlation analysis indicated that inhibition of growth was possibly due to down-regulation of expression of genes related to the growth hormone/insulin-like growth factor (GH/IGF) axis. Furthermore, exposure to TBOEP significantly increased thyroxine (T4) and 3,5,3'-triiodothyronine (T3) in whole larvae. In addition, changed expression of genes involved in the hypothalamic-pituitary-thyroid (HPT) axis was observed, indicating that perturbation of HPT axis might be responsible for the developmental damage and growth delay induced by TBOEP. The present study provides a new set of evidence that exposure of embryo-larval zebrafish to TBOEP can cause perturbation of GH/IGF axis and HPT axis, which could result in developmental impairment and growth inhibition. Copyright © 2017. Published by Elsevier B.V.

  19. Zebrafish embryos exposed to alcohol undergo abnormal development of motor neurons and muscle fibers.

    PubMed

    Sylvain, Nicole J; Brewster, Daniel L; Ali, Declan W

    2010-01-01

    Children exposed to alcohol in utero have significantly delayed gross and fine motor skills, as well as deficiencies in reflex development. The reasons that underlie the motor deficits caused by ethanol (EtOH) exposure remain to be fully elucidated. The present study was undertaken to investigate the effects of embryonic alcohol exposure (1.5%, 2% and 2.5% EtOH) on motor neuron and muscle fiber morphology in 3 days post fertilization (dpf) larval zebrafish. EtOH treated fish exhibited morphological deformities and fewer bouts of swimming in response to touch, compared with untreated fish. Immunolabelling with anti-acetylated tubulin indicated that fish exposed to 2.5% EtOH had significantly higher rates of motor neuron axon defects. Immunolabelling of primary and secondary motor neurons, using znp-1 and zn-8, revealed that fish exposed to 2% and 2.5% EtOH exhibited significantly higher rates of primary and secondary motor neuron axon defects compared to controls. Examination of red and white muscle fibers revealed that fish exposed to EtOH had significantly smaller fibers compared with controls. These findings indicate that motor neuron and muscle fiber morphology is affected by early alcohol exposure in zebrafish embryos, and that this may be related to deficits in locomotion. Copyright 2010 Elsevier Inc. All rights reserved.

  20. The effects of carbaryl on the development of zebrafish (Danio rerio) embryos.

    PubMed

    Schock, Elizabeth N; Ford, Windsor C; Midgley, Kirsten J; Fader, Joseph G; Giavasis, Michael N; McWhorter, Michelle L

    2012-12-01

    In the United States, Sevin(™) brand insecticide is one of the most commonly used insecticides. The active ingredient in Sevin(™), carbaryl (1-napthyl-N-methylcarbamate), is a known acetylcholinesterase (AChE) inhibitor that prevents the breakdown of acetylcholine to acetate and choline at the synapse. While carbaryl successfully causes the death of insects by paralysis, it has also been shown to have negative effects on the development of several nontarget species. To study the effects of carbaryl on nontarget species, zebrafish (Danio rerio) were used, as they are a good model for both toxicology and development studies. Our study suggests that carbaryl induces changes in morphology, specifically in embryo size and shape. Additionally, carbaryl causes defects in heart formation that is characterized by a decrease in heart rate and a developmental delay/defect in cardiac looping. A significant decrease in the number of spinal cord neurons present was also observed. Further investigation showed that there was an increase in cell death in carbaryl-treated embryos. The results indicate that carbaryl may have a greater environmental impact than initially intended. Our study, which was conducted solely by undergraduates at a liberal arts college, indicates that carbaryl may be detrimental to the development of nontarget species.

  1. Effects of metal exposure on motor neuron development, neuromasts and the escape response of zebrafish embryos.

    PubMed

    Sonnack, Laura; Kampe, Sebastian; Muth-Köhne, Elke; Erdinger, Lothar; Henny, Nicole; Hollert, Henner; Schäfers, Christoph; Fenske, Martina

    2015-01-01

    Low level metal contaminations are a prevalent issue with often unknown consequences for health and the environment. Effect-based, multifactorial test systems with zebrafish embryos to assess in particular developmental toxicity are beneficial but rarely used in this context. We therefore exposed wild-type embryos to the metals copper (CuSO4), cadmium (CdCl2) and cobalt (CoSO4) for 72 h to determine lethal as well as sublethal morphological effects. Motor neuron damage was investigated by immunofluorescence staining of primary motor neurons (PMNs) and secondary motor neurons (SMNs). In vivo stainings using the vital dye DASPEI were used to quantify neuromast development and damage. The consequences of metal toxicity were also assessed functionally, by testing fish behavior following tactile stimulation. The median effective concentration (EC50) values for morphological effects 72 h post fertilization (hpf) were 14.6 mg/L for cadmium and 0.018 mg/L for copper, whereas embryos exposed up to 45.8 mg/L cobalt showed no morphological effects. All three metals caused a concentration-dependent reduction in the numbers of normal PMNs and SMNs, and in the fluorescence intensity of neuromasts. The results for motor neuron damage and behavior were coincident for all three metals. Even the lowest metal concentrations (cadmium 2mg/L, copper 0.01 mg/L and cobalt 0.8 mg/L) resulted in neuromast damage. The results demonstrate that the neuromast cells were more sensitive to metal exposure than morphological traits or the response to tactile stimulation and motor neuron damage. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Zebrafish embryo developmental toxicology assay.

    PubMed

    Panzica-Kelly, Julieta M; Zhang, Cindy X; Augustine-Rauch, Karen

    2012-01-01

    A promising in vitro zebrafish developmental toxicology assay was generated to test compounds for their teratogenic potential. The assay's predictivity is approximately 87% in AB strain fish (Brannen KC et al., Birth Defects Res B Dev Reprod Toxicol 89:66-77, 2010). The procedure entails exposing dechorionated gastrulation-stage embryos to a range of compound concentrations for 5 days throughout embryonic and larva development. The larvae are evaluated for viability in order to identify an LC25 (the compound concentration in which 25% lethality is observed) and morphological anomalies using a numerical score system to identify the NOAEL (no observed adverse effect level). These values are used to calculate the teratogenic index (LC25/NOAEL ratio) of each compound. If the teratogenic index is equal to or greater than 10 then the compound is classified as a teratogen, and if the ratio is less than 10 then the compound is classified as a nonteratogen (Brannen KC et al., Birth Defects Res B Dev Reprod Toxicol 89:66-77, 2010).

  3. Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos.

    PubMed

    Berrun, A C; Stachura, D L

    2017-11-30

    Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

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

    SciT

    Hermsen, Sanne A.B., E-mail: Sanne.Hermsen@rivm.nl; Department of Toxicogenomics, Maastricht University, P.O. Box 616, 6200 MD, Maastricht; Institute for Risk Assessment Sciences

    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 andmore » 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.« less

  5. Zebrafish aussicht mutant embryos exhibit widespread overexpression of ace (fgf8) and coincident defects in CNS development.

    PubMed

    Heisenberg, C P; Brennan, C; Wilson, S W

    1999-05-01

    During the development of the zebrafish nervous system both noi, a zebrafish pax2 homolog, and ace, a zebrafish fgf8 homolog, are required for development of the midbrain and cerebellum. Here we describe a dominant mutation, aussicht (aus), in which the expression of noi and ace is upregulated. In aus mutant embryos, ace is upregulated at many sites in the embryo, while noi expression is only upregulated in regions of the forebrain and midbrain which also express ace. Subsequent to the alterations in noi and ace expression, aus mutants exhibit defects in the differentiation of the forebrain, midbrain and eyes. Within the forebrain, the formation of the anterior and postoptic commissures is delayed and the expression of markers within the pretectal area is reduced. Within the midbrain, En and wnt1 expression is expanded. In heterozygous aus embryos, there is ectopic outgrowth of neural retina in the temporal half of the eyes, whereas in putative homozygous aus embryos, the ventral retina is reduced and the pigmented retinal epithelium is expanded towards the midline. The observation that aus mutant embryos exhibit widespread upregulation of ace raised the possibility that aus might represent an allele of the ace gene itself. However, by crossing carriers for both aus and ace, we were able to generate homozygous ace mutant embryos that also exhibited the aus phenotype. This indicated that aus is not tightly linked to ace and is unlikely to be a mutation directly affecting the ace locus. However, increased Ace activity may underly many aspects of the aus phenotype and we show that the upregulation of noi in the forebrain of aus mutants is partially dependent upon functional Ace activity. Conversely, increased ace expression in the forebrain of aus mutants is not dependent upon functional Noi activity. We conclude that aus represents a mutation involving a locus normally required for the regulation of ace expression during embryogenesis.

  6. Comparison of the toxicity of silver, gold and platinum nanoparticles in developing zebrafish embryos.

    PubMed

    Asharani, P V; Lianwu, Yi; Gong, Zhiyuan; Valiyaveettil, Suresh

    2011-03-01

    Nanoparticles have diverse applications in electronics, medical devices, therapeutic agents and cosmetics. While the commercialization of nanoparticles is rapidly expanding, their health and environmental impact is not well understood. Toxicity assays of silver, gold, and platinum nanoparticles, using zebrafish embryos to study their developmental effects were carried out. Gold (Au-NP, 15-35 nm), silver (Ag-NP, 5-35 nm) and platinum nanoparticles (Pt-NP, 3-10 nm) were synthesized using polyvinyl alcohol (PVA) as a capping agent. Toxicity was recorded in terms of mortality, hatching delay, phenotypic defects and metal accumulation. The addition of Ag-NP resulted in a concentration-dependant increase in mortality rate. Both Ag-NP and Pt-NP induced hatching delays, as well as a concentration dependant drop in heart rate, touch response and axis curvatures. Ag-NP also induced other significant phenotypic changes including pericardial effusion, abnormal cardiac morphology, circulatory defects and absence or malformation of the eyes. In contrast, Au-NP did not show any indication of toxicity. Uptake and accumulation of nanoparticles in embryos was confirmed by inductively coupled plasma optical emission spectroscopy (ICP-OES), which revealed detectable levels in embryos within 72 hpf. Ag-NP and Au-NP were taken up by the embryos in relatively equal amounts whereas lower Pt concentrations were observed in embryos exposed to Pt-NP. This was probably due to the small size of the Pt nanoparticles compared to Ag-NP and Au-NP, thus resulting in fewer metal atoms being retained in the embryos. Among the nanoparticles studied, Ag-NPs were found to be the most toxic and Au-NPs the non-toxic. The toxic effects exhibited by the zebrafish embryos as a consequence of nanoparticle exposure, accompanied by the accumulation of metals inside the body calls for urgent further investigations in this field.

  7. Expression profiling identifies novel Hh/Gli regulated genes in developing zebrafish embryos.

    PubMed Central

    Bergeron, Sadie A.; Milla, Luis A.; Villegas, Rosario; Shen, Meng-Chieh; Burgess, Shawn M.; Allende, Miguel L.; Karlstrom, Rolf O.; Palma, Verónica

    2008-01-01

    The Hedgehog (Hh) signaling pathway plays critical instructional roles during embryonic development. Mis-regulation of Hh/Gli signaling is a major causative factor in human congenital disorders and in a variety of cancers. The zebrafish is a powerful genetic model for the study of Hh signaling during embryogenesis, as a large number of mutants have been identified affecting different components of the Hh/Gli signaling system. By performing global profiling of gene expression in different Hh/Gli gain- and loss-of-function scenarios we identified several known (e.g. ptc1 and nkx2.2a) as well as a large number of novel Hh regulated genes that are differentially expressed in embryos with altered Hh/Gli signaling function. By uncovering changes in tissue specific gene expression, we revealed new embryological processes that are influenced by Hh signaling. We thus provide a comprehensive survey of Hh/Gli regulated genes during embryogenesis and we identify new Hh-regulated genes that may be targets of mis-regulation during tumorogenesis. PMID:18055165

  8. Effect of PMA-induced protein kinase C activation on development and apoptosis in early zebrafish embryos.

    PubMed

    Hrubik, Jelena; Glisic, Branka; Samardzija, Dragana; Stanic, Bojana; Pogrmic-Majkic, Kristina; Fa, Svetlana; Andric, Nebojsa

    2016-12-01

    Protein kinase C (PKC) isoforms have been implicated in several key steps during early development, but the consequences of xenobiotic-induced PKC activation during early embryogenesis are still unknown. In this study, zebrafish embryos were exposed to a range of phorbol 12-myristate 13-acetate (PMA) concentrations (0-200μg/L) at different time points after fertilization. Results showed that 200μgPMA/L caused development of yolk bags, cardiac edema, slow blood flow, pulsating blood flow, slow pulse, elongated heart, lack of tail fins, curved tail, and coagulation. PMA exposure decreased survival rate of the embryos starting within the first 24h and becoming more pronounced after prolonged exposure (96h). PMA increased the number of apoptotic cells in the brain region as demonstrated by acridine orange staining and caused up-regulation of caspase 9 (casp9) and p53 up-regulated modulator of apoptosis (puma) mRNA in whole embryos. PMA caused oxidative stress in the embryos as demonstrated by decreased mRNA expression of catalase and superoxide dismutase 2. Inhibition of Pkc with GF109203X improved overall survival rate, reduced apoptosis in the brain and decreased expression of casp9 and puma in the PMA-exposed embryos. However, Pkc inhibition neither prevented development of deformities nor reversed oxidative stress in the PMA-exposed embryos. These data suggest that direct over-activation of Pkc during early embryogenesis of zebrafish is associated with apoptosis and decreased survival rate of the embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed

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

    2014-08-01

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

  10. Stimulus-triggered enhancement of chilling tolerance in zebrafish embryos

    PubMed Central

    Szabó, Katalin; Budai, Csilla; Losonczi, Eszter; Bernáth, Gergely; Csenki-Bakos, Zsolt; Urbányi, Béla; Pribenszky, Csaba; Horváth, Ákos; Cserepes, Judit

    2017-01-01

    Background Cryopreservation of zebrafish embryos is still an unsolved problem despite market demand and massive efforts to preserve genetic variation among numerous existing lines. Chilled storage of embryos might be a step towards developing successful cryopreservation, but no methods to date have worked. Methods In the present study, we applied a novel strategy to improve the chilling tolerance of zebrafish embryos by introducing a preconditioning hydrostatic pressure treatment to the embryos. In our experiments, 26-somites and Prim-5 stage zebrafish embryos were chilled at 0°C for 24 hours after preconditioning. Embryo survival rate, ability to reach maturation and fertilizing capacity were tested. Results Our results indicate that applied preconditioning technology made it possible for the chilled embryos to develop normally until maturity, and to produce healthy offspring as normal, thus passing on their genetic material successfully. Treated embryos had a significantly higher survival and better developmental rate, moreover the treated group had a higher ratio of normal morphology during continued development. While all controls from chilled embryos died by 30 day-post-fertilization, the treated group reached maturity (~90–120 days) and were able to reproduce, resulting in offspring in expected quantity and quality. Conclusions Based on our results, we conclude that the preconditioning technology represents a significant improvement in zebrafish embryo chilling tolerance, thus enabling a long-time survival. Furthermore, as embryonic development is arrested during chilled storage this technology also provides a solution to synchronize or delay the development. PMID:28166301

  11. Insulin-like growth factor (IGF) signalling is required for early dorso-anterior development of the zebrafish embryo.

    PubMed

    Eivers, Edward; McCarthy, Karena; Glynn, Catherine; Nolan, Catherine M; Byrnes, Lucy

    2004-12-01

    The insulin-like growth factor (IGF) signalling pathway has been highly conserved in animal evolution and, in mammals and Xenopus, plays a key role in embryonic growth and development, with the IGF-1 receptor (IGF-1R) being a crucial regulator of the signalling cascade. Here we report the first functional role for the IGF pathway in zebrafish. Expression of mRNA coding for a dominant negative IGF-1R resulted in embryos that were small in size compared to controls and had disrupted head and CNS development. At its most extreme, this phenotype was characterized by a complete loss of head and eye structures, an absence of notochord and the presence of abnormal somites. In contrast, up-regulation of IGF signalling following injection of IGF-1 mRNA, resulted in a greatly expanded development of anterior structures at the expense of trunk and tail. IGF-1R knockdown caused a significant decrease in the expression of Otx2, Rx3, FGF8, Pax6.2 and Ntl, while excess IGF signalling expanded Otx2 expression in presumptive forebrain tissue and widened the Ntl expression domain in the developing notochord. The observation that IGF-1R knockdown reduced expression of two key organizer genes (chordin and goosecoid) suggests that IGF signalling plays a role in regulating zebrafish organizer activity. This is supported by the expression of IGF-1, IGF-2 and IGF-1R in shield-stage zebrafish embryos and the demonstration that IGF signalling influences expression of BMP2b, a gene that plays an important role in zebrafish pattern formation. Our data is consistent with a common pathway for integration of IGF, FGF8 and anti-BMPs in early vertebrate development.

  12. 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

  13. 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

    2016-02-05

    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.

  14. Urban stormwater runoff negatively impacts lateral line development in larval zebrafish and salmon embryos.

    PubMed

    Young, Alexander; Kochenkov, Valentin; McIntyre, Jenifer K; Stark, John D; Coffin, Allison B

    2018-02-12

    After a storm, water often runs off of impervious urban surfaces directly into aquatic ecosystems. This stormwater runoff is a cocktail of toxicants that have serious effects on the ecological integrity of aquatic habitats. Zebrafish that develop in stormwater runoff suffer from cardiovascular toxicity and impaired growth, but the effects of stormwater on fish sensory systems are not understood. Our study investigated the effect of stormwater on hair cells of the lateral line in larval zebrafish and coho salmon. Our results showed that although toxicants in stormwater did not kill zebrafish hair cells, these cells did experience damage. Zebrafish developing in stormwater also experienced impaired growth, fewer neuromasts in the lateral line, and fewer hair cells per neuromast. A similar reduction in neuromast number was observed in coho salmon reared in stormwater. Bioretention treatment, intended to filter out harmful constituents of stormwater, rescued the lateral line defects in zebrafish but not in coho salmon, suggesting that not all of the harmful constituents were removed by the filtration media and that salmonids are particularly sensitive to aquatic toxicants. Collectively, these data demonstrate that sub-lethal exposure to stormwater runoff negatively impacts a fish sensory system, which may have consequences for organismal fitness.

  15. Toxicity evaluation of β-diketone antibiotics on the development of embryo-larval zebrafish (Danio rerio).

    PubMed

    Wang, Huili; Che, Baoguang; Duan, Ailian; Mao, Jingwen; Dahlgren, Randy A; Zhang, Minghua; Zhang, Hongqin; Zeng, Aibing; Wang, Xuedong

    2014-10-01

    This study evaluated the effects of β-diketone antibiotics (DKAs) on the development of embryo-larval zebrafish (Danio rerio). When exposure to DKAs, developmental malformations, such as hatching delay, curved body axis, pericardial edema, uninflated swim bladder and yolk sac edema, were observed at 120 h postfertilization (hpf). The estimated 120 hpf nominal concentrations of no observed effect concentration and lowest observed effect concentration for DKAs were 18.75 and 37.50 mg/L, respectively, suggesting that DKAs have much lower toxicity than other persistent pollutants. Following DKA exposure, embryonic heart rates were significantly reduced as compared to the controls at 48 and 60 hpf. The peak bending motion frequency appeared 1 h earlier than in control embryos. The 2.34 and 9.38-mg/L treatment groups had a higher basal swim rate than control groups at 120 hpf in both light and light-to-dark photoperiod experiments. The occurrence of high speed swim rates was enhanced approximately threefold to sevenfold in the 2.34 and 9.38 mg/L treatments compared to the control. Glutathione (GSH) concentrations in the 2.34 and 9.38-mg/L treatments were significantly higher than the control at 72 hpf, suggesting that GSH production was induced at the end of the hatching period. When exposed to DKAs, zebrafish superoxide dismutase enzyme (SOD) activities were significantly inhibited in the early embryonic period, demonstrating that the clearing ability in zebrafish was lower than the generation rate of free radicals. In summary, the combined DKAs were developmentally toxic to zebrafish in their early life stages and had the ability to impair individual behaviors that are of great importance in the assessment of their ecological fitness. Copyright © 2013 Wiley Periodicals, Inc., a Wiley company.

  16. Assessing biological effects of fluoxetine in developing zebrafish embryos using gas chromatography-mass spectrometry based metabolomics.

    PubMed

    Mishra, Priti; Gong, Zhiyuan; Kelly, Barry C

    2017-12-01

    Continuous low-dose exposure of pharmaceutically active compounds (PhACs) in aquatic ecosystems is a concern worldwide. In this study, we utilized a gas chromatography mass spectrometry (GC-MS) based metabolomics approach to assess endogenous metabolite changes in developing zebrafish embryos exposed to different concentrations of the widely used antidepressant, fluoxetine. Embryos were exposed from 2 h post fertilization (hpf) until 96 hpf. Using the Fiehn GC-MS library, a total of 31 metabolites were positively identified in embryos. Statistical analyses revealed significant dysregulation of 11 metabolites in fluoxetine exposed embryos. Metabolite classes that were significantly altered included, amino acids, monosaccharides, glycerophosphates, fatty acids, carboxylic acid derivatives and sugars. Concentrations of amino acids, maltose, d-malic acid, 3-phosphoglycerate and d-glucose were significantly reduced in exposed embryos. Conversely, concentrations of citric acid were in some cases significantly elevated in exposed embryos. Metabolic pathway analysis revealed perturbation of five main pathways, including (i) alanine, aspartate and glutamate metabolism, (ii) phenylalanine, tyrosine and tryptophan biosynthesis, (iii) phenylalanine metabolism. (iv) tyrosine metabolism and (v) starch and sucrose metabolism. The results indicate fluoxetine exposure causes perturbation of energy and amino acid metabolism, which may adversely impact embryogenesis due to depletion of energy reserves during this period. Also, the observed alterations in aspartic acid, phenylalanine and tyrosine in fluoxetine exposed embryos suggests potential disruption of normal neurobehavioral and liver function. The results further demonstrate that GC-MS based metabolomics is an effective approach for assessing toxicodynamics and threshold effect levels of environmental pollutants in aquatic organisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Inhibition of endogenous MTF-1 signaling in zebrafish embryos identifies novel roles for MTF-1 in development.

    PubMed

    O'Shields, Britton; McArthur, Andrew G; Holowiecki, Andrew; Kamper, Martin; Tapley, Jeffrey; Jenny, Matthew J

    2014-09-01

    The metal responsive element-binding transcription factor-1 (MTF-1) responds to changes in cellular zinc levels caused by zinc exposure or disruption of endogenous zinc homeostasis by heavy metals or oxygen-related stress. Here we report the functional characterization of a complete zebrafish MTF-1 in comparison with the previously identified isoform lacking the highly conserved cysteine-rich motif (Cys-X-Cys-Cys-X-Cys) found in all other vertebrate MTF-1 orthologs. In an effort to develop novel molecular tools, a constitutively nuclear dominant-negative MTF-1 (dnMTF-1) was generated as tool for inhibiting endogenous MTF-1 signaling. The in vivo efficacy of the dnMTF-1 was determined by microinjecting in vitro transcribed dnMTF-1 mRNA into zebrafish embryos (1-2 cell stage) followed by transcriptomic profiling using an Agilent 4x44K array on 28- and 36-hpf embryos. A total of 594 and 560 probes were identified as differentially expressed at 28hpf and 36hpf, respectively, with interesting overlaps between timepoints. The main categories of genes affected by the inhibition of MTF-1 signaling were: nuclear receptors and genes involved in stress signaling, neurogenesis, muscle development and contraction, eye development, and metal homeostasis, including novel observations in iron and heme homeostasis. Finally, we investigate both the transcriptional activator and transcriptional repressor role of MTF-1 in potential novel target genes identified by transcriptomic profiling during early zebrafish development. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Miniaturized Embryo Array for Automated Trapping, Immobilization and Microperfusion of Zebrafish Embryos

    PubMed Central

    Akagi, Jin; Khoshmanesh, Khashayar; Evans, Barbara; Hall, Chris J.; Crosier, Kathryn E.; Cooper, Jonathan M.; Crosier, Philip S.; Wlodkowic, Donald

    2012-01-01

    Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale. PMID:22606275

  19. Effects of nitric oxide on neuromuscular properties of developing zebrafish embryos.

    PubMed

    Jay, Michael; Bradley, Sophie; McDearmid, Jonathan Robert

    2014-01-01

    Nitric oxide is a bioactive signalling molecule that is known to affect a wide range of neurodevelopmental processes. However, its functional relevance to neuromuscular development is not fully understood. Here we have examined developmental roles of nitric oxide during formation and maturation of neuromuscular contacts in zebrafish. Using histochemical approaches we show that elevating nitric oxide levels reduces the number of neuromuscular synapses within the axial swimming muscles whilst inhibition of nitric oxide biosynthesis has the opposite effect. We further show that nitric oxide signalling does not change synapse density, suggesting that the observed effects are a consequence of previously reported changes in motor axon branch formation. Moreover, we have used in vivo patch clamp electrophysiology to examine the effects of nitric oxide on physiological maturation of zebrafish neuromuscular junctions. We show that developmental exposure to nitric oxide affects the kinetics of spontaneous miniature end plate currents and impacts the neuromuscular drive for locomotion. Taken together, our findings implicate nitrergic signalling in the regulation of zebrafish neuromuscular development and locomotor maturation.

  20. Automated image-based phenotypic analysis in zebrafish embryos

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2010-12-24

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

  2. Toxicogenomic analysis in the combined effect of tributyltin and benzo[a]pyrene on the development of zebrafish embryos.

    PubMed

    Huang, Lixing; Zuo, Zhenghong; Zhang, Youyu; Wang, Chonggang

    2015-01-01

    There is a growing recognition that the toxic effects of chemical mixtures are been an important issue in toxicological sciences. Tributyltin (TBT) and benzo[a]pyrene (BaP) are widespread pollutants that occur simultaneously in the aquatic environments. This study was designed to examine comprehensively the combined effects of TBT and BaP on zebrafish (Danio rerio) embryos using toxicogenomic approach combined with biochemical detection and morphological analysis, and tried to gain insight into the mechanisms underlying the combined effects of TBT and BaP. The results of toxicogenomic data indicated that: (1) TBT cotreatment rescued the embryos from decreased hatching ratio caused by BaP alone, while the alteration of gene expression (in this article the phrase gene expression is used as a synonym to gene transcription, although in is acknowledged that gene expression can also be regulated by, e.g., translation and mRNA or protein stability) relative to zebrafish hatching in the BaP groups was resumed by the cotreatment with TBT; (2) BaP cotreatment decreased TBT-mediated dorsal curvature, and alleviated the perturbation of Notch pathway caused by TBT alone; (3) cotreatment with TBT decreased BaP-mediated bradycardia, which might be due to that TBT cotreatment alleviated the perturbation in expression of genes related to cardiac muscle cell development and calcium handling caused by BaP alone; 4) TBT cotreatment brought an antagonistic effect on the BaP-mediated oxidative stress and DNA damage. These results suggested that toxicogenomic approach was available for analyzing combined toxicity with high sensitivity and accuracy, which might improve our understanding and predictability for the combined effects of chemicals. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Development of a flow-through system for the fish embryo toxicity test (FET) with the zebrafish (Danio rerio).

    PubMed

    Lammer, E; Kamp, H G; Hisgen, V; Koch, M; Reinhard, D; Salinas, E R; Wendler, K; Zok, S; Braunbeck, Th

    2009-10-01

    The acute fish test is still a mandatory component in chemical hazard and risk assessment. However, one of the objectives of the new European chemicals policy (REACH - Registration, Evaluation, Authorization and Restriction of Chemicals) is to promote non-animal testing. For whole effluent testing in Germany, the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) has been an accepted and mandatory replacement of the fish test since January 2005. For chemical testing, however, further optimization of the FET is required to improve the correlation between the acute fish test and the alternative FET. Since adsorption of the test chemical to surfaces may reduce available exposure concentrations, a flow-through system for the FET using modified commercially available polystyrene 24-well microtiter plates was developed, thus combining the advantages of the standard FET with those of continuous delivery of test substances. The advantages of the design presented include: small test footprint, availability of adequate volumes of test solution for subsequent chemical analysis, and sufficient flow to compensate for effects of non-specific adsorption within 24h. The flow-through test system can also be utilized to conduct longer-term embryo larval fish tests, thus offering the possibility for teratogenicity testing.

  4. 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

  5. Evaluating the Zebrafish Embryo Toxicity Test for Pesticide ...

    EPA Pesticide Factsheets

    Given the numerous chemicals used in society, it is critical to develop tools for accurate and efficient evaluation of potential risks to human and ecological receptors. Fish embryo acute toxicity tests are 1 tool that has been shown to be highly predictive of standard, more resource-intensive, juvenile fish acute toxicity tests. However, there is also evidence that fish embryos are less sensitive than juvenile fish for certain types of chemicals, including neurotoxicants. The utility of fish embryos for pesticide hazard assessment was investigated by comparing published zebrafish embryo toxicity data from pesticides with median lethal concentration 50% (LC50) data for juveniles of 3 commonly tested fish species: rainbow trout, bluegill sunfish, and sheepshead minnow. A poor, albeit significant, relationship (r2 = 0.28; p < 0.05) was found between zebrafish embryo and juvenile fish toxicity when pesticides were considered as a single group, but a much better relationship (r2 = 0.64; p < 0.05) when pesticide mode of action was factored into an analysis of covariance. This discrepancy is partly explained by the large number of neurotoxic pesticides in the dataset, supporting previous findings that commonly used fish embryo toxicity test endpoints are particularly insensitive to neurotoxicants. These results indicate that it is still premature to replace juvenile fish toxicity tests with embryo-based tests such as the Organisation for Economic Co-op

  6. Development of a new screening assay to identify proteratogenic substances using zebrafish danio rerio embryo combined with an exogenous mammalian metabolic activation system (mDarT).

    PubMed

    Busquet, François; Nagel, Roland; von Landenberg, Friedrich; Mueller, Stefan O; Huebler, Nicole; Broschard, Thomas H

    2008-07-01

    The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays, for example, in rats or rabbits. We have developed an in vitro teratogenicity assay using the zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide (CPA) and ethanol were used as proteratogens to test the efficiency of this assay. Briefly, the zebrafish embryos were cocultured at 2 hpf (hours postfertilization) with the test material at varying concentrations, induced male rat liver microsomes and nicotinamide adenine dinucleotide phosphate (reduced) for 60 min at 32 degrees C under moderate agitation in Tris-buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterward fish embryos were transferred individually into 24-well plates filled with fish medium for 48 h at 26 degrees C with a 12-h light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. No teratogenic effects were observed in fish embryos exposed to the proteratogens alone, that is, without metabolic activation. In contrast, CPA and ethanol induced abnormalities in fish embryos when coincubated with microsomes. The severity of malformations increased with increasing concentrations of the proteratogens. We conclude that the application of microsomes will improve and refine the D. rerio teratogenicity assay as a predictive and valuable alternative method to screen teratogenic substances.

  7. Myomaker mediates fusion of fast myocytes in zebrafish embryos

    SciT

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

    2014-09-05

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

  8. Evaluating the zebrafish embryo toxicity test for pesticide hazard screening.

    PubMed

    Glaberman, Scott; Padilla, Stephanie; Barron, Mace G

    2017-05-01

    Given the numerous chemicals used in society, it is critical to develop tools for accurate and efficient evaluation of potential risks to human and ecological receptors. Fish embryo acute toxicity tests are 1 tool that has been shown to be highly predictive of standard, more resource-intensive, juvenile fish acute toxicity tests. However, there is also evidence that fish embryos are less sensitive than juvenile fish for certain types of chemicals, including neurotoxicants. The utility of fish embryos for pesticide hazard assessment was investigated by comparing published zebrafish embryo toxicity data from pesticides with median lethal concentration 50% (LC50) data for juveniles of 3 commonly tested fish species: rainbow trout, bluegill sunfish, and sheepshead minnow. A poor, albeit significant, relationship (r 2  = 0.28; p < 0.05) was found between zebrafish embryo and juvenile fish toxicity when pesticides were considered as a single group, but a much better relationship (r 2  = 0.64; p < 0.05) when pesticide mode of action was factored into an analysis of covariance. This discrepancy is partly explained by the large number of neurotoxic pesticides in the dataset, supporting previous findings that commonly used fish embryo toxicity test endpoints are particularly insensitive to neurotoxicants. These results indicate that it is still premature to replace juvenile fish toxicity tests with embryo-based tests such as the Organisation for Economic Co-operation and Development Fish Embryo Acute Toxicity Test for routine pesticide hazard assessment, although embryo testing could be used with other screening tools for testing prioritization. Environ Toxicol Chem 2017;36:1221-1226. © 2016 SETAC. © 2016 SETAC.

  9. 2,3,7,8-Tetrachlorodibenzo-p-dioxin toxicity in the zebrafish embryo: altered regional blood flow and impaired lower jaw development.

    PubMed

    Teraoka, Hiroki; Dong, Wu; Ogawa, Shuji; Tsukiyama, Shusaku; Okuhara, Yuji; Niiyama, Masayoshi; Ueno, Naoto; Peterson, Richard E; Hiraga, Takeo

    2002-02-01

    The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on regional red blood cell (RBC) perfusion rate, as an index of blood flow, and lower jaw development were investigated quantitatively in zebrafish embryos (Danio rerio) during early development. As revealed by observation of live embryos and alcian-blue staining, TCDD retarded lower jaw development in a concentration-dependent manner with only a minor inhibitory effect on total body length. Both inhibitory effects were significant as early as 60 h postfertilization (hpf), at which time the area of goosecoid (gsc) mRNA expression was clearly reduced in the lower jaw. To examine effects of TCDD on RBC perfusion rate, time-lapse recording was performed using a digital video camera attached to a light microscope. TCDD did not show marked effects on RBC perfusion rate until 72 hpf, when vessel-specific effects emerged. TCDD severely inhibited RBC perfusion rate in intersegmental arteries of the trunk, but only modestly and slightly inhibited RBC perfusion rate in certain vessels of the head such as the central arteries and optic vein. Conversely, at both 72 and 84 hpf, TCDD significantly increased RBC perfusion rate in the hypobranchial artery branching to the lower jaw primordia, and then reduced it at 96 hpf. RBC perfusion rate in all vessels examined in TCDD-exposed embryos was inhibited at 96 hpf. The zebrafish aryl hydrocarbon receptor 2 (zfAhR2) mRNA was strongly expressed in the lower jaw primordia at 48 hpf, and expression of this transcript was augmented by TCDD treatment. Thus, TCDD exposure of the zebrafish embryo has a disruptive effect on local circulation and lower jaw cartilage growth. Initially, TCDD may act directly on the lower jaw primordia to impair lower jaw development. Reductions in hypobranchial RBC perfusion rate occurred well after the initial retardation in lower jaw development had become apparent, and may contribute further to the effect.

  10. The role of gastrulation brain homeobox 2 (gbx2) in the development of the ventral telencephalon in zebrafish embryos.

    PubMed

    Wang, Zhe; Nakayama, Yukiko; Tsuda, Sachiko; Yamasu, Kyo

    During vertebrate brain development, the gastrulation brain homeobox 2 gene (gbx2) is expressed in the forebrain, but its precise roles are still unknown. In this study, we addressed this issue in zebrafish (Danio rerio) first by carefully examining gbx2 expression in the developing forebrain. We showed that gbx2 was expressed in the telencephalon during late somitogenesis, from 18h post-fertilization (hpf) to 24 hpf, and in the thalamic primordium after 26 hpf. In contrast, another gbx gene, gbx1, was expressed in the anterior-most ventral telencephalon after 36 hpf. Thus, the expression patterns of these two gbx genes did not overlap, arguing against their redundant function in the forebrain. Two-color fluorescence in situ hybridization (FISH) showed close relationships between the telencephalic expression of gbx2 and other forebrain-forming genes, suggesting that their interactions contribute to the regionalization of the telencephalon. FISH further revealed that gbx2 is expressed in the ventricular region of the telencephalon. By using transgenic fish in which gbx2 can be induced by heat shock, we found that gbx2 induction at 16 hpf repressed the expression of emx3, dlx2a, and six3b in the ventral telencephalon. Among secreted factor genes, bmp2b and wnt1 were repressed in the vicinity of the gbx2 domain in the telencephalon. The expression of forebrain-forming genes was examined in mutant embryos lacking gbx2, showing emx3 and dlx2a to be upregulated in the subpallium at 24 hpf. Taken together, these findings indicate that gbx2 contributes to the development of the subpallium through its repressive activities against other telencephalon-forming genes. We further showed that inhibiting FGF signaling and activating Wnt signaling repressed gbx2 and affected the regionalization of the telencephalon, supporting a functional link between gbx2, intracellular signaling, and telencephalon development. Copyright © 2017 International Society of Differentiation

  11. Developmental Toxicity of Dextromethorphan in Zebrafish Embryos/Larvae

    PubMed Central

    Xu, Zheng; Williams, Frederick E.; Liu, Ming-Cheh

    2012-01-01

    Dextromethorphan is widely used in over-the-counter cough and cold medications. Its efficacy and safety for infants and young children remains to be clarified. The present study was designed to use the zebrafish as a model to investigate the potential toxicity of dextromethorphan during the embryonic and larval development. Three sets of zebrafish embryos/larvae were exposed to dextromethorphan at 24 hours post fertilization (hpf), 48 hpf, and 72 hpf, respectively, during the embryonic/larval development. Compared with the 48 and 72 hpf exposure sets, the embryos/larvae in the 24 hpf exposure set showed much higher mortality rates which increased in a dose-dependent manner. Bradycardia and reduced blood flow were observed for the embryos/larvae treated with increasing concentrations of dextromethorphan. Morphological effects of dextromethorphan exposure, including yolk sac and cardiac edema, craniofacial malformation, lordosis, non-inflated swim bladder, and missing gill, were also more frequent and severe among zebrafish embryos/larvae exposed to dextromethorphan at 24 hpf. Whether the more frequent and severe developmental toxicity of dextromethorphan observed among the embryos/larvae in the 24 hpf exposure set, as compared with the 48 and 72 hpf exposure sets, is due to the developmental expression of the Phase I and Phase II enzymes involved in the metabolism of dextromethorphan remains to be clarified. A reverse transcription-polymerase chain reaction (RT-PCR) analysis, nevertheless, revealed developmental stage-dependent expression of mRNAs encoding SULT3 ST1 and SULT3 ST3, two enzymes previously shown to be capable of sulfating dextrorphan, an active metabolite of dextromethorphan. PMID:20737414

  12. Developmental toxicity of dextromethorphan in zebrafish embryos/larvae.

    PubMed

    Xu, Zheng; Williams, Frederick E; Liu, Ming-Cheh

    2011-03-01

    Dextromethorphan is widely used in over-the-counter cough and cold medications. Its efficacy and safety for infants and young children remains to be clarified. The present study was designed to use zebrafish as a model to investigate the potential toxicity of dextromethorphan during embryonic and larval development. Three sets of zebrafish embryos/larvae were exposed to dextromethorphan at 24, 48 and 72 h post fertilization (hpf), respectively, during the embryonic/larval development. Compared with the 48 and 72 hpf exposure sets, the embryos/larvae in the 24 hpf exposure set showed much higher mortality rates which increased in a dose-dependent manner. Bradycardia and reduced blood flow were observed for the embryos/larvae treated with increasing concentrations of dextromethorphan. Morphological effects of dextromethorphan exposure, including yolk sac and cardiac edema, craniofacial malformation, lordosis, non-inflated swim bladder and missing gill, were also more frequent and severe among zebrafish embryos/larvae exposed to dextromethorphan at 24 hpf. Whether the more frequent and severe developmental toxicity of dextromethorphan observed among the embryos/larvae in the 24 hpf exposure set, as compared with the 48 and 72 hpf exposure sets, is due to the developmental expression of the phase I and phase II enzymes involved in the metabolism of dextromethorphan remains to be clarified. A reverse transcription-polymerase chain reaction analysis, nevertheless, revealed developmental stage-dependent expression of mRNAs encoding SULT3 ST1 and SULT3 ST3, two enzymes previously shown to be capable of sulfating dextrorphan, an active metabolite of dextromethorphan. Copyright © 2010 John Wiley & Sons, Ltd.

  13. Quantification of Estradiol Uptake in Zebrafish Embryos and Larvae.

    PubMed

    Souder, Jaclyn Paige; Gorelick, Daniel A

    2017-08-01

    Zebrafish are a powerful model system to assess the molecular and cellular effects of exposure to toxic chemicals during embryonic development. To study the effects of environmental endocrine disruptors, embryos and larvae are commonly exposed to supraphysiologic concentrations of these compounds in the water, but their bioavailability in zebrafish is largely unknown. One hypothesis is that supraphysiologic concentrations of estrogens in the water are required to achieve physiologic levels in vivo; however, this has not been directly tested. To test this hypothesis, we developed an assay using radiolabeled estradiol ([3H]E2) to measure uptake from water at multiple concentrations and exposure durations in developing zebrafish from 0 to 5 days postfertilization (dpf). We found that [3H]E2 uptake increased with increasing concentration, duration, and developmental stage. Percent uptake from the total volume of treatment solution increased with increasing exposure duration and developmental stage, but remained constant with increasing concentration. We also found that the chorion, an acellular envelope surrounding embryos through 3 dpf, did not substantially affect [3H]E2 uptake. Finally, we found that at 1 dpf, E2 was preferentially taken up by the yolk at multiple exposure durations, while at 2 dpf E2 was preferentially taken up into the embryonic body. Our results support the hypothesis that exposing zebrafish embryos and larvae to supraphysiologic concentrations of estrogens is required to achieve physiologically relevant doses in vivo. The isotopic assay reported here will provide a foundation for determining the uptake of other compounds for teratogenicity, toxicology and drug discovery studies. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. 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...

  15. Strategies for Analyzing Cardiac Phenotypes in the Zebrafish Embryo

    PubMed Central

    Houk, Andrew R.; Yelon, Deborah

    2017-01-01

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

  16. Comparative toxicity of metal oxide nanoparticles (CuO, ZnO and TiO2) to developing zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Vicario-Parés, Unai; Castañaga, Luis; Lacave, Jose Maria; Oron, Miriam; Reip, Paul; Berhanu, Deborah; Valsami-Jones, Eugenia; Cajaraville, Miren P.; Orbea, Amaia

    2014-08-01

    Increasing use of nanomaterials is resulting in their release into the environment, making necessary to determine the toxicity of these materials. With this aim, the effects of CuO, ZnO and TiO2 nanoparticles (NPs) on zebrafish development were assessed in comparison with the effects caused by the ionic forms (for copper and zinc), bulk counterparts and the stabilizer used for rutile TiO2 NPs. None of the NPs caused significant embryo mortality. CuO NPs were the most toxic affecting hatching and increasing malformation prevalence (≥1 mg Cu/L), followed by ZnO NPs that affected hatching at ≥5 mg Zn/L and stabilized TiO2 NPs that caused mortality and decreased hatching at 100 mg Ti/L. Exposure to the stabilizer alone provoked the same effect. Thus, toxicity of the TiO2 NP suspension can be linked to the surfactant. For all the endpoints, the greatest effects were exerted by the ionic forms, followed by the NPs and finally by the bulk compounds. By autometallography, metal-bearing deposits were observed in embryos exposed to CuO and ZnO NPs, being more abundant in the case of embryos exposed to CuO NPs. The largest and most abundant metal-bearing deposits were detected in embryos exposed to ionic copper. In conclusion, metal oxide NPs affected zebrafish development altering hatching and increasing the prevalence of malformations. Thus, the use and release of metal oxide NPs to the environment may pose a risk to aquatic organisms as a result of the toxicity caused by NPs themselves or by the additives used in their production.

  17. Characterization of neural development in zebrafish embryos using real-time quantitative PCR.

    EPA Science Inventory

    Chemicals adversely affecting the developing nervous system may cause long-term consequences on human health. Little information exists on a large number of environmental compounds to guide developmental neurotoxicity risk assessments. Because developmental neurotoxicity studies ...

  18. 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

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

    PubMed

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

    2014-06-01

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

  20. Network analysis reveals stage-specific changes in zebrafish embryo development using time course whole transcriptome profiling and prior biological knowledge.

    PubMed

    Zhang, Yuji

    2015-01-01

    Molecular networks act as the backbone of molecular activities within cells, offering a unique opportunity to better understand the mechanism of diseases. While network data usually constitute only static network maps, integrating them with time course gene expression information can provide clues to the dynamic features of these networks and unravel the mechanistic driver genes characterizing cellular responses. Time course gene expression data allow us to broadly "watch" the dynamics of the system. However, one challenge in the analysis of such data is to establish and characterize the interplay among genes that are altered at different time points in the context of a biological process or functional category. Integrative analysis of these data sources will lead us a more complete understanding of how biological entities (e.g., genes and proteins) coordinately perform their biological functions in biological systems. In this paper, we introduced a novel network-based approach to extract functional knowledge from time-dependent biological processes at a system level using time course mRNA sequencing data in zebrafish embryo development. The proposed method was applied to investigate 1α, 25(OH)2D3-altered mechanisms in zebrafish embryo development. We applied the proposed method to a public zebrafish time course mRNA-Seq dataset, containing two different treatments along four time points. We constructed networks between gene ontology biological process categories, which were enriched in differential expressed genes between consecutive time points and different conditions. The temporal propagation of 1α, 25-Dihydroxyvitamin D3-altered transcriptional changes started from a few genes that were altered initially at earlier stage, to large groups of biological coherent genes at later stages. The most notable biological processes included neuronal and retinal development and generalized stress response. In addition, we also investigated the relationship among

  1. 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

  2. Teratogenic responses of zebrafish embryos to decabromodiphenyl ether (BDE-209) in the presence of nano-SiO2 particles.

    PubMed

    Chao, Shu-Ju; Huang, Chin Pao; Chen, Pei-Chung; Huang, Chihpin

    2017-07-01

    This study investigated the influence of nano-SiO 2 particles (nSiO 2 ) on the teratogenic responses of zebrafish embryos to decabromodiphenyl ether (BDE-209). Zebrafish embryos were exposed to BDE-209 in the absence and presence of nSiO 2 for 96 h post fertilization (hpf). Results showed that formation of nSiO 2 -BDE-209 associates promoted both extracellular and intracellular uptake of BDE-209 by zebrafish embryos, thereby increasing the bioconcentration of BDE-209 on the chorion surface and the embryos. Results also showed embryos delay hatching temporarily when co-exposure to BDE-209 and nSiO 2 at 60 hpf. Furthermore, there was heartbeat decline (28.3 beats/10s) and increase in irregular heartbeat (45.8%) in zebrafish larvae at 96 hpf, compared to the sole exposure to BDE-209 (32.7 beats/10s and 0%). Malformation in terms of spinal curvature (SC), pericardial edema (PE) and yolk sac edema (YSE) were observed on zebrafish larvae at 33.9, 23.4, and 18%, respectively. Overall, abnormal development of zebrafish was apparent when co-exposure to BDE-209 and nSiO 2 . All relevant evidence considered, nSiO 2 could facilitate the transport of BDE-209 towards zebrafish embryos and negatively impact the development of zebrafish. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    PubMed

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

    2011-03-15

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

  4. Laser surgery of zebrafish (Danio rerio) embryos using femtosecond laser pulses: Optimal parameters for exogenous material delivery, and the laser's effect on short- and long-term development

    PubMed Central

    Kohli, Vikram; Elezzabi, Abdulhakem Y

    2008-01-01

    Background Femtosecond (fs) laser pulses have recently received wide interest as an alternative tool for manipulating living biological systems. In various model organisms the excision of cellular components and the intracellular delivery of foreign exogenous materials have been reported. However, the effect of the applied fs laser pulses on cell viability and development has yet to be determined. Using the zebrafish (Danio rerio) as our animal model system, we address both the short- and long-term developmental changes following laser surgery on zebrafish embryonic cells. Results An exogenous fluorescent probe, fluorescein isothiocyanate (FITC), was successfully introduced into blastomere cells and found to diffuse throughout all developing cells. Using the reported manipulation tool, we addressed whether the applied fs laser pulses induced any short- or long-term developmental effects in embryos reared to 2 and 7 days post-fertilization (dpf). Using light microscopy and scanning electron microscopy we compared key developmental features of laser-manipulated and control samples, including the olfactory pit, dorsal, ventral and pectoral fins, notochord, pectoral fin buds, otic capsule, otic vesicle, neuromast patterning, and kinocilia of the olfactory pit rim and cristae of the lateral wall of the ear. Conclusion In our study, no significant differences in hatching rates and developmental morphologies were observed in laser-manipulated samples relative to controls. This tool represents an effective non-destructive technique for potential medical and biological applications. PMID:18230185

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

    PubMed

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

    2017-08-01

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

  6. OpenSource lab-on-a-chip physiometer for accelerated zebrafish embryo biotests.

    PubMed

    Akagi, Jin; Hall, Chris J; Crosier, Kathryn E; Cooper, Jonathan M; Crosier, Philip S; Wlodkowic, Donald

    2014-01-02

    Zebrafish (Danio rerio) embryo assays have recently come into the spotlight as convenient experimental models in both biomedicine and ecotoxicology. As a small aquatic model organism, zebrafish embryo assays allow for rapid physiological, embryo-, and genotoxic tests of drugs and environmental toxins that can be simply dissolved in water. This protocol describes prototyping and application of an innovative, miniaturized, and polymeric chip-based device capable of immobilizing a large number of living fish embryos for real-time and/or time-lapse microscopic examination. The device provides a physical address designation to each embryo during analysis, continuous perfusion of medium, and post-analysis specimen recovery. Miniaturized embryo array is a new concept of immobilization and real-time drug perfusion of multiple individual and developing zebrafish embryos inside the mesofluidic device. The OpenSource device presented in this protocol is particularly suitable to perform accelerated fish embryo biotests in ecotoxicology and phenotype-based pharmaceutical screening. Copyright © 2014 John Wiley & Sons, Inc.

  7. 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

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

    PubMed

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

    2011-01-21

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

  9. 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.

  10. The role of apoptosis in MCLR-induced developmental toxicity in zebrafish embryos.

    PubMed

    Zeng, Cheng; Sun, Hong; Xie, Ping; Wang, Jianghua; Zhang, Guirong; Chen, Nan; Yan, Wei; Li, Guangyu

    2014-04-01

    We previously demonstrated that cyanobacteria-derived microcystin-leucine-arginine (MCLR) is able to induce developing toxicity, such as malformation, growth delay and also decreased heart rates in zebrafish embryos. However, the molecular mechanisms by which MCLR induces its toxicity during the development of zebrafish remain largely unknown. Here, we evaluate the role of apoptosis in MCLR-induced developmental toxicity. Zebrafish embryos were exposed to various concentrations of MCLR (0, 0.2, 0.5, 2, and 5.0 mg L(-1)) for 96 h, at which time reactive oxygen species (ROS) was significantly induced in the 2 and 5.0 mg L(-1) MCLR exposure groups. Acridine orange (AO) staining and terminal deoxynucleotide transferase-mediated deoxy-UTP nick end labelling (TUNEL) assay showed that MCLR exposure resulted in cell apoptosis. To test the apoptotic pathway, the expression pattern of several apoptotic-related genes was examined for the level of enzyme activity, gene and protein expression, respectively. The overall results demonstrate that MCLR induced ROS which consequently triggered apoptosis in the heart of developing zebrafish embryos. Our results also indicate that the p53-Bax-Bcl-2 pathway and the caspase-dependent apoptotic pathway play major roles in MCLR-induced apoptosis in the developing embryos. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Toxicity test of xanthone from mangosteen on zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Noordin, Muhammad Akram Mohd; Noor, Mahanem Mat; Kamaruddin, Wan Mohd Aizat Wan; Lazim, Azwan Mat; Fazry, Shazrul

    2016-11-01

    Xanthone is a chemical compound identified in mangosteen pericarp. A previous study showed that xanthone has anti-proliferating effect on cancer cells. In this study we investigate the toxicity level of xanthone in zebrafish embryo to for future reference on other animal model. We employed Fish Embryo Toxicity (FET) assay to determine the toxicity level of different concentrations of xanthone. Embryos were observed at 24, 48 and 72 hours post fertilization (hpf) under microscope at 4× magnification. The extract showed toxicity effect on embryo at concentrations of 250, 125 and 62.5 µg/mL. Concentrations at 15.63, 7.81 and 3.91 µg / mL of xanthone did not harm the embryos and showed 100% of survival.

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

    PubMed

    Raghunath, Azhwar; Perumal, Ekambaram

    2018-01-01

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

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

    SciT

    Bouwmeester, Manon C.; Ruiter, Sander; Lommelaars, Tobias

    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,more » 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. - Highlights: • Compound induced effects on DNA methylation in zebrafish embryos • Global methylation not an informative biomarker • Minimal

  14. Environmental concentrations of the cocaine metabolite benzoylecgonine induced sublethal toxicity in the development of plants but not in a zebrafish embryo-larval model.

    PubMed

    García-Cambero, J P; García-Cortés, H; Valcárcel, Y; Catalá, M

    2015-12-30

    Several studies have found cocaine and its main active metabolite benzoylecgonine (BE) in the aquatic environment and drinking water, derived from its consumption by humans as well as the inability of water treatment processes to eliminate it. A few studies have already investigated the ecotoxicology of BE to aquatic invertebrates, but none has still addressed the effects of BE on aquatic vertebrates or vascular plants. The goal of this publication is to provide information on the toxicity of environmental concentrations of BE during animal and vascular plant development, in order to contribute to a better understanding of the potential risk of this substance for the environment. BE induced alterations in mitochondrial activity and DNA levels of fern spores at environmental concentrations (1 ng L(-1)), which could disrupt gametophyte germination. However, BE at concentrations ranging from 1 ng L(-1) to 1 mg L(-1) did not disturb morphogenesis, hatching, heartbeat rate or larval motility in a zebrafish embryo-larval model. Adverse effects on ferns agree with the allelophathic role described for alkaloids and their unspecific interference with plant germination. Therefore, the anthropogenic dispersion of alkaloid allelochemicals may pose a risk for biodiversity and irrigated food production that should be further investigated. Copyright © 2015 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2016-01-01

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

  16. 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

  17. 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 4 days 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Zebrafish Pronephros Development.

    PubMed

    Naylor, Richard W; Qubisi, Sarah S; Davidson, Alan J

    The pronephros is the first kidney type to form in vertebrate embryos. The first step of pronephrogenesis in the zebrafish is the formation of the intermediate mesoderm during gastrulation, which occurs in response to secreted morphogens such as BMPs and Nodals. Patterning of the intermediate mesoderm into proximal and distal cell fates is induced by retinoic acid signaling with downstream transcription factors including wt1a, pax2a, pax8, hnf1b, sim1a, mecom, and irx3b. In the anterior intermediate mesoderm, progenitors of the glomerular blood filter migrate and fuse at the midline and recruit a blood supply. More posteriorly localized tubule progenitors undergo epithelialization and fuse with the cloaca. The Notch signaling pathway regulates the formation of multi-ciliated cells in the tubules and these cells help propel the filtrate to the cloaca. The lumenal sheer stress caused by flow down the tubule activates anterior collective migration of the proximal tubules and induces stretching and proliferation of the more distal segments. Ultimately these processes create a simple two-nephron kidney that is capable of reabsorbing and secreting solutes and expelling excess water-processes that are critical to the homeostasis of the body fluids. The zebrafish pronephric kidney provides a simple, yet powerful, model system to better understand the conserved molecular and cellular progresses that drive nephron formation, structure, and function.

  19. Kupffer's vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut.

    PubMed

    Essner, Jeffrey J; Amack, Jeffrey D; Nyholm, Molly K; Harris, Erin B; Yost, H Joseph

    2005-03-01

    Monocilia have been proposed to establish the left-right (LR) body axis in vertebrate embryos by creating a directional fluid flow that triggers asymmetric gene expression. In zebrafish, dorsal forerunner cells (DFCs) express a conserved ciliary dynein gene (left-right dynein-related1, lrdr1) and form a ciliated epithelium inside a fluid-filled organ called Kupffer's vesicle (KV). Here, videomicroscopy demonstrates that cilia inside KV are motile and create a directional fluid flow just prior to the onset of asymmetric gene expression in lateral cells. Laser ablation of DFCs and surgical disruption of KV provide direct evidence that ciliated KV cells are required during early somitogenesis for subsequent LR patterning in the brain, heart and gut. Antisense morpholinos against lrdr1 disrupt KV fluid flow and perturb LR development. Furthermore, lrdr1 morpholinos targeted to DFC/KV cells demonstrate that Lrdr1 functions in these ciliated cells to control LR patterning. This provides the first direct evidence, in any vertebrate, that impairing cilia function in derivatives of the dorsal organizer, and not in other cells that express ciliogenic genes, alters LR development. Finally, genetic analysis reveals novel roles for the T-box transcription factor no tail and the Nodal signaling pathway as upstream regulators of lrdr1 expression and KV morphogenesis. We propose that KV is a transient embryonic 'organ of asymmetry' that directs LR development by establishing a directional fluid flow. These results suggest that cilia are an essential component of a conserved mechanism that controls the transition from bilateral symmetry to LR asymmetry in vertebrates.

  20. In vivo imaging and quantitative analysis of changes in axon length using transgenic zebrafish embryos.

    PubMed

    Kanungo, Jyotshnabala; Lantz, Susan; Paule, Merle G

    2011-01-01

    We describe an imaging procedure to measure axon length in zebrafish embryos in vivo. Automated fluorescent image acquisition was performed with the ImageXpress Micro high content screening reader and further analysis of axon lengths was performed on archived images using AcuityXpress software. We utilized the Neurite Outgrowth Application module with a customized protocol (journal) to measure the axons. Since higher doses of ethanol (2-2.5%, v/v) have been shown to deform motor neurons and axons during development, here we used ethanol to treat transgenic [hb9:GFP (green fluorescent protein)] zebrafish embryos at 28 hpf (hours post-fertilization). These embryos express GFP in the motor neurons and their axons. Embryos after ethanol treatment were arrayed in 384-well plates for automated fluorescent image acquisition in vivo. Average axon lengths of high dose ethanol-treated embryos were significantly lower than the control. Another experiment showed that there was no significant difference in the axon lengths between the embryos grown for 24h at 22°C and 28.5°C. These test experiments demonstrate that using axon development as an end-point, compound screening can be performed in a time-efficient manner. Published by Elsevier Inc.

  1. Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

    PubMed Central

    Bernut, Audrey; Dupont, Christian; Sahuquet, Alain; Herrmann, Jean-Louis; Lutfalla, Georges; Kremer, Laurent

    2015-01-01

    Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions. The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model. In this respect, the present manuscript describes the use of zebrafish as an in vivo model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate. PMID:26382225

  2. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Chitosan nanoparticles and their Tween 80 modified counterparts disrupt the developmental profile of zebrafish embryos.

    PubMed

    Yuan, Zhongyue; Li, Ying; Hu, Yulan; You, Jian; Higashisaka, Kazuma; Nagano, Kazuya; Tsutsumi, Yasuo; Gao, Jianqing

    2016-12-30

    Chitosan nanoparticles (CS-NPs) and their Tween 80 modified counterparts (TmCS-NPs) are among the most commonly used brain-targeted vehicles. However, their potential developmental toxicity is poorly understood. In this study, zebrafish embryos are introduced as an in vivo platform. Both NPs showed a dose-dependent increase in developmental toxicity (decreased hatching rate, increased mortality and incidences of malformation). Neurobehavioral changes included decreased spontaneous movement in TmCS-NP treated embryos and hyperactive effect in CS-NP treated larvae. Both NPs remarkably inhibited axonal development of primary and secondary motor neurons, and affected the muscle structure. Overall, this study demonstrated that CS-NPs and TmCS-NPs could affect embryonic development, disrupt neurobehavior of zebrafish larvae and affect muscle and neuron development, suggesting more attention on biodegradable chitosan nanoparticles. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2013-01-01

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

  5. Toxic Effects of Silica Nanoparticles on Zebrafish Embryos and Larvae

    PubMed Central

    Shi, Huiqin; Tian, Linwei; Guo, Caixia; Huang, Peili; Zhou, Xianqing; Peng, Shuangqing; Sun, Zhiwei

    2013-01-01

    Silica nanoparticles (SiNPs) have been widely used in biomedical and biotechnological applications. Environmental exposure to nanomaterials is inevitable as they become part of our daily life. Therefore, it is necessary to investigate the possible toxic effects of SiNPs exposure. In this study, zebrafish embryos were treated with SiNPs (25, 50, 100, 200 µg/mL) during 4–96 hours post fertilization (hpf). Mortality, hatching rate, malformation and whole-embryo cellular death were detected. We also measured the larval behavior to analyze whether SiNPs had adverse effects on larvae locomotor activity. The results showed that as the exposure dosages increasing, the hatching rate of zebrafish embryos was decreased while the mortality and cell death were increased. Exposure to SiNPs caused embryonic malformations, including pericardial edema, yolk sac edema, tail and head malformation. The larval behavior testing showed that the total swimming distance was decreased in a dose-dependent manner. The lower dose (25 and 50 µg/mL SiNPs) produced substantial hyperactivity while the higher doses (100 and 200 µg/mL SiNPs) elicited remarkably hypoactivity in dark periods. In summary, our data indicated that SiNPs caused embryonic developmental toxicity, resulted in persistent effects on larval behavior. PMID:24058598

  6. Transcriptome analysis of zebrafish embryos exposed to deltamethrin.

    PubMed

    Chueh, Tsung-Cheng; Hsu, Li-Sung; Kao, Chin-Ming; Hsu, Tung-Wei; Liao, Hung-Yu; Wang, Kuan-Yi; Chen, Ssu Ching

    2017-05-01

    Deltamethrin (DTM), a type II pyrethroid, is one of the most commonly used insecticides. The increased use of pyrethroid leads to potential adverse effects, particularly in sensitive populations such as children and pregnant women. None of the related studies was focused on the transcriptome responses in zebrafish embryos after treatment with DTM; therefore, RNA-seq, a high-throughput method, was performed to analyze the global expression of differential expressed genes (DEGs) in zebrafish embryos treated with DTM (40 and 80 μg/L) from fertilization to 48 h postfertilization (hpf) as compared with that in the control group (without DTM treatment). Two cDNA libraries were generated from treated embryos and one cDNA library from nontreated embryos, respectively. Over 92% of reads mapped to the reference in these three libraries. It was observed that many differential genes were expressed in comparison with embryos before and after DTM. The 20 most differentially expressed upregulated or downregulated genes were majorly involved in the signaling transduction. Validation of selected nine genes expression using qRT-PCR confirmed RNA-seq results. The transcriptome sequences were further subjected to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, showing G-protein-coupled receptor signaling pathway and neuroactive ligand-receptor interaction, respectively, were most enriched. The data from this study contributed to a better understanding of the potential consequences of fish exposed to DTM, to an evaluation of the potential threat of DTM to fish populations in aquatic environments. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1548-1557, 2017. © 2016 Wiley Periodicals, Inc.

  7. Developmental neurotoxicity of pyrethroid insecticides in zebrafish embryos.

    PubMed

    DeMicco, Amy; Cooper, Keith R; Richardson, Jason R; White, Lori A

    2010-01-01

    Pyrethroid insecticides are one of the most commonly used residential and agricultural insecticides. Based on the increased use of pyrethroids and recent studies showing that pregnant women and children are exposed to pyrethroids, there are concerns over the potential for developmental neurotoxicity. However, there have been relatively few studies on the developmental neurotoxicity of pyrethroids. In this study, we sought to investigate the developmental toxicity of six common pyrethroids, three type I compounds (permethrin, resmethrin, and bifenthrin) and three type II compounds (deltamethrin, cypermethrin, and lambda-cyhalothrin), and to determine whether zebrafish embryos may be an appropriate model for studying the developmental neurotoxicity of pyrethroids. Exposure of zebrafish embryos to pyrethroids caused a dose-dependent increase in mortality and pericardial edema, with type II compounds being the most potent. At doses approaching the LC(50), permethrin and deltamethrin caused craniofacial abnormalities. These findings are consistent with mammalian studies demonstrating that pyrethroids are mildly teratogenic at very high doses. However, at lower doses, body axis curvature and spasms were observed, which were reminiscent of the classic syndromes observed with pyrethroid toxicity. Treatment with diazepam ameliorated the spasms, while treatment with the sodium channel antagonist MS-222 ameliorated both spasms and body curvature, suggesting that pyrethroid-induced neurotoxicity is similar in zebrafish and mammals. Taken in concert, these data suggest that zebrafish may be an appropriate alternative model to study the mechanism(s) responsible for the developmental neurotoxicity of pyrethroid insecticides and aid in identification of compounds that should be further tested in mammalian systems.

  8. Developmental Neurotoxicity of Pyrethroid Insecticides in Zebrafish Embryos

    PubMed Central

    DeMicco, Amy; Cooper, Keith R.; Richardson, Jason R.; White, Lori A.

    2010-01-01

    Pyrethroid insecticides are one of the most commonly used residential and agricultural insecticides. Based on the increased use of pyrethroids and recent studies showing that pregnant women and children are exposed to pyrethroids, there are concerns over the potential for developmental neurotoxicity. However, there have been relatively few studies on the developmental neurotoxicity of pyrethroids. In this study, we sought to investigate the developmental toxicity of six common pyrethroids, three type I compounds (permethrin, resmethrin, and bifenthrin) and three type II compounds (deltamethrin, cypermethrin, and λ-cyhalothrin), and to determine whether zebrafish embryos may be an appropriate model for studying the developmental neurotoxicity of pyrethroids. Exposure of zebrafish embryos to pyrethroids caused a dose-dependent increase in mortality and pericardial edema, with type II compounds being the most potent. At doses approaching the LC50, permethrin and deltamethrin caused craniofacial abnormalities. These findings are consistent with mammalian studies demonstrating that pyrethroids are mildly teratogenic at very high doses. However, at lower doses, body axis curvature and spasms were observed, which were reminiscent of the classic syndromes observed with pyrethroid toxicity. Treatment with diazepam ameliorated the spasms, while treatment with the sodium channel antagonist MS-222 ameliorated both spasms and body curvature, suggesting that pyrethroid-induced neurotoxicity is similar in zebrafish and mammals. Taken in concert, these data suggest that zebrafish may be an appropriate alternative model to study the mechanism(s) responsible for the developmental neurotoxicity of pyrethroid insecticides and aid in identification of compounds that should be further tested in mammalian systems. PMID:19861644

  9. [10]-Gingerdiols as the major metabolites of [10]-gingerol in zebrafish embryos and in humans and their hematopoietic effects in zebrafish embryos

    PubMed Central

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

    2013-01-01

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

  10. Developmental toxicity of 2,4-dichlorophenoxyacetic acid in zebrafish embryos.

    PubMed

    Li, Kang; Wu, Jia-Qi; Jiang, Ling-Ling; Shen, Li-Zhen; Li, Jian-Ying; He, Zhi-Heng; Wei, Ping; Lv, Zhuo; He, Ming-Fang

    2017-03-01

    2,4-Dichlorophenoxyacetic acid (2,4-D) is widely used in agriculture as herbicide/pesticide, plant growth regulator and fruit preservative agent. It progressively accumulates in the environment including surface water, air and soil. It could be detected in human food and urine, which poses great risk to the living organisms. In the present study, we investigated the developmental toxicity of 2,4-D on zebrafish (Danio rerio) embryo. 2,4-D exposure significantly decreased both the survival rate (LC 50  = 46.71 mg/L) and hatching rate (IC 50  = 46.26 mg/L) of zebrafish embryos. The most common developmental defect in 2,4-D treated embryos was pericardial edema. 2,4-D (25 mg/L) upregulated marker genes of cardiac development (vmhc, amhc, hand2, vegf, and gata1) and downregulated marker genes of oxidative stress (cat and gpx1a). Whole mount in situ hybridization confirmed the vmhc and amhc upregulation by 2,4-D treatment. LC/MS/MS showed that the bioaccumulation of 2,4-D in zebrafish embryos were increased in a time-dependent manner after 25 mg/L of 2,4-D treatment. Taken together, our study investigated the toxic effects of 2,4-D on zebrafish embryonic development and its potential molecular mechanisms, gave evidence for the full understanding of 2,4-D toxicity on living organisms and shed light on its environmental impact. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Fishing on chips: up-and-coming technological advances in analysis of zebrafish and Xenopus embryos.

    PubMed

    Zhu, Feng; Skommer, Joanna; Huang, Yushi; Akagi, Jin; Adams, Dany; Levin, Michael; Hall, Chris J; Crosier, Philip S; Wlodkowic, Donald

    2014-11-01

    Biotests performed on small vertebrate model organisms provide significant investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, or tissue samples. The main advantage offered by whole-organism approaches is that the effects under study occur in the context of intact physiological milieu, with all its intercellular and multisystem interactions. The gap between the high-throughput cell-based in vitro assays and low-throughput, disproportionally expensive and ethically controversial mammal in vivo tests can be closed by small model organisms such as zebrafish or Xenopus. The optical transparency of their tissues, the ease of genetic manipulation and straightforward husbandry, explain the growing popularity of these model organisms. Nevertheless, despite the potential for miniaturization, automation and subsequent increase in throughput of experimental setups, the manipulation, dispensing and analysis of living fish and frog embryos remain labor-intensive. Recently, a new generation of miniaturized chip-based devices have been developed for zebrafish and Xenopus embryo on-chip culture and experimentation. In this work, we review the critical developments in the field of Lab-on-a-Chip devices designed to alleviate the limits of traditional platforms for studies on zebrafish and clawed frog embryo and larvae. © 2014 International Society for Advancement of Cytometry. © 2014 International Society for Advancement of Cytometry.

  12. Toxicity of porcelain-fused-to-metal substrate to zebrafish (Danio rerio) embryos and larvae.

    PubMed

    Zhao, Libo; Si, Jing; Wei, Yuan; Li, Sirui; Jiang, Yanjiao; Zhou, Rong; Liu, Bin; Zhang, Hong

    2018-06-15

    Porcelain-fused-to-metal (PFM) crowns are a standard restoration technique in dentistry, but toxicity of PFM in vivo has not been systematically evaluated. The present study evaluated the effects of various metal alloy shells of PFM crowns on the development of zebrafish embryos and larvae in order to determine the safety of these materials. Gold palladium (Au-Pd), silver palladium (Ag-Pd), Nickel chromium (Ni-Cr), cobalt chromium (Co-Cr), titanium (Ti) alloy porcelain crowns were immersed in artificial saliva for 1, 4, and 7 weeks, and the leach solution was collected and used to treat zebrafish embryos at 4-144 h PFM. Toxicity was assessed based on mortality, spontaneous movement, heart rate, hatchability, malformation, and swimming behavior. The 1-week leachates of the five PFMs were not toxic to zebrafish. The rates of mortality and malformation of zebrafish in the Ni-Cr alloy group were increased whereas spontaneous movement, heart rate, and swimming behavior were decreased for 4- and 7-week leachates. Among metal substrates commonly used in dental work, Ni-Cr alloy was most toxic, followed by Co-Cr and Ag-Pd alloys. Ti and Au-Pd alloys showed good biocompatibility and are therefore the most suitable materials for clinical applications. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. Oxygen decline in biotesting of environmental samples--is there a need for consideration in the acute zebrafish embryo assay?

    PubMed

    Küster, Eberhard; Altenburger, Rolf

    2008-12-01

    Environmental samples such as groundwater, sediment pore water, native or freeze dried sediments may be difficult to analyze for toxic effects with organismic aquatic bioassays. These samples might evoke low oxygen concentration or oxygen depletion during the test. The toxicity assessment could thus be confounded by low oxygen concentrations. The acute zebrafish embryo assay was used to analyze the influence of oxygen deficit on the embryonic development in the first 48 h post fertilization. Embryos were exposed to varying oxygen concentrations ranging from <30 to >80% oxygen saturation of water. A clear concentration dependent retardation of fish embryo development was observed. Because of a retarded development toxic thresholds of environmental samples which might include substances slowing down the development will be altered. For the purpose of identification of critical contaminants in complex environmental samples, it is proposed to actively aerate environmental samples which are likely to be oxygen depleted during the duration of the zebrafish embryo bioassay. 2008 Wiley Periodicals, Inc.

  14. Zebrafish pancreas development.

    PubMed

    Tiso, Natascia; Moro, Enrico; Argenton, Francesco

    2009-11-27

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

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

    PubMed

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

    2016-12-28

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

  16. Flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization.

    PubMed

    Cheng, Christina N; Li, Yue; Marra, Amanda N; Verdun, Valerie; Wingert, Rebecca A

    2014-07-17

    The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

  17. Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

    PubMed Central

    Cheng, Christina N.; Li, Yue; Marra, Amanda N.; Verdun, Valerie; Wingert, Rebecca A.

    2014-01-01

    The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples. PMID:25078510

  18. Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos.

    PubMed

    Joseph, Shai R; Pálfy, Máté; Hilbert, Lennart; Kumar, Mukesh; Karschau, Jens; Zaburdaev, Vasily; Shevchenko, Andrej; Vastenhouw, Nadine L

    2017-04-20

    Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA-bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo.

  19. Screening of Toxic Effects of Bisphenol A and Products of Its Degradation: Zebrafish (Danio rerio) Embryo Test and Molecular Docking.

    PubMed

    Makarova, Katerina; Siudem, Pawel; Zawada, Katarzyna; Kurkowiak, Justyna

    2016-10-01

    Bisphenol A (BPA) acts as an endocrine-disrupting compound even at a low concentration. Degradation of BPA could lead to the formation of toxic products. In this study, we compare the toxicity of BPA and seven intermediate products of its degradation. The accuracy of three molecular docking programs (Surflex, Autodock, and Autodock Vina) in predicting the binding affinities of selected compounds to human (ERα, ERβ, and ERRγ) and zebrafish (ERα, ERRγA, and ERRγB) estrogen and estrogen-related receptors was evaluated. The docking experiments showed that 4-isopropylphenol could have similar toxicity to that of BPA due to its high affinity to ERRγ and ERRγB and high octanol-water partitioning coefficient. The least toxic compounds were hydroquinone and phenol. Those compounds as well as BPA were screened in the zebrafish (Danio rerio) embryo test. 4-isopropylphenol had the strongest toxic effect on zebrafish embryos and caused 100% lethality shortly after exposure. BPA caused the delay in development, multiple deformations, and low heartbeats (30 bps), whereas hydroquinone had no impact on the development of the zebrafish embryo. Thus, the results of zebrafish screening are in good agreement with our docking experiment. The molecular docking could be used to screen the toxicity of other xenoestrogens and their products of degradation.

  20. Acute toxicity of 353-nonylphenol and its metabolites for zebrafish embryos.

    PubMed

    Kammann, Ulrike; Vobach, Michael; Wosniok, Werner; Schäffer, Andreas; Telscher, Andreas

    2009-03-01

    Nonylphenol (NP) can be detected in the aquatic environment all over the world. It is applied as a technical mixture of isomers of which 353-NP is the most relevant both in terms of abundance (about 20% of total mass) and endocrine potential. 353-NP is metabolised in sewage sludge. The aims of the present study were to determine and to compare the acute toxicity of t-NP, 353-NP and its metabolites as well as to discuss if the toxicity of 353-NP changes during degradation. 353-NP and two of its metabolites were synthesised. The zebrafish embryo test was performed according to standard protocols. Several lethal and non-lethal endpoints during embryonal development were reported. NOEL, LOEL and EC50 were calculated. All tested compounds caused lethal as well as non-lethal malformations during embryo development. 353-NP showed a higher toxicity (EC50 for lethal endpoints 6.7 mg/L) compared to its metabolites 4-(3.5-dimethyl-3-heptyl)-2-nitrophenol (EC50 13.3 mg/L) and 4-(3,5-dimethyl-3-heptyl)-2-bromophenol (EC50 27.1 mg/L). In surface water, concentrations of NP are far below the NOEC identified by the zebrafish embryo test. However, in soils and sewage sludge, concentrations may reach or even exceed these concentrations. Therefore, sludge-treated sites close to surface waters should be analysed for NP and its metabolites in order to detect an unduly high contamination due to runoff events. The results of the present study point out that the toxicity of 353-NP probably declines during metabolisation in water, sediment and soil, but does not vanish since the major metabolites exhibit a clear toxic potential for zebrafish embryos. Metabolites of environmental pollutants should be included in the ecotoxicological test strategy for a proper risk assessment.

  1. Toxicity of multi-walled carbon nanotubes, graphene oxide, and reduced graphene oxide to zebrafish embryos.

    PubMed

    Liu, Xiao Tong; Mu, Xi Yan; Wu, Xiao Li; Meng, Li Xuan; Guan, Wen Bi; Ma, Yong Qiang; Sun, Hua; Wang, Cheng Ju; Li, Xue Feng

    2014-09-01

    This study was aimed to investigate the toxic effects of 3 nanomaterials, i.e. multi-walled carbon nanotubes (MWCNTs), graphene oxide (GO), and reduced graphene oxide (RGO), on zebrafish embryos. The 2-h post-fertilization (hpf) zebrafish embryos were exposed to MWCNTs, GO, and RGO at different concentrations (1, 5, 10, 50, 100 mg/L) for 96 h. Afterwards, the effects of the 3 nanomateria on spontaneous movement, heart rate, hatching rate, length of larvae, mortality, and malformations ls were evaluated. Statistical analysis indicated that RGO significantly inhibited the hatching of zebrafish embryos. Furthermore, RGO and MWCNTs decreased the length of the hatched larvae at 96 hpf. No obvious morphological malformation or mortality was observed in the zebrafish embryos after exposure to the three nanomaterials. MWCNTs, GO, and RGO were all toxic to zebrafish embryos to influence embryos hatching and larvae length. Although no obvious morphological malformation and mortality were observed in exposed zebrafish embryos, further studies on the toxicity of the three nanomaterials are still needed. Copyright © 2014 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  2. In vivo assessment of impact of titanium oxide nanoparticle on zebrafish embryo

    NASA Astrophysics Data System (ADS)

    Verma, Suresh K.; Mishra, Anurag K.; Suar, M.; Parashar, S. K. S.

    2017-05-01

    Technologies and innovations have attended a new height with recent development in nanotechnology in last few decades. With these developments there has a great raise in demand of metal oxides like TiO2, ZnO having versatile physical, chemical and biological application. However the great rise has raised concern over the effect of these nanoparticles in biological system. In this study, we have assessed the impact of titanium oxide nanoparticles synthesized by high energy ball milling (HEBM) by milling bulk TiO2 particles for 15h. The synthesized particles were characterized with XRD, UV-Visible spectroscopy and DLS for their physiochemical properties. Biological impact of these nanoparticles was then studied on zebrafish embryo as invivo model. Mortality and hatching rate were calculated for 48hpf and 96hpf treatment. To determine the mechanism of mortality effect, Reactive oxygen species (ROS) was determined with the help of flow cytometry. 15h nanoparticles were found to have a LC50 of ( ) for zebrafish embryo. However TiO2 nanoparticles were found to be a ROS scavenger for the treated Zebrafish cells.

  3. 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

  4. Perturbation of metabonome of embryo/larvae zebrafish after exposure to fipronil.

    PubMed

    Yan, Lu; Gong, Chenxue; Zhang, Xiaofeng; Zhang, Quan; Zhao, Meirong; Wang, Cui

    2016-12-01

    The escalating demand for fipronil by the increasing insects' resistance to synthetic pyrethroids placed a burden on aquatic vertebrates. Although awareness regarding the toxicity of fipronil to fish is arising, the integral alteration caused by fipronil remains unexplored. Here, we investigated on the development toxicity of fipronil and the metabolic physiology perturbation at 120h post fertilization through GC-MS metabolomics on zebrafish embryo. We observed that fipronil dose-dependently induced malformations including uninflated swim bladder and bent spine. Further, the "omic" technique hit 26 differential metabolites after exposure to fipronil and five significant signaling pathways. We speculated that changes in primary bile acid synthesis pathway and the content of saturated fatty acid in the chemical-related group indicated the liver toxicity. Pathway of Aminoacyl-tRNA biosynthesis changed by fipronil may relate to the macromolecular synthesis. Concurrently, methane metabolism pathway was also identified while the role in zebrafish needs further determination. Overall, this study revealed several new signaling pathways in fipronil-treated zebrafish embryo/larval. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. Imaging Subcellular Structures in the Living Zebrafish Embryo.

    PubMed

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

    2016-04-02

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

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

    SciT

    Shi Xiongjie; Graduate School of the Chinese Academy of Sciences, Beijing 100039; Du Yongbing

    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 tomore » 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

  7. AHR2 morpholino knockdown reduces the toxicity of total particulate matter to zebrafish embryos

    SciT

    Massarsky, Andrey, E-mail: andrey.massarsky@duke.e

    The zebrafish embryo has been proposed as a ‘bridge model’ to study the effects of cigarette smoke on early development. Previous studies showed that exposure to total particulate matter (TPM) led to adverse effects in developing zebrafish, and suggested that the antioxidant and aryl hydrocarbon receptor (AHR) pathways play important roles. This study investigated the roles of these two pathways in mediating TPM toxicity. The study consisted of four experiments. In experiment I, zebrafish embryos were exposed from 6 h post fertilization (hpf) until 96 hpf to TPM{sub 0.5} and TPM{sub 1.0} (corresponding to 0.5 and 1.0 μg/mL equi-nicotine units)more » in the presence or absence of an antioxidant (N-acetyl cysteine/NAC) or a pro-oxidant (buthionine sulfoximine/BSO). In experiment II, TPM exposures were performed in embryos that were microinjected with nuclear factor erythroid 2-related factor 2 (Nrf2), AHR2, cytochrome P450 1A (CYP1A), or CYP1B1 morpholinos, and deformities were assessed. In experiment III, embryos were exposed to TPM, and embryos/larvae were collected at 24, 48, 72, and 96 hpf to assess several genes associated with the antioxidant and AHR pathways. Lastly, experiment IV assessed the activity and protein levels of CYP1A and CYP1B1 after exposure to TPM. We demonstrate that the incidence of TPM-induced deformities was generally not affected by NAC/BSO treatments or Nrf2 knockdown. In contrast, AHR2 knockdown reduced, while CYP1A or CYP1B1 knockdowns elevated the incidence of some deformities. Moreover, as shown by gene expression the AHR pathway, but not the antioxidant pathway, was induced in response to TPM exposure, providing further evidence for its importance in mediating TPM toxicity. - Highlights: • Total particulate matter (TPM) is the particulate phase of cigarette smoke. • Zebrafish is proposed as a ‘bridge model’ to study the effects of TPM. • We investigate the roles of antioxidant and aryl hydrocarbon receptor (AHR

  8. Retinoic Acid Signaling Is Essential for Valvulogenesis by Affecting Endocardial Cushions Formation in Zebrafish Embryos.

    PubMed

    Li, Junbo; Yue, Yunyun; Zhao, Qingshun

    2016-02-01

    Retinoic acid (RA) plays important roles in many stages of heart morphogenesis. Zebrafish embryos treated with exogenous RA display defective atrio-ventricular canal (AVC) specification. However, whether endogenous RA signaling takes part in cardiac valve formation remains unknown. Herein, we investigated the role of RA signaling in cardiac valve development by knocking down aldh1a2, the gene encoding an enzyme that is mainly responsible for RA synthesis during early development, in zebrafish embryos. The results showed that partially knocking down aldh1a2 caused defective formation of primitive cardiac valve leaflets at 108 hpf (hour post-fertilization). Inhibiting endogenous RA signaling by 4-diethylaminobenzal-dehyde revealed that 16-26 hpf was a key time window when RA signaling affects the valvulogenesis. The aldh1a2 morphants had defective formation of endocardial cushion (EC) at 76 hpf though they had almost normal hemodynamics and cardiac chamber specification at early development. Examining the expression patterns of AVC marker genes including bmp4, bmp2b, nppa, notch1b, and has2, we found the morphants displayed abnormal development of endocardial AVC but almost normal development of myocardial AVC at 50 hpf. Being consistent with the reduced expression of notch1b in endocardial AVC, the VE-cadherin gene cdh5, the downstream gene of Notch signaling, was ectopically expressed in AVC of aldh1a2 morphants at 50 hpf, and overexpression of cdh5 greatly affected the formation of EC in the embryos at 76 hpf. Taken together, our results suggest that RA signaling plays essential roles in zebrafish cardiac valvulogenesis.

  9. Focal gene misexpression in zebrafish embryos induced by local heat shock using a modified soldering iron.

    PubMed

    Hardy, Melissa E; Ross, Louis V; Chien, Chi-Bin

    2007-11-01

    Misexpression of genes in a temporally and spatially controlled fashion is an important tool for assessing gene function during development. Because few tissue-specific promoters have been identified in zebrafish, inducible systems such as the Cre/LoxP and Tet repressor systems are of limited utility. Here we describe a new method of misexpression: local heat shock using a modified soldering iron. Zebrafish carrying transgenes under the control of a heat shock promoter (hsp70) are focally heated with the soldering iron to induce gene expression in a small area of the embryo. We have validated this method in three stable transgenic lines and at three developmental timepoints. Local heat shock is a fast, easy, and inexpensive method for gene misexpression. Copyright 2007 Wiley-Liss, Inc.

  10. Toxicity of Vascular Disrupting Chemicals to Developing Zebrafish

    EPA Science Inventory

    Vascular development is integral to proper embryonic development and disruption of that process can have serious developmental consequences. We performed static 48-hr exposures of transgenic TG(kdr:EGFP)s843 zebrafish (Danio rerio) embryos with the known vascular inhibitors Vatal...

  11. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. 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.

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

    PubMed Central

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

    2015-01-01

    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

  14. 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.

  15. 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.

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

    PubMed

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

    2017-03-09

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

  17. Expression of voltage-activated calcium channels in the early zebrafish embryo.

    PubMed

    Sanhueza, Dayán; Montoya, Andro; Sierralta, Jimena; Kukuljan, Manuel

    2009-05-01

    Increases in cytosolic calcium concentrations regulate many cellular processes, including aspects of early development. Calcium release from intracellular stores and calcium entry through non-voltage-gated channels account for signalling in non-excitable cells, whereas voltage-gated calcium channels (CaV) are important in excitable cells. We report the expression of multiple transcripts of CaV, identified by its homology to other species, in the early embryo of the zebrafish, Danio rerio, at stages prior to the differentiation of excitable cells. CaV mRNAs and proteins were detected as early as the 2-cell stages, which indicate that they arise from both maternal and zygotic transcription. Exposure of embryos to pharmacological blockers of CaV does not perturb early development significantly, although late effects are appreciable. These results suggest that CaV may have a role in calcium homeostasis and control of cellular process during early embryonic development.

  18. 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.

  19. Zebrafish embryo toxicity of anaerobic biotransformation products from the insensitive munitions compound 2,4-dinitroanisole.

    PubMed

    Olivares, Christopher I; Sierra-Alvarez, Reyes; Abrell, Leif; Chorover, Jon; Simonich, Michael; Tanguay, Robert L; Field, Jim A

    2016-11-01

    2,4-Dinitroanisole (DNAN) is an emerging insensitive munitions compound that readily undergoes anaerobic nitro-group reduction to 2-methoxy-5-nitroaniline (MENA) and 2,4-diaminoanisole (DAAN), followed by formation of unique azo dimers. Currently there is little knowledge on the ecotoxicity of DNAN (bio)transformation products. In the present study, mortality, development, and behavioral effects of DNAN (bio)transformation products were assessed using zebrafish (Danio rerio) embryos. The authors tested individual products, MENA and DAAN, as well as dimer and trimer surrogates. As pure compounds, 3-nitro-4-methoxyaniline and 2,2'-dimethoxy-4,4'-azodianiline caused statistically significant effects, with lowest-observable-adverse effect levels (LOAEL) at 6.4 μM on 1 or 2 developmental endpoints, respectively. The latter had 6 additional statistically significant developmental endpoints with LOAELs of 64 μM. Based on light-to-dark swimming behavioral tests, DAAN (640 μM) caused reduction in swimming, suggestive of neurotoxicity. No statistically significant mortality occurred (≤64 μM) for any of the individual compounds. However, metabolite mixtures formed during different stages of MENA (bio)transformation in soil were characterized using high-resolution mass spectrometry in parallel with zebrafish embryo toxicity assays, which demonstrated statistically significant mortality during the onset of azo-dimer formation. Overall the results indicate that several DNAN (bio)transformation products cause different types of toxicity to zebrafish embryos. Environ Toxicol Chem 2016;35:2774-2781. © 2016 SETAC. © 2016 SETAC.

  20. R-spondin 3 regulates dorsoventral and anteroposterior patterning by antagonizing Wnt/β-catenin signaling in zebrafish embryos.

    PubMed

    Rong, Xiaozhi; Chen, Chen; Zhou, Pin; Zhou, Yumei; Li, Yun; Lu, Ling; Liu, Yunzhang; Zhou, Jianfeng; Duan, Cunming

    2014-01-01

    The Wnt/β-catenin or canonical Wnt signaling pathway plays fundamental roles in early development and in maintaining adult tissue homeostasis. R-spondin 3 (Rspo3) is a secreted protein that has been implicated in activating the Wnt/β-catenin signaling in amphibians and mammals. Here we report that zebrafish Rspo3 plays a negative role in regulating the zygotic Wnt/β-catenin signaling. Zebrafish Rspo3 has a unique domain structure. It contains a third furin-like (FU3) domain. This FU3 is present in other four ray-finned fish species studied but not in elephant shark. In zebrafish, rspo3 mRNA is maternally deposited and has a ubiquitous expression in early embryonic stages. After 12 hpf, its expression becomes tissue-specific. Forced expression of rspo3 promotes dorsoanterior patterning and increases the expression of dorsal and anterior marker genes. Knockdown of rspo3 increases ventral-posterior development and stimulates ventral and posterior marker genes expression. Forced expression of rspo3 abolishes exogenous Wnt3a action and reduces the endogenous Wnt signaling activity. Knockdown of rspo3 results in increased Wnt/β-catenin signaling activity. Further analyses indicate that Rspo3 does not promote maternal Wnt signaling. Human RSPO3 has similar action when tested in zebrafish embryos. These results suggest that Rspo3 regulates dorsoventral and anteroposterior patterning by negatively regulating the zygotic Wnt/β-catenin signaling in zebrafish embryos.

  1. R-Spondin 3 Regulates Dorsoventral and Anteroposterior Patterning by Antagonizing Wnt/β-Catenin Signaling in Zebrafish Embryos

    PubMed Central

    Zhou, Pin; Zhou, Yumei; Li, Yun; Lu, Ling; Liu, Yunzhang; Zhou, Jianfeng; Duan, Cunming

    2014-01-01

    The Wnt/β-catenin or canonical Wnt signaling pathway plays fundamental roles in early development and in maintaining adult tissue homeostasis. R-spondin 3 (Rspo3) is a secreted protein that has been implicated in activating the Wnt/β-catenin signaling in amphibians and mammals. Here we report that zebrafish Rspo3 plays a negative role in regulating the zygotic Wnt/β-catenin signaling. Zebrafish Rspo3 has a unique domain structure. It contains a third furin-like (FU3) domain. This FU3 is present in other four ray-finned fish species studied but not in elephant shark. In zebrafish, rspo3 mRNA is maternally deposited and has a ubiquitous expression in early embryonic stages. After 12 hpf, its expression becomes tissue-specific. Forced expression of rspo3 promotes dorsoanterior patterning and increases the expression of dorsal and anterior marker genes. Knockdown of rspo3 increases ventral-posterior development and stimulates ventral and posterior marker genes expression. Forced expression of rspo3 abolishes exogenous Wnt3a action and reduces the endogenous Wnt signaling activity. Knockdown of rspo3 results in increased Wnt/β-catenin signaling activity. Further analyses indicate that Rspo3 does not promote maternal Wnt signaling. Human RSPO3 has similar action when tested in zebrafish embryos. These results suggest that Rspo3 regulates dorsoventral and anteroposterior patterning by negatively regulating the zygotic Wnt/β-catenin signaling in zebrafish embryos. PMID:24918770

  2. Identification of Estrogen Target Genes during Zebrafish Embryonic Development through Transcriptomic Analysis

    EPA Science Inventory

    Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio) as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 μM 17β-estradiol (E2) or vehicle from 3 hours to 4 days post...

  3. Cornelia de Lange Syndrome: NIPBL haploinsufficiency downregulates canonical Wnt pathway in zebrafish embryos and patients fibroblasts.

    PubMed

    Pistocchi, A; Fazio, G; Cereda, A; Ferrari, L; Bettini, L R; Messina, G; Cotelli, F; Biondi, A; Selicorni, A; Massa, V

    2013-10-17

    Cornelia de Lange Syndrome is a severe genetic disorder characterized by malformations affecting multiple systems, with a common feature of severe mental retardation. Genetic variants within four genes (NIPBL (Nipped-B-like), SMC1A, SMC3, and HDAC8) are believed to be responsible for the majority of cases; all these genes encode proteins that are part of the 'cohesin complex'. Cohesins exhibit two temporally separated major roles in cells: one controlling the cell cycle and the other involved in regulating the gene expression. The present study focuses on the role of the zebrafish nipblb paralog during neural development, examining its expression in the central nervous system, and analyzing the consequences of nipblb loss of function. Neural development was impaired by the knockdown of nipblb in zebrafish. nipblb-loss-of-function embryos presented with increased apoptosis in the developing neural tissues, downregulation of canonical Wnt pathway genes, and subsequent decreased Cyclin D1 (Ccnd1) levels. Importantly, the same pattern of canonical WNT pathway and CCND1 downregulation was observed in NIPBL-mutated patient-specific fibroblasts. Finally, chemical activation of the pathway in nipblb-loss-of-function embryos rescued the adverse phenotype and restored the physiological levels of cell death.

  4. Embryotoxic and genotoxic effects of sewage effluents in zebrafish embryo using multiple endpoint testing.

    PubMed

    Babić, Sanja; Barišić, Josip; Višić, Hrvoje; Sauerborn Klobučar, Roberta; Topić Popović, Natalija; Strunjak-Perović, Ivančica; Čož-Rakovac, Rozelindra; Klobučar, Göran

    2017-05-15

    Wastewater treatment plant (WWTP) effluents are often complex mixtures of various organic and inorganic substances. Quality control of wastewaters and sludges has been regulated with measuring several physico-chemical parameters and sometimes using biological methods with non-specific responses, while synergistic action mechanisms of contaminants in such complex mixtures is still unknown. Toxic effects of wastewaters within and downstream of the WWTP in City of Virovitica, Croatia, were tested on zebrafish Danio rerio using a set of biomarkers that enabled an insight in wastewaters toxic potential on embryos at the cellular, tissue and the whole organism level during an early ontogenesis (24 and 48 hpf). Exposure of embryos to the wastewater samples from WWTP Virovitica increased mortality and abnormality rate. Heart rate, spontaneous movements and pigmentation formation were also markedly affected. Biochemical markers confirmed the presence of MXR inhibitors in all tested wastewater samples, indicating the increase of pollutant accumulation in the cell/organism. Also, a tendency of DNA damage decrease measured with Comet assay was evident in wastewater samples downstream from WWTP although control levels were not reached in any environmental sample. Histopathological analysis showed that exposure to tested samples resulted in impaired muscle organization, notochord malformation and retardation in eye and brain development at embryos 48 hpf. Furthermore, semi-quantitative histopathology assessment indicated increased percentage of embryo defects in river water sampled several kilometers downstream from the WWTP, confirming toxic potential of WWTP effluents. Extension of the zebrafish embryotoxicity test (ZET) with biochemical and histopathological biomarkers could serve as a guiding principle in biomonitoring of wastewater contamination. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    SciT

    Teraoka, Hiroki; Urakawa, Satsuki; Nanba, Satomi

    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 ormore » 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.« less

  6. Developmental toxicity, oxidative stress and immunotoxicity induced by three strobilurins (pyraclostrobin, trifloxystrobin and picoxystrobin) in zebrafish embryos.

    PubMed

    Li, Hui; Cao, Fangjie; Zhao, Feng; Yang, Yang; Teng, Miaomiao; Wang, Chengju; Qiu, Lihong

    2018-05-25

    Strobilurins is the most widely used class of fungicides, but is reported highly toxic to some aquatic organisms. In this study, zebrafish embryos were exposed to a range concentrations of three strobilurins (pyraclostrobin, trifloxystrobin and picoxystrobin) for 96 h post-fertilization (hpf) to assess their aquatic toxicity. The 96-h LC 50 values of pyraclostrobin, trifloxystrobin and picoxystrobin to embryos were 61, 55, 86 μg/L, respectively. A series of symptoms were observed in developmental embryos during acute exposure, including decreased heartbeat, hatching inhibition, growth regression, and morphological deformities. Moreover, the three fungicides induced oxidative stress in embryos through increasing reactive oxygen species (ROS) and malonaldehyde (MDA) contents, inhibiting superoxide dismutase (SOD) activity and glutathione (GSH) content as well as differently changing catalase (CAT) activity and mRNA levels of genes related to antioxidant system (Mn-sod, Cu/Zn-sod, Cat, Nrf2, Ucp2 and Bcl2). In addition, exposure to the three strobilurins resulted in significant upregulation of IFN and CC-chem as well as differently changed expressions of TNFa, IL-1b, C1C and IL-8, which related to the innate immune system, suggesting that these fungicides caused immunotoxicity during zebrafish embryo development. The different response of enzymes and genes in embryos exposed to the three fungicides might be the cause that leads to the difference of their toxicity. This work made a comparison of the toxicity of three strobilurins to zebrafish embryos on multi-levels and would provide a better understanding of the toxic effects of strobilurins on aquatic organisms. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. Evaluating the Zebrafish Embryo Toxicity Test for Pesticide Hazard Screening

    EPA Science Inventory

    Given the numerous chemicals used in society, it is critical to develop tools for accurate and efficient evaluation of potential risks to human and ecological receptors. Fish embryo acute toxicity tests are 1 tool that has been shown to be highly predictive of standard, more reso...

  8. Comparative effects of zinc oxide nanoparticles and dissolved zinc on zebrafish embryos and eleuthero-embryos: importance of zinc ions.

    PubMed

    Brun, Nadja Rebecca; Lenz, Markus; Wehrli, Bernhard; Fent, Karl

    2014-04-01

    The increasing use of zinc oxide nanoparticles (nZnO) and their associated environmental occurrence make it necessary to assess their potential effects on aquatic organisms. Upon water contact, nZnO dissolve partially to zinc (Zn(II)). To date it is not yet completely understood, whether effects of nZnO are solely or partly due to dissolved Zn(II). Here we compare potential effects of 0.2, 1 and 5mg/L nZnO and corresponding concentrations of released Zn(II) by water soluble ZnCl2 to two development stages of zebrafish, embryos and eleuthero-embryos, by analysing expressional changes by RT-qPCR. Another objective was to assess uptake and tissue distribution of Zn(II). Laser ablation-ICP-MS analysis demonstrated that uptake and tissue distribution of Zn(II) were identical for nZnO and ZnCl2 in eleuthero-embryos. Zn(II) was found particularly in the retina/pigment layer of eyes and brain. Both nZnO and dissolved Zn(II) derived from ZnCl2 had similar inhibiting effects on hatching, and they induced similar expressional changes of target genes. At 72hours post fertilization (hpf), both nZnO and Zn(II) delayed hatching at all doses, and inhibited hatching at 1 and 5 mg/L at 96 hpf. Both nZnO and Zn(II) lead to induction of metallothionein (mt2) in both embryos and eleuthero-embryos at all concentrations. Transcripts of oxidative stress related genes cat and Cu/Zn sod were also altered. Moreover, we show for the first time that nZnO exposure results in transcriptional changes of pro-inflammatory cytokines IL-1β and TNFα. Overall, transcriptional alterations were higher in embryos than eleuthero-embryos. The similarities of the effects lead to the conclusion that effects of nZnO are mainly related to the release of Zn(II). Copyright © 2014 Elsevier B.V. All rights reserved.

  9. 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

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

    PubMed

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

    2016-02-01

    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.

  11. Triphasic low-dose response in zebrafish embryos irradiated by microbeam protons.

    PubMed

    Choi, Viann Wing Yan; Yum, Emily Hoi Wa; Konishi, Teruaki; Oikawa, Masakazu; Cheng, Shuk Han; Yu, Kwan Ngok

    2012-01-01

    The microbeam irradiation system (Single-Particle Irradiation System to Cell, acronym as SPICE) at the National Institute of Radiological Sciences (NIRS), Japan, was employed to irradiate dechorionated zebrafish embryos at the 2-cell stage at 0.75 h post fertilization (hpf) by microbeam protons. Either one or both of the cells of the embryos were irradiated with 10, 20, 40, 50, 80, 100, 160, 200, 300 and 2000 protons each with an energy of 3.37 MeV. The embryos were then returned back to the incubator until 24 hpf for analyses. The levels of apoptosis in zebrafish embryos at 25 hpf were quantified through terminal dUTP transferase-mediated nick end-labeling (TUNEL) assay, with the apoptotic signals captured by a confocal microscope. The results revealed a triphasic dose-response for zebrafish embryos with both cells irradiated at the 2-cell stage, namely, (1) increase in apoptotic signals for < 200 protons (< 30 mGy), (2) hormesis to reduce the apoptotic signals below the spontaneous number for 200-400 protons (at doses of 30-60 mGy), and (3) increase in apoptotic signals again for > 600 protons (at doses > 90 mGy). The dose response for zebrafish embryos with only one cell irradiated at the 2-cell stage was also likely a triphasic one, but the apoptotic signals in the first zone (< 200 protons or < 30 mGy) did not have significant differences from those of the background. At the same time, the experimental data were in line with induction of radiation-induced bystander effect as well as rescue effect in the zebrafish embryos, particular in those embryos with unirradiated cells.

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

    PubMed

    Rieger, Sandra; Wang, Fang; Sagasti, Alvaro

    2011-07-01

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

  13. The DVR-1 (Vg1) transcript of zebrafish is maternally supplied and distributed throughout the embryo.

    PubMed

    Helde, K A; Grunwald, D J

    1993-10-01

    It is not known how region- or tissue-specific differences are generated in the zebrafish embryo. To look at the potential role of maternal transcripts in generating cell diversity, we have isolated and characterized the zebrafish homologue of Xenopus DVR-1 (Vg1), a maternally supplied RNA that encodes a member of the transforming growth factor-beta superfamily. The zebrafish DVR-1 RNA is maternally supplied and its protein product shares a high degree of sequence identity with Xenopus DVR-1. These conserved features indicate that DVR-1 is likely to have an essential function in early embryogenesis. However, unlike the frog transcript, which is restricted to vegetal cells, DVR-1 RNA is distributed equally among all zebrafish blastomeres. We suggest that the ubiquitous distribution of DVR-1 RNA reflects a significant aspect of the developmental strategy of the zebrafish in which each blastomere retains an equivalent developmental potential throughout the cleavage period.

  14. Zebrafish Craniofacial Development: A Window into Early Patterning

    PubMed Central

    Mork, Lindsey; Crump, Gage

    2016-01-01

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

  15. Growth and recovery of zebrafish embryos after developmental exposure to raw and ozonated oil sands process-affected water.

    PubMed

    Lyons, Danielle D; Morrison, Christie; Philibert, Danielle A; Gamal El-Din, Mohamed; Tierney, Keith B

    2018-05-07

    Due to the increasing volume of oil sands process-affect water (OSPW) and its toxicity to aquatic organisms, it is important to fully understand its effects and study remediation processes that will enable its release to the environment. Ozone treatment is currently being considered as a tool to expedite remediation, as it is known to degrade toxic organic compounds present in OSPW. In this study, we aimed to measure the effects of OSPW exposure on the growth, development and recovery of zebrafish (Danio rerio) embryos. We also used ozone-treated OSPW to determine whether ozonation negated any effects of raw OSPW exposure. As biomarkers of exposure, we assessed the expression of genes involved in neurodevelopment (ngn1, neuroD), estrogenicity (vtg), oxidative stress (sod1), and biotransformation (cyp1a, cyp1b). Our study found that exposure to both raw and ozonated OSPW did not impair growth of zebrafish embryos, however, otoliths of exposed embryos were smaller than those of control embryos. The expression levels of both cyp1a and cyp1b were induced by raw OSPW exposure. However, after the exposure period, expression levels of these genes returned to control levels within two days of residence in clean water. We found no changes in the expression levels of ngn1, neuroD and vtg genes with exposure to treated or untreated OSPW. Overall, our study found that raw OSPW exposure did not have many negative effects on zebrafish embryos and embryos appeared to recover relatively quickly after exposure ended. Furthermore, ozone treatment decreased the induction of cyp1a and cyp1b. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. Assessment of the lethal and sublethal effects of 20 environmental chemicals in zebrafish embryos and larvae by using OECD TG 212.

    PubMed

    Horie, Yoshifumi; Yamagishi, Takahiro; Takahashi, Hiroko; Shintaku, Youko; Iguchi, Taisen; Tatarazako, Norihisa

    2017-10-01

    Fish embryo toxicity tests are used to assess the lethal and sublethal effects of environmental chemicals in aquatic organisms. Previously, we used a short-term toxicity test published by the Organization for Economic Co-operation and Development (test no. 212: Fish, Short-term Toxicity Test on Embryo and Sac-Fry Stages [OECD TG 212]) to assess the lethal and sublethal effects of aniline and several chlorinated anilines in zebrafish embryos and larvae. To expand upon this previous study, we used OECD TG 212 in zebrafish embryos and larvae to assess the lethal and sublethal effects of 20 additional environmental chemicals that included active pharmaceutical ingredients, pesticides, metals, aromatic compounds or chlorinated anilines. Zebrafish embryos (Danio rerio) were exposed to the test chemicals until 8 days post-fertilization. A delayed lethal effect was induced by 16 of the 20 test chemicals, and a positive correlation was found between heart rate turbulence and mortality. We also found that exposure to the test chemicals at concentrations lower than the lethal concentration induced the sublethal effects of edema, body curvature and absence of swim-bladder inflation. In conclusion, the environmental chemicals assessed in the present study induced both lethal and sublethal effects in zebrafish embryos and larvae, as assessed by using OECD TG 212. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  17. Development and origins of zebrafish ocular vasculature.

    PubMed

    Kaufman, Rivka; Weiss, Omri; Sebbagh, Meyrav; Ravid, Revital; Gibbs-Bar, Liron; Yaniv, Karina; Inbal, Adi

    2015-03-27

    The developing eye receives blood supply from two vascular systems, the intraocular hyaloid system and the superficial choroidal vessels. In zebrafish, a highly stereotypic and simple set of vessels develops on the surface of the eye prior to development of choroidal vessels. The origins and formation of this so-called superficial system have not been described. We have analyzed the development of superficial vessels by time-lapse imaging and identified their origins by photoconversion experiments in kdrl:Kaede transgenic embryos. We show that the entire superficial system is derived from a venous origin, and surprisingly, we find that the hyaloid system has, in addition to its previously described arterial origin, a venous origin for specific vessels. Despite arising solely from a vein, one of the vessels in the superficial system, the nasal radial vessel (NRV), appears to acquire an arterial identity while growing over the nasal aspect of the eye and this happens in a blood flow-independent manner. Our results provide a thorough analysis of the early development and origins of zebrafish ocular vessels and establish the superficial vasculature as a model for studying vascular patterning in the context of the developing eye.

  18. Effect of chilling and cryopreservation on expression of Pax genes in zebrafish (Danio rerio) embryos and blastomeres.

    PubMed

    Lin, C; Spikings, E; Zhang, T; Rawson, D M

    2009-08-01

    Cryopreservation is now common practice in the fields of aquaculture, conservation and biomedicine. However, there is a lack of information on the effect of chilling and cryopreservation at the molecular level. In the present study, we used real-time RT-PCR analysis to determine the effect of chilling and cryopreservation on expression of Pax2a, Pax2b, Pax5 and Pax8 which constitute one subgroup of the Pax gene family. As intact embryos of zebrafish have not yet been successfully cryopreserved, we have used two alternatives: chilling of intact embryos and cryopreservation of isolated blastomeres. Cryopreservation was found to affect the normal pattern of gene expression in zebrafish embryonic blastomeres. The trends, profile changes, in expression of Pax2a and Pax5 occurred to a lesser extent in frozen-thawed blastomeres than in fresh blastomeres whilst the opposite was true for Pax8. The trends in expression of Pax2b were delayed in frozen-thawed blastomeres compared to fresh blastomeres. Cryopreservation can therefore disrupt normal gene expression patterns in zebrafish embryonic blastomeres which could have a detrimental effect on embryo development.

  19. The threshold number of protons to induce an adaptive response in zebrafish embryos.

    PubMed

    Choi, V W Y; Konishi, Teruaki; Oikawa, Masakazu; Cheng, S H; Yu, K N

    2013-03-01

    In this study, microbeam protons were used to provide the priming dose to induce an in vivo radioadaptive response (RAR) in the embryos of zebrafish, Danio rerio, against subsequent challenging doses provided by x-ray photons. The microbeam irradiation system (Single-Particle Irradiation System to Cell, acronym SPICE) at the National Institute of Radiological Sciences (NIRS), Japan, was employed. The embryos were dechorionated at 4 h post fertilisation (hpf) and irradiated at 5 hpf by microbeam protons. For each embryo, one irradiation point was chosen, to which 5, 10, 20, 30, 40, 50, 100, 200, 300 and 500 protons each with an energy of 3.4 MeV were delivered. The embryos were returned to the incubator until 10 hpf to further receive the challenging exposure, which was achieved using 2 Gy of x-ray irradiation, and then again returned to the incubator until 24 hpf for analyses. The levels of apoptosis in zebrafish embryos at 25 hpf were quantified through terminal dUTP transferase-mediated nick end-labelling (TUNEL) assay. The results revealed that at least 200 protons (with average radiation doses of about 300 and 650 mGy absorbed by an irradiated epithelial and deep cell, respectively) would be required to induce RAR in the zebrafish embryos in vivo. Our previous investigation showed that 5 protons delivered at 10 points on an embryo would already be sufficient to induce RAR in the zebrafish embryos. The difference was explained in terms of the radiation-induced bystander effect as well as the rescue effect.

  20. Competition between histone and transcription factor binding regulates the onset of transcription in zebrafish embryos

    PubMed Central

    Joseph, Shai R; Pálfy, Máté; Hilbert, Lennart; Kumar, Mukesh; Karschau, Jens; Zaburdaev, Vasily; Shevchenko, Andrej; Vastenhouw, Nadine L

    2017-01-01

    Upon fertilization, the genome of animal embryos remains transcriptionally inactive until the maternal-to-zygotic transition. At this time, the embryo takes control of its development and transcription begins. How the onset of zygotic transcription is regulated remains unclear. Here, we show that a dynamic competition for DNA binding between nucleosome-forming histones and transcription factors regulates zebrafish genome activation. Taking a quantitative approach, we found that the concentration of non-DNA-bound core histones sets the time for the onset of transcription. The reduction in nuclear histone concentration that coincides with genome activation does not affect nucleosome density on DNA, but allows transcription factors to compete successfully for DNA binding. In agreement with this, transcription factor binding is sensitive to histone levels and the concentration of transcription factors also affects the time of transcription. Our results demonstrate that the relative levels of histones and transcription factors regulate the onset of transcription in the embryo. DOI: http://dx.doi.org/10.7554/eLife.23326.001 PMID:28425915

  1. Lethal and sublethal effects of aniline and chlorinated anilines on zebrafish embryos and larvae.

    PubMed

    Horie, Yoshifumi; Yamagishi, Takahiro; Koshio, Masaaki; Iguchi, Taisen; Tatarazako, Norihisa

    2017-07-01

    Environmental risk assessments show increased attention to the sublethal effects of chemicals on aquatic organisms. The Organization for Economic Cooperation and Development (OECD) established the "Fish, Short-term Toxicity Test on Embryo and Sac-fry Stages" (OECD test 212) to predict lethal effects. It is still unclear, however, whether this test can predict sublethal effects. Although their sublethal effects are still unknown, chlorinated anilines are widely used in various fields. The purpose of this study, therefore, is to investigate sublethal effects of chlorinated anilines using OECD test 212 with zebrafish, and to examine the correlation of several sublethal effects between embryo and larval stages. Embryos were exposed to aniline and nine chlorinated anilines until 8 days post-fertilization. A delayed lethal effect was observed from three of the 10 anilines tested. In the control group, the swim bladder inflated after hatching, but there was no swim-bladder inflation after exposure to the chlorinated anilines. Fertilized eggs exposed to lower concentrations of test chemicals showed effects during embryogenesis that did not affect mortality rates, such as changes in body curvature and edema. Our results show that chlorinated anilines induce not only lethal effects but also a variety of sublethal effects. Moreover, a detailed estimate of these effects requires study during both embryonic and larval stages. OECD test 212 may therefore prove useful as a method for screening chemicals for lethal and sublethal effects. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  2. 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.

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

    PubMed

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

    2018-05-07

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

  4. Combined toxicity of silica nanoparticles and methylmercury on cardiovascular system in zebrafish (Danio rerio) embryos.

    PubMed

    Duan, Junchao; Hu, Hejing; Li, Qiuling; Jiang, Lizhen; Zou, Yang; Wang, Yapei; Sun, Zhiwei

    2016-06-01

    This study was to investigate the combined toxicity of silica nanoparticles (SiNPs) and methylmercury (MeHg) on cardiovascular system in zebrafish (Danio rerio) embryos. Ultraviolet absorption analysis showed that the co-exposure system had high absorption and stability. The dosages used in this study were based on the NOAEL level. Zebrafish embryos exposed to the co-exposure of SiNPs and MeHg did not show any cardiovascular malformation or atrioventricular block, but had an inhibition effect on bradycardia. Using o-Dianisidine for erythrocyte staining, the cardiac output of zebrafish embryos was decreased gradually in SiNPs, MeHg, co-exposure groups, respectively. Co-exposure of SiNPs and MeHg enhanced the vascular endothelial damage in Tg(fli-1:EGFP) transgenic zebrafish line. Moreover, the co-exposure significantly activated the oxidative stress and inflammatory response in neutrophils-specific Tg(mpo:GFP) transgenic zebrafish line. This study suggested that the combined toxic effects of SiNPs and MeHg on cardiovascular system had more severe toxicity than the single exposure alone. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. 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.

  6. 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

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

    PubMed Central

    Amack, Jeffrey D.; Whipps, Christopher M.

    2016-01-01

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

  8. A reduced transcriptome approach to assess environmental toxicants using zebrafish embryo tests

    EPA Science Inventory

    This paper reports on the pilot testing of a new bioassay platform that monitors expression of 1600 genes in zebrafish embryos exposed to either single chemicals or complex water samples. The method provides a more cost effective, high throughput means to broadly evaluate the pot...

  9. 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...

  10. Spatiotemporal image correlation analysis of blood flow in branched vessel networks of zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Ceffa, Nicolo G.; Cesana, Ilaria; Collini, Maddalena; D'Alfonso, Laura; Carra, Silvia; Cotelli, Franco; Sironi, Laura; Chirico, Giuseppe

    2017-10-01

    Ramification of blood circulation is relevant in a number of physiological and pathological conditions. The oxygen exchange occurs largely in the capillary bed, and the cancer progression is closely linked to the angiogenesis around the tumor mass. Optical microscopy has made impressive improvements in in vivo imaging and dynamic studies based on correlation analysis of time stacks of images. Here, we develop and test advanced methods that allow mapping the flow fields in branched vessel networks at the resolution of 10 to 20 μm. The methods, based on the application of spatiotemporal image correlation spectroscopy and its extension to cross-correlation analysis, are applied here to the case of early stage embryos of zebrafish.

  11. Egfl6 is involved in zebrafish notochord development.

    PubMed

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

    2015-08-01

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

  12. 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

  13. Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish (Danio rerio).

    PubMed

    Henn, Kirsten; Braunbeck, Thomas

    2011-01-01

    Prior to hatching, the zebrafish embryo is surrounded by an acellular envelope, the chorion. Despite repeated speculations, it could not be clarified unequivocally whether the chorion represents an effective barrier and, thus, protects the embryo from exposure to distinct chemicals. Potentially, there is a risk of generating false negative results in developmental toxicity studies due to limited permeability of the chorion for some compounds. The simplest way to exclude this is to remove the chorion and expose the "naked" embryo. In the context of ecotoxicity testing, standardized protocols do not exist for fish embryo dechorionation, and survival rates of dechorionated embryos have usually not been subjected to statistical analysis. Since reproducibly high survival rates are of fundamental importance for chemical toxicity assessment, the present study was designed to develop and optimize a dechorionation procedure. With appropriate modifications of the fish embryo test protocol, embryos can be dechorionated at 24h post-fertilization (hpf) with survival rates of ≥90%. However, for fish embryo tests with dechorionated embryos, the standard positive control test substance, 3,4-dichloroaniline, should be replaced by another compound, e.g., acetone, since 3,4-dichloroaniline exerts its effects during the first 24h of development. Dechorionation of younger stages (<24 hpf) is generally possible, however with lower survival rates. The effect of dechorionation was demonstrated with the cationic polymer Luviquat HM 552, which is blocked by the chorion non-dechorionated embryos due to its molecular weight of ~400,000 Dalton, but becomes strongly toxic after dechorionation. Copyright © 2010 Elsevier Inc. All rights reserved.

  14. MicroXRF tomographic visualization of zinc and iron in the zebrafish embryo at the onset of the hatching period

    SciT

    Bourassa, Daisy; Gleber, Sophie-Charlotte; Vogt, Stefan

    2016-01-01

    Transition metals such as zinc, copper, and iron play key roles in cellular proliferation, cell differentiation, growth, and development. Over the past decade, advances in synchrotron X-ray fluorescence instrumentation presented new opportunities for the three-dimensional mapping of trace metal distributions within intact specimens. Taking advantage of microXRF tomography, we visualized the 3D distribution of zinc and iron in a zebrafish embryo at the onset of the hatching period. The reconstructed volumetric data revealed distinct differences in the elemental distributions, with zinc predominantly localized to the yolk and yolk extension, and iron to various regions of the brain as well asmore » the myotome extending along the dorsal side of the embryo. The data set complements an earlier tomographic study of an embryo at the pharyngula stage (24 hpf), thus offering new insights into the trace metal distribution at key stages of embryonic development.« less

  15. Development of the zebrafish mesonephros.

    PubMed

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

    2015-01-01

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

  16. Development of the zebrafish mesonephros

    PubMed Central

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

    2015-01-01

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

  17. Exposure to mercuric chloride induces developmental damage, oxidative stress and immunotoxicity in zebrafish embryos-larvae.

    PubMed

    Zhang, Qun-Fang; Li, Ying-Wen; Liu, Zhi-Hao; Chen, Qi-Liang

    2016-12-01

    Mercury (Hg) is a widespread environmental pollutant that can produce severe negative effects on fish even at very low concentrations. However, the mechanisms underlying inorganic Hg-induced oxidative stress and immunotoxicity in the early development stage of fish still need to be clarified. In the present study, zebrafish (Danio rerio) embryos were exposed to different concentrations of Hg 2+ (0, 1, 4 and 16μg/L; added as mercuric chloride, HgCl 2 ) from 2h post-fertilization (hpf) to 168hpf. Developmental parameters and total Hg accumulation were monitored during the exposure period, and antioxidant status and the mRNA expression of genes related to the innate immune system were examined at 168hpf. The results showed that increasing Hg 2+ concentration and time significantly increased total Hg accumulation in zebrafish embryos-larvae. Exposure to 16μg/L Hg 2+ caused developmental damage, including increased mortality and malformation, decreased body length, and delayed hatching period. Meanwhile, HgCl 2 exposure (especially in the 16μg/L Hg 2+ group) induced oxidative stress affecting antioxidant enzyme (CAT, GST and GPX) activities, endogenous GSH and MDA contents, as well as the mRNA levels of genes (cat1, sod1, gstr, gpx1a, nrf2, keap1, hsp70 and mt) encoding antioxidant proteins. Moreover, the transcription levels of several representative genes (il-1β, il-8, il-10, tnfα2, lyz and c3) involved in innate immunity were up-regulated by HgCl 2 exposure, suggesting that inorganic Hg had the potential to induce immunotoxicity. Taken together, the present study provides evidence that waterborne HgCl 2 exposure can induce developmental impairment, oxidative stress and immunotoxicity in the early development stage of fish, which brings insights into the toxicity mechanisms of inorganic Hg in fish. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. The Transcriptome of the Zebrafish Embryo After Chemical Exposure: A Meta-Analysis.

    PubMed

    Schüttler, Andreas; Reiche, Kristin; Altenburger, Rolf; Busch, Wibke

    2017-06-01

    Numerous studies have been published in the past years investigating the transcriptome of the zebrafish embryo (ZFE) upon being subjected to chemical stress. Aiming at a more mechanistic understanding of the results of such studies, knowledge about commonalities of transcript regulation in response to chemical stress is needed. Thus, our goal in this study was to identify and interpret genes and gene sets constituting a general response to chemical exposure. Therefore, we aggregated and reanalyzed published toxicogenomics data obtained with the ZFE. We found that overlap of differentially transcribed genes in response to chemical stress across independent studies is generally low and the most commonly differentially transcribed genes appear in less than 50% of all treatments across studies. However, effect size analysis revealed several genes showing a common trend of differential expression, among which genes related to calcium homeostasis emerged as key, especially in exposure settings up to 24 h post-fertilization. Additionally, we found that these and other downregulated genes are often linked to anatomical regions developing during the respective exposure period. Genes showing a trend of increased expression were, among others, linked to signaling pathways (e.g., Wnt, Fgf) as well as lysosomal structures and apoptosis. The findings of this study help to increase the understanding of chemical stress responses in the developing zebrafish embryo and provide a starting point to improve experimental designs for this model system. In future, improved time- and concentration-resolved experiments should offer better understanding of stress response patterns and access to mechanistic information. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology.

  19. Evaluation of 14 Organic Solvents and Carriers for Screening Applications in Zebrafish Embryos and Larvae

    PubMed Central

    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

  20. Evaluation of Common Use Brominated Flame Retardant (BFR) Toxicity Using a Zebrafish Embryo Model.

    PubMed

    Usenko, Crystal Y; Abel, Erika L; Hopkins, Aaron; Martinez, Gerardo; Tijerina, Jonathan; Kudela, Molly; Norris, Nick; Joudeh, Lana; Bruce, Erica D

    2016-09-02

    Brominated flame retardants (BFRs) are used to reduce the flammability of plastics, textiles, and electronics. BFRs vary in their chemical properties and structures, and it is expected that these differences alter their biological interactions and toxicity. Zebrafish were used as the model organism for assessing the toxicity of nine structurally-diverse BFRs. In addition to monitoring for overt toxicity, the rate of spontaneous movement, and acetylcholinesterase and glutathione- S -transferase (GST) activities were assessed following exposure. The toxicities of BFRs tested can be ranked by LC50 as tetrabromobisphenol A (TBBPA) < 4,4'-isopropylidenebis[2-(2,6-dibromophenoxyl)ethanol] (TBBPA-OHEE) < Pentabromochlorocyclohexane (PBCH) < 2-ethylhexyl 2,3,4,5-tetrabromobenzoate (TBB) < hexabromocyclododecane (HBCD) < hexabromobenzene (HBB) < Tetrabromophthalic anhydride (PHT4). No adverse effect was observed in di(2-ethylhexyl) tetrabromophthalate (TBPH) or dibromoneopentyl glycol (DBNPG)-treated embryos. The rate of spontaneous movement was decreased in a concentration-dependent manner following exposure to four of the nine compounds. GST activity was elevated following treatment with PBCH, TBBPA, HBCD, and HBB. The results indicate that exposure to several BFRs may activate an antioxidant response and alter behavior during early development. Some of the BFRs, such as TBBPA and TBBPA-OHEE, induced adverse effects at concentrations lower than chemicals that are currently banned. These results suggest that zebrafish are sensitive to exposure to BFRs and can be used as a comparative screening model, as well as to determine alterations in behavior following exposure and probe mechanisms of action.

  1. 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

  2. Comparative analysis of the transcriptome responses of zebrafish embryos after exposure to low concentrations of cadmium, cobalt and copper.

    PubMed

    Sonnack, Laura; Klawonn, Thorsten; Kriehuber, Ralf; Hollert, Henner; Schäfers, Christoph; Fenske, Martina

    2018-03-01

    Metal toxicity is a global environmental challenge. Fish are particularly prone to metal exposure, which can be lethal or cause sublethal physiological impairments. The objective of this study was to investigate how adverse effects of chronic exposure to non-toxic levels of essential and non-essential metals in early life stage zebrafish may be explained by changes in the transcriptome. We therefore studied the effects of three different metals at low concentrations in zebrafish embryos by transcriptomics analysis. The study design compared exposure effects caused by different metals at different developmental stages (pre-hatch and post-hatch). Wild-type embryos were exposed to solutions of low concentrations of copper (CuSO 4 ), cadmium (CdCl 2 ) and cobalt (CoSO 4 ) until 96h post-fertilization (hpf) and microarray experiments were carried out to determine transcriptome profiles at 48 and 96hpf. We found that the toxic metal cadmium affected the expression of more genes at 96hpf than 48hpf. The opposite effect was observed for the essential metals cobalt and copper, which also showed enrichment of different GO terms. Genes involved in neuromast and motor neuron development were significantly enriched, agreeing with our previous results showing motor neuron and neuromast damage in the embryos. Our data provide evidence that the response of the transcriptome of fish embryos to metal exposure differs for essential and non-essential metals. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Potential teratogenicity of methimazole: exposure of zebrafish embryos to methimazole causes similar developmental anomalies to human methimazole embryopathy.

    PubMed

    Komoike, Yuta; Matsuoka, Masato; Kosaki, Kenjiro

    2013-06-01

    While methimazole (MMI) is widely used in the therapy for hyperthyroidism, several groups have reported that maternal exposure to MMI results in a variety of congenital anomalies, including choanal and esophageal atresia, iridic and retinal coloboma, and delayed neurodevelopment. Thus, adverse effects of maternal exposure to MMI on fetal development have long been suggested; however, direct evidence for the teratogenicity of MMI has not been presented. Therefore, we studied the effects of MMI on early development by using zebrafish as a model organism. The fertilized eggs of zebrafish were collected immediately after spawning and grown in egg culture water containing MMI at various concentrations. External observation of the embryos revealed that exposure to high concentrations of MMI resulted in loss of pigmentation, hypoplastic hindbrain, turbid tissue in the forebrain, swelling of the notochord, and curly trunk. Furthermore, these effects occurred in a dose-dependent manner. Precise observation of the serial cross-sections of MMI-exposed embryos elucidated delayed development and hypoplasia of the whole brain and spinal cord, narrowing of the pharynx and esophagus, severe disruption of the retina, and aberrant structure of the notochord. These neuronal, pharyngeal, esophageal, and retinal anomalous morphologies have a direct analogy to the congenital anomalies observed in children exposed to MMI in utero. Here, we show the teratogenic effects of MMI on the development of zebrafish and provide the first experimental evidence for the connection between exposure to MMI and human MMI embryopathy. © 2013 Wiley Periodicals, Inc.

  4. Protective effects of puerarin against tetrabromobisphenol a-induced apoptosis and cardiac developmental toxicity in zebrafish embryo-larvae.

    PubMed

    Yang, Suwen; Wang, Shengrui; Sun, Fengchao; Zhang, Mengmeng; Wu, Fengchang; Xu, Fanfan; Ding, Zhishan

    2015-09-01

    Tetrabromobisphenol A (TBBPA), a brominated flame retardant, is detected commonly in aquatic environments, where it is thought to be highly toxic to the development of aquatic life. In this study, zebrafish embryos and larvae were used to investigate the protective effects of puerarin after exposure to TBBPA. Malformation, blood flow disorders, pericardial edema, and spawn coagulation rates increased, whereas survival decreased significantly after exposure to 0.5 and 1.0 mg L(-1) TBBPA. The measured indices of morphological toxicity improved after treatment with puerarin. TBBPA also induced reactive oxygen species (ROS) production in a dose-dependent manner. Acridine orange staining results revealed that TBBPA exposure caused cardiomyocyte apoptosis and induced the expression of three proapoptotic genes: P53, Bax, and Caspase9. In contrast, the expression of the antiapoptotic gene Bcl2 was down-regulated. When genes related to cardiac development were assessed, the expression of Tbx1, Raldh2, and Bmp2b changed after exposure to the combination of TBBPA and puerarin. These results suggest that TBBPA induces cardiomyocyte apoptosis and ROS production, resulting in cardiac developmental toxicity in zebrafish embryos or larvae. Therefore, puerarin regulates the expression of cardiac developmental genes, such as Tbx1, Bmp2b, and Raldh2 by inhibiting ROS production, and subsequently modulates cardiac development after the exposure of zebrafish larvae to TBBPA. © 2014 Wiley Periodicals, Inc.

  5. Aspp2 negatively regulates body growth but not developmental timing by modulating IRS signaling in zebrafish embryos.

    PubMed

    Liu, Chengdong; Luan, Jing; Bai, Yan; Li, Yun; Lu, Ling; Liu, Yunzhang; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Duan, Cunming; Zhou, Jianfeng

    2014-02-01

    The growth and developmental rate of developing embryos and fetus are tightly controlled and coordinated to maintain proper body shape and size. The insulin receptor substrate (IRS) proteins, key intracellular transducers of insulin and insulin-like growth factor signaling, play essential roles in the regulation of growth and development. A short isoform of apoptosis-stimulating protein of p53 2 (ASPP2) was recently identified as a binding partner of IRS-1 and IRS-2 in mammalian cells in vitro. However, it is unclear whether ASPP2 plays any role in vertebrate embryonic growth and development. Here, we show that zebrafish Aspp2a and Aspp2b negatively regulate embryonic growth without affecting developmental rate. Human ASPP2 had similar effects on body growth in zebrafish embryos. Aspp2a and 2b inhibit Akt signaling. This inhibition was reversed by coinjection of myr-Akt1, a constitutively active form of Akt1. Zebrafish Aspp2a and Aspp2b physically bound with Irs-1, and the growth inhibitory effects of ASPP2/Aspp2 depend on the presence of their ankyrin repeats and SH3 domains. These findings uncover a novel role of Aspp2 in regulating vertebrate embryonic growth. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Dechorionation of Zebrafish Embryos on Day 1 Post Fertilization Alters Response to an Acute Chemical Challenge at 6 Days Post Fertilization

    EPA Science Inventory

    Dechorionation is a method used to enable image acquisition in embryonic and larval zebrafish studies. As it is assumed that dechorionation has no long-term effects on fish embryo development, it is important to determine if that assumption is correct. The present study explored ...

  7. ApoA-II directs morphogenetic movements of zebrafish embryo by preventing chromosome fusion during nuclear division in yolk syncytial layer.

    PubMed

    Zhang, Ting; Yao, Shaohua; Wang, Ping; Yin, Chaoran; Xiao, Chun; Qian, Meilin; Liu, Donghui; Zheng, Lemin; Meng, Wentong; Zhu, Hongyan; Liu, Jin; Xu, Hong; Mo, Xianming

    2011-03-18

    The high density lipoprotein (HDL) represents a class of lipid- and protein-containing particles and consists of two major apolipoproteins apoA-I and apoA-II. ApoA-II has been shown to be involved in the pathogenesis of insulin resistance, adiposity, diabetes, and metabolic syndrome. In embryo, apoa2 mRNAs are abundant in the liver, brain, lung, placenta, and in fish yolk syncytial layer (YSL), suggesting that apoa2 may perform a function during embryonic development. Here we find out that apoa2 modulates zebrafish embryonic development by regulating the organization of YSL. Disruption of apoa2 function in zebrafish caused chromosome fusing, which strongly blocked YSL nuclear division, inducing disorders in YSL organization and finally disturbing the embryonic epiboly. Purified native human apoA-II was able specifically to rescue the defects and induced nuclear division in zebrafish embryos and in human HeLa cells. The C terminus of apoA-II was required for the proper chromosome separation during nuclear division of YSL in zebrafish embryos and in human HeLa cells. Our data indicate that organization of YSL is required for blastoderm patterning and morphogenesis and suggest that apolipoprotein apoA-II is a novel factor of nuclear division in YSL involved in the regulation of early zebrafish embryonic morphogenesis and in mammalian cells for proliferation.

  8. ApoA-II Directs Morphogenetic Movements of Zebrafish Embryo by Preventing Chromosome Fusion during Nuclear Division in Yolk Syncytial Layer*

    PubMed Central

    Zhang, Ting; Yao, Shaohua; Wang, Ping; Yin, Chaoran; Xiao, Chun; Qian, Meilin; Liu, Donghui; Zheng, Lemin; Meng, Wentong; Zhu, Hongyan; Liu, Jin; Xu, Hong; Mo, Xianming

    2011-01-01

    The high density lipoprotein (HDL) represents a class of lipid- and protein-containing particles and consists of two major apolipoproteins apoA-I and apoA-II. ApoA-II has been shown to be involved in the pathogenesis of insulin resistance, adiposity, diabetes, and metabolic syndrome. In embryo, apoa2 mRNAs are abundant in the liver, brain, lung, placenta, and in fish yolk syncytial layer (YSL), suggesting that apoa2 may perform a function during embryonic development. Here we find out that apoa2 modulates zebrafish embryonic development by regulating the organization of YSL. Disruption of apoa2 function in zebrafish caused chromosome fusing, which strongly blocked YSL nuclear division, inducing disorders in YSL organization and finally disturbing the embryonic epiboly. Purified native human apoA-II was able specifically to rescue the defects and induced nuclear division in zebrafish embryos and in human HeLa cells. The C terminus of apoA-II was required for the proper chromosome separation during nuclear division of YSL in zebrafish embryos and in human HeLa cells. Our data indicate that organization of YSL is required for blastoderm patterning and morphogenesis and suggest that apolipoprotein apoA-II is a novel factor of nuclear division in YSL involved in the regulation of early zebrafish embryonic morphogenesis and in mammalian cells for proliferation. PMID:21212265

  9. 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). Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Benomyl induction of brain aromatase and toxic effects in the zebrafish embryo.

    PubMed

    Kim, Dong-Jae; Seok, Seung-Hyeok; Baek, Min-Won; Lee, Hui-Young; Na, Yi-Rang; Park, Sung-Hoon; Lee, Hyun-Kyoung; Dutta, Noton Kumar; Kawakami, Koichi; Park, Jae-Hak

    2009-05-01

    Benomyl is a benzimidazole fungicide that has been widely used on a variety of food crops and ornamental plants. It is known to cause adverse effects on reproductive systems, including decreased testicular and epididymal weights and reduced epididymal sperm counts and fertility. The brain aromatase gene is up-regulated by estrogens and estrogen mimics and considered a target gene to screen estrogen mimics. This study was designed to test the estrogenic potential and toxic effects of benomyl in the zebrafish system, and validated this system as a model that may correspond to the effect of benomyl in rodents. Concentrations of 20 x 10(-6), 40 x 10(-6) and 80 x 10(-6) M of benomyl-treated embryos showed decreased survival, hatching and heart rates, and increased incidence of malformations, such as pericardial edema, spinal lordosis, elongated heart, head edema, eye lens protrusion and caudal fin disappearance. Benomyl induced enhanced green fluorescent protein (EGFP) expression in the mediobasal hypothalamus (MBH) in transient zebrafish embryos with a brain aromatase-based reporter gene. In this study, we determined that benomyl has estrogenic potential based on zebrafish brain aromatase gene induction, and that benomyl is toxic at 20 x 10(-6) M concentration and higher. These results demonstrate the usefulness of zebrafish embryos as an in vivo system to examine the estrogenic and developmental toxic potential of unknown compounds.

  11. Cynodon dactylon and Sida acuta extracts impact on the function of the cardiovascular system in zebrafish embryos.

    PubMed

    Kannan, Rajaretinam Rajesh; Vincent, Samuel Gnana Prakash

    2012-03-01

    The aim of the present study was to screen cardioactive herbs from Western Ghats of India. The heart beat rate (HBR) and blood flow during systole and diastole were tested in zebrafish embryos. We found that Cynodon dactylon (C. dactylon) induced increases in the HBR in zebrafish embryos with a HBR of (3.968±0.344) beats/s, which was significantly higher than that caused by betamethosone [(3.770±0.344) beats/s]. The EC50 value of C. dactylon was 3.738 µg/mL. The methanolic extract of Sida acuta (S. acuta) led to decreases in the HBR in zebrafish embryos [(1.877±0.079) beats/s], which was greater than that caused by nebivolol (positive control). The EC50 value of Sida acuta was 1.195 µg/mL. The untreated embryos had a HBR of (2.685±0.160) beats/s at 3 d post fertilization (dpf). The velocities of blood flow during the cardiac cycle were (2,291.667±72.169) µm/s for the control, (4,250±125.000) µm/s for C. dactylon and (1,083.333±72.169) µm/s for S. acuta. The LC50 values were 32.6 µg/mL for C. dactylon and 20.9 µg/mL for S. acuta. In addition, the extracts exhibited no chemical genetic effects in the drug dosage range tested. In conclusion, we developed an assay that can measure changes in cardiac function in response to herbal small molecules and determine the cardiogenic effects by microvideography.

  12. 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

  13. 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. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. 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

  15. 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. © The Author 2016. Published by Oxford University Press on behalf of The Japan Radiation Research Society and Japanese Society for Radiation Oncology.

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

    PubMed

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

    2011-05-01

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

  17. Exploring uptake and biodistribution of polystyrene (nano)particles in zebrafish embryos at different developmental stages.

    PubMed

    van Pomeren, M; Brun, N R; Peijnenburg, W J G M; Vijver, M G

    2017-09-01

    In ecotoxicology, it is continuously questioned whether (nano)particle exposure results in particle uptake and subsequent biodistribution or if particles adsorb to the epithelial layer only. To contribute to answering this question, we investigated different uptake routes in zebrafish embryos and how they affect particle uptake into organs and within whole organisms. This is addressed by exposing three different life stages of the zebrafish embryo in order to cover the following exposure routes: via chorion and dermal exposure; dermal exposure; oral and dermal exposure. How different nanoparticle sizes affect uptake routes was assessed by using polystyrene particles of 25, 50, 250 and 700nm. In our experimental study, we showed that particle uptake in biota is restricted to oral exposure, whereas the dermal route resulted in adsorption to the epidermis and gills only. Ingestion followed by biodistribution was observed for the tested particles of 25 and 50nm. The particles spread through the body and eventually accumulated in specific organs and tissues such as the eyes. Particles larger than 50nm were predominantly adsorbed onto the intestinal tract and outer epidermis of zebrafish embryos. Embryos exposed to particles via both epidermis and intestine showed highest uptake and eventually accumulated particles in the eye, whereas uptake of particles via the chorion and epidermis resulted in marginal uptake. Organ uptake and internal distribution should be monitored more closely to provide more in depth information of the toxicity of particles. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  18. 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

  19. Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

    PubMed Central

    Andersen, Erica; Asuri, Namrata; Clay, Matthew; Halloran, Mary

    2010-01-01

    The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons. Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages. Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1. Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work. PMID:20130524

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

    PubMed

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

    2018-03-06

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

  1. Embedding, serial sectioning and staining of zebrafish embryos using JB-4 resin.

    PubMed

    Sullivan-Brown, Jessica; Bisher, Margaret E; Burdine, Rebecca D

    2011-01-01

    Histological techniques are critical for observing tissue and cellular morphology. In this paper, we outline our protocol for embedding, serial sectioning, staining and visualizing zebrafish embryos embedded in JB-4 plastic resin-a glycol methacrylate-based medium that results in excellent preservation of tissue morphology. In addition, we describe our procedures for staining plastic sections with toluidine blue or hematoxylin and eosin, and show how to couple these stains with whole-mount RNA in situ hybridization. We also describe how to maintain and visualize immunofluorescence and EGFP signals in JB-4 resin. The protocol we outline-from embryo preparation, embedding, sectioning and staining to visualization-can be accomplished in 3 d. Overall, we reinforce that plastic embedding can provide higher resolution of cellular details and is a valuable tool for cellular and morphological studies in zebrafish.

  2. Biological response of zebrafish embryos after short-term exposure to thifluzamide

    NASA Astrophysics Data System (ADS)

    Yang, Yang; Liu, Wenxian; Mu, Xiyan; Qi, Suzhen; Fu, Bin; Wang, Chengju

    2016-12-01

    Thifluzamide is a new amide fungicide, and its extensive application may have toxic effects on zebrafish. To better understand the underlying mechanism, we investigated in detail the potential toxic effects of thifluzamide on zebrafish embryos. In the present study, embryos were exposed to 0, 0.19, 1.90, and 2.85 mg/L thifluzamide for 4 days. Obvious pathological changes were found upon a histological exam, and negative changes in mitochondrial structure were observed under Transmission Electron Microscopy (TEM), which qualitatively noted the toxic effects of thifluzamide on embryos. Moreover, we quantitatively evaluated the enzyme activities [succinate dehydrogenase (SDH), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), caspases], the contents of malonaldehyde (MDA) and interleukin-8 (IL-8) and the expression levels of the related genes. This study suggests that the negative changes in mitochondrial structure and SDH activity might be responsible for oxidative damage, cell apoptosis and inflammation, which would facilitate the action of these factors in cell death and might play a crucial role during toxic events. In addition to providing the first description of the mechanism of the toxic effects of thifluzamide on embryos, this study also represents a step towards using embryos to assess mitochondrial metabolism and disease.

  3. 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.

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

    PubMed

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

    2016-08-19

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

  5. In vivo screening and discovery of novel candidate thalidomide analogs in the zebrafish embryo and chicken embryo model systems

    PubMed Central

    Beedie, Shaunna L.; Rore, Holly M.; Barnett, Shelby; Chau, Cindy H.; Luo, Weiming; Greig, Nigel H.; Figg, William D.; Vargesson, Neil

    2016-01-01

    Thalidomide, a drug known for its teratogenic side-effects, is used successfully to treat a variety of clinical conditions including leprosy and multiple myeloma. Intense efforts are underway to synthesize and identify safer, clinically relevant analogs. Here, we conduct a preliminary in vivo screen of a library of new thalidomide analogs to determine which agents demonstrate activity, and describe a cohort of compounds with anti-angiogenic properties, anti-inflammatory properties and some compounds which exhibited both. The combination of the in vivo zebrafish and chicken embryo model systems allows for the accelerated discovery of new, potential therapies for cancerous and inflammatory conditions. PMID:27120781

  6. Development and Validation of an Automated High-Throughput System for Zebrafish In Vivo Screenings

    PubMed Central

    Virto, Juan M.; Holgado, Olaia; Diez, Maria; Izpisua Belmonte, Juan Carlos; Callol-Massot, Carles

    2012-01-01

    The zebrafish is a vertebrate model compatible with the paradigms of drug discovery. The small size and transparency of zebrafish embryos make them amenable for the automation necessary in high-throughput screenings. We have developed an automated high-throughput platform for in vivo chemical screenings on zebrafish embryos that includes automated methods for embryo dispensation, compound delivery, incubation, imaging and analysis of the results. At present, two different assays to detect cardiotoxic compounds and angiogenesis inhibitors can be automatically run in the platform, showing the versatility of the system. A validation of these two assays with known positive and negative compounds, as well as a screening for the detection of unknown anti-angiogenic compounds, have been successfully carried out in the system developed. We present a totally automated platform that allows for high-throughput screenings in a vertebrate organism. PMID:22615792

  7. Evaluation of the Biotoxicity of Tree Wood Ashes in Zebrafish Embryos.

    PubMed

    Consigli, Veronica; Guarienti, Michela; Bilo, Fabjola; Benassi, Laura; Depero, Laura E; Bontempi, Elza; Presta, Marco

    2016-10-01

    Ashes derived from biomass combustion and used as soil fertilizers can generate negative environmental and human health risks, related to leaching of heavy metals and other putative toxic elements. Tree wood ash composition may vary depending on geographical location and surrounding industrial processes. In this study, we evaluated the biotoxicity of lixiviated tree wood ash samples from trees of the Ash (Fraxinus), Cherry (Pronus), Hazel (Corylus), and Black locust (Robinia) genus collected in an industrialized region in Northern Italy. Elemental chemical analysis of the samples was performed by total reflection X-ray fluorescence technique and their biotoxicity was assessed in zebrafish (Danio rerio) embryos. Ashes from Ash, Cherry, and Hazel trees, but not Black locust trees, had a high concentration of heavy metals and other putative toxic elements. Accordingly, a dose-dependent increase in mortality rate and morphological and teratogenic defects was observed in zebrafish embryos treated with lixiviated Ash, Cherry, and Hazel tree wood samples, whereas the toxicity of Black locust tree wood ashes was negligible. In conclusion, lixiviated wood ashes from different plants show a different content of toxic elements that correlate with their biotoxic effects on zebrafish embryos. Tree wood ashes derived from biomass combustion may represent a potential risk for the environment and human health.

  8. 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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. 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. © 2016 Wiley Periodicals, Inc.

  10. Evaluation of Common Use Brominated Flame Retardant (BFR) Toxicity Using a Zebrafish Embryo Model

    PubMed Central

    Usenko, Crystal Y.; Abel, Erika L.; Hopkins, Aaron; Martinez, Gerardo; Tijerina, Jonathan; Kudela, Molly; Norris, Nick; Joudeh, Lana; Bruce, Erica D.

    2016-01-01

    Brominated flame retardants (BFRs) are used to reduce the flammability of plastics, textiles, and electronics. BFRs vary in their chemical properties and structures, and it is expected that these differences alter their biological interactions and toxicity. Zebrafish were used as the model organism for assessing the toxicity of nine structurally-diverse BFRs. In addition to monitoring for overt toxicity, the rate of spontaneous movement, and acetylcholinesterase and glutathione-S-transferase (GST) activities were assessed following exposure. The toxicities of BFRs tested can be ranked by LC50 as tetrabromobisphenol A (TBBPA) < 4,4′-isopropylidenebis[2-(2,6-dibromophenoxyl)ethanol] (TBBPA-OHEE) < Pentabromochlorocyclohexane (PBCH) < 2-ethylhexyl 2,3,4,5-tetrabromobenzoate (TBB) < hexabromocyclododecane (HBCD) < hexabromobenzene (HBB) < Tetrabromophthalic anhydride (PHT4). No adverse effect was observed in di(2-ethylhexyl) tetrabromophthalate (TBPH) or dibromoneopentyl glycol (DBNPG)-treated embryos. The rate of spontaneous movement was decreased in a concentration-dependent manner following exposure to four of the nine compounds. GST activity was elevated following treatment with PBCH, TBBPA, HBCD, and HBB. The results indicate that exposure to several BFRs may activate an antioxidant response and alter behavior during early development. Some of the BFRs, such as TBBPA and TBBPA-OHEE, induced adverse effects at concentrations lower than chemicals that are currently banned. These results suggest that zebrafish are sensitive to exposure to BFRs and can be used as a comparative screening model, as well as to determine alterations in behavior following exposure and probe mechanisms of action. PMID:29051424

  11. Toxic effects of strychnine and strychnine N-oxide on zebrafish embryos.

    PubMed

    Li, Yu; Qi, Xu; Yang, Yu-Wei; Pan, Yang; Bian, Hui-Min

    2014-10-01

    The application of strychnine (S) is limited due to its toxicity; strychnine N-oxide (SNO) is a derivative of strychnine. The aim was to employ zebrafish embryos to investigate and compare the developmental toxicity induced by S and SNO. The toxicity of S and SNO was examined through the hatching rate and survival rate. Morphological changes of the zebrafish were observed with a dissecting microscope. Apoptosis was detected through acridine orange (AO) staining and flow cytometry. Apoptotic genes were measured by RT-PCR. Embryo malformation was observed in the embryos exposed to S at 200 μmol·L(-1). When SNO concentration was increased to 1 mmol·L(-1), scoliolosis, and pericardial edema could be seen in some embryos. Results from fluorescence microscopy and flow cytometry analysis showed that S at 200 μmol·L(-1) induced apoptosis, whereas the apoptotic rate in the SNO-treated group (200 μmol·L(-1)) was much lower than that in the S group. RT-PCR analysis showed that p53 mRNA expression and the ratio of Bax/Bcl-2 in the S group were significantly altered compared with the control group (*P < 0.05). Moreover, Bax mRNA expression in both S and SNO group were significantly different from that in the control group (**P < 0.01). These results lead to the conclusion that SNO has significantly lower toxicity than S in zebrafish embryos. Copyright © 2014 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  12. Gene expression analysis in zebrafish embryos: a potential approach to predict effect concentrations in the fish early life stage test.

    PubMed

    Weil, Mirco; Scholz, Stefan; Zimmer, Michaela; Sacher, Frank; Duis, Karen

    2009-09-01

    Based on the hypothesis that analysis of gene expression could be used to predict chronic fish toxicity, the zebrafish (Danio rerio) embryo test (DarT), developed as a replacement method for the acute fish test, was expanded to a gene expression D. rerio embryo test (Gene-DarT). The effects of 14 substances on lethal and sublethal endpoints of the DarT and on expression of potential marker genes were investigated: the aryl hydrocarbon receptor 2, cytochrome P450 1A (cypla), heat shock protein 70, fizzy-related protein 1, the transcription factors v-maf musculoaponeurotic fibrosarcoma oncogene family protein g (avian) 1 and NF-E2-p45-related factor, and heme oxygenase 1 (hmox1). After exposure of zebrafish embryos for 48 h, differential gene expression was evaluated using reverse transcriptase-polymerase chain reaction, gel electrophoresis, and densitometric analysis of the gels. All tested compounds significantly affected the expression of at least one potential marker gene, with cyp1a and hmox1 being most sensitive. Lowest-observed-effect concentrations (LOECs) for gene expression were below concentrations resulting in 10% lethal effects in the DarT. For 10 (3,4- and 3,5-dichloroaniline, 1,4-dichlorobenzene, 2,4-dinitrophenol, atrazine, parathion-ethyl, chlorotoluron, genistein, 4-nitroquinoline-1-oxide, and cadmium) out of the 14 tested substances, LOEC values derived with the Gene-DarT differ by a factor of less than 10 from LOEC values of fish early life stage tests with zebrafish. For pentachloroaniline and pentachlorobenzene, the Gene-DarT showed a 23- and 153-fold higher sensitivity, respectively, while for lindane, it showed a 13-fold lower sensitivity. For ivermectin, the Gene-DarT was by a factor of more than 1,000 less sensitive than the acute fish test. The results of the present study indicate that gene expression analysis in zebrafish embryos could principally be used to predict effect concentrations in the fish early life stage test.

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

    PubMed

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

    2016-02-19

    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.

  14. TBBPA induces developmental toxicity, oxidative stress, and apoptosis in embryos and zebrafish larvae (Danio rerio).

    PubMed

    Wu, Shengmin; Ji, Guixiang; Liu, Jining; Zhang, Shenghu; Gong, Yang; Shi, Lili

    2016-10-01

    Tetrabromobisphenol A (TBBPA) is currently one of the most frequently used brominated flame retardants and can be considered as a high production volume chemical. In this study, zebrafish embryos and larvae served as a biological model to evaluate TBBPA-induced developmental toxicity, oxidative stress, oxidant-associated gene expression, and cell apoptosis. Abnormalities, including hyperemia and pericardial edema, were induced in zebrafish larvae. The results showed that toxicity endpoints such as hatching rate, survival rate, malformation rate, and growth rate had a significant dose-response relationship with TBBPA. Further studies revealed that TBBPA did not alter the enzyme activities of Copper/Zinc Superoxide dismutase (Cu/Zn-SOD), catalase (CAT), and glutathioneperoxidase (GPx) at 0.10 mg/L, but decreased activities following exposure to 0.40, 0.70, and 1.00 mg/L. Despite the significantly decreased gene expression of Cu/Zn-SOD, CAT, and GPx1a in the 1.00 mg/L treatment group, other treatments (0.10, 0.40, 0.70 mg/L) did not alter gene expression. Moreover, Acridine orange staining results showed that apoptotic cells mainly accumulated in the brain, heart, and tail, indicating possible TBBPA-induced brain, cardiac, and blood circulation system impairment in zebrafish embryos and larvae. Histological analysis also showed evidence of obvious heart impairment in TBBPA-treated groups. This study provides new evidence on the developmental toxicity, oxidative stress, and apoptosis of embryos and zebrafish larvae, which is important for the evaluation of environmental toxicity and chemical risk. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1241-1249, 2016. © 2015 Wiley Periodicals, Inc.

  15. Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

    PubMed Central

    Baars, Destiny L.; Pelegri, Francisco

    2016-01-01

    Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species. PMID:28060351

  16. The toxicity potential of pharmaceuticals found in the Douro River estuary (Portugal)--experimental assessment using a zebrafish embryo test.

    PubMed

    Madureira, Tânia Vieira; Cruzeiro, Catarina; Rocha, Maria João; Rocha, Eduardo

    2011-09-01

    Fish embryos are a particularly vulnerable stage of development, so they represent optimal targets for screening toxicological effects of waterborne xenobiotics. Herein, the toxicity potential of two mixtures of pharmaceuticals was evaluated using a zebrafish embryo test. One of the mixtures corresponds to an environmentally realistic scenario and both have carbamazepine, fenofibric acid, propranolol, trimethoprim and sulfamethoxazole. The results evidenced morphological alterations, such as spinal deformities and yolk-sac oedemas. Moreover, heart rates decreased after both mixture exposures, e.g., at 48hpf, highest mixture versus blank control (47.8±4.9 and 55.8±3.7 beats/30s, respectively). The tail lengths also diminished significantly from 3208±145μm in blank control to 3130±126μm in highest mixture. The toxicological effects were concentration dependent. Mortality, hatching rate and the number of spontaneous movements were not affected. However, the low levels of pharmaceuticals did interfere with the normal development of zebrafish, which indicates risks for wild organisms. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  18. Concentration dependent transcriptome responses of zebrafish embryos after exposure to cadmium, cobalt and copper.

    PubMed

    Sonnack, Laura; Klawonn, Thorsten; Kriehuber, Ralf; Hollert, Henner; Schäfers, Christoph; Fenske, Martina

    2017-12-01

    Environmental metals are known to cause harmful effects to fish of which many molecular mechanisms still require elucidation. Particularly concentration dependence of gene expression effects is unclear. Focusing on this matter, zebrafish embryo toxicity tests were used in combination with transcriptomics. Embryos were exposed to three concentrations of copper (CuSO 4 ), cadmium (CdCl 2 ) and cobalt (CoSO 4 ) from just after fertilization until the end of the 48hpf pre- and 96hpf post-hatch stage. The RNA was then analyzed on Agilent's Zebrafish (V3, 4×44K) arrays. Enrichment for GO terms of biological processes illustrated for cadmium that most affected GO terms were represented in all three concentrations, while for cobalt and copper most GO terms were represented in the lowest test concentration only. This suggested a different response to the non-essential cadmium than cobalt and copper. In cobalt and copper treated embryos, many developmental and cellular processes as well as the Wnt and Notch signaling pathways, were found significantly enriched. Also, different exposure concentrations affected varied functional networks. In contrast, the largest clusters of enriched GO terms for all three concentrations of cadmium included responses to cadmium ion, metal ion, xenobiotic stimulus, stress and chemicals. However, concentration dependence of mRNA levels was evident for several genes in all metal exposures. Some of these genes may be indicative of the mechanisms of action of the individual metals in zebrafish embryos. Real-time quantitative RT-PCR (qRT-PCR) verified the microarray data for mmp9, mt2, cldnb and nkx2.2a. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Aryl hydrocarbon receptor 2 mediates the toxicity of Paclobutrazol on the digestive system of zebrafish embryos.

    PubMed

    Wang, Wen-Der; Chen, Guan-Ting; Hsu, Hwei-Jan; Wu, Chang-Yi

    2015-02-01

    Paclobutrazol (PBZ), a trazole-containing fungicide and plant growth retardant, has been widely used for over 30 years to regulate plant growth and promote early fruit setting. Long-term usage of PBZ in agriculture and natural environments has resulted in residual PBZ in the soil and water. Chronic exposure to waterborne PBZ can cause various physiological effects in fish, including hepatic steatosis, antioxidant activity, and disruption of spermatogenesis. We have previously shown that PBZ also affects the rates of zebrafish embryonic survival and hatching, and causes developmental failure of the head skeleton and eyes; here, we further show that PBZ has embryonic toxic effects on digestive organs of zebrafish, and describe the underlying mechanisms. PBZ treatment of embryos resulted in dose-dependent morphological and functional abnormalities of the digestive organs. Real-time RT-PCR and in situ hybridization were used to show that PBZ strongly induces cyp1a1 expression in the digestive system, and slightly induces ahr2 expression in zebrafish embryos. Knockdown of ahr2 with morpholino oligonucleotides prevents PBZ toxicity. Thus, the toxic effect of PBZ on digestive organs is mediated by AhR2, as was previously reported for retene and TCDD. These findings have implications for understanding the potential toxicity of PBZ during embryogenesis, and thus the potential impact of fungicides on public health and the environment. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2014-01-01

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

  1. Biological impacts of glyphosate on morphology, embryo biomechanics and larval behavior in zebrafish (Danio rerio).

    PubMed

    Zhang, Shuhui; Xu, Jia; Kuang, Xiangyu; Li, Shibao; Li, Xiang; Chen, Dongyan; Zhao, Xin; Feng, Xizeng

    2017-08-01

    All of these days, residues of herbicides such as glyphosate are widely distributed in the environment. The ubiquitous use of glyphosate has drawn extensive attention to its toxicity as an organic pollutant. In this study, we employed larval zebrafish as an animal model to evaluate the effect of different concentrations of glyphosate on early development via morphological, biomechanics, behavioral and physiological analyses. Morphological results showed that an obvious delay occurred in the epiboly process and body length, eye and head area were reduced at concentrations higher than 10 mg/L. The expression of ntl (no tail) shortened and krox20 (also known as Egr2b, early growth response 2b) changed as the glyphosate concentration increased, but there was no change in the expression of shh (sonic hedgehog). In addition, biomechanical analysis of the elasticity of chorion indicated that treated embryos' surface tension was declined. Furthermore, a 48-h locomotion test revealed that embryonic exposure to glyphosate significantly elevated locomotor activities, which is probably attributed to motoneuronal damage. The decreased surface tension of chorion and the increased locomotive activities may contribute to the hatching rates after glyphosate treatment. Our study enriches the researches of evaluating glyphosate toxicity and probablely plays a warning role in herbicides used in farming. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. 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

  3. Embryonic exposure to sodium arsenite perturbs vascular development in zebrafish.

    PubMed

    McCollum, Catherine W; Hans, Charu; Shah, Shishir; Merchant, Fatima A; Gustafsson, Jan-Åke; Bondesson, Maria

    2014-07-01

    Exposure to arsenic in its inorganic form, arsenite, causes adverse effects to many different organs and tissues. Here, we have investigated arsenite-induced adverse effects on vascular tissues in the model organism zebrafish, Danio rerio. Zebrafish embryos were exposed to arsenite at different exposure windows and the susceptibility to vascular tissue damage was recorded at 72hours post fertilization (hpf). Intersegmental vessel sprouting and growth was most perturbed by exposure to arsenite during the 24-48hpf window, while disruption in the condensation of the caudal vein plexus was more often observed at the 48-72hpf exposure window, reflecting when these structures develop during normal embryogenesis. The vascular growth rate was decreased by arsenite exposure, and deviated from that of control embryos at around 24-26.5hpf. We further mapped changes in expression of key regulators of angiogenesis and vasculogenesis. Downregulation of vascular endothelial growth factor receptor 1/fms-related tyrosine kinase 1 (vegfr1/flt1) expression was evident already at 24hpf, coinciding with the decreased vascular growth rate. At later time points, matrix metalloproteinase 9 (mmp9) expression was upregulated, suggesting that arsenite affects the composition of the extracellular matrix. In total, the expression of eight key factors involved in different aspects of vascularization was significantly altered by arsenic exposure. In conclusion, our results show that arsenite is a potent vascular disruptor in the developing zebrafish embryo, a finding that calls for an evaluation of arsenite as a developmental vascular toxicant in mammalian model systems. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. High-Resolution Magic Angle Spinning Nuclear Magnetic Resonance of Intact Zebrafish Embryos Detects Metabolic Changes Following Exposure to Teratogenic Polymethoxyalkenes from Algae

    PubMed Central

    Roy, Upasana; Jaja-Chimedza, Asha; Sanchez, Kristel; Matysik, Joerg

    2016-01-01

    Abstract Techniques based on nuclear magnetic resonance (NMR) for imaging and chemical analyses of in vivo, or otherwise intact, biological systems are rapidly emerging and finding diverse applications within a wide range of fields. Very recently, several NMR-based techniques have been developed for the zebrafish as a model animal system. In the current study, the novel application of high-resolution magic angle spinning (HR-MAS) NMR is presented as a means of metabolic profiling of intact zebrafish embryos. Toward investigating the utility of HR-MAS NMR as a toxicological tool, these studies specifically examined metabolic changes of embryos exposed to polymethoxy-1-alkenes (PMAs)—a recently identified family of teratogenic compounds from freshwater algae—as emerging environmental contaminants. One-dimensional and two-dimensional HR-MAS NMR analyses were able to effectively identify and quantify diverse metabolites in early-stage (≤36 h postfertilization) embryos. Subsequent comparison of the metabolic profiles between PMA-exposed and control embryos identified several statistically significant metabolic changes associated with subacute exposure to the teratogen, including (1) elevated inositol as a recognized component of signaling pathways involved in embryo development; (2) increases in several metabolites, including inositol, phosphoryl choline, fatty acids, and cholesterol, which are associated with lipid composition of cell membranes; (3) concomitant increase in glucose and decrease in lactate; and (4) decreases in several biochemically related metabolites associated with central nervous system development and function, including γ-aminobutyric acid, glycine, glutamate, and glutamine. A potentially unifying model/hypothesis of PMA teratogenicity based on the data is presented. These findings, taken together, demonstrate that HR-MAS NMR is a promising tool for metabolic profiling in the zebrafish embryo, including toxicological applications. PMID

  5. The effect of MRN complex and ATM kinase inhibitors on Zebrafish embryonic development

    NASA Astrophysics Data System (ADS)

    Kumaran, Malina; Fazry, Shazrul

    2018-04-01

    Zebrafish is an ideal animal model to study developmental biology due to its transparent embryos and rapid development stages of embryogenesis. Here we investigate the role of DNA damage proteins, specifically Mre11/Rad50/NBN (MRN) complex and ataxia-telangiectasia mutated (ATM) kinase during embryogenesis by inhibiting its function using specific MRN complex (Mirin) and ATM Kinase inhibitors (Ku60019 and Ku55933). Zebrafish embryos at midblastula transition (MBT) stage are treated with Mirin, Ku60019 and Ku55933. The embryonic development of the embryos was monitored at 24 hours-post fertilisation (hpf), 48 hpf and 72 hpf. We observed that at the lowest concentrations (3 µM of Mirin, 1.5 nM of Ku60019 and 3 nM of Ku55933), the inhibitors treated embryos have 100% survivability. However, with increasing inhibitor concentration, the survivability drops. Control or mock treatment of all embryos shows 100 % survivability rate. This study suggests that DNA damage repair proteins may be crucial for normal zebrafish embryo development and survival.

  6. Smoc2 modulates embryonic myelopoiesis during zebrafish development.

    PubMed

    Mommaerts, Hendrik; Esguerra, Camila V; Hartmann, Ursula; Luyten, Frank P; Tylzanowski, Przemko

    2014-11-01

    SMOC2 is a member of the BM-40 (SPARC) family of matricellular proteins, reported to influence signaling in the extracellular compartment. In mice, Smoc2 is expressed in many different tissues and was shown to enhance the response to angiogenic growth factors, mediate cell adhesion, keratinocyte migration, and metastasis. Additionally, SMOC2 is associated with vitiligo and craniofacial and dental defects. The function of Smoc2 during early zebrafish development has not been determined to date. In pregastrula zebrafish embryos, smoc2 is expressed ubiquitously. As development progresses, the expression pattern becomes more anteriorly restricted. At the onset of blood cell circulation, smoc2 morphants presented a mild ventralization of posterior structures. Molecular analysis of the smoc2 morphants indicated myelopoietic defects in the rostral blood islands during segmentation stages. Hemangioblast development and further specification of the myeloid progenitor cells were shown to be impaired. Additional experiments indicated that Bmp target genes were down-regulated in smoc2 morphants. Our findings reveal that Smoc2 is an essential player in the development of myeloid cells of the anterior lateral plate mesoderm during embryonic zebrafish development. Furthermore, our data show that Smoc2 affects the transcription of Bmp target genes without affecting initial dorsoventral patterning or mesoderm development. Copyright © 2014 Wiley Periodicals, Inc.

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

    SciT

    Bodewein, Lambert

    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 96 h and human cancer cell lines for 24 h, to assess and compare developmental in vivo toxicity with cytotoxicity. The zebrafish embryo toxicity of cationic PAMAM and PPI dendrimers increased over time, withmore » EC50 values ranging from 0.16 to just below 1.7 μM at 24 and 48 hpf. 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 ≥ 402 μM (PAMAMs) and ≤ 240 μM (PPIs) for HepG2 and ≤ 13.24 μM (PAMAMs) and ≤ 12.84 μM (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. - Highlights: • Zebrafish embryo toxicity of cationic PAMAM and PPI dendrimers increased over time. • Zebrafish embryo toxicity of cationic dendrimers did not increase with generation. • Cationic dendrimers induced apoptosis in zebrafish embryos. • Toxicity of cationic dendrimers was lower in HepG2 and DU145 than zebrafish

  8. Combination effects of AHR agonists and Wnt/β-catenin modulators in zebrafish embryos: Implications for physiological and toxicological AHR functions

    SciT

    Wincent, Emma; Institute of Environmental Medicine, Karolinska Institutet, 17177 Stockholm; Stegeman, John J.

    2015-04-15

    Wnt/β-catenin signaling regulates essential biological functions and acts in developmental toxicity of some chemicals. The aryl hydrocarbon receptor (AHR) is well-known to mediate developmental toxicity of persistent dioxin-like compounds (DLCs). Recent studies indicate a crosstalk between β-catenin and the AHR in some tissues. However the nature of this crosstalk in embryos is poorly known. We observed that zebrafish embryos exposed to the β-catenin inhibitor XAV939 display effects phenocopying those of the dioxin-like 3,3′,4,4′,5-pentachlorobiphenyl (PCB126). This led us to investigate the AHR interaction with β-catenin during development and ask whether developmental toxicity of DLCs involves antagonism of β-catenin signaling. We examinedmore » phenotypes and transcriptional responses in zebrafish embryos exposed to XAV939 or to a β-catenin activator, 1-azakenpaullone, alone or with AHR agonists, either PCB126 or 6-formylindolo[3,2-b]carbazole (FICZ). Alone 1-azakenpaullone and XAV939 both were embryo-toxic, and we found that in the presence of FICZ, the toxicity of 1-azakenpaullone decreased while the toxicity of XAV939 increased. This rescue of 1-azakenpaullone effects occurred in the time window of Ahr2-mediated toxicity and was reversed by morpholino-oligonucleotide knockdown of Ahr2. Regarding PCB126, addition of either 1-azakenpaullone or XAV939 led to lower mortality than with PCB126 alone but surviving embryos showed severe edemas. 1-Azakenpaullone induced transcription of β-catenin-associated genes, while PCB126 and FICZ blocked this induction. The data indicate a stage-dependent antagonism of β-catenin by Ahr2 in zebrafish embryos. We propose that the AHR has a physiological role in regulating β-catenin during development, and that this is one point of intersection linking toxicological and physiological AHR-governed processes.« less

  9. Toxicity of single-wall carbon nanotubes functionalized with polyethylene glycol in zebrafish (Danio rerio) embryos.

    PubMed

    Girardi, Felipe A; Bruch, Gisele E; Peixoto, Carolina S; Dal Bosco, Lidiane; Sahoo, Sangram K; Gonçalves, Carla O F; Santos, Adelina P; Furtado, Clascídia A; Fantini, Cristiano; Barros, Daniela M

    2017-02-01

    Single-wall carbon nanotubes functionalized with polyethylene glycol (SWCNT-PEG) are promising materials for biomedical applications such as diagnostic devices and controlled drug-release systems. However, several questions about their toxicological profile remain unanswered. Thus, the aim of this study was to investigate the action of SWCNT-PEG in Danio rerio zebrafish embryos at the molecular, physiological and morphological levels. The SWCNT used in this study were synthesized by the high-pressure carbon monoxide process, purified and then functionalized with distearoyl phosphatidylethanolamine block copolymer-PEG (molecular weight 2 kDa). The characterization process was carried out with low-resolution transmission electron microscopy, thermogravimetric analysis and Raman spectroscopy. Individual zebrafish embryos were exposed to the SWCNT-PEG. Toxic effects occurred only at the highest concentration tested (1 ppm) and included high mortality rates, delayed hatching and decreased total larval length. For all the concentrations tested, the alkaline comet assay revealed no genotoxicity, and Raman spectroscopy measurements on the histological slices revealed no intracellular nanotubes. The results shown here demonstrate that SWCNT-PEG has low toxicity in zebrafish embryos, but more studies are needed to understand what mechanisms are involved. However, the presence of residual metals is possibly among the primary mechanisms responsible for the toxic effects observed, because the purification process was not able to remove all metal contamination, as demonstrated by the thermogravimetric analysis. More attention must be given to the toxicity of these nanomaterials before they are used in biomedical applications. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  10. Localization of BDNF expression in the developing brain of zebrafish

    PubMed Central

    De Felice, E; Porreca, I; Alleva, E; De Girolamo, P; Ambrosino, C; Ciriaco, E; Germanà, A; Sordino, P

    2014-01-01

    The brain-derived neurotrophic factor (BDNF) gene is expressed in differentiating and post-mitotic neurons of the zebrafish embryo, where it has been implicated in Huntington's disease. Little is known, however, about the full complement of neuronal cell types that express BDNF in this important vertebrate model. Here, we further explored the transcriptional profiles during the first week of development using real-time quantitative polymerase chain reaction (RT-qPCR) and whole-mount in situ hybridization (WISH). RT-qPCR results revealed a high level of maternal contribution followed by a steady increase of zygotic transcription, consistent with the notion of a prominent role of BDNF in neuronal maturation and maintenance. Based on WISH, we demonstrate for the first time that BDNF expression in the developing brain of zebrafish is structure specific. Anatomical criteria and co-staining with genetic markers (shh, pax2a, emx1, krox20, lhx2b and lhx9) visualized major topological domains of BDNF-positive cells in the pallium, hypothalamus, posterior tuberculum and optic tectum. Moreover, the relative timing of BDNF transcription in the eye and tectum may illustrate a mechanism for coordinated development of the retinotectal system. Taken together, our results are compatible with a local delivery and early role of BDNF in the developing brain of zebrafish, adding basic knowledge to the study of neurotrophin functions in neural development and disease. PMID:24588510

  11. Localization of BDNF expression in the developing brain of zebrafish.

    PubMed

    De Felice, E; Porreca, I; Alleva, E; De Girolamo, P; Ambrosino, C; Ciriaco, E; Germanà, A; Sordino, P

    2014-05-01

    The brain-derived neurotrophic factor (BDNF) gene is expressed in differentiating and post-mitotic neurons of the zebrafish embryo, where it has been implicated in Huntington's disease. Little is known, however, about the full complement of neuronal cell types that express BDNF in this important vertebrate model. Here, we further explored the transcriptional profiles during the first week of development using real-time quantitative polymerase chain reaction (RT-qPCR) and whole-mount in situ hybridization (WISH). RT-qPCR results revealed a high level of maternal contribution followed by a steady increase of zygotic transcription, consistent with the notion of a prominent role of BDNF in neuronal maturation and maintenance. Based on WISH, we demonstrate for the first time that BDNF expression in the developing brain of zebrafish is structure specific. Anatomical criteria and co-staining with genetic markers (shh, pax2a, emx1, krox20, lhx2b and lhx9) visualized major topological domains of BDNF-positive cells in the pallium, hypothalamus, posterior tuberculum and optic tectum. Moreover, the relative timing of BDNF transcription in the eye and tectum may illustrate a mechanism for coordinated development of the retinotectal system. Taken together, our results are compatible with a local delivery and early role of BDNF in the developing brain of zebrafish, adding basic knowledge to the study of neurotrophin functions in neural development and disease. © 2014 Anatomical Society.

  12. Shp2–Mitogen-Activated Protein Kinase Signaling Drives Proliferation during Zebrafish Embryo Caudal Fin Fold Regeneration

    PubMed Central

    Hale, Alexander James

    2017-01-01

    ABSTRACT Regeneration of the zebrafish caudal fin following amputation occurs through wound healing, followed by formation of a blastema, which produces cells to replace the lost tissue in the final phase of regenerative outgrowth. We show that ptpn11a−/− ptpn11b−/− zebrafish embryos, lacking functional Shp2, fail to regenerate their caudal fin folds. Rescue experiments indicated that Shp2a has a functional signaling role, requiring its catalytic activity and SH2 domains but not the two C-terminal tyrosine phosphorylation sites. Surprisingly, expression of Shp2a variants with increased and reduced catalytic activity, respectively, rescued caudal fin fold regeneration to similar extents. Expression of mmp9 and junbb, indicative of formation of the wound epidermis and distal blastema, respectively, suggested that these processes occurred in ptpn11a−/− ptpn11b−/− zebrafish embryos. However, cell proliferation and MAPK phosphorylation were reduced. Pharmacological inhibition of MEK1 in wild-type zebrafish embryos phenocopied loss of Shp2. Our results suggest an essential role for Shp2a–mitogen-activated protein kinase (MAPK) signaling in promoting cell proliferation during zebrafish embryo caudal fin fold regeneration. PMID:29203641

  13. The zebrafish as a model system to study cardiovascular development.

    PubMed

    Stainier, D Y; Fishman, M C

    1994-01-01

    The zebrafish, Brachydanio rerio, is rapidly becoming a system of choice for vertebrate developmental biologists. It presents unique embryological attributes and is amenable to saturation style mutagenesis, a powerful approach that, in invertebrates, has already led to the identification of a large number of key developmental genes. Since fertilization is external, the zebrafish embryo develops in the dish and is thus accessible for continued observation and manipulation at all stages of development. Furthermore, because the embryo is transparent, the developing heart and vessels can be resolved at the single-cell level. A large number of mutations that affect the development of cardiovascular form and function have recently been isolated from large-scale genetic screens for zygotic embryonic lethals. Our further understanding of the development of the cardiovascular system is important not only because of the high incidence, and familial inheritance, of congenital abnormalities, but also because it should lead to novel, differentiation-based strategies for the analysis and therapy of the diseased state. Copyright © 1994. Published by Elsevier Inc.

  14. Vitamin D receptor deficiency impairs inner ear development in zebrafish

    SciT

    Kwon, Hye-Joo; Biology Department, Princess Nourah University, Riyadh 11671

    The biological actions of vitamin D are largely mediated through binding to the vitamin D receptor (VDR), a member of the nuclear hormone receptor family, which regulates gene expression in a wide variety of tissues and cells. Mutations in VDR gene have been implicated in ear disorders (hearing loss and balance disorder) but the mechanisms are not well established. In this study, to investigate the role of VDR in inner ear development, morpholino-mediated gene knockdown approaches were used in zebrafish model system. Two paralogs for VDR, vdra and vdrb, have been identified in zebrafish. Knockdown of vdra had no effectmore » on ear development, whereas knockdown of vdrb displayed morphological ear defects including smaller otic vesicles with malformed semicircular canals and abnormal otoliths. Loss-of-vdrb resulted in down-regulation of pre-otic markers, pax8 and pax2a, indicating impairment of otic induction. Furthermore, zebrafish embryos lacking vdrb produced fewer sensory hair cells in the ears and showed disruption of balance and motor coordination. These data reveal that VDR signaling plays an important role in ear development. - Highlights: • VDR signaling is involved in ear development. • Knockdown of vdrb causes inner ear malformations during embryogenesis. • Knockdown of vdrb affects otic placode induction. • Knockdown of vdrb reduces the number of sensory hair cells in the inner ear. • Knockdown of vdrb disrupts balance and motor coordination.« less

  15. CTCF knockout reveals an essential role for this protein during the zebrafish development.

    PubMed

    Carmona-Aldana, Francisco; Zampedri, Cecilia; Suaste-Olmos, Fernando; Murillo-de-Ozores, Adrián; Guerrero, Georgina; Arzate-Mejía, Rodrigo; Maldonado, Ernesto; Navarro, Rosa; Chimal-Monroy, Jesús; Recillas-Targa, Félix

    2018-05-01

    Chromatin regulation and organization are essential processes that regulate gene activity. The CCCTC-binding factor (CTCF) is a protein with different and important molecular functions related with chromatin dynamics. It is conserved since invertebrates to vertebrates, posing it as a factor with an important role in the physiology. In this work, we aimed to understand the distribution and functional relevance of CTCF during the embryonic development of the zebrafish (Danio rerio). We generated a zebrafish specific anti-Ctcf antibody, and found this protein to be ubiquitous, through different stages and tissues. We used the CRISPR-Cas9 system to induce molecular alterations in the locus. This resulted in early lethality. We delayed the lethality performing knockdown morpholino experiments, and found an aberrant embryo morphology involving malformations in structures through all the length of the embryo. These phenotypes were rescued with human CTCF mRNA injections, showing the specificity of the morpholinos and a partial functional conservation between the fish and the human proteins. Lastly, we found that the pro-apoptotic genes p53 and bbc3/PUMA are deregulated in the ctcf morpholino-injected embryos. In conclusion, CTCF is a ubiquitous factor during the zebrafish development, which regulates the correct formation of different structures of the embryo, and its deregulation impacts on essential cell survival genes. Overall, this work provides a basis to look for the particular functions of CTCF in the different developing tissues and organs of the zebrafish. Copyright © 2018. Published by Elsevier B.V.

  16. 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. Copyright © 2015 John Wiley & Sons, Ltd.

  17. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Tributyltin induces premature hatching and reduces locomotor activity in zebrafish (Danio rerio) embryos/larvae at environmentally relevant levels.

    PubMed

    Liang, Xuefang; Souders, Christopher L; Zhang, Jiliang; Martyniuk, Christopher J

    2017-12-01

    Tributyltin (TBT) is an organotin compound that is the active ingredient of many biocides and antifouling agents. In addition to its well established role as an endocrine disruptor, TBT is also associated with adverse effects on the nervous system and behavior. In this study, zebrafish (Danio rerio) embryos were exposed to environmentally relevant concentrations of TBT (0.01, 0.1, 1 nM) to determine how low levels affected development and behavior. Fish exposed to 1 nM TBT hatched earlier when compared to controls. Following a 96-h exposure, total swimming distance, velocity, and activity of zebrafish larvae were reduced compared to controls. To identify putative mechanisms for these altered endpoints, we assessed embryo bioenergetics and gene expression. We reasoned that the accelerated hatch time could be related to ATP production and energy, thus embryos were exposed to TBT for 24 and 48-h exposure prior to hatch. There were no differences among groups for endpoints related to bioenergetics (i.e. basal, ATP-dependent, and maximal respiration). To address mechanisms related to changes in behavioral activity, we measured transcripts associated with muscle function (myf6, myoD, and myoG) and dopamine signaling (th, dat, dopamine receptors) as dopamine regulates behavior. No transcript was altered in expression by TBT in larvae, suggesting that other mechanisms exist that may explain changes in higher level endpoints. These results suggest that endpoints related to the whole animal (i.e. timing of hatch and locomotor behavior) are more sensitive to environmentally-relevant concentrations of TBT compared to the molecular and metabolic endpoints examined here. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. 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.

  20. Vitamin D receptor deficiency impairs inner ear development in zebrafish.

    PubMed

    Kwon, Hye-Joo

    2016-09-16

    The biological actions of vitamin D are largely mediated through binding to the vitamin D receptor (VDR), a member of the nuclear hormone receptor family, which regulates gene expression in a wide variety of tissues and cells. Mutations in VDR gene have been implicated in ear disorders (hearing loss and balance disorder) but the mechanisms are not well established. In this study, to investigate the role of VDR in inner ear development, morpholino-mediated gene knockdown approaches were used in zebrafish model system. Two paralogs for VDR, vdra and vdrb, have been identified in zebrafish. Knockdown of vdra had no effect on ear development, whereas knockdown of vdrb displayed morphological ear defects including smaller otic vesicles with malformed semicircular canals and abnormal otoliths. Loss-of-vdrb resulted in down-regulation of pre-otic markers, pax8 and pax2a, indicating impairment of otic induction. Furthermore, zebrafish embryos lacking vdrb produced fewer sensory hair cells in the ears and showed disruption of balance and motor coordination. These data reveal that VDR signaling plays an important role in ear development. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Transcriptome alterations in zebrafish embryos after exposure to environmental estrogens and anti-androgens can reveal endocrine disruption.

    PubMed

    Schiller, Viktoria; Wichmann, Arne; Kriehuber, Ralf; Schäfers, Christoph; Fischer, Rainer; Fenske, Martina

    2013-12-01

    Exposure to environmental chemicals known as endocrine disruptors (EDs) is in many cases associated with an unpredictable hazard for wildlife and human health. The identification of endocrine disruptive properties of chemicals certain to enter the aquatic environment relies on toxicity tests with fish, assessing adverse effects on reproduction and sexual development. The demand for quick, reliable ED assays favored the use of fish embryos as alternative test organisms. We investigated the application of a transcriptomics-based assay for estrogenic and anti-androgenic chemicals with zebrafish embryos. Two reference compounds, 17α-ethinylestradiol and flutamide, were tested to evaluate the effects on development and the transcriptome after 48h-exposures. Comparison of the transcriptome response with other estrogenic and anti-androgenic compounds (genistein, bisphenol A, methylparaben, linuron, prochloraz, propanil) showed commonalities and differences in regulated pathways, enabling us to classify the estrogenic and anti-androgenic potencies. This demonstrates that different mechanism of ED can be assessed already in fish embryos. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. Initial specification of the epibranchial placode in zebrafish embryos depends on the fibroblast growth factor signal.

    PubMed

    Nikaido, Masataka; Doi, Kazunao; Shimizu, Takashi; Hibi, Masahiko; Kikuchi, Yutaka; Yamasu, Kyo

    2007-02-01

    In vertebrates, cranial sensory ganglia are mainly derived from ectodermal placodes, which are focal thickenings at characteristic positions in the embryonic head. Here, we provide the first description of the early development of the epibranchial placode in zebrafish embryos using sox3 as a molecular marker. By the one-somite stage, we saw a pair of single sox3-expressing domains appear lateral to the future hindbrain. The sox3 domain, which is referred to here as the early lateral placode, is segregated during the early phase of segmentation to form a pax2a-positive medial area and a pax2a-negative lateral area. The medial area subsequently developed to form the otic placode, while the lateral area was further segregated along the anteroposterior axis, giving rise to four sox3-positive subdomains by 26 hr postfertilization. Given their spatial relationship with the expression of the markers for the epibranchial ganglion, as well as their positions and temporal changes, we propose that these four domains correspond to the facial, glossopharyngeal, vagal, and posterior lateral line placodes in an anterior-to-posterior order. The expression of sox3 in the early lateral placode was absent in mutants lacking functional fgf8, while implantation of fibroblast growth factor (FGF) beads restored the sox3 expression. Using SU5402, which inhibits the FGF signal, we were able to demonstrate that formation of both the early lateral domains and later epibranchial placodes depends on the FGF signal operating at the beginning of somitogenesis. Together, these data provide evidence for the essential role of FGF signals in the development of the epibranchial placodes.

  3. Label-free imaging of developing vasculature in zebrafish with phase variance optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Yu; Fingler, Jeff; Trinh, Le A.; Fraser, Scott E.

    2016-03-01

    A phase variance optical coherence microscope (pvOCM) has been created to visualize blood flow in the vasculature of zebrafish embryos, without using exogenous labels. The pvOCM imaging system has axial and lateral resolutions of 2 μm in tissue, and imaging depth of more than 100 μm. Imaging of 2-5 days post-fertilization zebrafish embryos identified the detailed structures of somites, spinal cord, gut and notochord based on intensity contrast. Visualization of the blood flow in the aorta, veins and intersegmental vessels was achieved with phase variance contrast. The pvOCM vasculature images were confirmed with corresponding fluorescence microscopy of a zebrafish transgene that labels the vasculature with green fluorescent protein. The pvOCM images also revealed functional information of the blood flow activities that is crucial for the study of vascular development.

  4. The paracrine effect of exogenous growth hormone alleviates dysmorphogenesis caused by tbx5 deficiency in zebrafish (Danio rerio) embryos

    PubMed Central

    2012-01-01

    Background Dysmorphogenesis and multiple organ defects are well known in zebrafish (Danio rerio) embryos with T-box transcription factor 5 (tbx5) deficiencies, mimicking human Holt-Oram syndrome. Methods Using an oligonucleotide-based microarray analysis to study the expression of special genes in tbx5 morphants, we demonstrated that GH and some GH-related genes were markedly downregulated. Zebrafish embryos microinjected with tbx5-morpholino (MO) antisense RNA and mismatched antisense RNA in the 1-cell stage served as controls, while zebrafish embryos co-injected with exogenous growth hormone (GH) concomitant with tbx5-MO comprised the treatment group. Results The attenuating effects of GH in tbx5-MO knockdown embryos were quantified and observed at 24, 30, 48, 72, and 96 h post-fertilization. Though the understanding of mechanisms involving GH in the tbx5 functioning complex is limited, exogenous GH supplied to tbx5 knockdown zebrafish embryos is able to enhance the expression of downstream mediators in the GH and insulin-like growth factor (IGF)-1 pathway, including igf1, ghra, and ghrb, and signal transductors (erk1, akt2), and eventually to correct dysmorphogenesis in various organs including the heart and pectoral fins. Supplementary GH also reduced apoptosis as determined by a TUNEL assay and decreased the expression of apoptosis-related genes and proteins (bcl2 and bad) according to semiquantitative reverse-transcription polymerase chain reaction and immunohistochemical analysis, respectively, as well as improving cell cycle-related genes (p27 and cdk2) and cardiomyogenetic genes (amhc, vmhc, and cmlc2). Conclusions Based on our results, tbx5 knockdown causes a pseudo GH deficiency in zebrafish during early embryonic stages, and supplementation of exogenous GH can partially restore dysmorphogenesis, apoptosis, cell growth inhibition, and abnormal cardiomyogenesis in tbx5 knockdown zebrafish in a paracrine manner. PMID:22776023

  5. Embryotoxicity of poorly soluble nanoparticles at various stages of Zebrafish development

    NASA Astrophysics Data System (ADS)

    Morgaleva, Tamara; Morgalev, Yuri; Gosteva, Irina; Morgalev, Sergey; Nesterenya, Daria

    2017-11-01

    The biological effects of the poorly soluble nanoparticles (NPs) of different chemical nature and structural characteristics were evaluated. It was established that the Zebrafish test response to contamination of aqueous medium with nickel NPs (nNi), platinum (nPt), zinc oxide (nZnO) and cerium oxide (nCeO2) depends on the physicochemical properties of the NPs and embryo development stage. The concentrations of NPs not causing disruptions in embryonic development of Zebrafish were determined. The smallest impact on embryogenesis was exerted by nCeO2: coagulation of a small number of embryos was observed only at C = 20.0 mg/L. The same effect was observed when exposed to lower concentrations of nPt (C = 5.0 mg/L) and nNi (C = 0.1 mg/L). The greatest number of coagulated embryos was observed when grown in the DS nZnO: 37.5% of embryos died at the DS concentration of C = 0.1 mg/L. Zebrafish cultivation in the DS with low concentrations (C ≤ LC10) of nNi and nZnO caused distortions in the development of embryos: development of scoliosis, malformation of somites, inhibited mobility.

  6. Multilayer mounting enables long-term imaging of zebrafish development in a light sheet microscope.

    PubMed

    Kaufmann, Anna; Mickoleit, Michaela; Weber, Michael; Huisken, Jan

    2012-09-01

    Light sheet microscopy techniques, such as selective plane illumination microscopy (SPIM), are ideally suited for time-lapse imaging of developmental processes lasting several hours to a few days. The success of this promising technology has mainly been limited by the lack of suitable techniques for mounting fragile samples. Embedding zebrafish embryos in agarose, which is common in conventional confocal microscopy, has resulted in severe growth defects and unreliable results. In this study, we systematically quantified the viability and mobility of zebrafish embryos mounted under more suitable conditions. We found that tubes made of fluorinated ethylene propylene (FEP) filled with low concentrations of agarose or methylcellulose provided an optimal balance between sufficient confinement of the living embryo in a physiological environment over 3 days and optical clarity suitable for fluorescence imaging. We also compared the effect of different concentrations of Tricaine on the development of zebrafish and provide guidelines for its optimal use depending on the application. Our results will make light sheet microscopy techniques applicable to more fields of developmental biology, in particular the multiview long-term imaging of zebrafish embryos and other small organisms. Furthermore, the refinement of sample preparation for in toto and in vivo imaging will promote other emerging optical imaging techniques, such as optical projection tomography (OPT).

  7. Mechanisms of cadmium-caused eye hypoplasia and hypopigmentation in zebrafish embryos.

    PubMed

    Zhang, Ting; Zhou, Xin-Ying; Ma, Xu-Fa; Liu, Jing-Xia

    2015-10-01

    Cadmium-caused head and eye hypoplasia and hypopigmentation has been recognized for a long time, but knowledge of the underlying mechanisms is limited. In this study, we found that high mortality occurred in exposed embryos after 24 hpf, when cadmium (Cd) dosage was above 17.8 μM. Using high-throughput in situ hybridization screening, we found that genes labelling the neural crest and its derivative pigment cells exhibited obviously reduced expression in Cd-exposed embryos from 24 hpf, 2 days earlier than head and eye hypoplasia and hypopigmentation occurred. Moreover, based on expression of crestin, a neural crest marker, we found that embryos before the gastrula stage were more sensitive to cadmium toxicity and that damage caused by Cd on embryogenesis was dosage dependent. In addition, by phenotype observation and detection of neural crest and pigment cell markers, we found that BIO and retinoic acid (RA) could neutralize the toxic effects of Cd on zebrafish embryogenesis. In this study, we first determined that Cd blocked the formation of the neural crest and inhibited specification of pigment cells, which might contribute to the molecular mechanisms underlying the phenotype defects of head and eye hypoplasia and hypopigmentation in Cd-exposed embryos. Moreover, we found that compounds BIO or RA could neutralize the toxic effects of Cd. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Activin- and Nodal-related factors control antero-posterior patterning of the zebrafish embryo.

    PubMed

    Thisse, B; Wright, C V; Thisse, C

    2000-01-27

    Definition of cell fates along the dorso-ventral axis depends on an antagonistic relationship between ventralizing transforming growth factor-beta superfamily members, the bone morphogenetic proteins and factors secreted from the dorsal organizer, such as Noggin and Chordin. The extracellular binding of the last group to the bone morphogenetic proteins prevents them from activating their receptors, and the relative ventralizer:antagonist ratio is thought to specify different dorso-ventral cell fates. Here, by taking advantage of a non-genetic interference method using a specific competitive inhibitor, the Lefty-related gene product Antivin, we provide evidence that cell fate along the antero-posterior axis of the zebrafish embryo is controlled by the morphogenetic activity of another transforming growth factor-beta superfamily subgroup--the Activin and Nodal-related factors. Increasing antivin doses progressively deleted posterior fates within the ectoderm, eventually resulting in the removal of all fates except forebrain and eyes. In contrast, overexpression of activin or nodal-related factors converted ectoderm that was fated to be forebrain into more posterior ectodermal or mesendodermal fates. We propose that modulation of intercellular signalling by Antivin/Activin and Nodal-related factors provides a mechanism for the graded establishment of cell fates along the antero-posterior axis of the zebrafish embryo.

  9. Developmental toxicity of CdTe QDs in zebrafish embryos and larvae

    NASA Astrophysics Data System (ADS)

    Duan, Junchao; Yu, Yongbo; Li, Yang; Yu, Yang; Li, Yanbo; Huang, Peili; Zhou, Xianqing; Peng, Shuangqing; Sun, Zhiwei

    2013-07-01

    Quantum dots (QDs) have widely been used in biomedical and biotechnological applications. However, few studies focus on the assessing toxicity of QDs exposure in vivo. In this study, zebrafish embryos were treated with CdTe QDs (4 nm) during 4-96 h post-fertilization (hpf). Mortality, hatching rate, malformation, heart rate, and QDs uptake were detected. We also measured the larval behavior to analyze whether QDs had persistent effects on larvae locomotor activity at 144 hpf. The results showed that as the exposure dosages increased, the hatching rate and heart rate of zebrafish embryos were decreased, while the mortality increased. Exposure to QDs caused embryonic malformations, including head malformation, pericardial edema, yolk sac edema, bent spine, and yolk not depleted. QDs fluorescence was mainly localized in the intestines region. The larval behavior testing showed that the total swimming distance was decreased in a dose-dependent manner. The lowest dose (2.5 nM QDs) produced substantial hyperactivity while the higher doses groups (5, 10, and 20 nM QDs) elicited remarkably hypoactivity in dark periods. In summary, the data of this article indicated that QDs caused embryonic developmental toxicity, resulted in persistent effects on larval behavior.

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

    PubMed

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

    2011-08-01

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

  11. Type-IV antifreeze proteins are essential for epiboly and convergence in gastrulation of zebrafish embryos.

    PubMed

    Xiao, Qing; Xia, Jian-Hong; Zhang, Xiao-Juan; Li, Zhi; Wang, Yang; Zhou, Li; Gui, Jian-Fang

    2014-01-01

    Many organisms in extremely cold environments such as the Antarctic Pole have evolved antifreeze molecules to prevent ice formation. There are four types of antifreeze proteins (AFPs). Type-IV antifreeze proteins (AFP4s) are present also in certain temperate and even tropical fish, which has raised a question as to whether these AFP4s have important functions in addition to antifreeze activity. Here we report the identification and functional analyses of AFP4s in cyprinid fish. Two genes, namely afp4a and afp4b coding for AFP4s, were identified in gibel carp (Carassius auratus gibelio) and zebrafish (Danio rerio). In both species, afp4a and afp4b display a head-to-tail tandem arrangement and share a common 4-exonic gene structure. In zebrafish, both afp4a and afp4b were found to express specifically in the yolk syncytial layer (YSL). Interestingly, afp4a expression continues in YSL and digestive system from early embryos to adults, whereas afp4b expression is restricted to embryogenesis. Importantly, we have shown by using afp4a-specific and afp4b-specifc morpholino knockdown and cell lineage tracing approaches that AFP4a participates in epiboly progression by stabilizing yolk cytoplasmic layer microtubules, and AFP4b is primarily related to convergence movement. Therefore, both AFP4 proteins are essential for gastrulation of zebrafish embryos. Our current results provide first evidence that AFP such as AFP4 has important roles in regulating developmental processes besides its well-known function as antifreeze factors.

  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.

  13. New isocoumarins from a cold-adapted fungal strain mucor sp. and their developmental toxicity to zebrafish embryos.

    PubMed

    Feng, Chun-Chi; Chen, Guo-Dong; Zhao, Yan-Qiu; Xin, Sheng-Chang; Li, Song; Tang, Jin-Shan; Li, Xiao-Xia; Hu, Dan; Liu, Xing-Zhong; Gao, Hao

    2014-07-01

    Three new isocoumarin derivatives, mucorisocoumarins A-C (1-3, resp.), together with seven known compounds, 4-10, were isolated from the cold-adapted fungal strain Mucor sp. (No. XJ07027-5). The structures of the new compounds were identified by detailed IR, MS, and 1D- and 2D-NMR analyses. It was noteworthy that compounds 1, 2, 4, and 5 were successfully resolved by chiral HPLC, indicating that 1-7 should exist as enantiomers. In an embryonic developmental toxicity assay using a zebrafish model, compound 3 produced developmental abnormalities in the zebrafish embryos. This is the first report of isocoumarins with developmental toxicity to zebrafish embryos. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

  14. Ketamine induction of p53-dependent apoptosis and oxidative stress in zebrafish (Danio rerio) embryos.

    PubMed

    Félix, Luís M; Vidal, Ana M; Serafim, Cindy; Valentim, Ana M; Antunes, Luís M; Monteiro, Sandra M; Matos, Manuela; Coimbra, Ana M

    2018-06-01

    Ketamine is a widely used pharmaceutical that has been detected in water sources worldwide. Zebrafish embryos were used in this study to investigate the oxidative stress and apoptotic signals following a 24h exposure to different ketamine concentrations (0, 50, 70 and 90 mg L -1 ). Early blastula embryos (∼2 h post fertilisation-hpf) were exposed for 24 h and analysed at 8 and 26 hpf. Reactive oxygen species and apoptotic cells were identified in vivo, at 26 hpf. Enzymatic activities (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), lactate dehydrogenase (LDH) and acetylcholinesterase (AChE)), glutathione levels (oxidised (GSSG) and reduced (GSH)), oxidative damage (lipid peroxidation (LPO) and protein carbonyls (CO)) as well as oxidative stress (gclc, gstp1, sod1 and cat), apoptosis (casp3a, casp6, casp8, casp9, aifm1 and tp53) and cell proliferation (pcna) related-genes were evaluated at 8 and 26 hpf. Caspase (3 and 9) activity was also determined at both time-points by colorimetric methods. Superoxide dismutase (SOD), catalase (CAT), glutathione levels (GSSG), caspase-9 and reactive oxygen species (ROS) were shown to be affected by ketamine exposure while in vivo analysis showed no difference in ROS. A significant up-regulation of superoxide dismutase (sod1) and catalase (cat) genes expression was also perceived. Ketamine-induced apoptosis was observed in vivo and confirmed by the apoptotic-related genes up-regulation. The overall results suggest that ketamine induced oxidative stress and apoptosis through the involvement of p53-dependent pathways in zebrafish embryos which could be important for the evaluation of the overall risk of ketamine in aquatic environments. Copyright © 2018 Elsevier Ltd. All rights reserved.

  15. Silver nanoparticles: in vivo toxicity in zebrafish embryos and a comparison to silver nitrate

    NASA Astrophysics Data System (ADS)

    Mosselhy, Dina A.; He, Wei; Li, Dan; Meng, Yaping; Feng, Qingling

    2016-08-01

    The wide antimicrobial administration of silver nanoparticles (AgNPs) has raised the risks associated with their exposure. However, there is lack of robust toxicological data for the applied AgNPs to be in line with their wide antimicrobial applications. This study therefore set out to assess the in vivo toxicity of two different sizes of AgNPs using zebrafish embryos ( Danio rerio) as a brilliant in vivo model. The pivotal role of size of AgNPs in the toxicity was highlighted, wherein the smaller AgNPs (Ag-9 nm) exhibited more embryo toxicities than the larger particles (Ag-30 nm). Much uncertainty still exists about whether the cause of in vivo toxicity of AgNPs is the physicochemical properties of AgNPs or the released silver ions (Ag+). Therefore, another purpose of this study is to compare the toxicity of AgNPs with silver nitrate (AgNO3) in terms of mortality, hatchability and cardiac rates, and a series of phenotypic endpoints of zebrafish embryos. Collectively, the present results point towards the remarkable size-dependent toxicity of AgNPs. Wherein, the smaller AgNPs (9 ± 2 nm) induce increased mortality rates and decreased hatchability rates than the larger particles (30 ± 5 nm) in a dose-dependent manner. Besides, AgNPs and AgNO3 induce holistic different toxic mortality and hatchability rates. We have also found striking discrepancies in the phenotypic defects that were induced by AgNPs and AgNO3. The significant phenotypic defect induced by AgNPs is the axial deformity, while it is the deposition of Ag+ on the embryonic chorion for AgNO3. Therefore, it is proposed that AgNPs and AgNO3 induce different in vivo toxicities.

  16. Novel liquid chromatography-mass spectrometry method shows that vitamin E deficiency depletes arachidonic and docosahexaenoic acids in zebrafish (Danio rerio) embryos.

    PubMed

    Lebold, Katie M; Kirkwood, Jay S; Taylor, Alan W; Choi, Jaewoo; Barton, Carrie L; Miller, Galen W; La Du, Jane; Jump, Donald B; Stevens, Jan Frederik; Tanguay, Robert L; Traber, Maret G

    2013-01-01

    To test the hypothesis that embryogenesis depends upon α-tocopherol (E) to protect embryo polyunsaturated fatty acids (PUFAs) from lipid peroxidation, new methodologies were applied to measure α-tocopherol and fatty acids in extracts from saponified zebrafish embryos. A solid phase extraction method was developed to separate the analyte classes, using a mixed mode cartridge (reverse phase, π-π bonding, strong anion exchange), then α-tocopherol and cholesterol were measured using standard techniques, while the fatty acids were quantitated using a novel, reverse phase liquid chromatography-mass spectrometry (LC-MS) approach. We also determined if α-tocopherol status alters embryonic lipid peroxidation products by analyzing 24 different oxidized products of arachidonic or docosahexaenoic (DHA) acids in embryos using LC with hybrid quadrupole-time of flight MS. Adult zebrafish were fed E- or E+ diets for 4 months, and then were spawned to obtain E- and E+ embryos. Between 24 and 72 hours post-fertilization (hpf), arachidonic acid decreased 3-times faster in E- (21 pg/h) compared with E+ embryos (7 pg/h, P<0.0001), while both α-tocopherol and DHA concentrations decreased only in E- embryos. At 36 hpf, E- embryos contained double the 5-hydroxy-eicosatetraenoic acids and 7-hydroxy-DHA concentrations, while other hydroxy-lipids remained unchanged. Vitamin E deficiency during embryogenesis depleted DHA and arachidonic acid, and increased hydroxy-fatty acids derived from these PUFA, suggesting that α-tocopherol is necessary to protect these critical fatty acids.

  17. Comparison of the In Vivo Biotransformation of Two Emerging Estrogenic Contaminants, BP2 and BPS, in Zebrafish Embryos and Adults

    PubMed Central

    Le Fol, Vincent; Brion, François; Hillenweck, Anne; Perdu, Elisabeth; Bruel, Sandrine; Aït-Aïssa, Selim; Cravedi, Jean-Pierre; Zalko, Daniel

    2017-01-01

    Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using 3H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances. PMID:28346357

  18. Comparison of the In Vivo Biotransformation of Two Emerging Estrogenic Contaminants, BP2 and BPS, in Zebrafish Embryos and Adults.

    PubMed

    Le Fol, Vincent; Brion, François; Hillenweck, Anne; Perdu, Elisabeth; Bruel, Sandrine; Aït-Aïssa, Selim; Cravedi, Jean-Pierre; Zalko, Daniel

    2017-03-25

    Zebrafish embryo assays are increasingly used in the toxicological assessment of endocrine disruptors. Among other advantages, these models are 3R-compliant and are fit for screening purposes. Biotransformation processes are well-recognized as a critical factor influencing toxic response, but major gaps of knowledge exist regarding the characterization of functional metabolic capacities expressed in zebrafish. Comparative metabolic studies between embryos and adults are even scarcer. Using ³H-labeled chemicals, we examined the fate of two estrogenic emerging contaminants, benzophenone-2 (BP2) and bisphenol S (BPS), in 4-day embryos and adult zebrafish. BPS and BP2 were exclusively metabolized through phase II pathways, with no major qualitative difference between larvae and adults except the occurrence of a BP2-di-glucuronide in adults. Quantitatively, the biotransformation of both molecules was more extensive in adults. For BPS, glucuronidation was the predominant pathway in adults and larvae. For BP2, glucuronidation was the major pathway in larvae, but sulfation predominated in adults, with ca. 40% conversion of parent BP2 and an extensive release of several conjugates into water. Further larvae/adults quantitative differences were demonstrated for both molecules, with higher residue concentrations measured in larvae. The study contributes novel data regarding the metabolism of BPS and BP2 in a fish model and shows that phase II conjugation pathways are already functional in 4-dpf-old zebrafish. Comparative analysis of BP2 and BPS metabolic profiles in zebrafish larvae and adults further supports the use of zebrafish embryo as a relevant model in which toxicity and estrogenic activity can be assessed, while taking into account the absorption and fate of tested substances.

  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.

  20. Screening Estrogenic Activities of Chemicals or Mixtures In Vivo Using Transgenic (cyp19a1b-GFP) Zebrafish Embryos

    PubMed Central

    Brion, François; Le Page, Yann; Piccini, Benjamin; Cardoso, Olivier; Tong, Sok-Keng; Chung, Bon-chu; Kah, Olivier

    2012-01-01

    The tg(cyp19a1b-GFP) transgenic zebrafish expresses GFP (green fluorescent protein) under the control of the cyp19a1b gene, encoding brain aromatase. This gene has two major characteristics: (i) it is only expressed in radial glial progenitors in the brain of fish and (ii) it is exquisitely sensitive to estrogens. Based on these properties, we demonstrate that natural or synthetic hormones (alone or in binary mixture), including androgens or progestagens, and industrial chemicals induce a concentration-dependent GFP expression in radial glial progenitors. As GFP expression can be quantified by in vivo imaging, this model presents a very powerful tool to screen and characterize compounds potentially acting as estrogen mimics either directly or after metabolization by the zebrafish embryo. This study also shows that radial glial cells that act as stem cells are direct targets for a large panel of endocrine disruptors, calling for more attention regarding the impact of environmental estrogens and/or certain pharmaceuticals on brain development. Altogether these data identify this in vivo bioassay as an interesting alternative to detect estrogen mimics in hazard and risk assessment perspective. PMID:22586461

  1. Cocaine Modulates the Expression of Opioid Receptors and miR-let-7d in Zebrafish Embryos

    PubMed Central

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

    2012-01-01

    Prenatal exposure to cocaine, in mammals, has been shown to interfere with the expression of opioid receptors, which can have repercussions in its activity. Likewise, microRNAs, such as let-7, have been shown to regulate the expression of opioid receptors and hence their functions in mammals and in vitro experiments. In light of this, using the zebrafish embryos as a model our aim here was to evaluate the actions of cocaine in the expression of opioid receptors and let-7d miRNA during embryogenesis. In order to determine the effects produced by cocaine on the opioid receptors (zfmor, zfdor1 and zfdor2) and let-7d miRNA (dre-let-7d) and its precursors (dre-let-7d-1 and dre-let-7d-2), embryos were exposed to 1.5 µM cocaine hydrochloride (HCl). Our results revealed that cocaine upregulated dre-let-7d and its precursors, and also increased the expression of zfmor, zfdor1 and zfdor2 during early developmental stages and decreased them in late embryonic stages. The changes observed in the expression of opioid receptors might occur through dre-let-7d, since DNA sequences and the morpholinos of opioid receptors microinjections altered the expression of dre-let-7d and its precursors. Likewise, opioid receptors and dre-let-7d showed similar distributions in the central nervous system (CNS) and at the periphery, pointing to a possible interrelationship between them. In conclusion, the silencing and overexpression of opioid receptors altered the expression of dre-let-7d, which points to the notion that cocaine via dre-let-7 can modulate the expression of opioid receptors. Our study provides new insights into the actions of cocaine during zebrafish embryogenesis, indicating a role of miRNAs, let-7d, in development and its relationship with gene expression of opioid receptors, related to pain and addiction process. PMID:23226419

  2. Alterations in zebrafish development induced by simvastatin: Comprehensive morphological and physiological study, focusing on muscle

    PubMed Central

    Campos, Laise M; Rios, Eduardo A; Guapyassu, Livia; Midlej, Victor; Atella, Georgia C; Herculano-Houzel, Suzana; Benchimol, Marlene; Mermelstein, Claudia

    2016-01-01

    The cholesterol synthesis inhibitor simvastatin, which is used to treat cardiovascular diseases, has severe collateral effects. We decided to comprehensively study the effects of simvastatin in zebrafish development and in myogenesis, because zebrafish has been used as a model to human diseases, due to its handling easiness, the optical clarity of its embryos, and the availability of physiological and structural methodologies. Furthermore, muscle is an important target of the drug. We used several simvastatin concentrations at different zebrafish developmental stages and studied survival rate, morphology, and physiology of the embryos. Our results show that high levels of simvastatin induce structural damage whereas low doses induce minor structural changes, impaired movements, and reduced heart beating. Morphological alterations include changes in embryo and somite size and septa shape. Physiological changes include movement reduction and slower heartbeat. These effects could be reversed by the addition of exogenous cholesterol. Moreover, we quantified the total cell number during zebrafish development and demonstrated a large reduction in cell number after statin treatment. Since we could classify the alterations induced by simvastatin in three distinct phenotypes, we speculate that simvastatin acts through more than one mechanism and could affect both cell replication and/or cell death and muscle function. Our data can contribute to the understanding of the molecular and cellular basis of the mechanisms of action of simvastatin. PMID:27444151

  3. Alterations in zebrafish development induced by simvastatin: Comprehensive morphological and physiological study, focusing on muscle.

    PubMed

    Campos, Laise M; Rios, Eduardo A; Guapyassu, Livia; Midlej, Victor; Atella, Georgia C; Herculano-Houzel, Suzana; Benchimol, Marlene; Mermelstein, Claudia; Costa, Manoel L

    2016-11-01

    The cholesterol synthesis inhibitor simvastatin, which is used to treat cardiovascular diseases, has severe collateral effects. We decided to comprehensively study the effects of simvastatin in zebrafish development and in myogenesis, because zebrafish has been used as a model to human diseases, due to its handling easiness, the optical clarity of its embryos, and the availability of physiological and structural methodologies. Furthermore, muscle is an important target of the drug. We used several simvastatin concentrations at different zebrafish developmental stages and studied survival rate, morphology, and physiology of the embryos. Our results show that high levels of simvastatin induce structural damage whereas low doses induce minor structural changes, impaired movements, and reduced heart beating. Morphological alterations include changes in embryo and somite size and septa shape. Physiological changes include movement reduction and slower heartbeat. These effects could be reversed by the addition of exogenous cholesterol. Moreover, we quantified the total cell number during zebrafish development and demonstrated a large reduction in cell number after statin treatment. Since we could classify the alterations induced by simvastatin in three distinct phenotypes, we speculate that simvastatin acts through more than one mechanism and could affect both cell replication and/or cell death and muscle function. Our data can contribute to the understanding of the molecular and cellular basis of the mechanisms of action of simvastatin. © 2016 by the Society for Experimental Biology and Medicine.

  4. Transcriptomic changes in zebrafish embryos and larvae following benzo[a]pyrene exposure

    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...

  5. A Zebrafish Embryo Culture System Defines Factors that Promote Vertebrate Myogenesis across Species

    PubMed Central

    Ciarlo, Christie; Liu, Jingxia; Castiglioni, Alessandra; Price, Emily; Liu, Min; Barton, Elisabeth R.; Kahn, C. Ronald; Wagers, Amy J.; Zon, Leonard I.

    2013-01-01

    SUMMARY Ex vivo expansion of satellite cells and directed differentiation of pluripotent cells to mature skeletal muscle have proved difficult challenges for regenerative biology. Using a zebrafish embryo culture system with reporters of early and late skeletal muscle differentiation, we examined the influence of 2,400 chemicals on myogenesis and identified six that expanded muscle progenitors, including three GSK3β inhibitors, two calpain inhibitors and one adenylyl cyclase activator, forskolin. Forskolin also enhanced proliferation of mouse satellite cells in culture and maintained their ability to engraft muscle in vivo. A combination of bFGF, forskolin and the GSK3β inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs) and produced engraftable myogenic progenitors that contributed to muscle repair in vivo. In summary, these studies reveal functionally conserved pathways regulating myogenesis across species and identify chemical compounds that expand mouse satellite cells and differentiate human iPSCs into engraftable muscle. PMID:24209627

  6. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Danio rerio embryos on Prozac - Effects on the detoxification mechanism and embryo development.

    PubMed

    Cunha, V; Rodrigues, P; Santos, M M; Moradas-Ferreira, P; Ferreira, M

    2016-09-01

    In the past decade the presence of psychopharmaceuticals, including fluoxetine (FLU), in the aquatic environment has been associated with the increasing trend in human consumption of these substances. Aquatic organisms are usually exposed to chronic low doses and, therefore, risk assessments should evaluate the effects of these compounds in non-target organisms. Teleost fish possess an array of active defence mechanisms to cope with the deleterious effects of xenobiotics. These include ABC transporters, phase I and II of cellular detoxification and oxidative stress enzymes. Hence, the present study aimed at characterising the effect of FLU on embryo development of the model teleost zebrafish (Danio rerio) concomitantly with changes in the detoxification mechanisms during early developmental phases. Embryos were exposed to different concentrations of FLU (0.0015, 0.05, 0.1, 0.5 and 0.8μM) for 80hours post fertilization. Development was screened and the impact in the transcription of key genes, i.e., abcb4, abcc1, abcc2, abcg2, cyp1a, cyp3a65, gst, sod, cat, ahr, pxr, pparα, pparβ, pparγ, rxraa, rxrab, rxrbb, rxrga, rxrgb, raraa, rarab, rarga evaluated. In addition, accumulation assays were performed to measure the activity of ABC proteins and antioxidant enzymes (CAT and Cu/ZnSOD) after exposure to FLU. Embryo development was disrupted at the lowest FLU concentration tested (0.0015μM), which is in the range of concentrations found in WWTP effluents. Embryos exposed to higher concentrations of FLU decreased Cu/Zn SOD, and increased CAT (0.0015 and 0.5μM) enzymatic activity. Exposure to higher concentrations of FLU decreased the expression of most genes belonging to the detoxification system and upregulated cat at 0.0015μM of FLU. Most of the tested concentrations downregulated pparα, pparβ, pparγ, and raraa, rxraa, rxrab, rxrbb rxrgb and ahr gene expression while pxr was significantly up regulated at all tested concentrations. In conclusion, this study

  8. 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.

  9. Proteomic Signatures of the Zebrafish (Danio rerio) Embryo: Sensitivity and Specificity in Toxicity Assessment of Chemicals.

    PubMed

    Hanisch, Karen; Küster, Eberhard; Altenburger, Rolf; Gündel, Ulrike

    2010-01-01

    Studies using embryos of the zebrafish Danio rerio (DarT) instead of adult fish for characterising the (eco-) toxic potential of chemicals have been proposed as animal replacing methods. Effect analysis at the molecular level might enhance sensitivity, specificity, and predictive value of the embryonal studies. The present paper aimed to test the potential of toxicoproteomics with zebrafish eleutheroembryos for sensitive and specific toxicity assessment. 2-DE-based toxicoproteomics was performed applying low-dose (EC(10)) exposure for 48 h with three-model substances Rotenone, 4,6-dinitro-o-cresol (DNOC) and Diclofenac. By multivariate "pattern-only" PCA and univariate statistical analyses, alterations in the embryonal proteome were detectable in nonetheless visibly intact organisms and treatment with the three substances was distinguishable at the molecular level. Toxicoproteomics enabled the enhancement of sensitivity and specificity of the embryonal toxicity assay and bear the potency to identify protein markers serving as general stress markers and early diagnosis of toxic stress.

  10. Proteomic Signatures of the Zebrafish (Danio rerio) Embryo: Sensitivity and Specificity in Toxicity Assessment of Chemicals

    PubMed Central

    Hanisch, Karen; Küster, Eberhard; Altenburger, Rolf; Gündel, Ulrike

    2010-01-01

    Studies using embryos of the zebrafish Danio rerio (DarT) instead of adult fish for characterising the (eco-) toxic potential of chemicals have been proposed as animal replacing methods. Effect analysis at the molecular level might enhance sensitivity, specificity, and predictive value of the embryonal studies. The present paper aimed to test the potential of toxicoproteomics with zebrafish eleutheroembryos for sensitive and specific toxicity assessment. 2-DE-based toxicoproteomics was performed applying low-dose (EC10) exposure for 48 h with three-model substances Rotenone, 4,6-dinitro-o-cresol (DNOC) and Diclofenac. By multivariate “pattern-only” PCA and univariate statistical analyses, alterations in the embryonal proteome were detectable in nonetheless visibly intact organisms and treatment with the three substances was distinguishable at the molecular level. Toxicoproteomics enabled the enhancement of sensitivity and specificity of the embryonal toxicity assay and bear the potency to identify protein markers serving as general stress markers and early diagnosis of toxic stress. PMID:22084678

  11. 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

  12. Effect of Photon Hormesis on Dose Responses to Alpha Particles in Zebrafish Embryos.

    PubMed

    Ng, Candy Yuen Ping; Cheng, Shuk Han; Yu, Kwan Ngok

    2017-02-11

    Photon hormesis refers to the phenomenon where the biological effect of ionizing radiation with a high linear energy transfer (LET) value is diminished by photons with a low LET value. The present paper studied the effect of photon hormesis from X-rays on dose responses to alpha particles using embryos of the zebrafish ( Danio rerio ) as the in vivo vertebrate model. The toxicity of these ionizing radiations in the zebrafish embryos was assessed using the apoptotic counts at 20, 24, or 30 h post fertilization (hpf) revealed through acridine orange (AO) staining. For alpha-particle doses ≥ 4.4 mGy, the additional X-ray dose of 10 mGy significantly reduced the number of apoptotic cells at 24 hpf, which proved the presence of photon hormesis. Smaller alpha-particle doses might not have inflicted sufficient aggregate damages to trigger photon hormesis. The time gap T between the X-ray (10 mGy) and alpha-particle (4.4 mGy) exposures was also studied. Photon hormesis was present when T ≤ 30 min, but was absent when T = 60 min, at which time repair of damage induced by alpha particles would have completed to prevent their interactions with those induced by X-rays. Finally, the drop in the apoptotic counts at 24 hpf due to photon hormesis was explained by bringing the apoptotic events earlier to 20 hpf, which strongly supported the removal of aberrant cells through apoptosis as an underlying mechanism for photon hormesis.

  13. Xenografts in zebrafish embryos as a rapid functional assay for breast cancer stem-like cell identification.

    PubMed

    Eguiara, Arrate; Holgado, Olaia; Beloqui, Izaskun; Abalde, Leire; Sanchez, Yolanda; Callol, Carles; Martin, Angel G

    2011-11-01

    The cancer stem cell is defined by its capacity to self-renew, the potential to differentiate into all cells of the tumor and the ability to proliferate and drive the expansion of the tumor. Thus, targeting these cells may provide novel anti-cancer treatment strategies. Breast cancer stem cells have been isolated according to surface marker expression, ability to efflux fluorescent dyes, increased activity of aldehyde dehydrogenase or the capacity to form spheres in non-adherent culture conditions. In order to test novel drugs directed towards modulating self-renewal of cancer stem cells, rapid, easy and inexpensive assays must be developed. Using 2 days-post-fertilization (dpf) zebrafish embryos as transplant recipients, we show that cells grown in mammospheres from breast carcinoma cell lines migrate to the tail of the embryo and form masses with a significantly higher frequency than parental monolayer populations. When stem-like self-renewal was targeted in the parental population by the use of the dietary supplement curcumin, cell migration and mass formation were reduced, indicating that these effects were associated with stem-like cell content. This is a proof of principle report that proposes a rapid and inexpensive assay to target in vivo cancer stem-like cells, which may be used to unravel basic cancer stem cell biology and for drug screening.

  14. Teratological Effects of a Panel of Sixty Water-Soluble Toxicants on Zebrafish Development

    PubMed Central

    Ali, Shaukat; Aalders, Jeffrey

    2014-01-01

    Abstract The zebrafish larva is a promising whole-animal model for safety pharmacology, environmental risk assessment, and developmental toxicity. This model has been used for the high-throughput toxicity screening of various compounds. Our aim here is to identify possible phenotypic markers of teratogenicity in zebrafish embryos that could be used for the assaying compounds for reproductive toxicity. We have screened a panel of 60 water-soluble toxicants to examine their effects on zebrafish development. A total of 22,080 wild-type zebrafish larvae were raised in 250 μL defined buffer in 96-well plates at a plating density of one embryo per well. They were exposed for a 96-h period starting at 24 h post-fertilization. A logarithmic concentration series was used for range-finding, followed by a narrower geometric series for developmental toxicity assessment. A total of 9017 survivors were analyzed at 5 days post-fertilization for nine phenotypes, namely, (1) normal, (2) pericardial oedema, (3) yolk sac oedema, (4) melanophores dispersed, (5) bent tail tip, (6) bent body axis, (7) abnormal Meckel's cartilage, (8) abnormal branchial arches, and (9) uninflated swim bladder. For each toxicant, the EC50 (concentration required to produce one or more of these abnormalities in 50% of embryos) was also calculated. For the majority of toxicants (55/60) there was, at the population level, a statistically significant, concentration-dependent increase in the incidence of abnormal phenotypes among survivors. The commonest abnormalities were pericardial oedema, yolk sac oedema, dispersed melanophores, and uninflated swim bladder. It is possible therefore that these could prove to be general indicators of reproductive toxicity in the zebrafish embryo assay. PMID:24650241

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

    PubMed

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

    2016-12-01

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

  16. Photon hormesis deactivates alpha-particle induced bystander effects between zebrafish embryos

    NASA Astrophysics Data System (ADS)

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

    2017-04-01

    In the present work, we studied the effects of low-dose X-ray photons on the alpha-particle induced bystander effects between embryos of the zebrafish, Danio rerio. The effects on the naive whole embryos were studied through quantification of apoptotic signals (amounts of cells undergoing apoptosis) at 24 h post fertilization (hpf) using vital dye acridine orange staining, followed by counting the stained cells under a fluorescent microscope. We report data showing that embryos at 5 hpf subjected to a 4.4 mGy alpha-particle irradiation could release a stress signal into the medium, which could induce bystander effect in partnered naive embryos sharing the same medium. We also report that the bystander effect was deactivated when the irradiated embryos were subjected to a concomitant irradiation of 10 or 14 mGy of X-rays, but no such deactivation was achieved if the concomitant X-ray dose dropped to 2.5 or 5 mGy. In the present study, the significant drop in the amount of apoptotic signals on the embryos having received 4.4 mGy alpha particles together X-rays irradiation from 2.5 or 5 mGy to 10 or 14 mGy, together with the deactivation of RIBE with concomitant irradiation of 10 or 14 mGy of X-rays supported the participation of photon hormesis with an onset dose between 5 and 10 mGy, which might lead to removal of aberrant cells through early apoptosis or induction of high-fidelity DNA repair. As we found that photons and alpha particles could have opposite biological effects when these were simultaneously irradiated onto living organisms, these ionizing radiations could be viewed as two different environmental stressors, and the resultant effects could be regarded as multiple stressor effects. The present work presented the first study on a multiple stressor effect which occurred on bystander organisms. In other words, this was a non-targeted multiple stressor effect. The photon hormesis could also explain some failed attempts to observe neutron-induced bystander

  17. Alternative methods for toxicity assessments in fish: comparison of the fish embryo toxicity and the larval growth and survival tests in zebrafish and fathead minnows.

    PubMed

    Jeffries, Marlo K Sellin; Stultz, Amy E; Smith, Austin W; Rawlings, Jane M; Belanger, Scott E; Oris, James T

    2014-11-01

    An increased demand for chemical toxicity evaluations has resulted in the need for alternative testing strategies that address animal welfare concerns. The fish embryo toxicity (FET) test developed for zebrafish (Danio rerio) is one such alternative, and the application of the FET test to other species such as the fathead minnow (Pimephales promelas) has been proposed. In the present study, the performances of the FET test and the larval growth and survival (LGS; a standard toxicity testing method) test in zebrafish and fathead minnows were evaluated. This required that testing methods for the fathead minnow FET and zebrafish LGS tests be harmonized with existing test methods and that the performance of these testing strategies be evaluated by comparing the median lethal concentrations of 2 reference toxicants, 3,4-dicholoraniline and ammonia, obtained via each of the test types. The results showed that procedures for the zebrafish FET test can be adapted and applied to the fathead minnow. Differences in test sensitivity were observed for 3,4-dicholoraniline but not ammonia; therefore, conclusions regarding which test types offer the least or most sensitivity could not be made. Overall, these results show that the fathead minnow FET test has potential as an alternative toxicity testing strategy and that further analysis with other toxicants is warranted in an effort to better characterize the sensitivity and feasibility of this testing strategy. © 2014 SETAC.

  18. 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

  19. Zebrafish cardiac development requires a conserved secondary heart field

    PubMed Central

    Hami, Danyal; Grimes, Adrian C.; Tsai, Huai-Jen; Kirby, Margaret L.

    2011-01-01

    The secondary heart field is a conserved developmental domain in avian and mammalian embryos that contributes myocardium and smooth muscle to the definitive cardiac arterial pole. This field is part of the overall heart field and its myocardial component has been fate mapped from the epiblast to the heart in both mammals and birds. In this study we show that the population that gives rise to the arterial pole of the zebrafish can be traced from the epiblast, is a discrete part of the mesodermal heart field, and contributes myocardium after initial heart tube formation, giving rise to both smooth muscle and myocardium. We also show that Isl1, a transcription factor associated with undifferentiated cells in the secondary heart field in other species, is active in this field. Furthermore, Bmp signaling promotes myocardial differentiation from the arterial pole progenitor population, whereas inhibiting Smad1/5/8 phosphorylation leads to reduced myocardial differentiation with subsequent increased smooth muscle differentiation. Molecular pathways required for secondary heart field development are conserved in teleosts, as we demonstrate that the transcription factor Tbx1 and the Sonic hedgehog pathway are necessary for normal development of the zebrafish arterial pole. PMID:21558385

  20. Combinatorial effects of zinc deficiency and arsenic exposure on zebrafish (Danio rerio) development

    PubMed Central

    Truong, Lisa; Barton, Carrie L.; Chase, Tyler T.; Gonnerman, Greg D.; Wong, Carmen P.; Tanguay, Robert L.; Ho, Emily

    2017-01-01

    Zinc deficiency and chronic low level exposures to inorganic arsenic in drinking water are both significant public health concerns that affect millions of people including pregnant women. These two conditions can co-exist in the human population but little is known about their interaction, and in particular, whether zinc deficiency sensitizes individuals to arsenic exposure and toxicity, especially during critical windows of development. To address this, we utilized the Danio rerio (zebrafish) model to test the hypothesis that parental zinc deficiency sensitizes the developing embryo to low-concentration arsenic toxicity, leading to altered developmental outcomes. Adult zebrafish were fed defined zinc deficient and zinc adequate diets and were spawned resulting in zinc adequate and zinc deficient embryos. The embryos were treated with environmentally relevant concentrations of 0, 50, and 500 ppb arsenic. Arsenic exposure significantly reduced the amount of zinc in the developing embryo by ~7%. The combination of zinc deficiency and low-level arsenic exposures did not sensitize the developing embryo to increased developmental malformations or mortality. The combination did cause a 40% decline in physical activity of the embryos, and this decline was significantly greater than what was observed with zinc deficiency or arsenic exposure alone. Significant changes in RNA expression of genes that regulate zinc homeostasis, response to oxidative stress and insulin production (including zip1, znt7, nrf2, ogg1, pax4, and insa) were found in zinc deficient, or zinc deficiency and arsenic exposed embryos. Overall, the data suggests that the combination of zinc deficiency and arsenic exposure has harmful effects on the developing embryo and may increase the risk for developing chronic diseases like diabetes. PMID:28837703

  1. Myosin-1 inhibition by PClP affects membrane shape, cortical actin distribution and lipid droplet dynamics in early Zebrafish embryos

    PubMed Central

    Gupta, Prabuddha; Martin, René; Knölker, Hans-Joachim; Nihalani, Deepak; Kumar Sinha, Deepak

    2017-01-01

    Myosin-1 (Myo1) represents a mechanical link between the membrane and actin-cytoskeleton in animal cells. We have studied the effect of Myo1 inhibitor PClP in 1–8 cell Zebrafish embryos. Our results indicate a unique involvement of Myo1 in early development of Zebrafish embryos. Inhibition of Myo1 (by PClP) and Myo2 (by Blebbistatin) lead to arrest in cell division. While Myo1 isoforms appears to be important for both the formation and the maintenance of cleavage furrows, Myo2 is required only for the formation of furrows. We found that the blastodisc of the embryo, which contains a thick actin cortex (~13 μm), is loaded with cortical Myo1. Myo1 appears to be crucial for maintaining the blastodisc morphology and the actin cortex thickness. In addition to cell division and furrow formation, inhibition of Myo1 has a drastic effect on the dynamics and distribution of lipid droplets (LDs) in the blastodisc near the cleavage furrow. All these results above are effects of Myo1 inhibition exclusively; Myo2 inhibition by blebbistatin does not show such phenotypes. Therefore, our results demonstrate a potential role for Myo1 in the maintenance and formation of furrow, blastodisc morphology, cell-division and LD organization within the blastodisc during early embryogenesis. PMID:28678859

  2. A Versatile Mounting Method for Long Term Imaging of Zebrafish Development.

    PubMed

    Hirsinger, Estelle; Steventon, Ben

    2017-01-26

    Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

  3. Progesterone and norgestrel alter transcriptional expression of genes along the hypothalamic-pituitary-thyroid axis in zebrafish embryos-larvae.

    PubMed

    Liang, Yan-Qiu; Huang, Guo-Yong; Ying, Guang-Guo; Liu, Shuang-Shuang; Jiang, Yu-Xia; Liu, Shan

    2015-01-01

    The aim of this study was to investigate the effects of progestins on the hypothalamic-pituitary-thyroid (HPT) axis in the early stage of zebrafish. Zebrafish embryos were exposed to progesterone (P4) or norgestrel (NGT) at 5, 50 and 100 ng L(-1) for 144 h post fertilization (hpf), and the transcriptional levels of target genes along the hypothalamic-pituitary-thyroid axis were determined daily. The results showed that P4 had only minor effects on the mRNA expression of thyroglobulin (Tg), iodothyronine deiodinase type Ι (Dio1) and thyroid hormone receptor β (Thrb) genes. Similarly, the effects of NGT on transcripts of thyrotropin-releasing hormone (Trh), Dio1, iodothyronine deiodinase type II (Dio2) and thyroid hormone receptor α (Thra) genes were generally low. In addition, NGT resulted in some alterations of Tg and Thrb transcripts at different time points. However, a strong induction of Nis mRNA by P4 and NGT was observed in zebrafish embryos-larvae. The overall results showed that besides Nis no effects on the hypothalamic-pituitary-thyroid (HPT) axis are observed following exposure to P4 and NGT, which imply that both P4 and NGT have potential effects on the thyroid endocrine system by inducing transcript of Nis gene during the early stage of zebrafish. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Light-controlled cellular internalization and cytotoxicity of nucleic acid-binding agents. Studies in vitro and in zebrafish embryos

    PubMed Central

    Penas, Cristina; Sánchez, Mateo I.; Guerra-Varela, Jorge; Sanchez-Piñón, Laura; Vázquez, M. Eugenio; Mascareñas, José L.

    2016-01-01

    We have synthesized oligoarginine conjugates of selected DNA-binding agents (a bisbenzamidine, acridine and thiazole orange) and demonstrated that the DNA binding and cell internalization properties of such conjugates can be inhibited by appending a negatively charged oligoglutamic tail through a photolabile linker. Irradiation with UV light releases the parent octaarginine conjugates, thus restoring their cell internalization and biological activity. Preliminary assays using zebrafish embryos demonstrates the potential of this prodrug strategy for controlling in vivo cytotoxicity. PMID:26534774

  5. The transcriptional activator ZNF143 is essential for normal development in zebrafish

    PubMed Central

    2012-01-01

    Background ZNF143 is a sequence-specific DNA-binding protein that stimulates transcription of both small RNA genes by RNA polymerase II or III, or protein-coding genes by RNA polymerase II, using separable activating domains. We describe phenotypic effects following knockdown of this protein in developing Danio rerio (zebrafish) embryos by injection of morpholino antisense oligonucleotides that target znf143 mRNA. Results The loss of function phenotype is pleiotropic and includes a broad array of abnormalities including defects in heart, blood, ear and midbrain hindbrain boundary. Defects are rescued by coinjection of synthetic mRNA encoding full-length ZNF143 protein, but not by protein lacking the amino-terminal activation domains. Accordingly, expression of several marker genes is affected following knockdown, including GATA-binding protein 1 (gata1), cardiac myosin light chain 2 (cmlc2) and paired box gene 2a (pax2a). The zebrafish pax2a gene proximal promoter contains two binding sites for ZNF143, and reporter gene transcription driven by this promoter in transfected cells is activated by this protein. Conclusions Normal development of zebrafish embryos requires ZNF143. Furthermore, the pax2a gene is probably one example of many protein-coding gene targets of ZNF143 during zebrafish development. PMID:22268977

  6. The transcriptional activator ZNF143 is essential for normal development in zebrafish.

    PubMed

    Halbig, Kari M; Lekven, Arne C; Kunkel, Gary R

    2012-01-23

    ZNF143 is a sequence-specific DNA-binding protein that stimulates transcription of both small RNA genes by RNA polymerase II or III, or protein-coding genes by RNA polymerase II, using separable activating domains. We describe phenotypic effects following knockdown of this protein in developing Danio rerio (zebrafish) embryos by injection of morpholino antisense oligonucleotides that target znf143 mRNA. The loss of function phenotype is pleiotropic and includes a broad array of abnormalities including defects in heart, blood, ear and midbrain hindbrain boundary. Defects are rescued by coinjection of synthetic mRNA encoding full-length ZNF143 protein, but not by protein lacking the amino-terminal activation domains. Accordingly, expression of several marker genes is affected following knockdown, including GATA-binding protein 1 (gata1), cardiac myosin light chain 2 (cmlc2) and paired box gene 2a (pax2a). The zebrafish pax2a gene proximal promoter contains two binding sites for ZNF143, and reporter gene transcription driven by this promoter in transfected cells is activated by this protein. Normal development of zebrafish embryos requires ZNF143. Furthermore, the pax2a gene is probably one example of many protein-coding gene targets of ZNF143 during zebrafish development.

  7. A novel one-pot green synthesis of selenium nanoparticles and evaluation of its toxicity in zebrafish embryos.

    PubMed

    Kalishwaralal, Kalimuthu; Jeyabharathi, Subhaschandrabose; Sundar, Krishnan; Muthukumaran, Azhaguchamy

    2016-01-01

    Over the last 50 years, compelling evidence has accumulated on the beneficial role of selenium in human health. In the present study, different proteins were evaluated as reducing agents for the eco-friendly synthesis of selenium nanoparticles from an aqueous solution of sodium selenite. This method is a simple, low cost green synthesis alternative to chemical synthesis. The high conversion of selenium ions to selenium nanoparticles (SeNPs) was achieved by a reaction mixture of 0.1 g bovine serum albumin and 0.1 g sodium selenite at a reaction temperature of 121°C for 20 min duration. The selenium nanoparticles were characterized by fourier transform infrared (FTIR), scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy. The FTIR spectral bands were sharp with strong absorption peaks at 1649 and 1551 cm(-1). SEM analysis of the synthesized selenium nanoparticles clearly showed the spherical shape with an average size ranging from 500 to 600 nm. The toxicity of SeNPs was evaluated using zebrafish embryos as a model system. SeNPs induced malformations in zebrafish embryos in a concentration-dependent manner. Selenium nanoparticles at 15-25 μg/ml concentration caused pericardial edema, tail malformation and decrease in heart rate in zebrafish embryos. Treatments with lower concentrations did not alter the heart rate or display any heart abnormalities. This study underlines the importance of identifying optimal SeNP concentration that could have potential therapeutic applications.

  8. Effects of probiotic administration on zebrafish development and reproduction.

    PubMed

    Carnevali, O; Avella, M A; Gioacchini, G

    2013-07-01

    As the consumption of probiotics increases worldwide, scientists focus on identifying bacterial strains able to improve human life quality and evidence the biological pathways affected by probiotic treatment. In this review, some recent observations on the effects of changes of microbiota on zebrafish metabolism were discussed. In addition, the effects of Lactobacillus rhamnosus - a component of the human gut microflora - as a diet supplement on Danio rerio were presented. When administered chronically, L. rhamnosus may affect larval development and the physiology of reproductive system in the zebrafish model. It was hypothesized exogenous L. rhamnosus accelerates larval growth and backbone development by acting on insulin-like growth factors-I (igfI) and -II (igfII), peroxisome proliferator activated receptors-α and -β, (pparα,β) vitamin D receptor-α (vdrα) and retinoic acid receptor-γ (rarγ). Gonadal differentiation was anticipated at 6weeks together with a higher expression of gnrh3 at the larval stage when L. rhamnosus was administered throughout development. Moreover, brood stock alimented with a L. rhamnosus-supplemented diet showed better reproductive performances as per follicles development, ovulated oocytes quantification and embryos quality. A plausible involvement of factors such as leptin, and kiss1 and 2 in the improvements was concluded. The observations made on the physiology of female reproduction were correlated with the gene expression of a gigantic number of factors as the aromatase cytochrome p 19 (cyp19a), the vitellogenin (vtg) and the α isoform of the E2 receptor (erα), luteinizing hormone receptor (lhr), 20-β hydroxysteroid dehydrogenase (20β-hsd), membrane progesterone receptors α and β, cyclin B, activinβA1, smad2, transforming growth factor β1 (tgfβ1), growth differentiation factor9 (gdf9) and bone morphogenetic protein15 (bmp15.) A model in which the exogenous L. rhamnosus in the digestive tract of zebrafish from the

  9. Teratogenic and toxic effects of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt.:Fr.) P. Karst. (higher Basidiomycetes), on zebrafish embryo as model.

    PubMed

    Dulay, Rich Milton R; Kalaw, Sofronio P; Reyes, Renato G; Alfonso, Noel F; Eguchi, Fumio

    2012-01-01

    This paper highlights the teratogenic and toxic effects of Ganoderma lucidum (Lingzhi or Reishi mushroom) extract on zebrafish embryos. Hatchability, malformations, and lethality rate of zebrafish embryos were assessed to provide valuable information regarding the potential teratogenic activity of G. lucidum. Hatching was completed 48 h post treatment application (hpta) at 1% or lower concentrations of extract and embryo water. The hatching rate of embryos treated with 5% or higher concentrations was significantly lower (p> 0.05) than the control. Tail malformation was the most marked morphological abnormality in embryos at 72 hpta, which was obviously caused by 1% extract (55.56% tail malformation) and was observed in all embryos exposed to 5% of extract. Growth retardation was evident in embryos exposed to 5%, 10%, and 20%. However, lethal effect of extract of G. lucidum was dependent on dose and time of exposure. Mortality rates of embryos treated with 5% (44.44%) or higher concentrations of the extract was significantly higher (p > 0.05) than that of the control embryos at 72 hpta. These results suggest that G. lucidum extract has lethal and sub-lethal effects on zebrafish embryos.

  10. In vivo cell biology in zebrafish - providing insights into vertebrate development and disease.

    PubMed

    Vacaru, Ana M; Unlu, Gokhan; Spitzner, Marie; Mione, Marina; Knapik, Ela W; Sadler, Kirsten C

    2014-02-01

    Over the past decades, studies using zebrafish have significantly advanced our understanding of the cellular basis for development and human diseases. Zebrafish have rapidly developing transparent embryos that allow comprehensive imaging of embryogenesis combined with powerful genetic approaches. However, forward genetic screens in zebrafish have generated unanticipated findings that are mirrored by human genetic studies: disruption of genes implicated in basic cellular processes, such as protein secretion or cytoskeletal dynamics, causes discrete developmental or disease phenotypes. This is surprising because many processes that were assumed to be fundamental to the function and survival of all cell types appear instead to be regulated by cell-specific mechanisms. Such discoveries are facilitated by experiments in whole animals, where zebrafish provides an ideal model for visualization and manipulation of organelles and cellular processes in a live vertebrate. Here, we review well-characterized mutants and newly developed tools that underscore this notion. We focus on the secretory pathway and microtubule-based trafficking as illustrative examples of how studying cell biology in vivo using zebrafish has broadened our understanding of the role fundamental cellular processes play in embryogenesis and disease.

  11. A novel TRIM family member, Trim69, regulates zebrafish development through p53-mediated apoptosis.

    PubMed

    Han, Ruiqin; Zhao, Qing; Zong, Shudong; Miao, Shiying; Song, Wei; Wang, Linfang

    2016-05-01

    Trim69 contains the hallmark domains of a tripartite motif (TRIM) protein, including a Ring-finger domain, B-box domain, and coiled-coil domain. Trim69 is structurally and evolutionarily conserved in zebrafish, mouse, rat, human, and chimpanzee. The role of this protein is unclear, however, so we investigated its function in zebrafish development. Trim69 is extensively expressed in zebrafish adults and developing embryos-particularly in the testis, brain, ovary, and heart-and its expression decreases in a time- and stage-dependent manner. Loss of trim69 in zebrafish induces apoptosis and activates apoptosis-related processes; indeed, the tp53 pathway was up-regulated in response to the knockdown. Expression of human trim69 rescued the apoptotic phenotype, while overexpression of trim69 does not increase cellular apoptosis. Taken together, our results suggest that trim69 participates in tp53-mediated apoptosis during zebrafish development. Mol. Reprod. Dev. 83: 442-454, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  12. In vivo cell biology in zebrafish – providing insights into vertebrate development and disease

    PubMed Central

    Vacaru, Ana M.; Unlu, Gokhan; Spitzner, Marie; Mione, Marina; Knapik, Ela W.; Sadler, Kirsten C.

    2014-01-01

    ABSTRACT Over the past decades, studies using zebrafish have significantly advanced our understanding of the cellular basis for development and human diseases. Zebrafish have rapidly developing transparent embryos that allow comprehensive imaging of embryogenesis combined with powerful genetic approaches. However, forward genetic screens in zebrafish have generated unanticipated findings that are mirrored by human genetic studies: disruption of genes implicated in basic cellular processes, such as protein secretion or cytoskeletal dynamics, causes discrete developmental or disease phenotypes. This is surprising because many processes that were assumed to be fundamental to the function and survival of all cell types appear instead to be regulated by cell-specific mechanisms. Such discoveries are facilitated by experiments in whole animals, where zebrafish provides an ideal model for visualization and manipulation of organelles and cellular processes in a live vertebrate. Here, we review well-characterized mutants and newly developed tools that underscore this notion. We focus on the secretory pathway and microtubule-based trafficking as illustrative examples of how studying cell biology in vivo using zebrafish has broadened our understanding of the role fundamental cellular processes play in embryogenesis and disease. PMID:24481493

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

    PubMed

    Bagatto, Brian

    2009-06-01

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

  14. Ca2+ signalling and early embryonic patterning during zebrafish development.

    PubMed

    Webb, Sarah E; Miller, Andrew L

    2007-09-01

    1. It has been proposed that Ca2+ signalling, in the form of pulses, waves and steady gradients, may play a crucial role in key pattern-forming events during early vertebrate development. 2. With reference to the embryo of the zebrafish (Danio rerio), herein we review the Ca2+ transients reported from the cleavage to segmentation periods. This time-window includes most of the major pattern-forming events of early development, which transform a single-cell zygote into a complex multicellular embryo with established primary germ layers and body axes. 3. Data are presented to support our proposal that intracellular Ca2+ waves are an essential feature of embryonic cytokinesis and that propagating intercellular Ca2+ waves (both long and short range) may play a crucial role in: (i) the establishment of the embryonic periderm and the coordination of cell movements during epiboly, convergence and extension; (ii) the establishment of the basic embryonic axes and germ layers; and (iii) definition of the morphological boundaries of specific tissue domains and embryonic structures, including future organ anlagen. 4. The potential downstream targets of these Ca2+ transients are also discussed, as well as how they may integrate with other pattern-forming signalling pathways known to modulate early developmental events.

  15. 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. Copyright © 2016 Elsevier Inc. All rights reserved.

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  18. Predicting adult fish acute lethality with the zebrafish embryo: relevance of test duration, endpoints, compound properties, and exposure concentration analysis.

    PubMed

    Knöbel, Melanie; Busser, Frans J M; Rico-Rico, Angeles; Kramer, Nynke I; Hermens, Joop L M; Hafner, Christoph; Tanneberger, Katrin; Schirmer, Kristin; Scholz, Stefan

    2012-09-04

    The zebrafish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, which is required by various regulations for environmental risk assessment of chemicals. We investigated the reliability of the embryo test by probing organic industrial chemicals with a wide range of physicochemical properties, toxicities, and modes of toxic action. Moreover, the relevance of using measured versus nominal (intended) exposure concentrations, inclusion of sublethal endpoints, and different exposure durations for the comparability with reported fish acute toxicity was explored. Our results confirm a very strong correlation of zebrafish embryo to fish acute toxicity. When toxicity values were calculated based on measured exposure concentrations, the slope of the type II regression line was 1 and nearly passed through the origin (1 to 1 correlation). Measured concentrations also explained several apparent outliers. Neither prolonged exposure (up to 120 h) nor consideration of sublethal effects led to a reduced number of outliers. Yet, two types of compounds were less lethal to embryos than to adult fish: a neurotoxic compound acting via sodium channels (permethrin) and a compound requiring metabolic activation (allyl alcohol).

  19. Visualizing morphogenesis in transgenic zebrafish embryos using BODIPY TR methyl ester dye as a vital counterstain for GFP.

    PubMed

    Cooper, Mark S; Szeto, Daniel P; Sommers-Herivel, Greg; Topczewski, Jacek; Solnica-Krezel, Lila; Kang, Hee-Chol; Johnson, Iain; Kimelman, David

    2005-02-01

    Green fluorescent protein (GFP) technology is rapidly advancing the study of morphogenesis, by allowing researchers to specifically focus on a subset of labeled cells within the living embryo. However, when imaging GFP-labeled cells using confocal microscopy, it is often essential to simultaneously visualize all of the cells in the embryo using dual-channel fluorescence to provide an embryological context for the cells expressing GFP. Although various counterstains are available, part of their fluorescence overlaps with the GFP emission spectra, making it difficult to clearly identify the cells expressing GFP. In this study, we report that a new fluorophore, BODIPY TR methyl ester dye, serves as a versatile vital counterstain for visualizing the cellular dynamics of morphogenesis within living GFP transgenic zebrafish embryos. The fluorescence of this photostable synthetic dye is spectrally separate from GFP fluorescence, allowing dual-channel, three-dimensional (3D) and four-dimensional (4D) confocal image data sets of living specimens to be easily acquired. These image data sets can be rendered subsequently into uniquely informative 3D and 4D visualizations using computer-assisted visualization software. We discuss a variety of immediate and potential applications of BODIPY TR methyl ester dye as a vital visualization counterstain for GFP in transgenic zebrafish embryos. Copyright 2004 Wiley-Liss, Inc.

  20. Developmental and metabolic responses of zebrafish (Danio rerio) embryos and larvae to short-chain chlorinated paraffins (SCCPs) exposure.

    PubMed

    Ren, Xiaoqian; Zhang, Haijun; Geng, Ningbo; Xing, Liguo; Zhao, Yu; Wang, Feidi; Chen, Jiping

    2018-05-01

    Short-chain chlorinated paraffins (SCCPs) are highly toxic to aquatic organisms, but their toxicity is yet not well characterized. In this study, the developmental toxicity of SCCPs to zebrafish embryos/larvae was evaluated, and a metabolomics approach was adopted to explore the impact of SCCPs exposure on the metabolism in zebrafish embryos. Exposure to SCCPs at concentrations of 1-200μg/L did not produce an observable effect on the hatching rate and morphological deformities of zebrafish embryos/larvae. However, the survival rate of zebrafish larvae in SCCPs exposure groups decreased in a concentration-dependent manner. The 13-day 50% lethal concentration (LC 50 ) value of SCCPs was calculated to be 34.4μg/L. Exposure to SCCPs induced a significant change of overall metabolism, even at environmentally relevant concentrations (1-5μg/L). The most relevant pathways affected by SCCPs exposure were glycerophospholipid metabolism, fatty acid metabolism and purine metabolism. Exposure to SCCPs at concentrations of 1-5μg/L had begun to accelerate the β-oxidation of unsaturated fatty acids and very long chain fatty acids, and affect the transformation of guanine to xanthine in the pathway of purine metabolism. Furthermore, when the exposure concentrations of SCCPs were increased to 50-200μg/L, the levels of phospholipids and amino acids were significantly raised; whereas the levels of fatty acids, carnitines and inosine were significantly decreased. In view of the significant effect on metabolism, the sub-chronic and chronic toxicity of SCCPs to fish should be concerned. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Physiological roles of glucocorticoids during early embryonic development of the zebrafish (Danio rerio)

    PubMed Central

    Wilson, K S; Matrone, G; Livingstone, D E W; Al-Dujaili, E A S; Mullins, J J; Tucker, C S; Hadoke, P W F; Kenyon, C J; Denvir, M A

    2013-01-01

    While glucocorticoids (GCs) are known to be present in the zebrafish embryo, little is known about their physiological roles at this stage. We hypothesised that GCs play key roles in stress response, hatching and swim activity during early development. To test this, whole embryo cortisol (WEC) and corticosteroid-related genes were measured in embryos from 6 to 120 h post fertilisation (hpf) by enzyme linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). Stress response was assessed by change in WEC following stirring, hypoxia or brief electrical impulses applied to the bathing water. The impact of pharmacological and molecular GC manipulation on the stress response, spontaneous hatching and swim activity at different stages of development was also assessed. WEC levels demonstrated a biphasic pattern during development with a decrease from 0 to 36 hpf followed by a progressive increase towards 120 hpf. This was accompanied by a significant and sustained increase in the expression of genes encoding cyp11b1 (GC biosynthesis), hsd11b2 (GC metabolism) and gr (GC receptor) from 48 to 120 hpf. Metyrapone (Met), an inhibitor of 11β-hydroxylase (encoded by cyp11b1), and cyp11b1 morpholino (Mo) knockdown significantly reduced basal and stress-induced WEC levels at 72 and 120 hpf but not at 24 hpf. Spontaneous hatching and swim activity were significantly affected by manipulation of GC action from approximately 48 hpf onwards. We have identified a number of key roles of GCs in zebrafish embryos contributing to adaptive physiological responses under adverse conditions. The ability to alter GC action in the zebrafish embryo also highlights its potential value for GC research. PMID:24167225

  2. Methylparaben induces malformations and alterations on apoptosis, oxidant-antioxidant status, ccnd1 and myca expressions in zebrafish embryos.

    PubMed

    Ateş, Perihan Seda; Ünal, İsmail; Üstündağ, Ünsal Veli; Alturfan, Ahmet Ata; Yiğitbaşı, Türkan; Emekli-Alturfan, Ebru

    2018-03-01

    Methylparabens (MP) are widely used as preservatives in cosmetics, pharmacy, and food industry. Although acute toxicity studies in animals indicated that parabens are not significantly toxic, the effects of chronic exposure under sublethal doses are still unknown and the number of related studies is limited. Our aim was to evaluate the effects of MP on the development of zebrafish embryos focusing on development, locomotor activity, oxidant-antioxidant status, apoptosis, and ccnd1 and myca expressions. The expressions of ccnd1 and myca were determined by RT-PCR. Lipid peroxidation (LPO), nitric oxide (NO), and glutathione-S-transferase (GST) activities were determined spectrophotometrically. Apoptosis was determined using acridine orange staining. Locomotor activity was measured using touch-evoked movement test. MP exposure increased malformations, LPO, apoptosis, ccnd1 and myca expressions, and decreased GST activities and NO levels compared with the control group. Our findings will lead to further understanding of the mechanism of MP toxicity, and merit further research. © 2018 Wiley Periodicals, Inc.

  3. A novel contact assay for testing aryl hydrocarbon receptor (AhR)-mediated toxicity of chemicals and whole sediments in zebrafish (Danio rerio) embryos.

    PubMed

    Schiwy, Sabrina; Bräunig, Jennifer; Alert, Henriette; Hollert, Henner; Keiter, Steffen H

    2015-11-01

    The European Water Framework Directive aims to achieve a good ecological and chemical status in surface waters until 2015. Sediment toxicology plays a major role in this intention as sediments can act as a secondary source of pollution. In order to fulfill this legal obligation, there is an urgent need to develop whole-sediment exposure protocols, since sediment contact assays represent the most realistic scenario to simulate in situ exposure conditions. Therefore, in the present study, a vertebrate sediment contact assay to determine aryl hydrocarbon receptor (AhR)-mediated activity of particle-bound pollutants was developed. Furthermore, the activity and the expression of the CYP1 family in early life stages of zebrafish after exposure to freeze-dried sediment samples were investigated. In order to validate the developed protocol, effects of β-naphthoflavone and three selected sediment on zebrafish embryos were investigated. Results documented clearly AhR-mediated toxicity after exposure to β-naphthoflavone (β-NF) and to the sediment from the Vering canal. Upregulation of mRNA levels was observed for all investigated sediment samples. The highest levels of all investigated cyp genes (cyp1a, cyp1b1, cyp1c1, and cyp1c2) were recorded after exposure to the sediment sample of the Vering canal. In conclusion, the newly developed sediment contact assay can be recommended for the investigation of dioxin-like activities of single substances and the bioavailable fraction of complex environmental samples. Moreover, the exposure of whole zebrafish embryos to native (freeze-dried) sediment samples represents a highly realistic and ecologically relevant exposure scenario.

  4. In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences.

    PubMed

    Dudczig, Stefanie; Currie, Peter D; Poggi, Lucia; Jusuf, Patricia R

    2017-03-22

    The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments. In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

  5. The paired domain-containing nuclear factor pax[b] is expressed in specific commissural interneurons in zebrafish embryos.

    PubMed

    Mikkola, I; Fjose, A; Kuwada, J Y; Wilson, S; Guddal, P H; Krauss, S

    1992-10-01

    The zebrafish paired box (Pax) genes are expressed in the early neural tube and are thought to be transcription factors that regulate the differentiation of cells in the central nervous system (CNS). The protein product of one of these Pax genes, pax[b], is detectable as a nuclear antigen in all the regions of the embryo that transcribe the gene including the posterior midbrain, the nephritic primordium, the Wolffian duct, the optic stalk, and, in specific neurons, in the hindbrain and spinal cord. The timing and pattern of axonal outgrowth by the early pax[b]-positive neurons suggest that they are the commissural secondary ascending (CoSA) interneurons in the spinal cord; the primary commissural interneurons (MiD2c and MiD3c) in hindbrain rhombomeres mi2 and mi3; and a previously unclassified set of commissural interneurons that we termed the commissural caudalrhombomere ascending (CoCaA) interneurons in the caudal hindbrain. In contrast, the Mauthner interneurons do not express pax[b] early in development. Shortly after the appearance of the first pax[b]-positive interneurons, additional nuclei adjacent to the first pax[b]-positive interneurons become pax[b] positive. This pattern of expression suggests that the pax[b] protein may be involved in determining the identity of specific commissural interneurons.

  6. Impacts of 17beta-estradiol, including environmentally relevant concentrations, on reproduction after exposure during embryo-larval-, juvenile- and adult-life stages in zebrafish (Danio rerio).

    PubMed

    Brion, F; Tyler, C R; Palazzi, X; Laillet, B; Porcher, J M; Garric, J; Flammarion, P

    2004-06-24

    Zebrafish (Danio rerio) were exposed for 3 weeks to low concentrations of estradiol including environmentally relevant concentrations (5, 25 and 100 ng/l), encompassing either their embryo-larvae (from fertilization to 21 day post-fertilization (dpf)), juvenile (from 21 to 42 dpf) or adult life stages (>200 dpf) with a view to investigating the most sensitive life stage of the zebrafish to 17beta-estradiol (E2). At all sampling points, whole-body vitellogenin concentrations and gonadal development were analyzed in order to investigate the effects of estrogen exposure on these endpoint in the zebrafish. In the adult stage, additional endpoints were measured including secondary sexual characteristics (manifestation of the uro-genital papillae (UGP) in males), gonadal growth (the gonado-somatic index (GSI)) and sex ratio. For all the different life stage exposures, reproductive performance of the F0 generation was assessed (egg production) and survival and development of the F1 embryo-larvae. Exposure to low concentrations of E2 resulted in vitellogenin induction whatever the life stage exposed but these effects were reversible after depuration. The effective concentration for vitellogenin induction in zebrafish early life stages was 100 ng E2/l, and in adult male zebrafish the effective concentration for vitellogenin induction (between 5 and 25 ng/l) was lower than for the early life stage fish. Exposure to E2 prior to (from fertilization to 21 dpf) and during the time of sex differentiation (from 21 to 42 dpf) also caused disruptions in the process of sexual differentiation (resulting in formation of a retrogonadal cavity in presumptive male, germ cell development and leading to a significant change of the sex ratio towards the female sex at the dose of 100 ng E2/l for the fish exposure as embryo-larvae) and altered patterns of egg production in the subsequent adults. Exposure of adult fish to E2 resulted in a modification of the secondary sexual characteristic in

  7. Effects of ammonium-based ionic liquids and 2,4-dichlorophenol on the phospholipid fatty acid composition of zebrafish embryos.

    PubMed

    Piotrowska, Aleksandra; Syguda, Anna; Wyrwas, Bogdan; Chrzanowski, Lukasz; Luckenbach, Till; Heipieper, Hermann J

    2018-01-01

    Ionic liquids consisting of a combination of herbicidal anions with a quaternary ammonium cation act as efficient herbicides, which are under consideration to be used in the agriculture. In the present study, we used embryos of the zebrafish (Danio rerio) as a model to assess the toxic potential of ammonium-based ionic liquids for aquatic organisms. As we assumed interference of the partially hydrophobic ionic liquid cation with lipids, we investigated the adaptation response in the lipid composition of the zebrafish embryos, triggered by the ionic compound. Therefore, the impact of ammonium-based ionic liquids with different lengths of the alkyl chain ([C6,C6,C1,C1N][Br], [C8,C8,C1,C1N][Br]) on the phospholipid fatty acid (PLFA) profile of zebrafish embryos up to 72 hours post fertilization (hpf) was examined. Furthermore, the changes in the unsaturation index (UI) of PLFAs, as the sum parameter of membrane fluidity in eukaryotic cells, were presented. The PLFA's UI in the zebrafish embryos upon exposure to quaternary ammonium salts was compared to the UI of the embryos upon exposure to nonionic 2,4-dichlorophenol, which has a similar hydrophobicity but is structurally different to [C8,C8,C1,C1N][Br]. It was shown that for ammonium-based ionic liquid precursors non-specific mode of action occurs and the toxic effect on lipid composition of zebrafish embryos can be well predicted based on chemical properties, like hydrophobicity. Furthermore, the changes in PLFAs, expressed by the UI, can be useful to study toxic effects of organic contamination. However, for zebrafish embryos, after ionic liquids and 2,4-DCP exposure, the changes were observed at high lethal concentrations, which caused the incidence of lethality of 30 and 50% of a group of test animals.

  8. Effects of ammonium-based ionic liquids and 2,4-dichlorophenol on the phospholipid fatty acid composition of zebrafish embryos

    PubMed Central

    Piotrowska, Aleksandra; Syguda, Anna; Wyrwas, Bogdan; Chrzanowski, Lukasz; Luckenbach, Till

    2018-01-01

    Ionic liquids consisting of a combination of herbicidal anions with a quaternary ammonium cation act as efficient herbicides, which are under consideration to be used in the agriculture. In the present study, we used embryos of the zebrafish (Danio rerio) as a model to assess the toxic potential of ammonium-based ionic liquids for aquatic organisms. As we assumed interference of the partially hydrophobic ionic liquid cation with lipids, we investigated the adaptation response in the lipid composition of the zebrafish embryos, triggered by the ionic compound. Therefore, the impact of ammonium-based ionic liquids with different lengths of the alkyl chain ([C6,C6,C1,C1N][Br], [C8,C8,C1,C1N][Br]) on the phospholipid fatty acid (PLFA) profile of zebrafish embryos up to 72 hours post fertilization (hpf) was examined. Furthermore, the changes in the unsaturation index (UI) of PLFAs, as the sum parameter of membrane fluidity in eukaryotic cells, were presented. The PLFA’s UI in the zebrafish embryos upon exposure to quaternary ammonium salts was compared to the UI of the embryos upon exposure to nonionic 2,4-dichlorophenol, which has a similar hydrophobicity but is structurally different to [C8,C8,C1,C1N][Br]. It was shown that for ammonium-based ionic liquid precursors non-specific mode of action occurs and the toxic effect on lipid composition of zebrafish embryos can be well predicted based on chemical properties, like hydrophobicity. Furthermore, the changes in PLFAs, expressed by the UI, can be useful to study toxic effects of organic contamination. However, for zebrafish embryos, after ionic liquids and 2,4-DCP exposure, the changes were observed at high lethal concentrations, which caused the incidence of lethality of 30 and 50% of a group of test animals. PMID:29342167

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

    PubMed

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

    2018-06-18

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

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

    NASA Technical Reports Server (NTRS)

    Moorman, S. J.

    2001-01-01

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

  11. Long-term in vivo harmonics imaging of zebrafish embryonic development based on a femtosecond Cr:forsterite laser

    NASA Astrophysics Data System (ADS)

    Chen, S.-Y.; Tsai, T.-H.; Hsieh, C.-S.; Tai, S.-P.; Lin, C.-Y.; Ko, C.-Y.; Chen, Y.-C.; Tsai, H.-J.; Hu, C.-H.; Sun, C.-K.

    2005-03-01

    Based on a femtosecond Cr:forsterite laser, harmonics optical microscopy (HOM) provides a truly "noninvasive" tool for in vivo and long-term study of vertebrate embryonic development. Based on optical nonlinearity, HOM provides sub-micrometer 3D spatial resolution and high 3D optical-sectioning power without using invasive and toxic fluorophores. Since only virtual-level-transition is involved, HOM is known to leave no energy deposition and no photodamage. Combined with second harmonic generation, which is sensitive to specific structure such as nerve and muscle fibers, HOM can perform functional studies of early developmental dynamics of many vertebrate physiological systems. Recently, zebrafish has become a standard model for many biological and medical studies of vertebrates, due to the similarity between embryonic development of zebrafish and human being. Here we demonstrate in vivo HOM studies of developmental dynamics of several important embryonic physiological systems in live zebrafish embryos, with focuses on the developments of brains, eyes, ears, and hearts. Based on a femtosecond Cr:forsterite laser, which provides the deepest penetration (~1.5mm) and least photodamage in the zebrafish embryo, complete developing processes of different physiological systems within a period of time longer than 20 hours can be non-invasively observed inside the same embryo.

  12. Genotoxic and teratogenic effect of freshwater sediment samples from the Rhine and Elbe River (Germany) in zebrafish embryo using a multi-endpoint testing strategy.

    PubMed

    Garcia-Käufer, M; Gartiser, S; Hafner, C; Schiwy, S; Keiter, S; Gründemann, C; Hollert, H

    2015-11-01

    The embryotoxic potential of three model sediment samples with a distinct and well-characterized pollutant burden from the main German river basins Rhine and Elbe was investigated. The Fish Embryo Contact Test (FECT) in zebrafish (Danio rerio) was applied and submitted to further development to allow for a comprehensive risk assessment of such complex environmental samples. As particulate pollutants are constructive constituents of sediments, they underlay episodic source-sink dynamics, becoming available to benthic organisms. As bioavailability of xenobiotics is a crucial factor for ecotoxicological hazard, we focused on the direct particle-exposure pathway, evaluating throughput-capable endpoints and considering toxicokinetics. Fish embryo and larvae were exposed toward reconstituted (freeze-dried) sediment samples on a microcosm-scale experimental approach. A range of different developmental embryonic stages were considered to gain knowledge of potential correlations with metabolic competence during the early embryogenesis. Morphological, physiological, and molecular endpoints were investigated to elucidate induced adverse effects, placing particular emphasis on genomic instability, assessed by the in vivo comet assay. Flow cytometry was used to investigate the extent of induced cell death, since cytotoxicity can lead to confounding effects. The implementation of relative toxicity indices further provides inter-comparability between samples and related studies. All of the investigated sediments represent a significant ecotoxicological hazard by disrupting embryogenesis in zebrafish. Beside the induction of acute toxicity, morphological and physiological embryotoxic effects could be identified in a concentration-response manner. Increased DNA strand break frequency was detected after sediment contact in characteristic non-monotonic dose-response behavior due to overlapping cytotoxic effects. The embryonic zebrafish toxicity model along with the in vivo comet

  13. Microcystin-LR affects the hypothalamic-pituitary-inter-renal (HPI) axis in early life stages (embryos and larvae) of zebrafish.

    PubMed

    Chen, Liang; Wang, Yeke; Giesy, John P; Chen, Feng; Shi, Ting; Chen, Jun; Xie, Ping

    2018-05-22

    Frequencies and durations of blooms of cyanobacteria are increasing. Some cyanobacteria can produce cyanotoxins including microcystins (MCs). MCs are the most common toxic products of hazardous algal blooms (HABs), with the greatest potential for exposure and to cause toxicity. Recently, MCs have been shown to disrupt endocrine functions. In this study, for the first time, effects of MC-LR on the hypothalamic-pituitary-inter-renal (HPI) axis during early embryonic development (embryos/larvae) of zebrafish (Danio rerio), were investigated. Embryos/larvae of zebrafish were exposed to 1, 10, 100, or 300 μg MC-LR/L during the period of 4-168 h post-fertilization (hpf). Exposure to 300 μg MC-LR/L resulted in significantly greater concentrations of whole-body cortisol than those in controls. Expressions of genes along the HPI axis and mineralocorticoid receptor (MR-) and glucocorticoid receptor (GR-) centered gene networks were evaluated by use of quantitative real-time PCR. Expression of mRNA for crh was significantly down-regulated by exposure to 300 μg MC-LR/L, while expressions of crhbp, crhr1, and crhr2 were significantly up-regulated, relative to controls. MC-LR caused significantly lesser levels of mRNA for steroidogenic genes including hmgra, star, and cyp17, but expression of mRNA for hsd20b was significantly greater than that of controls. Treatment with MC-LR also altered profiles of transcription of MR- and GR-centered gene networks, which might result in multiple responses. Taken together, these results demonstrated that MC-LR affected the corticosteroid-endocrine system of larvae of zebrafish. This study provided valuable insights into molecular mechanisms behind potential toxicity and endocrine disruption of MCs. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Aquaporin 1 Is Involved in Acid Secretion by Ionocytes of Zebrafish Embryos through Facilitating CO2 Transport

    PubMed Central

    Horng, Jiun-Lin; Chao, Pei-Lin; Chen, Po-Yen; Shih, Tin-Han; Lin, Li-Yih

    2015-01-01

    Mammalian aquaporin 1 (AQP1) is well known to function as a membrane channel for H2O and CO2 transport. Zebrafish AQP1a.1 (the homologue of mammalian AQP1) was recently identified in ionocytes of embryos; however its role in ionocytes is still unclear. In this study, we hypothesized that zebrafish AQP1a.1 is involved in the acid secretion by ionocytes through facilitating H2O and CO2 diffusion. A real-time PCR showed that mRNA levels of AQP1a.1 in embryos were induced by exposure to 1% CO2 hypercapnia for 3 days. In situ hybridization and immunohistochemistry showed that the AQP1a.1 transcript was highly expressed by acid-secreting ionocytes, i.e., H+-ATPase-rich (HR) cells. A scanning ion-selective electrode technique (SIET) was applied to analyze CO2-induced H+ secretion by individual ionocytes in embryos. H+ secretion by HR cells remarkably increased after a transient loading of CO2 (1% for 10 min). AQP1a.1 knockdown with morpholino oligonucleotides decreased the H+ secretion of HR cells by about half and limited the CO2 stimulated increase. In addition, exposure to an AQP inhibitor (PCMB) for 10 min also suppressed CO2-induced H+ secretion. Results from this study support our hypothesis and provide in vivo evidence of the physiological role of AQP1 in CO2 transport. PMID:26287615

  15. The Zebrafish G12 Gene is required for Nuclear Positioning and Cell Migrations during Early Development

    NASA Technical Reports Server (NTRS)

    Reinsch, S. S.; Conway, G. C.

    2003-01-01

    After fertilization Zebrafish embryos undergo synchronous cleavage to form a blastula of cells sitting upon a single multinucleate yolk cell. At the beginning of gastrulation these cells undergo extensive cell migrations to form the major body axes. We have discovered a gene, G12, which is required for cell migrations and positioning of nuclei in the large syncytial yolk cell. Overexpression of a G12-GFP fusion protein is not toxic and shows that the protein localizes inside the yolk cell to the yolk nuclei, microtubules, and to the margin between the blastomeres and the large yolk cell. Morpholino (MO) injection into the 1-cell embryo or into just the yolk syncytium conipletely inhibits cell migrations, doming of the yolk cell, and positioning of nuclei around the margin. This effect can be partially rescued by injection of G12-GFP encoding RNA. Given the known role of microtubules in nuclear positioning of yolk nuclei in Zebrafish, we investigated the microtubules in morpholiiio injected and rescued embryos. We find that microtubules are sparse and disorganized in MO-injected embryos and are restored to normal organization upon G12-GFP rescue. G12 plays a pivotal role in organization of inicrotubules during early development. G12 is highly conserved in vertebrates and two homologues exist in the human genome. One of the human hoinologues is amplified in aggressive breast tumors.

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

    PubMed Central

    Gamse, Joshua T.

    2016-01-01

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

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

    PubMed

    Gamse, Joshua T; Gorelick, Daniel A

    2016-10-01

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

  18. Histone deacetylase 1 is required for the development of the zebrafish inner ear

    PubMed Central

    He, Yingzi; Tang, Dongmei; Li, Wenyan; Chai, Renjie; Li, Huawei

    2016-01-01

    Histone deacetylase 1 (HDAC1) has been reported to be important for multiple aspects of normal embryonic development, but little is known about its function in the development of mechanosensory organs. Here, we first confirmed that HDAC1 is expressed in the developing otic vesicles of zebrafish by whole-mount in situ hybridization. Knockdown of HDAC1 using antisense morpholino oligonucleotides in zebrafish embryos induced smaller otic vesicles, abnormal otoliths, malformed or absent semicircular canals, and fewer sensory hair cells. HDAC1 loss of function also caused attenuated expression of a subset of key genes required for otic vesicle formation during development. Morpholino-mediated knockdown of HDAC1 resulted in decreased expression of members of the Fgf family in the otic vesicles, suggesting that HDAC1 is involved in the development of the inner ear through regulation of Fgf signaling pathways. Taken together, our results indicate that HDAC1 plays an important role in otic vesicle formation. PMID:26832938

  19. DMSO modifies the permeability of the zebrafish (Danio rerio) chorion-implications for the fish embryo test (FET).

    PubMed

    Kais, B; Schneider, K E; Keiter, S; Henn, K; Ackermann, C; Braunbeck, T

    2013-09-15

    Since 2007, when REACH came into force, the fish embryo test has received increasing attention as a potential alternative for the acute fish test. Due to its low toxicity and the ability to permeate biological membranes without significant damage to their structural integrity, dimethyl sulfoxide (DMSO) is a commonly used solvent in the fish embryo test. Little is known, however, about the membrane penetration properties of DMSO, the impact of different concentrations of DMSO on the potential barrier function of the zebrafish chorion and on changes in the uptake of chemicals into the embryo. Therefore, in the present study, the fluorescent dyes fluorescein (mol wt 332; Pow 3.4) and 2,7-dichlorofluorescein (mol wt 401; Pow 4.7), both substances with limited water solubility, were used to visualize the uptake into the egg as well as the accumulation in the embryo of the zebrafish depending on different concentrations of DMSO. The distribution of fluorescein within the egg compartments varied with DMSO concentration: When dissolved in 0.01% DMSO, fluorescein did not pass the chorion. In contrast, concentrations ≥ 0.1% DMSO increasingly facilitated the uptake into the perivitelline space. In contrast, the uptake of 2,7-dichlorofluorescein was not substantially increased with rising DMSO concentrations, indicating the importance of factors other than the solvent (e.g. mol wt). With respect to the fish embryo test, results indicate that DMSO may be used without complications as a solvent, however, only at a maximum concentration of 0.01% (0.1 mL/L) as already indicated in the OECD difficult substances paper (OECD, 2000). Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Zinc oxide nanoparticles induce oxidative DNA damage and ROS-triggered mitochondria-mediated apoptosis in zebrafish embryos.

    PubMed

    Zhao, Xuesong; Ren, Xin; Zhu, Rong; Luo, Zhouying; Ren, Baixiang

    2016-11-01

    Zinc oxide nanoparticles (nano-ZnO) are one of the most important nanoparticles in the industry. The objectives of this study were (1) to investigate the effects of nano-ZnO on oxidative damage to DNA and on apoptosis in zebrafish (Danio rerio) embryos, and (2) to identify the underlying molecular mechanism affecting theapoptotic process. In addition to nano-ZnO, we also investigated the toxic effects of the Zn 2+ ion. Zebrafish embryos were exposed to 10, 30, 60, 90, or 120mg/L nano-ZnO for 96h postfertilization. Nano-ZnO (at concentrations between 10 and 120mg/L) significantly reduced the rate of embryo hatching. Embryos/larvae exposed to 120mg/L nano-ZnO had significantly higher heart rates. Increased heart rates could be a physiological mechanism compensating for body hypoxia. Embryos/larvae exposed to nano-ZnO exhibited oxidative stress, due to an excessive generation of reactive oxygen species (ROS). Oxidative stress was evidenced by increased levels of superoxide dismutase, by increased lipid peroxidation, and by increased expression of genes related to the antioxidant defense system (sod1, cat, gpx1a, and pparα), which were altered at different degrees. Upon exposure to nano-ZnO, the percentage of apoptotic cells increased in a dose-dependent manner (0.41% to 4.21%). In addition, altered transcriptional regulation of pro-apoptotic genes (bax, puma, and apaf-1) and anti-apoptotic genes (bcl-2) provided further evidence of the activation of apoptosis. In this study, exposure of zebrafish embryos to nano-ZnO triggered an excessive production of ROS, which was followed by several phenomena: the up-regulation of p53, a reduction in the bcl-2/bax ratio,a reduction in the mitochondrial membrane potential (ψ m ), the release of cytochrome c into the cytosolic fraction, and the activation of caspases 9 and 3. Collectively, our data imply that nano-ZnO induce an excessive production of ROS which then activate the apoptosis pathway mediated by mitochondria and

  1. Maternal Cortisol Mediates Hypothalamus-Pituitary-Interrenal Axis Development in Zebrafish

    PubMed Central

    Nesan, Dinushan; Vijayan, Mathilakath M.

    2016-01-01

    In zebrafish (Danio rerio), de novo synthesis of cortisol in response to stressor exposure commences only after hatch. Maternally deposited cortisol is present during embryogenesis, but a role for this steroid in early development is unclear. We tested the hypothesis that maternal cortisol is essential for the proper development of hypothalamus-pituitary-interrenal (HPI) axis activity and the onset of the stressor-induced cortisol response in larval zebrafish. In this study, zygotic cortisol content was manipulated by microinjecting antibody to sequester this steroid, thereby making it unavailable during embryogenesis. This was compared with embryos containing excess cortisol by microinjection of exogenous steroid. The resulting larval phenotypes revealed distinct treatment effects, including deformed mesoderm structures when maternal cortisol was unavailable and cardiac edema after excess cortisol. Maternal cortisol unavailability heightened the cortisol stress response in post-hatch larvae, whereas excess cortisol abolished the stressor-mediated cortisol elevation. This contrasting hormonal response corresponded with altered expression of key HPI axis genes, including crf, 11B hydroxylase, pomca, and star, which were upregulated in response to reduced cortisol availability and downregulated when embryos had excess cortisol. These findings for the first time underscore a critical role for maternally deposited cortisol in programming HPI axis development and function in zebrafish. PMID:26940285

  2. 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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Cardio-respirometry disruption in zebrafish (Danio rerio) embryos exposed to hydraulic fracturing flowback and produced water.

    PubMed

    Folkerts, Erik J; Blewett, Tamzin A; He, Yuhe; Goss, Greg G

    2017-12-01

    Hydraulic fracturing to extract oil and natural gas reserves is an increasing practice in many international energy sectors. Hydraulic fracturing flowback and produced water (FPW) is a hyper saline wastewater returned to the surface from a fractured well containing chemical species present in the initial fracturing fluid, geogenic contaminants, and potentially newly synthesized chemicals formed in the fracturing well environment. However, information on FPW toxicological mechanisms of action remain largely unknown. Both cardiotoxic and respirometric responses were explored in zebrafish (Danio rerio) embryos after either an acute sediment-free (FPW-SF) or raw/sediment containing (FPW-S) fraction exposure of 24 and 48 h at 2.5% and 5% dilutions. A 48 h exposure to either FPW fraction in 24-72 h post fertilization zebrafish embryos significantly increased occurrences of pericardial edema, yolk-sac edema, and tail/spine curvature. In contrast, larval heart rates significantly decreased after FPW fraction exposures. FPW-S, but not FPW-SF, at 2.5% doses significantly reduced embryonic respiration/metabolic rates (MO 2 ), while for 5% FPW, both fractions reduced MO 2 . Expression of select cardiac genes were also significantly altered in each FPW exposure group, implicating a cardiovascular system compromise as the potential cause for reduced embryonic MO 2 . Collectively, these results support our hypothesis that organics are major contributors to cardiac and respiratory responses to FPW exposure in zebrafish embryos. Our study is the first to investigate cardiac and respiratory sub-lethal effects of FPW exposure, demonstrating that FPW effects extend beyond initial osmotic stressors and verifies the use of respirometry as a potential marker for FPW exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Production of a compound against methicillin resistant Staphylococcus aureus (MRSA) from Streptomyces rubrolavendulae ICN3 & its evaluation in zebrafish embryos

    PubMed Central

    Kannan, Rajaretinam Rajesh; Iniyan, Appadurai Muthamil; Vincent, Samuel Gnana Prakash

    2014-01-01

    Background & objectives: Antibiotic resistance in pathogens has become a serious problem worldwide. Therefore, the search for new antibiotics for drug resistanct pathogens is an important endeavor. The present study deals with the production of anti-methicillin resistant Staphylococcus aureus (MRSA) potential of Streptomyces rubrolavendulae ICN3 and evaluation of anti-MRSA compound in zebrafish embryos. Methods: The antibiotic production from S. rubrolavendulae ICN3 was optimized in solid state fermentation and extracted. The antagonistic activity was confirmed against MRSA and purified in silica gel column and reverse phase - HPLC with an absorption maximum at 215 nm. Minimal inhibitory concentration of the compound was determined by broth microdilution method. Zebrafish embryos were used to evaluate the extract/compound for its minimal inhibition studies, influences on heart beat rates, haematopoietic blood cell count and lethal dose values. Results: Streptomyces rubrolavendulae ICN3 showed potent antagonistic activity against MRSA with a zone of 42 mm. The minimum inhibitory concentration was calculated as 500 μg/ml of the crude extract and the purified C23 exhibited 2.5 μg/ml in in vitro assay. The LC50 value of the anti MRSA compound C23 was calculated as 60.49 μg/ml and the MRSA treated embryos survived in the presence of purified compound C23 at a dose of 10 μg/ml. Interpretation & conclusions: Our results suggested that the compound was potent with less toxic effects in zebrafish embryonic model system for MRSA infection. Further structural evaluation and analysis in higher mammalian model system may lead to a novel drug candidate for drug resistant Staphylococcus aureus. PMID:25109726

  5. Integrated toxic evaluation of sulfamethazine on zebrafish: Including two lifespan stages (embryo-larval and adult) and three exposure periods (exposure, post-exposure and re-exposure).

    PubMed

    Yan, Zhengyu; Yang, Qiulian; Jiang, Weili; Lu, Jilai; Xiang, Zhongrun; Guo, Ruixin; Chen, Jianqiu

    2018-03-01

    Persistence of antibiotics in aquatic environment may pose a risk to the non-target aquatic organisms. This study provided an integrated evaluation to analyze the toxic stress of sulfamethazine (SMZ) on zebrafish in two lifespan stages (embryo-larval and adult) and three exposure periods (exposure, post-exposure and re-exposure). Zebrafish embryos and adult zebrafish were exposed to SMZ at 0.2, 20 and 2000 μg/L, respectively. The results showed that SMZ at any given concentration inhibited the hatching of embryos at 58-96 hpf (hours post-fertilization). Our result also indicated that two major kinds of the malformation, which was induced by the antibiotic, were edema and spinal curvature. Additionally, the antibiotic stimulated the heartbeat while reduced the body length of the embryo at 72 hpf. Superoxide dismutase (SOD) activities and malondialdehyde (MDA) contents significantly increased at 120 hpf when the embryos were exposed to the lowest concentration (0.2 μg/L) of the antibiotic. On the other hand, the antibiotic induced SOD activities and MDA contents in adult zebrafish in the exposure and re-exposure periods. The MDA contents could recover while SOD activities still increased in 2 d after the exposure. Both SOD activities and MDA contents could recover in 7 d after the exposure. Levels of SOD and MDA in the re-exposure were higher than those in the first exposure. Our results suggested that SMZ had toxic effects on both embryos and adult zebrafish, and provided an integrated evaluation of the toxic effects of SMZ on zebrafish at a new perspective. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. The Influence of Hydroxyapatite Nanoparticle Morphology on Embryonic Development in a Zebrafish Exposure Model

    PubMed Central

    Pujari-Palmer, Shiuli; Lu, Xi; Karlsson Ott, Marjam

    2017-01-01

    Nanomaterials are used in many different industries such as cosmetics, food, clothing, and electronics. There is increasing concern that exposure to nanoparticles (NPs) during pregnancy can adversely affect fetal development. It is well known that the size, charge, and chemistry of a nanoparticle can modulate embryological development. The role that particle morphology plays on early development, however, is still widely unknown. The present study aims to investigate the effect of hydroxyapatite nanoparticle (HANP) morphology on embryological development in a zebrafish exposure model. Four distinct HANP morphologies (dots, long rods, sheets, and fibers) were fabricated and characterized. Zebrafish embryos were exposed to HANPs (0–100 mg/L), and viability and developmental deformities were evaluated for up to 5 days post-fertilization (dpf). Malformations such as pericardial edema and axial curvature were apparent in embryos as early as 1 dpf, following exposure to the dot and fiber particles, and developed in embryos by 3 dpf in the sheet and long rod particle groups. Minimal death was observed in response to dot, long rod, and sheet particles (≤25%), while fiber particles induced overwhelming toxicity (≤60%) after 1 dpf, and complete toxicity during all subsequent time points. Collectively, these results suggest that nanoparticle morphology can significantly impact embryological development and should be a required consideration when designing nanomaterials for commercial use. PMID:28441729

  7. Egr1 gene knockdown affects embryonic ocular development in zebrafish.

    PubMed

    Hu, Chao-Yu; Yang, Chang-Hao; Chen, Wei-Yu; Huang, Chiu-Ju; Huang, Hsing-Yen; Chen, Muh-Shy; Tsai, Huai-Jen

    2006-10-26

    To identify the changes in zebrafish embryonic ocular development after early growth response factor 1 (Egr1) gene knockdown by Egr1-specific translation inhibitor, morpholino oligonucleotides (MO). Two kinds of Egr1-MO were microinjected separately with various dosages into one to four celled zebrafish embryos to find an optimal dose generating an acceptable mortality rate and high frequency of specific phenotype. Chordin-MO served as the positive control; a 5 mismatch MO of Egr1-MO1 and a nonspecific MO served as negative controls. We graded the Egr1 morphants according to their gross abnormalities, and measured their ocular dimensions accordingly. Western blot analysis and synthetic Egr1 mRNA rescue experiments confirmed whether the deformities were caused by Egr1 gene knockdown. Histological examination and three kinds of immunohistochemical staining were applied to identify glutamate receptor one expression in retinal ganglion cells and amacrine cells, to recognize acetylated alpha-tubulin expression which indicated axonogenesis, and to label photoreceptor cells with zpr-1 antibody. After microinjection of 8 ng Egr1-MO1 or 2 ng Egr1-MO2, 81.8% and 97.3% of larvae at 72 h postfertilization had specific defects, respectively. The gross phenotype included string-like heart, flat head, and deformed tail. The more severely deformed larvae had smaller eyes and pupils. Co-injection of 8 ng Egr1-MO1 and supplementary 12 pg synthetic Egr1 mRNA reduced the gross abnormality rate from 84.4% to 29.7%, and decreased the severity of deformities. Egr1 protein appeared in the wildtype and rescued morphants, but was lacking in the Egr1 morphants with specific deformities. Lenses of Egr1 morphants were smaller and had some residual nucleated lens fiber cells. Morphants' retinal cells arranged disorderly and compactly with thin plexiform layers. Immunohistochemical studies showed that morphants had a markedly decreased number of mature retinal ganglion cells, amacrine cells, and

  8. Joint acute and endocrine disruptive toxicities of malathion, cypermethrin and prochloraz to embryo-larval zebrafish, Danio rerio.

    PubMed

    Guo, Dongmei; Wang, Yanhua; Qian, Yongzhong; Chen, Chen; Jiao, Bining; Cai, Leiming; Wang, Qiang

    2017-01-01

    It remains a daunting challenge to determine ecotoxicological risks of exposure to mixtures of endocrine disrupting chemicals (EDCs) in environmental toxicology. In the present study, we investigated acute and endocrine disruptive toxicities of cypermethrin (CPM), malathion (MAL), prochloraz (PRO) and their binary mixtures of MAL + CPM and MAL + PRO to the early life stages of zebrafish. In the acute lethal toxicity test, three pesticides exhibited different levels of toxicity to zebrafish larvae, and the order of toxicity was as follows: CPM > PRO > MAL. The binary mixture of MAL + CPM displayed a synergistic effect on zebrafish larvae after exposure for 24, 48, 72 and 96 h. However, binary mixture of MAL + PRO showed an antagonistic effect. To evaluate the estrogenic effect, the expression of genes in the hypothalamic-pituitary-gonadal axis was assessed after zebrafish embryos were exposed to CPM, MAL, PRO and their binary mixtures from blastula stage (1 h post-fertilization, 1 hpf) to 14 dpf (14 d post-fertilization). Our data indicated that the transcription patterns of many key genes (vtg1, vtg2, era, erβ1, erβ2, cyp19a1a and cyp19a1b) were affected in hatched zebrafish after exposure to CPM, MAL and PRO. Moreover, following exposure to binary mixtures of 1000 μg/L MAL +4 μg/L CPM and 1000 μg/L MAL +900 μg/L PRO, the gene expressions were significantly changed compared with the individual pesticides. Our data provided a better understanding of bidirectional interactions of toxic response induced by these pesticides. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. PhOTO Zebrafish: A Transgenic Resource for In Vivo Lineage Tracing during Development and Regeneration

    PubMed Central

    Dempsey, William P.; Fraser, Scott E.; Pantazis, Periklis

    2012-01-01

    Background Elucidating the complex cell dynamics (divisions, movement, morphological changes, etc.) underlying embryonic development and adult tissue regeneration requires an efficient means to track cells with high fidelity in space and time. To satisfy this criterion, we developed a transgenic zebrafish line, called PhOTO, that allows photoconvertible optical tracking of nuclear and membrane dynamics in vivo. Methodology PhOTO zebrafish ubiquitously express targeted blue fluorescent protein (FP) Cerulean and photoconvertible FP Dendra2 fusions, allowing for instantaneous, precise targeting and tracking of any number of cells using Dendra2 photoconversion while simultaneously monitoring global cell behavior and morphology. Expression persists through adulthood, making the PhOTO zebrafish an excellent tool for studying tissue regeneration: after tail fin amputation and photoconversion of a ∼100µm stripe along the cut area, marked differences seen in how cells contribute to the new tissue give detailed insight into the dynamic process of regeneration. Photoconverted cells that contributed to the regenerate were separated into three distinct populations corresponding to the extent of cell division 7 days after amputation, and a subset of cells that divided the least were organized into an evenly spaced, linear orientation along the length of the newly regenerating fin. Conclusions/Significance PhOTO zebrafish have wide applicability for lineage tracing at the systems-level in the early embryo as well as in the adult, making them ideal candidate tools for future research in development, traumatic injury and regeneration, cancer progression, and stem cell behavior. PMID:22431986

  10. Quantitative in vivo optical tomography of cancer progression & vasculature development in adult zebrafish

    PubMed Central

    Kumar, Sunil; Lockwood, Nicola; Ramel, Marie-Christine; Correia, Teresa; Ellis, Matthew; Alexandrov, Yuriy; Andrews, Natalie; Patel, Rachel; Bugeon, Laurence; Dallman, Margaret J.; Brandner, Sebastian; Arridge, Simon; Katan, Matilda; McGinty, James; Frankel, Paul; French, Paul M.W.

    2016-01-01

    We describe a novel approach to study tumour progression and vasculature development in vivo via global 3-D fluorescence imaging of live non-pigmented adult zebrafish utilising angularly multiplexed optical projection tomography with compressive sensing (CS-OPT). This “mesoscopic” imaging method bridges a gap between established ~μm resolution 3-D fluorescence microscopy techniques and ~mm-resolved whole body planar imaging and diffuse tomography. Implementing angular multiplexing with CS-OPT, we demonstrate the in vivo global imaging of an inducible fluorescently labelled genetic model of liver cancer in adult non-pigmented zebrafish that also present fluorescently labelled vasculature. In this disease model, addition of a chemical inducer (doxycycline) drives expression of eGFP tagged oncogenic K-RASV12 in the liver of immune competent animals. We show that our novel in vivo global imaging methodology enables non-invasive quantitative imaging of the development of tumour and vasculature throughout the progression of the disease, which we have validated against established methods of pathology including immunohistochemistry. We have also demonstrated its potential for longitudinal imaging through a study of vascular development in the same zebrafish from early embryo to adulthood. We believe that this instrument, together with its associated analysis and data management tools, constitute a new platform for in vivo cancer studies and drug discovery in zebrafish disease models. PMID:27259259

  11. Embryo density and medium volume effects on early murine embryo development.

    PubMed

    Canseco, R S; Sparks, A E; Pearson, R E; Gwazdauskas, F C

    1992-10-01

    One-cell mouse embryos were used to determine the effects of drop size and number of embryos per drop for optimum development in vitro. Embryos were collected from immature C57BL6 female mice superovulated with pregnant mare serum gonadotropin and human chorionic gonadotropin and mated by CD1 males. Groups of 1, 5, 10, or 20 embryos were cultured in 5-, 10-, 20-, or 40-microliters drops of CZB under silicon oil at 37.5 degrees C in a humidified atmosphere of 5% CO2 and 95% air. Development score for embryos cultured in 10 microliters was higher than that of embryos cultured in 20 or 40 microliters. Embryos cultured in groups of 5, 10, or 20 had higher development scores than embryos cultured singly. The highest development score was obtained by the combination of 5 embryos per 10-microliters drop. The percentage of live embryos in 20 or 40 microliters was lower than that of embryos cultured in 10 microliters. Additionally, the percentage of live embryos cultured singly was lower than that of embryos cultured in groups. Our results suggest that a stimulatory interaction occurs among embryos possibly exerted through the secretion of growth factors. This effect can be diluted if the embryos are cultured in large drops or singly.

  12. Smad4 is required for the development of cardiac and skeletal muscle in zebrafish.

    PubMed

    Yang, Jie; Wang, Junnai; Zeng, Zhen; Qiao, Long; Zhuang, Liang; Jiang, Lijun; Wei, Juncheng; Ma, Quanfu; Wu, Mingfu; Ye, Shuangmei; Gao, Qinglei; Ma, Ding; Huang, Xiaoyuan

    Transforming growth factor-beta (TGF-beta) regulates cellular functions and plays key roles in development and carcinogenesis. Smad4 is the central intracellular mediator of TGF-beta signaling and plays crucial roles in tissue regeneration, cell differentiation, embryonic development, regulation of the immune system and tumor progression. To clarify the role of smad4 in development, we examined both the pattern of smad4 expression in zebrafish embryos and the effect of smad4 suppression on embryonic development using smad4-specific antisense morpholino-oligonucleotides. We show that smad4 is expressed in zebrafish embryos at all developmental stages examined and that embryonic knockdown of smad4 results in pericardial edema, decreased heartbeat and defects in the trunk structure. Additionally, these phenotypes were associated with abnormal expression of the two heart-chamber markers, cmlc2 and vmhc, as well as abnormal expression of three makers of myogenic terminal differentiation, mylz2, smyhc1 and mck. Furthermore, a notable increase in apoptosis was apparent in the smad4 knockdown embryos, while no obvious reduction in cell proliferation was observed. Collectively, these data suggest that smad4 plays an important role in heart and skeletal muscle development. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  13. Sema4d is required for the development of the hindbrain boundary and skeletal muscle in zebrafish

    SciT

    Yang, Jie; Zeng, Zhen; Wei, Juncheng

    2013-04-05

    Highlights: ► Sema4d was expressed at all developmental stages of zebrafish. ► Knockdown of sema4d in embryos resulted in defects in the hindbrain and the trunk structure. ► Knockdown of sema4d in embryos upregulated the expression of three hindbrain rhombomere markers. ► Knockdown of sema4d in embryos increased the expression of myogenic regulatory factors. ► Knockdown of sema4d in embryos resulted in an obvious increase of cell apoptosis. -- Abstract: Semaphorin4d (SEMA4D), also known as CD100, an oligodendrocyte secreted R-Ras GTPase-activating protein (GAP), affecting axonal growth is involved in a range of processes including cell adhesion, motility, angiogenesis, immune responsesmore » and tumour progression. However, its actual physiological mechanisms and its role in development remain unclear. This study has focused on the role of sema4d in the development and expression patterns in zebrafish embryos and the effect of its suppression on development using sema4d-specific antisense morpholino-oligonucleotides. In this study the knockdown of sema4d, expressed at all developmental stages, lead to defects in the hindbrain and trunk structure of zebrafish embryos. In addition, these phenotypes appeared to be associated with the abnormal expression of three hindbrain rhombomere boundary markers, wnt1, epha4a and foxb1.2, and two myogenic regulatory factors, myod and myog. Further, a notable increase of cell apoptosis appeared in the sema4d knockdown embryos, while no obvious reduction in cell proliferation was observed. Collectively, these data suggest that sema4d plays an important role in the development of the hindbrain and skeletal muscle.« less

  14. Transcription of fgf8 is regulated by activating and repressive cis-elements at the midbrain-hindbrain boundary in zebrafish embryos.

    PubMed

    Inoue, Fumitaka; Parvin, Mst Shahnaj; Yamasu, Kyo

    2008-04-15

    Fgf8 is expressed in the isthmic region of the developing brain, serving an organizing function in vertebrate embryos. We previously identified S4.2 downstream to the zebrafish fgf8 gene as a regulatory region that drives transcription in the anterior hindbrain. Here, we investigated the mechanism of fgf8 regulation by the S4.2 region during development. Reporter analyses in embryos revealed that S4.2 closely recapitulates fgf8 expression in the anteriormost hindbrain during somitogenesis. This region contains a sequence highly conserved in fgf8 of diverse vertebrates. Further analyses of S4.2 revealed a 342-bp core region composed of three subregions (#2, #3, and #4). Regions #3 and #4 drove expression broadly in the brain from the midbrain to r5 of the hindbrain, whereas a 28-bp sequence in #2 repressed ectopic expression in the midbrain and in r2 to r5. The enhancer function of S4.2 was absent in pax2a mutant embryos, while it was activated ectopically by pax2a misexpression in the hindbrain. We identified two sites in the core region that are bound by Pax2a in vitro and in vivo, the disruption of which abrogated the S4.2 activity. Thus, fgf8 expression in the anteriormost hindbrain involves activation and repression, with Pax2a as a pivotal regulator.

  15. Inhibitory Effects of Red Wine on Lipid Oxidation in Fish Oil Emulsion and Angiogenesis in Zebrafish Embryo.

    PubMed

    Sun, Haiyan; Zhang, Yulin; Shen, Yixiao; Zhu, Yongchao; Wang, Hua; Xu, Zhimin

    2017-03-01

    The capabilities of red wine against lipid oxidation and angiogenesis were evaluated by using a fish oil emulsion system and an in vivo zebrafish embryos model, respectively. The red wine contained 12 different antioxidant phenolics which levels were led by anthocyanins (140.46 mg/L), catechin (55.08 mg/L), and gallic acid (46.76 mg/L). The diversity of the phenolics in red wine was greater than the tea, coffee, or white wine selected as a peer control in this study. The total phenolics concentration of red wine was 305.53 mg/L, although the levels of tea, coffee, and white wine were 85.59, 76.85, and 26.57 mg/L, respectively. The activity of red wine in scavenging DPPH (2,2-diphenyl-1-picrylhydrazyl) free radicals was approximately 4 times higher than the tea and 8 times than the coffee or white wine. The red wine showed the highest capability in preventing long chain PUFA oxidation in the fish oil emulsion. Because of the outstanding antioxidant activity of red wine, the red wine dried extract was used to monitor its inhibitory effect against angiogenesis by using transgenic zebrafish embryos (Tg[fli1:egfp] y1 ) with fluorescent blood vessels. After incubated in 100 μg/mL of the extract solution for 26 h pf, each of the embryos had a lower number of intersegmental vessel than the control embryo. The inhibition rate of red wine extract against growing of angiogenic blood vessel reached 100%. © 2017 Institute of Food Technologists®.

  16. Hedgehog signaling is required at multiple stages of zebrafish tooth development.

    PubMed

    Jackman, William R; Yoo, James J; Stock, David W

    2010-11-30

    The accessibility of the developing zebrafish pharyngeal dentition makes it an advantageous system in which to study many aspects of tooth development from early initiation to late morphogenesis. In mammals, hedgehog signaling is known to be essential for multiple stages of odontogenesis; however, potential roles for the pathway during initiation of tooth development or in later morphogenesis are incompletely understood. We have identified mRNA expression of the hedgehog ligands shha and the receptors ptc1 and ptc2 during zebrafish pharyngeal tooth development. We looked for, but did not detect, tooth germ expression of the other known zebrafish hedgehog ligands shhb, dhh, ihha, or ihhb, suggesting that as in mammals, only Shh participates in zebrafish tooth development. Supporting this idea, we found that morphological and gene expression evidence of tooth initiation is eliminated in shha mutant embryos, and that morpholino antisense oligonucleotide knockdown of shha, but not shhb, function prevents mature tooth formation. Hedgehog pathway inhibition with the antagonist compound cyclopamine affected tooth formation at each stage in which we applied it: arresting development at early stages and disrupting mature tooth morphology when applied later. These results suggest that hedgehog signaling is required continuously during odontogenesis. In contrast, over-expression of shha had no effect on the developing dentition, possibly because shha is normally extensively expressed in the zebrafish pharyngeal region. We have identified previously unknown requirements for hedgehog signaling for early tooth initiation and later morphogenesis. The similarity of our results with data from mouse and other vertebrates suggests that despite gene duplication and changes in the location of where teeth form, the roles of hedgehog signaling in tooth development have been largely conserved during evolution.

  17. Temporal cohesion of the structural, functional and molecular characteristics of the developing zebrafish heart.

    PubMed

    Matrone, Gianfranco; Wilson, Kathryn S; Mullins, John J; Tucker, Carl S; Denvir, Martin A

    2015-06-01

    Heart formation is a complex, dynamic and highly coordinated process of molecular, morphogenetic and functional factors with each interacting and contributing to formation of the mature organ. Cardiac abnormalities in early life can be lethal in mammals but not in the zebrafish embryo which has been widely used to study the developing heart. While early cardiac development in the zebrafish has been well characterized, functional changes during development and how these relate to architectural, cellular and molecular aspects of development have not been well described previously. To address this we have carefully characterised cardiac structure, function, cardiomyocyte proliferation and cardiac-specific gene expression between 48 and 120 hpf in the zebrafish. We show that the zebrafish heart increases in volume and changes shape significantly between 48 and 72 hpf accompanied by a 40% increase in cardiomyocyte number. Between 96 and 120 hpf, while external heart expansion slows, there is rapid formation of a mature and extensive trabecular network within the ventricle chamber. While ejection fraction does not change during the course of development other determinants of contractile function increase significantly particularly between 72 and 96 hpf leading to an increase in cardinal vein blood flow. This study has revealed a number of novel aspects of cardiac developmental dynamics with striking temporal orchestration of structure and function within the first few days of development. These changes are associated with changes in expression of developmental and maturational genes. This study provides important insights into the complex temporal relationship between structure and function of the developing zebrafish heart. Copyright © 2015 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  18. Silica nanoparticles inhibit macrophage activity and angiogenesis via VEGFR2-mediated MAPK signaling pathway in zebrafish embryos.

    PubMed

    Duan, Junchao; Hu, Hejing; Feng, Lin; Yang, Xiaozhe; Sun, Zhiwei

    2017-09-01

    The safety evaluation of silica nanoparticles (SiNPs) are getting great attention due to its widely-used in food sciences, chemical industry and biomedicine. However, the adverse effect and underlying mechanisms of SiNPs on cardiovascular system, especially on angiogenesis is still unclear. This study was aimed to illuminate the possible mechanisms of SiNPs on angiogenesis in zebrafish transgenic lines, Tg(fli-1:EGFP) and Albino. SiNPs caused the cardiovascular malformations in a dose-dependent manner via intravenous microinjection. The incidences of cardiovascular malformations were observed as: Pericardial edema > Bradycardia > Blood deficiency. The area of subintestinal vessels (SIVs) was significant reduced in SiNPs-treated groups, accompanied with the weaken expression of vascular endothelial cells in zebrafish embryos. Using neutral red staining, the quantitative number of macrophage was declined; whereas macrophage inhibition rate was elevated in a dose-dependent way. Furthermore, SiNPs significantly decreased the mRNA expression of macrophage activity related gene, macrophage migration inhibitory factor (MIF) and the angiogenesis related gene, vascular endothelial growth factor receptor 2 (VEGFR2). The protein levels of p-Erk1/2 and p-p38 MAPK were markedly decreased in zebrafish exposed to SiNPs. Our results implicate that SiNPs inhibited the macrophage activity and angiogenesis via the downregulation of MAPK singaling pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Assessment of cardiotoxicity and effects of malathion on the early development of zebrafish (Danio rerio) using computer vision for heart rate quantification.

    PubMed

    Simoneschi, Daniele; Simoneschi, Francesco; Todd, Nancy E

    2014-06-01

    Malathion, a common organophosphate insecticide, is a proven acetylcholinesterase inhibitor and is the most applied organophosphate insecticide in the United States. The use of zebrafish as a model to study the effects of pesticides on development is an innovative approach yielding relevant implications for determining the potential toxic effects of these pesticides on humans. In this study, a simple noninvasive technique was developed to investigate the cardiotoxicity of malathion on Danio rerio embryos, and to detect and quantify its effect on heart rate. Videos were recorded under a stereomicroscope and examined with our custom-made software (FishBeat) to determine the heart rate of the embryos. The pixel average intensity frequency (PI) of the videos was computed at its maximum probability to indicate the average number of heartbeats per second. Experimental observations successfully demonstrated that this method was able to detect the heart rate of zebrafish embryos as compared with manual stopwatch counting, with no significant difference. Embryos were treated acutely with increasing malathion concentrations (33.3 and 50 μg/mL malathion) at 52, 76, and 96 hpf. Embryos treated with 33.3 μg/mL malathion had significant bradycardia at 52 and 76 hpf, whereas embryos treated with 50 μg/mL malathion presented bradycardia at all hpf. These novel observations confirmed that malathion, acting as an acetylcholinesterase inhibitor, induced heartbeat irregularity in zebrafish embryos.

  20. Acetyl L-carnitine targets adenosine triphosphate synthase in protecting zebrafish embryos from toxicities induced by verapamil and ketamine: An in vivo assessment.

    PubMed

    Guo, Xiaoqing; Dumas, Melanie; Robinson, Bonnie L; Ali, Syed F; Paule, Merle G; Gu, Qiang; Kanungo, Jyotshna

    2017-02-01

    Verapamil is a Ca 2 + channel blocker and is highly prescribed as an anti-anginal, antiarrhythmic and antihypertensive drug. Ketamine, an antagonist of the Ca 2 + -permeable N-methyl-d-aspartate-type glutamate receptors, is a pediatric anesthetic. Previously we have shown that acetyl l-carnitine (ALCAR) reverses ketamine-induced attenuation of heart rate and neurotoxicity in zebrafish embryos. Here, we used 48 h post-fertilization zebrafish embryos that were exposed to relevant drugs for 2 or 4 h. Heart beat and overall development were monitored in vivo. In 48 h post-fertilization embryos, 2 mm ketamine reduced heart rate in a 2 or 4 h exposure and 0.5 mm ALCAR neutralized this effect. ALCAR could reverse ketamine's effect, possibly through a compensatory mechanism involving extracellular Ca 2 + entry through L-type Ca 2 + channels that ALCAR is known to activate. Hence, we used verapamil to block the L-type Ca 2 + channels. Verapamil was more potent in attenuating heart rate and inducing morphological defects in the embryos compared to ketamine at specific times of exposure. ALCAR reversed cardiotoxicity and developmental toxicity in the embryos exposed to verapamil or verapamil plus ketamine, even in the presence of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, an inhibitor of intracellular Ca 2 + release suggesting that ALCAR acts via effectors downstream of Ca 2 + . In fact, ALCAR's protective effect was blunted by oligomycin A, an inhibitor of adenosine triphosphate synthase that acts downstream of Ca 2 + during adenosine triphosphate generation. We have identified, for the first time, using in vivo studies, a downstream effector of ALCAR that is critical in abrogating ketamine- and verapamil-induced developmental toxicities. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  1. The PBDE metabolite 6-OH-BDE 47 affects melanin pigmentation and THRβ MRNA expression in the eye of zebrafish embryos

    PubMed Central

    Dong, Wu; Macaulay, Laura J; Kwok, Kevin WH; Hinton, David E; Ferguson, P Lee; Stapleton, Heather M

    2015-01-01

    Polybrominated diphenyl ethers and their hydroxyl-metabolites (OH-BDEs) are commonly detected contaminants in human serum in the US population. They are also considered to be endocrine disruptors, and are specifically known to affect thyroid hormone regulation. In this study, we investigated and compared the effects of a PBDE and its OH-BDE metabolite on developmental pathways regulated by thyroid hormones using zebrafish as a model. Exposure to 6-OHBDE 47 (10–100 nM), but not BDE 47 (1–50 μM), led to decreased melanin pigmentation and increased apoptosis in the retina of zebrafish embryos in a concentration-dependent manner in short-term exposures (4 – 30 hours). Six-OH-BDE 47 exposure also significantly decreased thyroid hormone receptor β (THRβ) mRNA expression, which was confirmed using both RT-PCR and in situ hybridization (whole mount and paraffin- section). Interestingly, exposure to the native thyroid hormone, triiodothyronine (T3) also led to similar responses: decreased THRβ mRNA expression, decreased melanin pigmentation and increased apoptosis, suggesting that 6-OH-BDE 47 may be acting as a T3 mimic. To further investigate short-term effects that may be regulated by THRβ, experiments using a morpholino gene knock down and THRβ mRNA over expression were conducted. Knock down of THRβ led to decreases in melanin pigmentation and increases in apoptotic cells in the eye of zebrafish embryos, similar to exposure to T3 and 6-OH-BDE 47, but THRβ mRNA overexpression rescued these effects. Histological analysis of eyes at 22 hpf from each group revealed that exposure to T3 or to 6-OH-BDE 47 was associated with a decrease of melanin and diminished proliferation of cells in layers of retina near the choroid. This study suggests that 6-OH-BDE 47 disrupts the activity of THRβ in early life stages of zebrafish, and warrants further studies on effects in developing humans. PMID:25767823

  2. Intraspinal serotonergic neurons consist of two, temporally distinct populations in developing zebrafish

    PubMed Central

    Montgomery, Jacob E.; Wiggin, Timothy D.; Rivera-Perez, Luis M.; Lillesaar, Christina; Masino, Mark A.

    2015-01-01

    Zebrafish intraspinal serotonergic neuron (ISN) morphology and distribution have been examined in detail at different ages; however, some aspects of the development of these cells remain unclear. Although antibodies to serotonin (5-HT) have detected ISNs in the ventral spinal cord of embryos, larvae, and adults, the only tryptophan hydroxylase (tph) transcript that has been described in the spinal cord is tph1a. Paradoxically, spinal tph1a is expressed transiently in embryos, which brings the source of 5-HT in the ISNs of larvae and adults into question. Because the pet1 and tph2 promoters drive transgene expression in the spinal cord, we hypothesized that tph2 is expressed in spinal cords of zebrafish larvae. We confirmed this hypothesis through in situ hybridization. Next, we used 5-HT antibody labeling and transgenic markers of tph2-expressing neurons to identify a transient population of ISNs in embryos that was distinct from ISNs that appeared later in development. The existence of separate ISN populations may not have been recognized previously due to their shared location in the ventral spinal cord. Finally, we used transgenic markers and immunohistochemical labeling to identify the transient ISN population as GABAergic Kolmer-Agduhr double-prime (KA″) neurons. Altogether, this study revealed a novel developmental paradigm in which KA″ neurons are transiently serotonergic before the appearance of a stable population of tph2-expressing ISNs. PMID:26437856

  3. Zebrafish larvae require specific strains of bacteria for neurobehavioral development

    EPA Science Inventory

    There is an increasing appreciation of the relationship between gut microbiota and nervous system development and function. We previously showed that axenic (microbe-free) larvae are hyperactive at 10 days post fertilization (dpf) relative to colonized zebrafish larvae. Interesti...

  4. Dioxin inhibition of swim bladder development in zebrafish: is it secondary to heart failure?

    PubMed

    Yue, Monica S; Peterson, Richard E; Heideman, Warren

    2015-05-01

    The swim bladder is a gas-filled organ that is used for regulating buoyancy and is essential for survival in most teleost species. In zebrafish, swim bladder development begins during embryogenesis and inflation occurs within 5 days post fertilization (dpf). Embryos exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) before 96 h post fertilization (hpf) developed swim bladders normally until the growth/elongation phase, at which point growth was arrested. It is known that TCDD exposure causes heart malformations that lead to heart failure in zebrafish larvae, and that blood circulation is a key factor in normal development of the swim bladder. The adverse effects of TCDD exposure on the heart occur during the same period of time that swim bladder development and growth occurs. Based on this coincident timing, and the dependence of swim bladder development on proper circulatory development, we hypothesized that the adverse effects of TCDD on swim bladder development were secondary to heart failure. We compared swim bladder development in TCDD-exposed embryos to: (1) silent heart morphants, which lack cardiac contractility, and (2) transiently transgenic cmlc2:caAHR-2AtRFP embryos, which mimic TCDD-induced heart failure via heart-specific, constitutive activation of AHR signaling. Both of these treatment groups, which were not exposed to TCDD, developed hypoplastic swim bladders of comparable size and morphology to those found in TCDD-exposed embryos. Furthermore, in all treatment groups swim bladder development was arrested during the growth/elongation phase. Together, these findings support a potential role for heart failure in the inhibition of swim bladder development caused by TCDD. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. The effects of triclosan on pluripotency factors and development of mouse embryonic stem cells and zebrafish.

    PubMed

    Chen, Xiaojiao; Xu, Bo; Han, Xiumei; Mao, Zhilei; Chen, Minjian; Du, Guizhen; Talbot, Prue; Wang, Xinru; Xia, Yankai

    2015-04-01

    Triclosan (TCS) poses potential risks to reproduction and development due to its endocrine-disrupting properties. However, the mechanism of TCS's effects on early embryonic development is little known. Embryonic stem cells (ESC) and zebrafish embryos provide valuable models for testing the toxic effects of environmental chemicals on early embryogenesis. In this study, mouse embryonic stem cells (mESC) were acutely exposed to TCS for 24 h, and general cytotoxicity and the effect of TCS on pluripotency were then evaluated. In addition, zebrafish embryos were exposed to TCS from 2- to 24-h post-fertilization (hpf), and their morphology was evaluated. In mESC, alkaline phosphatase staining was significantly decreased after treatment with the highest concentration of TCS (50 μM). Although the expression levels of Sox2 mRNA were not changed, the mRNA levels of Oct4 and Nanog in TCS-treated groups were significantly decreased compared to controls. In addition, the protein levels of Oct4, Sox2 and Nanog were significantly reduced in response to TCS treatment. MicroRNA (miR)-134, an expression inhibitor of pluripotency markers, was significantly increased in TCS-treated mESC. In zebrafish experiments, after 24 hpf of treatment, the controls had developed to the late stage of somitogenesis, while embryos exposed to 300 μg/L of TCS were still at the early stage of somitogenesis, and three genes (Oct4, Sox2 and Nanog) were upregulated in treated groups when compared with the controls. The two models demonstrated that TCS may affect early embryonic development by disturbing the expression of the pluripotency markers (Oct4, Sox2 and Nanog).

  6. Biosensors for detecting stress in developing embryos

    NASA Astrophysics Data System (ADS)

    Purdey, Malcolm S.; Saini, Avishkar; McLennan, Hanna J.; Pullen, Benjamin J.; Schartner, Erik P.; Sutton-McDowall, Melanie L.; Thompson, Jeremy G.; Monro, Tanya M.; Nicholls, Stephen J.; Abell, Andrew D.

    2016-12-01

    Reactive Oxygen Species (ROS) cause DNA damage and defective function in sperm and also affects the developmental competence of embryos. It is therefore critical to monitor ROS in sperm, oocytes and developing embryos. In particular, hydrogen peroxide (H2O2) is a ROS important to normal cell function and signalling as well as its role in oxidative stress. Here we report the development of a fluorescent sensor for H2O2 using carboxyperoxyfluor-1 (CPF1) in solution and attached to a glass slide or multi-mode optical fibre. CPF1 increases in fluorescence upon reaction with H2O2 to non-invasively detect H2O2 near developing embryos. These probes are constructed by immobilising CPF1 to the optical fibre tip a polyacrylamide layer. Also reported is a new dual optical fibre sensor for detecting both H2O2 and pH that is functional at biologically concentrations of H2O2 and can sense pH to 0.1 units. This research shows promise for the use of optical fibre sensors for monitoring the health of developing embryos. Furthermore, these sensors are applicable for use beyond embryos such as detecting stress in endothelial cells involved in cardiovascular dysfunction.

  7. Effects of oxytetracycline and amoxicillin on development and biomarkers activities of zebrafish (Danio rerio).

    PubMed

    Oliveira, Rhaul; McDonough, Sakchai; Ladewig, Jessica C L; Soares, Amadeu M V M; Nogueira, António J A; Domingues, Inês

    2013-11-01

    Antibiotics have been widely used in human and veterinary medicine to treat or prevent diseases. Residues of antibiotics have been found in aquatic environments, but their effects on fish have been not properly investigated. This work aimed to assess the sub-lethal effects of oxytetracycline and amoxicillin on zebrafish development and biomarkers. Embryos and adults were exposed during 96 h to amoxicillin and oxytetracycline following OECD guidelines. Tissues of adults and pools of embryos were used for catalase, glutathione-S-transferases and lactate dehydrogenase determinations. Amoxicillin caused premature hatching (48 h-EC50=132.4 mg/l) whereas oxytetracycline cause delayed hatching of embryos (72 h-EC50=127.6 mg/l). Moreover, both antibiotics inhibited catalase and induced glutathione-S-transferases in zebrafish adults. However, only oxytetracycline induced lactate dehydrogenase. Short-term effects of antibiotics were observed at high doses (mg/l) indicating that physiological impairment in fish populations is unlike to occur. However, effects of chronic exposures to low doses of ABs must be investigated. Copyright © 2013 Elsevier B.V. All rights reserved.

  8. Parental exposure to natural mixtures of persistent organic pollutants (POP) induced changes in transcription of apoptosis-related genes in offspring zebrafish embryos.

    PubMed

    Lyche, Jan L; Grześ, Irena M; Karlsson, Camilla; Nourizadeh-Lillabadi, Rasoul; Aleström, Peter; Ropstad, Erik

    2016-01-01

    Apoptosis is an integral element of development that may also be initiated by environmental contaminants. The aim of the present study was to assess potential changes in the regulation of apoptotic genes in zebrafish embryos following parental exposure to two natural mixtures of persistent organic pollutants (POP). The mixture from Lake Mjøsa contained exceptionally high concentrations of polybrominated diphenyl ethers (PBDE), as well as relatively high levels of polychlorinated biphenyls (PCB) and dichlorodiphenyltrichloroethane (DDT). The mixture from Lake Losna contained background concentrations of POP. Genes involved in the apoptotic machinery were screened for their expression profile at four time points during embryonic development. Thirteen and 15 genes involved in apoptosis were found to be significantly upregulated in the high-exposure and background exposure groups, respectively, compared with controls. Modulation of apoptotic genes was restricted only to the first time point, which corresponds with the blastula stage. Although there were substantial differences in POP concentrations between mixtures, genes underlying the apoptosis process showed almost similar responses to the two mixtures. In both exposure groups the main executors of apoptosis p53, casp 2, casp 6, cassp 8, and BAX displayed upregulation compared to controls, suggesting that these POP induce apoptosis via a p53-dependent mechanism. Upregulation of genes that play a critical role in apoptosis suggests that disturbance of normal apoptotic signaling during gametogenesis and embryogenesis may be one of the central mechanisms involved in adverse reproductive effects produced by POP in zebrafish.

  9. Multiple bio-analytical methods to reveal possible molecular mechanisms of developmental toxicity in zebrafish embryos/larvae exposed to tris(2-butoxyethyl) phosphate.

    PubMed

    Han, Zhihua; Wang, Qiangwei; Fu, Jie; Chen, Hongshan; Zhao, Ye; Zhou, Bingsheng; Gong, Zhiyuan; Wei, Si; Li, Jun; Liu, Hongling; Zhang, Xiaowei; Liu, Chunsheng; Yu, Hongxia

    2014-05-01

    The flame retardant tris(2-butoxyethyl) phosphate (TBEP) is a frequently detected contaminant in the environment, wildlife and human milk. The potentially toxic effects of TBEP and their underlying molecular mechanisms have not been elucidated. Here, zebrafish embryos were exposed to different concentrations of TBEP from 4 hours of post-fertilization (hpf) to 120 hpf, and effects on embryonic development and global protein expression patterns examined. Our results demonstrate that treatment with TBEP (0.8-100mg/L) causes a concentration- and time-dependent decrease in embryonic survival and the hatching percentage. The median lethal concentration was 10.7 mg/L at 120 hpf. Furthermore, exposure to 150 or 800 μg/L TBEP inhibited the degradation and utilization of vitellogenins and down-regulated the expression of proteins related to cation binding, and lipid transport, uptake and metabolism, accompanied by a decrease in heart rate and body length. Exposure to TBEP (150 or 800 μg/L) also decreased the expression of proteins involved in cell proliferation and DNA repair, and led to an increased number of apoptotic cells in the tail region. Collectively, our results suggest that exposure to TBEP causes toxicity in the developing zebrafish by inhibiting the degradation and utilization of nutrients from the mother and inducing apoptosis. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Teratogenic, bioenergetic, and behavioral effects of exposure to total particulate matter on early development of zebrafish (Danio rerio) are not mimicked by nicotine

    PubMed Central

    Massarsky, Andrey; Jayasundara, Nishad; Bailey, Jordan M.; Oliveri, Anthony N.; Levin, Edward D.; Prasad, G.L.; Di Giulio, Richard T.

    2016-01-01

    Cigarette smoke has been associated with a number of pathologies; however, the mechanisms leading to developmental effects are yet to be fully understood. The zebrafish embryo is regarded as a ‘bridge model’; however, not many studies examined its applicability to cigarette smoke toxicity. This study examined the effects of total particulate matter (TPM) from 3R4F reference cigarettes on the early development of zebrafish (Danio rerio). Zebrafish embryos were exposed to two concentrations of TPM (0.4 and 1.4 μg/mL equi-nicotine units) or nicotine at equivalent doses. The exposures began at 2 h post-fertilization (hpf) and lasted until 96 hpf. Several physiological parameters were assessed during or after the exposure. We show that TPM increased mortality, delayed hatching, and increased the incidence of deformities in zebrafish. TPM exposure also increased the incidence of hemorrhage and disrupted the angiogenesis of the major vessels in the brain. Moreover, TPM exposure reduced the larval body length, decreased the heart rate, and reduced the metabolic rate. Biomarkers of xenobiotic metabolism and oxidative stress were also affected. TPM-exposed zebrafish also differed behaviorally: at 24 hpf the embryos had a higher frequency of spontaneous contractions and at 144 hpf the larvae displayed swimming hyperactivity. This study demonstrates that TPM disrupts several aspects of early development in zebrafish. The effects reported for TPM were not attributable to nicotine, since embryos treated with nicotine alone did not differ significantly from the control group. Collectively, our work illustrates the utility of zebrafish as an alternative model to evaluate the toxic effects of cigarette smoke constituents. PMID:26391568

  11. Teratogenic, bioenergetic, and behavioral effects of exposure to total particulate matter on early development of zebrafish (Danio rerio) are not mimicked by nicotine.

    PubMed

    Massarsky, Andrey; Jayasundara, Nishad; Bailey, Jordan M; Oliveri, Anthony N; Levin, Edward D; Prasad, G L; Di Giulio, Richard T

    2015-01-01

    Cigarette smoke has been associated with a number of pathologies; however, the mechanisms leading to developmental effects are yet to be fully understood. The zebrafish embryo is regarded as a 'bridge model'; however, not many studies examined its applicability to cigarette smoke toxicity. This study examined the effects of total particulate matter (TPM) from 3R4F reference cigarettes on the early development of zebrafish (Danio rerio). Zebrafish embryos were exposed to two concentrations of TPM (0.4 and 1.4 μg/mL equi-nicotine units) or nicotine at equivalent doses. The exposures began at 2h post-fertilization (hpf) and lasted until 96 hpf. Several physiological parameters were assessed during or after the exposure. We show that TPM increased mortality, delayed hatching, and increased the incidence of deformities in zebrafish. TPM exposure also increased the incidence of hemorrhage and disrupted the angiogenesis of the major vessels in the brain. Moreover, TPM exposure reduced the larval body length, decreased the heart rate, and reduced the metabolic rate. Biomarkers of xenobiotic metabolism and oxidative stress were also affected. TPM-exposed zebrafish also differed behaviorally: at 24 hpf the embryos had a higher frequency of spontaneous contractions and at 144 hpf the larvae displayed swimming hyperactivity. This study demonstrates that TPM disrupts several aspects of early development in zebrafish. The effects reported for TPM were not attributable to nicotine, since embryos treated with nicotine alone did not differ significantly from the control group. Collectively, our work illustrates the utility of zebrafish as an alternative model to evaluate the toxic effects of cigarette smoke constituents. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. The roles of the FGF signal in zebrafish embryos analyzed using constitutive activation and dominant-negative suppression of different FGF receptors.

    PubMed

    Ota, Satoshi; Tonou-Fujimori, Noriko; Yamasu, Kyo

    2009-01-01

    The roles of the FGF family growth factors and their receptors (FGFRs) in zebrafish embryos were examined using variously modified versions of the four FGFR genes (fgfr1-4). Constitutively active forms of all of the examined FGFRs (ca-FGFRs) caused dorsalization, brain caudalization, and secondary axis formation, indicating that the main FGF signal transduction downstream of the receptor is highly similar among FGFRs. All of the membrane-bound type of dominant-negative FGFRs (mdn-FGFRs) derived from the four fgfr genes, which interfere with endogenous FGFRs, produced posterior truncation, as previously reported in both Xenopus and zebrafish. mdn-FGFR3c had the strongest effects on embryos, progressively disrupting the posterior structure as the dose increased. At the highest dose, only the forebrain was formed. At lower doses, mdn-FGFR3c mainly suppressed the paraxial mesoderm. The co-injection of mRNA for different mdn-FGFRs and FGFs resulted in diverse suppression spectra of the respective FGFRs against FGFs. Only mdn-FGFR3c severely suppressed all of the FGFs examined. We also examined the effects of the secretory type of dominant-negative FGFRs (sdn-FGFRs), which are released from cells and trap FGF ligands. Only sdn-FGFR3c resulted in the characteristic effect of selectively disrupting the isthmic development, as well as the tailbud. The co-injection of the mRNA for sdn-FGFRs and FGFs suggested that sdn-FGFR3c inhibits FGFs of the FGF8 subfamily, which is consistent with its specific effects on development. We discuss the implications of our findings obtained in the present study.

  13. GRG5/AES Interacts with T-Cell Factor 4 (TCF4) and Downregulates Wnt Signaling in Human Cells and Zebrafish Embryos

    PubMed Central

    Costa, Ângela M. Sousa; Pereira-Castro, Isabel; Ricardo, Elisabete; Spencer, Forrest; Fisher, Shannon; da Costa, Luís Teixeira

    2013-01-01

    Transcriptional control by TCF/LEF proteins is crucial in key developmental processes such as embryo polarity, tissue architecture and cell fate determination. TCFs associate with β-catenin to activate transcription in the presence of Wnt signaling, but in its absence act as repressors together with Groucho-family proteins (GRGs). TCF4 is critical in vertebrate intestinal epithelium, where TCF4-β-catenin complexes are necessary for the maintenance of a proliferative compartment, and their abnormal formation initiates tumorigenesis. However, the extent of TCF4-GRG complexes’ roles in development and the mechanisms by which they repress transcription are not completely understood. Here we characterize the interaction between TCF4 and GRG5/AES, a Groucho family member whose functional relationship with TCFs has been controversial. We map the core GRG interaction region in TCF4 to a 111-amino acid fragment and show that, in contrast to other GRGs, GRG5/AES-binding specifically depends on a 4-amino acid motif (LVPQ) present only in TCF3 and some TCF4 isoforms. We further demonstrate that GRG5/AES represses Wnt-mediated transcription both in human cells and zebrafish embryos. Importantly, we provide the first evidence of an inherent repressive function of GRG5/AES in dorsal-ventral patterning during early zebrafish embryogenesis. These results improve our understanding of TCF-GRG interactions, have significant implications for models of transcriptional repression by TCF-GRG complexes, and lay the groundwork for in depth direct assessment of the potential role of Groucho-family proteins in both normal and abnormal development. PMID:23840876

  14. GRG5/AES interacts with T-cell factor 4 (TCF4) and downregulates Wnt signaling in human cells and zebrafish embryos.

    PubMed

    Costa, Angela M Sousa; Pereira-Castro, Isabel; Ricardo, Elisabete; Spencer, Forrest; Fisher, Shannon; da Costa, Luís Teixeira

    2013-01-01

    Transcriptional control by TCF/LEF proteins is crucial in key developmental processes such as embryo polarity, tissue architecture and cell fate determination. TCFs associate with β-catenin to activate transcription in the presence of Wnt signaling, but in its absence act as repressors together with Groucho-family proteins (GRGs). TCF4 is critical in vertebrate intestinal epithelium, where TCF4-β-catenin complexes are necessary for the maintenance of a proliferative compartment, and their abnormal formation initiates tumorigenesis. However, the extent of TCF4-GRG complexes' roles in development and the mechanisms by which they repress transcription are not completely understood. Here we characterize the interaction between TCF4 and GRG5/AES, a Groucho family member whose functional relationship with TCFs has been controversial. We map the core GRG interaction region in TCF4 to a 111-amino acid fragment and show that, in contrast to other GRGs, GRG5/AES-binding specifically depends on a 4-amino acid motif (LVPQ) present only in TCF3 and some TCF4 isoforms. We further demonstrate that GRG5/AES represses Wnt-mediated transcription both in human cells and zebrafish embryos. Importantly, we provide the first evidence of an inherent repressive function of GRG5/AES in dorsal-ventral patterning during early zebrafish embryogenesis. These results improve our understanding of TCF-GRG interactions, have significant implications for models of transcriptional repression by TCF-GRG complexes, and lay the groundwork for in depth direct assessment of the potential role of Groucho-family proteins in both normal and abnormal development.

  15. 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

    SciT

    Teraoka, Hiroki; Kubota, Akira; Dong, Wu

    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 veinmore » 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.« less

  16. Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

    SciT

    Ju, Yan; Liu, Chunying; Lu, Wenwen

    The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysismore » revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.« less

  17. The UV-absorber benzophenone-4 alters transcripts of genes involved in hormonal pathways in zebrafish (Danio rerio) eleuthero-embryos and adult males

    SciT

    Zucchi, Sara; Bluethgen, Nancy; University of Basel, Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, Klingelbergstrasse 50, CH-4056 Basel

    Benzophenone-4 (BP-4) is frequently used as UV-absorber in cosmetics and materials protection. Despite its frequent detection in the aquatic environment potential effects on aquatic life are unknown. In this study, we evaluate the effects of BP-4 in eleuthero-embryos and in the liver, testis and brain of adult male fish on the transcriptional level by focusing on target genes involved in hormonal pathways to provide a more complete toxicological profile of this important UV-absorber. Eleuthero-embryos and males of zebrafish were exposed up to 3 days after hatching and for 14 days, respectively, to BP-4 concentrations between 30 and 3000 {mu}g/L. Inmore » eleuthero-embryos transcripts of vtg1, vtg3, esr1, esr2b, hsd17ss3, cyp19b cyp19a, hhex and pax8 were induced at 3000 {mu}g/L BP-4, which points to a low estrogenic activity and interference with early thyroid development, respectively. In adult males BP-4 displayed multiple effects on gene expression in different tissues. In the liver vtg1, vtg3, esr1 and esr2b were down-regulated, while in the brain, vtg1, vtg3 and cyp19b transcripts were up-regulated. In conclusion, the transcription profile revealed that BP-4 interferes with the expression of genes involved in hormonal pathways and steroidogenesis. The effects of BP-4 differ in life stages and adult tissues and point to an estrogenic activity in eleuthero-embryos and adult brain, and an antiestrogenic activity in the liver. The results indicate that BP-4 interferes with the sex hormone system of fish, which is important for the risk assessment of this UV-absorber.« less

  18. The classification of motor neuron defects in the zebrafish embryo toxicity test (ZFET) as an animal alternative approach to assess developmental neurotoxicity.

    PubMed

    Muth-Köhne, Elke; Wichmann, Arne; Delov, Vera; Fenske, Martina

    2012-07-01

    Rodents are widely used to test the developmental neurotoxicity potential of chemical substances. The regulatory test procedures are elaborate and the requirement of numerous animals is ethically disputable. Therefore, non-animal alternatives are highly desirable, but appropriate test systems that meet regulatory demands are not yet available. Hence, we have developed a new developmental neurotoxicity assay based on specific whole-mount immunostainings of primary and secondary motor neurons (using the monoclonal antibodies znp1 and zn8) in zebrafish embryos. By classifying the motor neuron defects, we evaluated the severity of the neurotoxic damage to individual primary and secondary motor neurons caused by chemical exposure and determined the corresponding effect concentration values (EC₅₀). In a proof-of-principle study, we investigated the effects of three model compounds thiocyclam, cartap and disulfiram, which show some neurotoxicity-indicating effects in vertebrates, and the positive controls ethanol and nicotine and the negative controls 3,4-dichloroaniline (3,4-DCA) and triclosan. As a quantitative measure of the neurotoxic potential of the test compounds, we calculated the ratios of the EC₅₀ values for motor neuron defects and the cumulative malformations, as determined in a zebrafish embryo toxicity test (zFET). Based on this index, disulfiram was classified as the most potent and thiocyclam as the least potent developmental neurotoxin. The index also confirmed the control compounds as positive and negative neurotoxicants. Our findings demonstrate that this index can be used to reliably distinguish between neurotoxic and non-neurotoxic chemicals and provide a sound estimate for the neurodevelopmental hazard potential of a chemical. The demonstrated method can be a feasible approach to reduce the number of animals used in developmental neurotoxicity evaluation procedures. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Znrg, a novel gene expressed mainly in the developing notochord of zebrafish.

    PubMed

    Zhou, Yaping; Xu, Yan; Li, Jianzhen; Liu, Yao; Zhang, Zhe; Deng, Fengjiao

    2010-06-01

    The notochord, a defining characteristic of the chordate embryo is a critical midline structure required for axial skeletal formation in vertebrates, and acts as a signaling center throughout embryonic development. We utilized the digital differential display program of the National Center for Biotechnology Information, and identified a contig of expressed sequence tags (no. Dr. 83747) from the zebrafish ovary library in Genbank. Full-length cDNA of the identified gene was cloned by 5'- and 3'- RACE, and the resulting sequence was confirmed by polymerase chain reaction and sequencing. The cDNA clone contains 2,505 base pairs and encodes a novel protein of 707 amino acids that shares no significant homology with any known proteins. This gene was expressed in mature oocytes and at the one-cell stage, and persisted until the 5th day of development, as determined by RT-PCR. Transcripts were detected by whole-mount RNA in situ hybridization from the two-cell stage to 72 h of embryonic development. This gene was uniformly distributed from the cleavage stage up to the blastula stage. During early gastrulation, it was present in the dorsal region, and became restricted to the notochord and pectoral fin at 48 and 72 h of embryonic development. Based on its abundance in the notochord, we hypothesized that the novel gene may play an important role in notochord development in zebrafish; we named this gene, zebrafish notochord-related gene, or znrg.

  20. Effects of exposure to BPF on development and sexual differentiation during early life stages of zebrafish (Danio rerio).

    PubMed

    Yang, Qian; Yang, Xianhai; Liu, Jining; Chen, Yingwen; Shen, Shubao

    2018-05-16

    Bisphenol F (BPF) has become a predominant bisphenol contaminant in recent years. It has significant estrogenic properties in both in vivo and in vitro studies. We have previously studied the disrupting mechanisms of BPF on the hypothalamic-pituitary-gonadal axis of adult zebrafish. However, the effects of BPF exposure on development and sexual differentiation of zebrafish embryos/larvae remain unclear. To determine the effects of BPF on the critical stage of sex differentiation in zebrafish, zebrafish embryos/larvae were exposed to 1, 10, 100, and 1000 μg/L BPF from fertilization to 60 days post-fertilization (dpf). Developmental malformations were induced by exposure to BPF from 2 h post-fertilization (hpf), with a LC 50 of 10,030 μg/L at 96 hpf and 9391 μg/L at 120 hpf. Long-term exposure during sex differentiation tended to result in a female sex ratio bias. Histological analyses at 60 dpf indicated that the development of ovo-testes and immature ovaries was induced by 100 and 1000 μg/L BPF. Homogenate testosterone levels decreased and 17β-estradiol levels increased in zebrafish in a concentration-dependent manner. BPF exposure suppressed gene expression of double sex, Mab3-related transcription factor 1(dmrt1), fushi tarazu factor 1d (ff1d), sry-box containing gene 9a (sox9a) and anti-Mullerian hormone (amh); induced expression of the forkhead box L2 transcription factor (foxl2), leading to increased expression of aromatase (cyp19a1a), which promoted production of estrogens, and further caused phenotypic feminization of zebrafish. These results suggest that developmental exposure to BPF has adverse effects on sexual differentiation, and the results were useful for a BPF risk assessment. Copyright © 2018. Published by Elsevier Inc.

  1. Three enhancer regions regulate gbx2 gene expression in the isthmic region during zebrafish development.

    PubMed

    Islam, Md Ekramul; Kikuta, Hiroshi; Inoue, Fumitaka; Kanai, Maiko; Kawakami, Atsushi; Parvin, Mst Shahnaj; Takeda, Hiroyuki; Yamasu, Kyo

    2006-12-01

    In vertebrate embryos, positioning of the boundary between the midbrain and hindbrain (MHB) and subsequent isthmus formation are dependent upon the interaction between the Otx2 and Gbx genes. In zebrafish, sequential expression of gbx1 and gbx2 in the anterior hindbrain contributes to this process, whereas in mouse embryos, a single Gbx gene (Gbx2) is responsible for MHB development. In the present study, to investigate the regulatory mechanism of gbx2 in the MHB/isthmic region of zebrafish embryos, we cloned the gene and showed that its organization is conserved among different vertebrates. Promoter analyses revealed three enhancers that direct reporter gene expression after the end of epiboly in the anterior-most hindbrain, which is a feature of the zebrafish gbx2 gene. One of the enhancers is located upstream of gbx2 (AMH1), while the other two enhancers are located downstream of gbx2 (AMH2 and AMH3). Detailed analysis of the AMH1 enhancer showed that it directs expression in the rhombomere 1 (r1) region and the dorsal thalamus, as has been shown for gbx2, whereas no expression was induced by the AMH1 enhancer in other embryonic regions in which gbx2 is expressed. The AMH1 enhancer is composed of multiple regulatory subregions that share the same spatial specificity. The most active of the regulatory subregions is a 291-bp region that contains at least two Pax2-binding sites, both of which are necessary for the function of the main component (PB1-A region) of the AMH1 enhancer. In accordance with these results, enhancer activity in the PB1-A region, as well as gbx2 expression in r1, was missing in no isthmus mutant embryos that lacked functional pax2a. In addition, we identified an upstream conserved sequence of 227bp that suppresses the enhancer activity of AMH1. Taken together, these findings suggest that gbx2 expression during the somitogenesis stage in zebrafish is regulated by a complex mechanism involving Pax2 as well as activators and suppressors in the

  2. Chitosan coated carbon fiber microelectrode for selective in vivo detection of neurotransmitters in live zebrafish embryos

    PubMed Central

    Özel, Rıfat Emrah; Wallace, Kenneth N.; Andreescu, Silvana

    2011-01-01

    We report the development of a chitosan modified carbon fiber microelectrode for in vivo detection of serotonin. We find that chitosan has the ability to reject physiological levels of ascorbic acid interferences and facilitate selective and sensitive detection of in vivo levels of serotonin, a common catecholamine neurotransmitter. Presence of chitosan on the microelectrode surface was investigated using scanning electron microscopy (SEM) and cyclic voltammetry (CV). The electrode was characterized using differential pulse voltammetry (DPV). A detection limit of 1.6 nM serotonin with a sensitivity of 5.12 nA/µM, a linear range from 2 to 100 nM and a reproducibility of 6.5 % for n=6 electrodes were obtained. Chitosan modified microelectrodes selectively measure serotonin in presence of physiological levels of ascorbic acid. In vivo measurements were performed to measure concentration of serotonin in the live embryonic zebrafish intestine. The sensor quantifies in vivo intestinal levels of serotonin while successfully rejecting ascorbic acid interferences. We demonstrate that chitosan can be used as an effective coating to reject ascorbic acid interferences at carbon fiber microelectrodes, as an alternative to Nafion, and that chitosan modified microelectrodes are reliable tools for in vivo monitoring of changes in neurotransmitter levels. PMID:21601035

  3. Histone deacetylase is required for the activation of Wnt/β-catenin signaling crucial for heart valve formation in zebrafish embryos.

    PubMed

    Kim, Young-Seop; Kim, Myoung-Jin; Koo, Tae-Hee; Kim, Jun-Dae; Koun, Soonil; Ham, Hyung Jin; Lee, You Mie; Rhee, Myungchull; Yeo, Sang-Yeob; Huh, Tae-Lin

    2012-06-22

    During vertebrate heart valve formation, Wnt/β-catenin signaling induces BMP signals in atrioventricular canal (AVC) myocardial cells and underlying AVC endocardial cells then undergo endothelial-mesenchymal transdifferentiation (EMT) by receiving this BMP signals. Histone deacetylases (HDACs) have been implicated in numerous developmental processes by regulating gene expression. However, their specific roles in controlling heart valve development are largely unexplored. To investigate the role of HDACs in vertebrate heart valve formation, we treated zebrafish embryos with trichostatin A (TSA), an inhibitor of class I and II HDACs, from 36 to 48 h post-fertilization (hpf) during which heart looping and valve formation occur. Following TSA treatment, abnormal linear heart tube development was observed. In these embryos, expression of AVC myocardial bmp4 and AVC endocardial notch1b genes was markedly reduced with subsequent failure of EMT in the AVC endocardial cells. However, LiCl-mediated activation of Wnt/β-catenin signaling was able to rescue defective heart tube formation, bmp4 and notch1b expression, and EMT in the AVC region. Taken together, our results demonstrated that HDAC activity plays a pivotal role in vertebrate heart tube formation by activating Wnt/β-catenin signaling which induces bmp4 expression in AVC myocardial cells. Copyright © 2012 Elsevier Inc. All rights reserved.

  4. Loss of DDB1 Leads to Transcriptional p53 Pathway Activation in Proliferating Cells, Cell Cycle Deregulation, and Apoptosis in Zebrafish Embryos.

    PubMed

    Hu, Zhilian; Holzschuh, Jochen; Driever, Wolfgang

    2015-01-01

    DNA damage-binding protein 1 (DDB1) is a large subunit of the heterodimeric DDB complex that recognizes DNA lesions and initiates the nucleotide excision repair process. DDB1 is also a component of the CUL4 E3 ligase complex involved in a broad spectrum of cellular processes by targeted ubiquitination of key regulators. Functions of DDB1 in development have been addressed in several model organisms, however, are not fully understood so far. Here we report an ENU induced mutant ddb1 allele (ddb1m863) identified in zebrafish (Danio rerio), and analyze its effects on development. Zebrafish ddb1 is expressed broadly, both maternally and zygotically, with enhanced expression in proliferation zones. The (ddb1m863 mutant allele affects the splice acceptor site of exon 20, causing a splicing defect that results in truncation of the 1140 amino acid protein after residue 800, lacking part of the β-propeller domain BPC and the C-terminal helical domain CTD. ddb1m863 zygotic mutant embryos have a pleiotropic phenotype, including smaller and abnormally shaped brain, head skeleton, eyes, jaw, and branchial arches, as well as reduced dopaminergic neuron groups. However, early forming tissues develop normally in zygotic ddb1m863 mutant embryos, which may be due to maternal rescue. In ddb1m863 mutant embryos, pcna-expressing proliferating cell populations were reduced, concurrent with increased apoptosis. We also observed a concomitant strong up-regulation of transcripts of the tumor suppressor p53 (tp53) and the cell cycle inhibitor cdkn1a (p21a/bCIP1/WAF1) in proliferating tissues. In addition, transcription of cyclin genes ccna2 and ccnd1 was deregulated in ddb1m863 mutants. Reduction of p53 activity by anti-sense morpholinos alleviated the apoptotic phenotype in ddb1m863 mutants. These results imply that Ddb1 may be involved in maintaining proper cell cycle progression and viability of dividing cells during development through transcriptional mechanisms regulating genes

  5. Lipidomics and H218O labeling techniques reveal increased remodeling of DHA-containing membrane phospholipids associated with abnormal locomotor responses in α-tocopherol deficient zebrafish (danio rerio) embryos

    PubMed Central

    McDougall, Melissa Q.; Choi, Jaewoo; Stevens, Jan F.; Truong, Lisa; Tanguay, Robert L.; Traber, Maret G.

    2016-01-01

    We hypothesized that vitamin E (α-tocopherol) is required by the developing embryonic brain to prevent depletion of highly polyunsaturated fatty acids, especially docosahexaenoic acid (DHA, 22:6), the loss of which we predicted would underlie abnormal morphological and behavioral outcomes. Therefore, we fed adult 5D zebrafish (Danio rerio) defined diets without (E−) or with added α-tocopherol (E+, 500 mg RRR-α-tocopheryl acetate/kg diet) for a minimum of 80 days, and then spawned them to obtain E− and E+ embryos. The E− compared with E+ embryos were 82% less responsive (p<0.01) to a light/dark stimulus at 96 h post-fertilization (hpf), demonstrating impaired locomotor behavior, even in the absence of gross morphological defects. Evaluation of phospholipid (PL) and lysophospholipid (lyso-PL) composition using untargeted lipidomics in E− compared with E+ embryos at 24, 48, 72, and 120 hpf showed that four PLs and three lyso-PLs containing docosahexaenoic acid (DHA), including lysophosphatidylcholine (LPC 22:6, required for transport of DHA into the brain, p<0.001), were at lower concentrations in E− at all time-points. Additionally, H218O labeling experiments revealed enhanced turnover of LPC 22:6 (p<0.001) and three other DHA-containing PLs in the E− compared with the E+ embryos, suggesting that increased membrane remodeling is a result of PL depletion. Together, these data indicate that α-tocopherol deficiency in the zebrafish embryo causes the specific depletion and increased turnover of DHA-containing PL and lyso-PLs, which may compromise DHA delivery to the brain and thereby contribute to the functional impairments observed in E− embryos. PMID:26774753

  6. The effect of silver nanoparticles on zebrafish embryonic development and toxicology.

    PubMed

    Xia, Guangqing; Liu, Tiantian; Wang, Zhenwei; Hou, Yi; Dong, Lihong; Zhu, Junyi; Qi, Jie

    2016-06-01

    The unique physical and chemical characteristics of nanomaterials, such as the effects of their small size, surface effects, very high rates of reaction, and quantum tunnel effect, have aroused great interest among scholars. However, improper usage has led to an increasing number of nanomaterials entering the environment through various channels, greatly threatening the security of the ecological environment and human health. The urgent need for a scientific assessment of their biosafety can enable nanomaterials to truly benefit humanity. However, the current research in this field is extremely limited with regard to safety standards and waste disposal. In this study, we used silver nanoparticles (nano-Ag) and zebrafish embryos as experimental subjects, and we have reported the deleterious effect on zebrafish embryos treated with different concentrations of nano-Ag, with respect to morphological features (mortality, deformity rate, and heartbeat) and the analysis of expression of relevant genes (sox17, gsc, ntl, otx2); we found a dose-dependent increase in mortality and hatching delay. The results of in situ hybridization indicated that nano-Ag causes a dose-dependent toxicity in embryonic development, and would affect their development and lead to deformity, delayed development, and even death. The safety limit for the concentration of nano-Ag was found to be less than 5 mg/L.

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

    SciT

    Strecker, Ruben, E-mail: Ruben.Strecker@cos.uni-heidelberg.de; Weigt, Stefan, E-mail: stefan.weigt@merckgroup.com; Braunbeck, Thomas, E-mail: braunbeck@uni-hd.de

    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 fracturesmore » 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.« less

  8. Spatiotemporal manipulation of retinoic acid activity in zebrafish hindbrain development via photo-isomerization.

    PubMed

    Xu, Lijun; Feng, Zhiping; Sinha, Deepak; Ducos, Bertrand; Ebenstein, Yuval; Tadmor, Arbel D; Gauron, Carole; Le Saux, Thomas; Lin, Shuo; Weiss, Shimon; Vriz, Sophie; Jullien, Ludovic; Bensimon, David

    2012-09-01

    All-trans retinoic acid (RA) is a key player in many developmental pathways. Most methods used to study its effects in development involve continuous all-trans RA activation by incubation in a solution of all-trans RA or by implanting all-trans RA-soaked beads at desired locations in the embryo. Here we show that the UV-driven photo-isomerization of 13-cis RA to the trans-isomer (and vice versa) can be used to non-invasively and quantitatively control the concentration of all-trans RA in a developing embryo in time and space. This facilitates the global or local perturbation of developmental pathways with a pulse of all-trans RA of known concentration or its inactivation by UV illumination. In zebrafish embryos in which endogenous synthesis of all-trans RA is impaired, incubation for as little as 5 minutes in 1 nM all-trans RA (a pulse) or 5 nM 13-cis RA followed by 1-minute UV illumination is sufficient to rescue the development of the hindbrain if performed no later than bud stage. However, if subsequent to this all-trans RA pulse the embryo is illuminated (no later than bud stage) for 1 minute with UV light (to isomerize, i.e. deactivate, all-trans RA), the rescue of hindbrain development is impaired. This suggests that all-trans RA is sequestered in embryos that have been transiently exposed to it. Using 13-cis RA isomerization with UV light, we further show that local illumination at bud stage of the head region (but not the tail) is sufficient to rescue hindbrain formation in embryos whose all-trans RA synthetic pathway has been impaired.

  9. Dithiocarbamates are teratogenic to developing zebrafish through inhibition of lysyl oxidase activity

    SciT

    Boxtel, Antonius L. van, E-mail: thijs.van.boxtel@ivm.vu.n; Kamstra, Jorke H.; Fluitsma, Donna M.

    2010-04-15

    Dithiocarbamates (DTCs) are a class of compounds that are extensively used in agriculture as pesticides. As such, humans and wildlife are undoubtedly exposed to these chemicals. Although DTCs are thought to be relatively safe due to their short half lives, it is well established that they are teratogenic to vertebrates, especially to fish. In zebrafish, these teratogenic effects are characterized by distorted notochord development and shortened anterior to posterior axis. DTCs are known copper (Cu) chelators but this does not fully explain the observed teratogenic effects. We show here that DTCs cause malformations in zebrafish that highly resemble teratogenic effectsmore » observed by direct inhibition of a group of cuproenzymes termed lysyl oxidases (LOX). Additionally, we demonstrate that partial knockdown of three LOX genes, lox, loxl1 and loxl5b, sensitizes the developing embryo to DTC exposure. Finally, we show that DTCs directly inhibit zebrafish LOX activity in an ex vivo amine oxidase assay. Taken together, these results provide the first evidence that DTC induced teratogenic effects are, at least in part, caused by direct inhibition of LOX activity.« less

  10. Grouper tshβ Promoter-Driven Transgenic Zebrafish Marks Proximal Kidney Tubule Development

    PubMed Central

    Wang, Yang; Sun, Zhi-Hui; Zhou, Li; Li, Zhi; Gui, Jian-Fang

    2014-01-01

    Kidney tubule plays a critical role in recovering or secreting solutes, but the detailed morphogenesis remains unclear. Our previous studies have found that grouper tshβ (gtshβ) is also expressed in kidney, however, the distribution significance is still unknown. To understand the gtshβ role and kidney tubule morphogenesis, here, we have generated a transgenic zebrafish line Tg(gtshβ:GFP) with green fluorescent protein driven by the gtshβ promoter. Similar to the endogenous tshβ in zebrafish or in grouper, the gtshβ promoter-driven GFP is expressed in pituitary and kidney, and the developing details of proximal kidney tubule are marked in the transgenic zebrafish line. The gfp initially transcribes at 16 hours post fertilization (hpf) above the dorsal mesentery, and partially co-localizes with pronephric tubular markers slc20a1a and cdh17. Significantly, the GFP specifically localizes in proximal pronephric segments during embryogenesis and resides at kidney duct epithelium in adult fish. To test whether the gtshβ promoter-driven GFP may serve as a readout signal of the tubular development, we have treated the embryos with retinoic acid signaing (RA) reagents, in which exogenous RA addition results in a distal extension of the proximal segments, while RA inhibition induces a weakness and shortness of the proximal segments. Therefore, this transgenic line provides a useful tool for genetic or chemical analysis of kidney tubule. PMID:24905828

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

    PubMed

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

    2010-12-01

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

  12. Using the zebrafish to understand tendon development and repair

    PubMed Central

    Chen, Jessica W.; Galloway, Jenna L.

    2017-01-01

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

  13. A zebrafish embryo behaves both as a "cortical shell-liquid core" structure and a homogeneous solid when experiencing mechanical forces.

    PubMed

    Liu, Fei; Wu, Dan; Chen, Ken

    2014-12-01

    Mechanical properties are vital for living cells, and various models have been developed to study the mechanical behavior of cells. However, there is debate regarding whether a cell behaves more similarly to a "cortical shell-liquid core" structure (membrane-like) or a homogeneous solid (cytoskeleton-like) when experiencing stress by mechanical forces. Unlike most experimental methods, which concern the small-strain deformation of a cell, we focused on the mechanical behavior of a cell undergoing small to large strain by conducting microinjection experiments on zebrafish embryo cells. The power law with order of 1.5 between the injection force and the injection distance indicates that the cell behaves as a homogenous solid at small-strain deformation. The linear relation between the rupture force and the microinjector radius suggests that the embryo behaves as membrane-like when subjected to large-strain deformation. We also discuss the possible reasons causing the debate by analyzing the mechanical properties of F-actin filaments.

  14. Application of Caenorhabditis elegans (nematode) and Danio rerio embryo (zebrafish) as model systems to screen for developmental and reproductive toxicity of Piperazine compounds.

    PubMed

    Racz, Peter I; Wildwater, Marjolein; Rooseboom, Martijn; Kerkhof, Engelien; Pieters, Raymond; Yebra-Pimentel, Elena Santidrian; Dirks, Ron P; Spaink, Herman P; Smulders, Chantal; Whale, Graham F

    2017-10-01

    To enable selection of novel chemicals for new processes, there is a recognized need for alternative toxicity screening assays to assess potential risks to man and the environment. For human health hazard assessment these screening assays need to be translational to humans, have high throughput capability, and from an animal welfare perspective be harmonized with the principles of the 3Rs (Reduction, Refinement, Replacement). In the area of toxicology a number of cell culture systems are available but while these have some predictive value, they are not ideally suited for the prediction of developmental and reproductive toxicology (DART). This is because they often lack biotransformation capacity, multicellular or multi- organ complexity, for example, the hypothalamus pituitary gonad (HPG) axis and the complete life cycle of whole organisms. To try to overcome some of these limitations in this study, we have used Caenorhabditis elegans (nematode) and Danio rerio embryos (zebrafish) as alternative assays for DART hazard assessment of some candidate chemicals being considered for a new commercial application. Nematodes exposed to Piperazine and one of the analogs tested showed a slight delay in development compared to untreated animals but only at high concentrations and with Piperazine as the most sensitive compound. Total brood size of the nematodes was also reduced primarily by Piperazine and one of the analogs. In zebrafish Piperazine and analogs showed developmental delays. Malformations and mortality in individual fish were also scored. Significant malformations were most sensitively identified with Piperazine, significant mortality was only observed in Piperazine and only at the higest dose. Thus, Piperazine seemed the most toxic compound for both nematodes and zebrafish. The results of the nematode and zebrafish studies were in alignment with data obtained from conventional mammalian toxicity studies indicating that these have potential as developmental

  15. Zebrafish pit1 mutants lack three pituitary cell types and develop severe dwarfism.

    PubMed

    Nica, Gabriela; Herzog, Wiebke; Sonntag, Carmen; Hammerschmidt, Matthias

    2004-05-01

    The Pou domain transcription factor Pit-1 is required for lineage determination and cellular commitment processes during mammalian adenohypophysis development. Here we report the cloning and mutational analysis of a pit1 homolog from zebrafish. Compared with mouse, zebrafish pit1 starts to be expressed at a much earlier stage of adenohypophysis development. However, as in the mouse, expression is restricted to a subset of pituitary cell types, excluding proopiomelanocortin (pomc)-expressing cells (corticotropes, melanotropes) and possibly gonadotropes. We could identify two N-ethyl-N-nitrosourea-induced zebrafish pit1 null mutants. Most mutants die during larval stages, whereas survivors develop severe dwarfism. Mutant larvae lack lactotropes, somatotropes, and thyrotropes, although the adenohypophysis is of normal size, without any sign of increased apoptosis rates. Instead, mutant embryos initiate ectopic expression of pomc in pit1-positive cells, leading to an expansion of the Pomc lineage. Similarly, the number of gonadotropes seems increased, as indicated by the expression of gsualpha, a marker for thyrotropes and gonadotropes. In pit1 mutants, the total number of gsualpha-positive cells is normal despite the loss of gsualpha and tshbeta coexpressing cells. Together, these data suggest a transfating of the Pit1 lineage to the Pomc and possibly the gonadotroph lineages in the mutant, and a pomc- and gonadotropin-repressive role of Pit1 during normal zebrafish development. This is different from mouse, for which a repressive role of Pit-1 has only been reported for the gonadotropin Lhbeta, but not for Pomc. In sum, our data point to both conserved and class-specific aspects of Pit1 function during pituitary development in different vertebrate species.

  16. ZebraBeat: a flexible platform for the analysis of the cardiac rate in zebrafish embryos

    NASA Astrophysics Data System (ADS)

    de Luca, Elisa; Zaccaria, Gian Maria; Hadhoud, Marwa; Rizzo, Giovanna; Ponzini, Raffaele; Morbiducci, Umberto; Santoro, Massimo Mattia

    2014-05-01

    Heartbeat measurement is important in assesssing cardiac function because variations in heart rhythm can be the cause as well as an effect of hidden pathological heart conditions. Zebrafish (Danio rerio) has emerged as one of the most useful model organisms for cardiac research. Indeed, the zebrafish heart is easily accessible for optical analyses without conducting invasive procedures and shows anatomical similarity to the human heart. In this study, we present a non-invasive, simple, cost-effective process to quantify the heartbeat in embryonic zebrafish. To achieve reproducibility, high throughput and flexibility (i.e., adaptability to any existing confocal microscope system and with a user-friendly interface that can be easily used by researchers), we implemented this method within a software program. We show here that this platform, called ZebraBeat, can successfully detect heart rate variations in embryonic zebrafish at various developmental stages, and it can record cardiac rate fluctuations induced by factors such as temperature and genetic- and chemical-induced alterations. Applications of this methodology may include the screening of chemical libraries affecting heart rhythm and the identification of heart rhythm variations in mutants from large-scale forward genetic screens.

  17. Glutathione redox dynamics and expression of glutathione-related genes in the developing embryo

    PubMed Central

    Timme-Laragy, Alicia R.; Goldstone, Jared V.; Imhoff, Barry R.; Stegeman, John J.; Hahn, Mark E.; Hansen, Jason M.

    2013-01-01

    Embryonic development involves dramatic changes in cell proliferation and differentiation that must be highly coordinated and tightly regulated. Cellular redox balance is critical for cell fate decisions, but it is susceptible to disruption by endogenous and exogenous sources of oxidative stress. The most abundant endogenous non-protein antioxidant defense molecule is the tri-peptide glutathione (γ-glutamyl-cysteinylglycine, GSH), but the ontogeny of GSH concentration and redox state during early life stages is poorly understood. Here, we describe the GSH redox dynamics during embryonic and early larval development (0–5 days post-fertilization) in the zebrafish (Danio rerio), a model vertebrate embryo. We measured reduced and oxidized glutathione (GSH, GSSG) using HPLC, and calculated the whole embryo total glutathione (GSHT) concentrations and redox potentials (Eh) over 0–120 hours of zebrafish development (including mature oocytes, fertilization, mid-blastula transition, gastrulation, somitogenesis, pharyngula, pre-hatch embryos, and hatched eleutheroembryos). GSHT concentration doubled between 12 hours post fertilization (hpf) and hatching. The GSH Eh increased, becoming more oxidizing during the first 12 h, and then oscillated around −190 mV through organogenesis, followed by a rapid change, associated with hatching, to a more negative (more reducing) Eh (−220 mV). After hatching, Eh stabilized and remained steady through 120 hpf. The dynamic changes in GSH redox status and concentration defined discrete windows of development: primary organogenesis, organ differentiation, and larval growth. We identified the set of zebrafish genes involved in the synthesis, utilization, and recycling of GSH, including several novel paralogs, and measured how expression of these genes changes during development. Ontogenic changes in the expression of GSH-related genes support the hypothesis that GSH redox state is tightly regulated early in development. This study

  18. The sensitivity and reproducibility of the zebrafish (Danio rerio) embryo test for the screening of waste water quality and for testing the toxicity of chemicals.

    PubMed

    Lahnsteiner, Franz

    2008-07-01

    The sensitivity of the zebrafish embryo test, a test proposed for routine waste water control, was compared with the acute fish toxicity test, in the determination of six types of waste water and ten different chemicals. The waste water was sampled from the following industrial processes: paper and cardboard production, hide tanning, metal galvanisation, carcass treatment and utilisation, and sewage treatment. The chemicals tested were: dimethylacetamide, dimethylsulphoxide, cadmium chloride, cyclohexane, hydroquinone, mercuric chloride, nickel chloride, nonylphenol, resmethrin and sodium nitrite. For many of the test substances, the zebrafish embryo test and the acute fish toxicity test results showed high correlations. However, there were certain environmentally-relevant substances for which the results of the zebrafish embryo test and the acute fish toxicity test differed significantly, up to 10,000-fold (Hg(2+) > 150-fold difference; NO(2)(-) > 300-fold; Cd(2+) > 200-fold; resmethrin > 10,000-fold). For the investigated waste water samples and chemicals, the survival rate of the zebrafish embryos showed high variations between different egg samples, within the range of the EC50 concentration. Subsequently, 5-6 parallel assays were deemed to be the appropriate number necessary for the precise evaluation of the toxicity of the test substances. Also, it was found that the sensitivities of different ontogenetic stages to chemical exposure differed greatly. During the first 12 hours after fertilisation (4-cell stage to the 5-somite stage), the embryos reacted most sensitively to test substance exposure, whereas the later ontogenetic stages showed only slight or no response, indicating that the test is most sensitive during the first 24 hours post-fertilisation.

  19. A conserved role of αA-crystallin in the development of the zebrafish embryonic lens.

    PubMed

    Zou, Ping; Wu, Shu-Yu; Koteiche, Hanane A; Mishra, Sanjay; Levic, Daniel S; Knapik, Ela; Chen, Wenbiao; Mchaourab, Hassane S

    2015-09-01

    αA- and αB-crystallins are small heat shock proteins that bind thermodynamically destabilized proteins thereby inhibiting their aggregation. Highly expressed in the mammalian lens, the α-crystallins have been postulated to play a critical role in the maintenance of lens optical properties by sequestering age-damaged proteins prone to aggregation as well as through a multitude of roles in lens epithelial cells. Here, we have examined the role of α-crystallins in the development of the vertebrate zebrafish lens. For this purpose, we have carried out morpholino-mediated knockdown of αA-, αBa- and αBb-crystallin and characterized the gross morphology of the lens. We observed lens abnormalities, including increased reflectance intensity, as a consequence of the interference with expression of these proteins. These abnormalities were less frequent in transgenic zebrafish embryos expressing rat αA-crystallin suggesting a specific role of α-crystallins in embryonic lens development. To extend and confirm these findings, we generated an αA-crystallin knockout zebrafish line. A more consistent and severe lens phenotype was evident in maternal/zygotic αA-crystallin mutants compared to those observed by morpholino knockdown. The penetrance of the lens phenotype was reduced by transgenic expression of rat αA-crystallin and its severity was attenuated by maternal αA-crystallin expression. These findings demonstrate that the role of α-crystallins in lens development is conserved from mammals to zebrafish and set the stage for using the embryonic lens as a model system to test mechanistic aspects of α-crystallin chaperone activity and to develop strategies to fine-tune protein-protein interactions in aging and cataracts. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. MicroRNA Signaling in Embryo Development

    PubMed Central

    Gross, Nicole; Khatib, Hasan

    2017-01-01

    Expression of microRNAs (miRNAs) is essential for embryonic development and serves important roles in gametogenesis. miRNAs are secreted into the extracellular environment by the embryo during the preimplantation stage of development. Several cell types secrete miRNAs into biological fluids in the extracellular environment. These fluid-derived miRNAs have been shown to circulate the body. Stable transport is dependent on proper packaging of the miRNAs into extracellular vesicles (EVs), including exosomes. These vesicles, which also contain RNA, DNA and proteins, are on the forefront of research on cell-to-cell communication. Interestingly, EVs have been identified in many reproductive fluids, such as uterine fluid, where their miRNA content is proposed to serve as a mechanism of crosstalk between the mother and conceptus. Here, we review the role of miRNAs in molecular signaling and discuss their transport during early embryo development and implantation. PMID:28906477

  1. Effects of particle size and coating on nanoscale Ag and TiO₂ exposure in zebrafish (Danio rerio) embryos.

    PubMed

    Osborne, Olivia J; Johnston, Blair D; Moger, Julian; Balousha, Mohammed; Lead, Jamie R; Kudoh, Tetsuhiro; Tyler, Charles R

    2013-12-01

    Manufactured metal (oxide) nanoparticles are entering the aquatic environment with little understanding on their potential health impacts for exposed organisms. Adopting an integrative approach, we investigated effects of particle size and coating on biological responses for two of the most commonly used metal (oxide) nanoscale particles, silver (Ag) and titanium dioxide (TiO₂) in zebrafish embryos. Titanium dioxide nanoparticles (nominally, 4 nm, 10 nm, 30 nm and 134 nm) had little or no toxicity on the endpoints measured. Ag both in nano form (10 nm and 35 nm) and its larger counterpart (600-1600 nm) induced dose-dependent lethality and morphological defects, occurring predominantly during gastrula stage. Of the silver material tested 10 nm nanoparticles appeared to be the most toxic. Coating Ag nanoparticles with citrate or fulvic acid decreased toxicity significantly. In situ hybridisation analysis identified the yolk syncytial layer (YSL) as a target tissue for Ag-nano toxicity where there was a significant induction of the heavy metal stress response gene, metallothionein 2 (Mt2) at sub-lethal exposures. Coherent Anti-stroke Raman Scattering (CARS) microscopy provided no evidence for silver particles crossing the chorionic membrane in exposed embryos. Collectively, our data suggest that silver ions play a major role in the toxicity of Ag nanoparticles.

  2. DarT: The embryo test with the Zebrafish Danio rerio--a general model in ecotoxicology and toxicology.

    PubMed

    Nagel, Roland

    2002-01-01

    The acute fish test is an animal test whose ecotoxicological relevance is worthy of discussion. The primary aim of protection in ecotoxicology is the population and not the individual. Furthermore the concentration of pollutants in the environment is normally not in the lethal range. Therefore the acute fish test covers solely the situation after chemical spills. Nevertheless, acute fish toxicity data still belong to the base set used for the assessment of chemicals. The embryo test with the zebrafish Danio rerio (DarT) is recommended as a substitute for the acute fish test. For validation an international laboratory comparison test was carried out. A summary of the results is presented in this paper. Based on the promising results of testing chemicals and waste water the test design was validated by the DIN-working group "7.6 Fischei-Test". A normed test guideline for testing waste water with fish is available. The test duration is short (48 h) and within the test different toxicological endpoints can be examined. Endpoints from the embryo test are suitable for QSAR-studies. Besides the use in ecotoxicology the introduction as a toxicological model was investigated. Disturbance of pigmentation and effects on the frequency of heart-beat were examined. A further important application is testing of teratogenic chemicals. Based on the results DarT could be a screening test within preclinical studies.

  3. Monodispersed spherical shaped selenium nanoparticles (SeNPs) synthesized by Bacillus subtilis and its toxicity evaluation in zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Chandramohan, Subburaman; Sundar, Krishnan; Muthukumaran, Azhaguchamy

    2018-02-01

    Selenium is one of the essential elements involved in antioxidative and antiinflammatory effects in human body. By naturally, selenium ions are metabolised and converted into nano selenium. Now a days there is an increasing attention on applications of nanoparticles in therapeutic field. In the present study Bacillus subtilis was used to convert sodium selenite to SeNPs. The synthesized SeNPs were characterized by Fourier Transform Infrared Spectroscopy (FTIR), X Ray Diffraction (XRD) and Scanning Electron Microscopy (SEM) coupled with Energy Dispersive X ray spectroscopy (EDX). The presence of SeNPs was confirmed by the formation of red colour. The bands were sharp with broad absorption peaks at 3562 cm-1 and 1678-1 cm in FTIR which showed that the bacterial proteins were responsible for the reduction of sodium selenite to SeNPs. The average size of the SeNPs was 334 nm and were spherical in shape with uniform distribution. The XRD data confirmed that SeNPs were of amorphous in nature. The zeta potential of SeNPs was negative in charge which indicated high stability. In the present study zebrafish embryos were used to study the toxicity of SeNPs and the results showed that the concentration beyond 10 μg ml-1 leads to toxic effects in embryos/hatchlings. The lesser concentration of SeNPs can be useful in various biomedical applications.

  4. Effects of ibuprofen, diclofenac and paracetamol on hatch and motor behavior in developing zebrafish (Danio rerio).

    PubMed

    Xia, Liang; Zheng, Liang; Zhou, Jun Liang

    2017-09-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) which are widely used as pain relief medicines are causing increasing environmental concern due to their incomplete removal in wastewater treatment plant and potential toxicity on endocrine, kidney and reproduction in teleost fish. This study focused on the effects of widely used ibuprofen, diclofenac and paracetamol on the hatch and motor ability of early-stage zebrafish, by exposing embryos to the target chemicals at 5, 50 and 500 μg/L starting from 6 h postfertilization (hpf). A significant reduction in hatch rate at 55 hpf was caused by both ibuprofen (-63%) and diclofenac (-58%) at 500 μg/L. Exposure to high concentration of ibuprofen significantly decreased the spontaneous movement by 25%, and reduced the free swimming distance, duration and speed under dark condition by 41%, 29% and 30%, respectively. High concentration of diclofenac also caused 23% decrease in spontaneous movement, and reduced the swimming distance as well as active duration by 17% and 13% under light stimulation. In comparison, the exposure to paracetamol did not cause any notable effect. Among neuron related genes tested, the expression of neurog1 was down-regulated from ibuprofen and diclofenac exposure by 19% and 26%, while the expression of neurod1 was up-regulated only by ibuprofen (31%). These findings indicated that ibuprofen and diclofenac significantly affected embryo locomotivity and were potentially neurotoxic, thus posing threats to zebrafish development. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. [Association of human chorionic gonadotropin level in embryo culture media with early embryo development].

    PubMed

    Wang, Haiying; Zhang, Renli; Han, Dong; Liu, Caixia; Cai, Jiajie; Bi, Yanling; Wen, Anmin; Quan, Song

    2014-06-01

    To investigate the association of human chorionic gonadotropin (HCG) level on day 3 of embryo culture with embryo development. Spent culture media were collected from individually cultured embryos on day 3 of in vitro fertilization and embryo transfer (IVF-ET) cycles. HCG concentration in the culture media was measured using an ELISA kit and its association with embryo development was assessed. In the 163 samples of embryo culture media from 60 patients, HCG was positive in 153 sample (93.8%) with a mean level of 0.85 ± 0.43 mIU/ml. The concentration of hCG in the culture media increased gradually as the number of blastomeres increased (F=2.273, P=0.03), and decreased as the morphological grade of the embryo was lowered (F=3.900, P=0.02). ELISA is capable of detecting HCG levels in spent culture media of embryos on day 3 of in vitro culture. The concentration of HCG in spent culture media is positively correlated with the status of early embryo development and implantation rate and thus serves as a useful marker for embryo selection in IVF-ET procedure.

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

    EPA Science Inventory

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

  7. Next generation mothers: Maternal control of germline development in zebrafish.

    PubMed

    Dosch, Roland

    2015-01-01

    In many animals, factors deposited by the mother into the egg control the earliest events in development of the zygote. These maternal RNAs and proteins play critical roles in oocyte development and the earliest steps of embryogenesis such as fertilization, cell division and embryonic patterning. Here, this article summarizes recent discoveries made on the maternal control of germline specification in zebrafish. Moreover, this review will discuss the major gaps remaining in our understanding of this process and highlight recent technical innovations in zebrafish, which allow tackling some of these questions in the near future.

  8. Venous-derived angioblasts generate organ-specific vessels during zebrafish embryonic development.

    PubMed

    Hen, Gideon; Nicenboim, Julian; Mayseless, Oded; Asaf, Lihee; Shin, Masahiro; Busolin, Giorgia; Hofi, Roy; Almog, Gabriella; Tiso, Natascia; Lawson, Nathan D; Yaniv, Karina

    2015-12-15

    Formation and remodeling of vascular beds are complex processes orchestrated by multiple signaling pathways. Although it is well accepted that vessels of a particular organ display specific features that enable them to fulfill distinct functions, the embryonic origins of tissue-specific vessels and the molecular mechanisms regulating their formation are poorly understood. The subintestinal plexus of the zebrafish embryo comprises vessels that vascularize the gut, liver and pancreas and, as such, represents an ideal model in which to investigate the early steps of organ-specific vessel formation. Here, we show that both arterial and venous components of the subintestinal plexus originate from a pool of specialized angioblasts residing in the floor of the posterior cardinal vein (PCV). Using live imaging of zebrafish embryos, in combination with photoconvertable transgenic reporters, we demonstrate that these angioblasts undergo two phases of migration and differentiation. Initially, a subintestinal vein forms and expands ventrally through a Bone Morphogenetic Protein-dependent step of collective migration. Concomitantly, a Vascular Endothelial Growth Factor-dependent shift in the directionality of migration, coupled to the upregulation of arterial markers, is observed, which culminates with the generation of the supraintestinal artery. Together, our results establish the zebrafish subintestinal plexus as an advantageous model for the study of organ-specific vessel development and provide new insights into the molecular mechanisms controlling its formation. More broadly, our findings suggest that PCV-specialized angioblasts contribute not only to the formation of the early trunk vasculature, but also to the establishment of late-forming, tissue-specific vascular beds. © 2015. Published by The Company of Biologists Ltd.

  9. Cells, embryos and development in space

    NASA Technical Reports Server (NTRS)

    Krikorian, A. D.

    1984-01-01

    Work continues to focus on the demonstrable totipotency of cultured somatic cells of various higher plants and has examined the conditions which regulate this propensity to be controllably released. This was done with special reference to cells obtained from cultured explants of daylily and carrot. For purposes of identifying the variables in question, work was carried out almost exclusively in liquid media. The events that intervene between the aseptic isolation of tissue explants, the culture of small derived units and free cells and the propagation in large numbers of adventive or somatic embryos to plantlets were traced and certain definitive stages at which control is exercised were identified. In daylily, morphologically competent units are now propagated with a high degree of precision in rotated liquid cultures in bulk, and under the conditions of continuous neutralized gravity, the development progresses so that embryo-plantlets are obtained.

  10. Molecular aspect of silver nanoparticles regulated embryonic development in Zebrafish (Danio rerio) by Oct-4 expression.

    PubMed

    Sarkar, Biplab; Verma, Suresh K; Akhtar, Javed; Netam, Surya Prakash; Gupta, Sanjay Kr; Panda, Pritam Kumar; Mukherjee, Koel

    2018-09-01

    With the enhancement of commercial manifestation of silver nanoparticles, concerned has risen on their accumulation in aquatic system and consequent effects on fish development and metabolism. In this study, experiments were conducted to assess the impacts of silver nanoparticles on early life cycles of fish considering Zebrafish (Danio rerio) as experimental model. Silver nanoparticles were synthesized through chemical reduction method and characterized through UV-visible spectroscopy, dynamic light scattering (DLS), and HR-TEM. Different sub lethal doses of nanosilver were applied (13.6, 21.6, 42.4, 64, and 128 μgL -1 ) to post-fertilization phases of Zebrafish embryos and their interaction effects were monitored up to six days period. No significant morphological variations were observed at 13.6, 21.6, 42.4 μgL -1 dose of silver nanoparticles, whereas 64 and 128 μgL -1 exposure dose exhibited bending in myotome, deformity in tail region, somites, notochord and swelling in anterior and posterior region of embryos and larva. Hatching performances analysis elicited highest hatching success in 13.6 and 21.6 μgL -1 doses of silver nanoparticles followed by positive and negative control, whereas exposure dose of 64 and 128 μgL -1 exhibited comparatively lower success. Western blot analysis were conducted on developing hatchlings with Oct4 antibody and at 13.6 and 21.6 μgL -1 dose,it showed over expression elucidating stimulatory role of nanosilver in these mentioned doses. In silico analysis depicted a firm interaction of nanosilver with Oct4 revealing their key role in growth stimulation of developing embryos. The study demonstrates the function of nanosilver as a growth promoter rather only as a toxicant in fish metabolic system. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. Comparison of the in vitro and in vivo toxic effects of three sizes of zinc oxide (ZnO) particles using flounder gill (FG) cells and zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Han, Li; Zhai, Yanan; Liu, Yang; Hao, Linhua; Guo, Huarong

    2017-02-01

    Nano-sized zinc oxide (nZnO) particles are one kind of the most commonly used metal oxide nanoparticles (NPs). This study compared the cytotoxic and embryotoxic effects of three increasing sized ZnO particles (ϕ 30 nm, 80-150 nm and 2 μm) in the flounder gill (FG) cells and zebrafish embryos, and analyzed the contribution of size, agglomeration and released Zn2+ to the toxic effects. All the tested ZnO particles were found to be highly toxic to both FG cells and zebrafish embryos. They induced growth inhibition, LDH release, morphological changes and apoptosis in FG cells in a concentration-, size- and time-dependent manner. Moreover, the release of LDH from the exposed FG cells into the medium occurred before the observable morphological changes happened. The ultrasonication treatment and addition of serum favored the dispersion of ZnO particles and alleviated the agglomeration, thus significantly increased the corresponding cytotoxicity. The released Zn2+ ions from ZnO particles into the extracellular medium only partially contributed to the cytotoxicity. All the three sizes of ZnO particles tested induced developmental malformations, decrease of hatching rates and lethality in zebrafish embryos, but size- and concentration- dependent toxic effects were not so obvious as in FG cells possibly due to the easy aggregation of ZnO particles in freshwater. In conclusion, both FG cells and zebrafish embryos are sensitive bioassay systems for safety assessment of ZnO particles and the environmental release of ZnO particles should be closely monitored as far as the safety of aquatic organisms is concerned.

  12. Transcription factor COUP-TFII is indispensable for venous and lymphatic development in zebrafish and Xenopus laevis

    SciT

    Aranguren, Xabier L., E-mail: xabier.lopezaranguren@med.kuleuven.be; Beerens, Manu, E-mail: manu.beerens@med.kuleuven.be; Vandevelde, Wouter, E-mail: woutervandevelde@gmail.com

    Highlights: {yields} COUP-TFII deficiency in zebrafish affects arterio-venous EC specification. {yields} COUP-TFII is indispensable for lymphatic development in zebrafish. {yields} COUP-TFII knockdown in Xenopus disrupts lymphatic EC differentiation and migration. {yields} COUP-TFII's role in EC fate decisions is evolutionary conserved. -- Abstract: Transcription factors play a central role in cell fate determination. Gene targeting in mice revealed that Chicken Ovalbumin Upstream Promoter-Transcription Factor II (COUP-TFII, also known as Nuclear Receptor 2F2 or NR2F2) induces a venous phenotype in endothelial cells (ECs). More recently, NR2F2 was shown to be required for initiating the expression of Prox1, responsible for lymphatic commitment ofmore » venous ECs. Small animal models like zebrafish embryos and Xenopus laevis tadpoles have been very useful to elucidate mechanisms of (lymph) vascular development. Therefore, the role of NR2F2 in (lymph) vascular development was studied by eliminating its expression in these models. Like in mice, absence of NR2F2 in zebrafish resulted in distinct vascular defects including loss of venous marker expression, major trunk vessel fusion and vascular leakage. Both in zebrafish and Xenopus the development of the main lymphatic structures was severely hampered. NR2F2 knockdown significantly decreased prox1 expression in zebrafish ECs and the same manipulation affected lymphatic (L)EC commitment, migration and function in Xenopus tadpoles. Therefore, the role of NR2F2 in EC fate determination is evolutionary conserved.« less

  13. Relative developmental toxicity of short-chain chlorinated paraffins in Zebrafish (Danio rerio) embryos.

    PubMed

    Liu, Lihua; Li, Yifan; Coelhan, Mehmet; Chan, Hing Man; Ma, Wanli; Liu, Liyan

    2016-12-01

    Short-chain chlorinated paraffins (SCCPs) are ubiquitous in the environment and might cause adverse environmental and human health effects. Little is known about the relative toxicity of different SCCP compounds especially during development. The objective of this study was to characterize and compare effects of seven SCCP groups at environmentally relevant levels, using a zebrafish (Danio rerio) model. Observations on malformation, survival rates at 96 h post fertilization (hpf), and hatching rates at 72 hpf indicated that the C 10- groups (C 10 H 18 Cl 4 , 1,2,5,6,9,10-C 10 H 16 Cl 6 and C 10 H 15 Cl 7 ) were more toxic than the C 12- groups (C 12 H 22 Cl 4 , C 12 H 19 Cl 7 and 1,1,1,3,10,12,12,12-C 12 H 18 Cl 8 ) and Cereclor 63L. The C 10- groups were also more potent than C 12- groups and Cereclor 63L in decreasing thyroid hormone levels. Among the three compounds within the C 10- group, the compounds with less chlorine content had stronger effects on sub-lethal malformations but less effects on triiodothyronine (T3) and tetraiodothyronine (T4). Only C 10 H 18 Cl 4 significantly decreased the mRNA expression of tyr, ttr, dio2 and dio3 at a dose-dependent manner suggesting that the specific mode of actions differ with different congeners. The mechanisms of disruption of thyroid status by different SCCPs could be different. C 10 H 18 Cl 4 might inhibit T3 production through the inhibition effect on dio2. These results indicate that SCCP exposure could alter gene expression in the hypothalamic-pituitary-thyroid (HPT) axis and thyroid hormone levels. The mechanisms of disruption of thyroid status by different SCCPs could be different. Our results on the relative developmental toxicities of SCCPs will be useful to reach a better understanding of SCCP toxicity supporting environmental risk evaluation and regulation and used as a guidance for environmental monitoring of SCCPs in the future. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Morphologic analysis of the zebrafish digestive system.

    PubMed

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

    2009-01-01

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

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

    PubMed Central

    Buske, Christine; Gerlai, Robert

    2010-01-01

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

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

    EPA Science Inventory

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

  17. Egg embryo development detection with hyperspectral imaging

    NASA Astrophysics Data System (ADS)

    Lawrence, Kurt C.; Smith, Douglas P.; Windham, William R.; Heitschmidt, Gerald W.; Park, Bosoon

    2006-10-01

    In the U. S. egg industry, anywhere from 130 million to over one billion infertile eggs are incubated each year. Some of these infertile eggs explode in the hatching cabinet and can potentially spread molds or bacteria to all the eggs in the cabinet. A method to detect the embryo development of incubated eggs was developed. Twelve brown-shell hatching eggs from two replicates (n=24) were incubated and imaged to identify embryo development. A hyperspectral imaging system was used to collect transmission images from 420 to 840 nm of brown-shell eggs positioned with the air cell vertical and normal to the camera lens. Raw transmission images from about 400 to 900 nm were collected for every egg on days 0, 1, 2, and 3 of incubation. A total of 96 images were collected and eggs were broken out on day 6 to determine fertility. After breakout, all eggs were found to be fertile. Therefore, this paper presents results for egg embryo development, not fertility. The original hyperspectral data and spectral means for each egg were both used to create embryo development models. With the hyperspectral data range reduced to about 500 to 700 nm, a minimum noise fraction transformation was used, along with a Mahalanobis Distance classification model, to predict development. Days 2 and 3 were all correctly classified (100%), while day 0 and day 1 were classified at 95.8% and 91.7%, respectively. Alternatively, the mean spectra from each egg were used to develop a partial least squares regression (PLSR) model. First, a PLSR model was developed with all eggs and all days. The data were multiplicative scatter corrected, spectrally smoothed, and the wavelength range was reduced to 539 - 770 nm. With a one-out cross validation, all eggs for all days were correctly classified (100%). Second, a PLSR model was developed with data from day 0 and day 3, and the model was validated with data from day 1 and 2. For day 1, 22 of 24 eggs were correctly classified (91.7%) and for day 2, all eggs

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

    PubMed

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

    2017-01-01

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

  19. Subacute developmental exposure of zebrafish to the organophosphate pesticide metabolite, chlorpyrifos-oxon, results in defects in Rohon-Beard sensory neuron development

    PubMed Central

    Jacobson, Saskia M.; Birkholz, Denise A.; McNamara, Marcy L.; Bharate, Sandip B.; George, Kathleen M.

    2010-01-01

    Organophosphate pesticides (OPs) are environmental toxicants known to inhibit the catalytic activity of acetylcholinesterase (AChE) resulting in hypercholinergic toxicity symptoms. In developing embryos, OPs have been hypothesized to affect both cholinergic and non-cholinergic pathways. In order to understand the neurological pathways affected by OP exposure during embryogenesis, we developed a subacute model of OP developmental exposure in zebrafish by exposing embryos to a dose of the OP metabolite chlorpyrifos oxon (CPO) that is non-lethal and significantly inhibited AChE enzymatic activity compared to control embryos (43% at 1 day post-fertilization (dpf) and 11% at 2 dpf). Phenotypic analysis of CPO-exposed embryos demonstrated that embryonic growth, as analyzed by gross morphology, was normal in 85% of treated embryos. Muscle fiber formation was similar to control embryos as analyzed by birefringence, and nicotinic acetylcholine receptor (nAChR) cluster formation was quantitatively similar to control embryos as analyzed by α-bungarotoxin staining. These results indicate that partial AChE activity during the early days of zebrafish development is sufficient for general development, muscle fiber, and nAChR development. Rohon-Beard (RB) sensory neurons exhibited aberrant peripheral axon extension and gene expression profiling suggests that several genes responsible for RB neurogenesis are down-regulated. Stability of CPO in egg water at 28.5 °C was determined by HPLC-UV-MS analysis which revealed that the CPO concentration used in our studies hydrolyzes in egg water with a half-life of one day. The result that developmental CPO exposure affected RB neurogenesis without affecting muscle fiber or nAChR cluster formation demonstrates that zebrafish are a strong model system for characterizing subtle neurological pathologies resulting from environmental toxicants. PMID:20701988

  20. Effect of embryo source and recipient progesterone environment on embryo development in cattle.

    PubMed

    Lonergan, P; Woods, A; Fair, T; Carter, F; Rizos, D; Ward, F; Quinn, K; Evans, A

    2007-01-01

    The aim of the present study was to examine the effect of embryo source (in vivo v. in vitro) and the progesterone environment into which it was transferred on Day 7 on embryo survival and size on Day 13. Day 7 blastocysts were produced either in vivo using superovulation, artificial insemination and non-surgical embryo recovery or in vitro using in vitro maturation, fertilisation and culture. In order to produce animals with divergent progesterone concentrations, following synchronisation recipients were either superovulated (High progesterone; n = 10) or not (Control progesterone; n = 10). Ten blastocysts, produced either in vivo or in vitro, were transferred to each recipient on Day 7. Both groups were killed on Day 13. The mean progesterone concentration from Day 7 to Day 13 (the period when the embryos were in the uterus) in the High and Control progesterone recipients was 36.32 +/- 1.28 and 10.30 +/- 0.51 ng mL(-1), respectively. Of the in vivo embryos transferred, the overall recovery rate at Day 13 was 64%, which was higher (P < 0.001) than that of 20% for the in vitro embryos transferred. The mean area of embryos recovered from High progesterone recipients was 3.86 +/- 0.45 mm(2) (n = 28) compared with 1.66 +/- 0.38 mm(2) (n = 24) for embryos recovered from Control progesterone recipients (P < 0.001). Similarly, the origin of the embryo used for transfer affected embryo size on Day 13. In summary, the recovery rate of blastocysts was higher for in vivo- than in vitro-derived embryos. Blastocyst size was approximately 2.3-fold greater in recipients with high compared with normal progesterone. The present study lends strong support to the hypothesis that an earlier rise in progesterone after conception stimulates blastocyst growth and the development of competent embryos.

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

    PubMed

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

    2015-12-01

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

  2. Erk-Creb pathway suppresses glutathione-S-transferase pi expression under basal and oxidative stress conditions in zebrafish embryos.

    PubMed

    Hrubik, Jelena; Glisic, Branka; Fa, Svetlana; Pogrmic-Majkic, Kristina; Andric, Nebojsa

    2016-01-05

    Transcriptional activation of phase II enzymes including glutathione-S-transferase pi class (Gst Pi) is important for redox regulation and defense from xenobiotics. The role of extracellular signal-regulated kinase (Erk) and protein kinase B (Akt) in regulation of Gst Pi expression has been described using adult mammalian cells. Whether these signaling pathways contribute to Gst Pi expression during embryogenesis is unknown. Using zebrafish embryo model, we provide novel evidence that Erk signaling acts as a specific suppressor of gstp1-2 mRNA during early embryogenesis. Addition of Erk inhibitor U0126 enhanced gstp1-2 mRNA expression during transition from blastula to the segmentation stage and from pharyngula until the hatching stage. Basal Erk activity did not affect gstp1-2 expression in tert-butylhydroquinone-exposed embryos. Addition of phorbol 12-myristate 13-acetate increased Erk activity leading to suppression of gstp1-2 mRNA. Activation of cAMP/Creb pathway by forskolin prevented gstp1-2 expression, whereas U0126 suppressed Creb phosphorylation, thus setting up Creb as a proximal transmitter of Erk inhibitory effect. Collectively, these findings suggest that Erk-Creb pathway exerts suppressive effect on gstp1-2 mRNA in a narrow developmental window. This study also provides a novel link between Erk and gstp1-2 expression, setting apart a possible differential regulation of gstp1-2 in adult and embryonic cells. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  3. Redundant roles of PRDM family members in zebrafish craniofacial development.

    PubMed

    Ding, Hai-Lei; Clouthier, David E; Artinger, Kristin B

    2013-01-01

    PRDM proteins are evolutionary conserved Zn-Finger transcription factors that share a characteristic protein domain organization. Previous studies have shown that prdm1a is required for the specification and differentiation of neural crest cells in the zebrafish. Here we examine other members of this family, specifically prdm3, 5, and 16, in the differentiation of the zebrafish craniofacial skeleton. prdm3 and prdm16 are strongly expressed in the pharyngeal arches, while prdm5 is expressed specifically in the area of the forming neurocranium. Knockdown of prdm3 and prdm16 results in a reduction in the neural crest markers dlx2a and barx1 and defects in both the viscerocranium and the neurocranium. The knockdown of prdm3 and prdm16 in combination is additive in the neurocranium, but not in the viscerocranium. Injection of sub-optimal doses of prdm1a with prdm3 or prdm16 Morpholinos together leads to more severe phenotypes in the viscerocranium and neurocranium. prdm5 mutants have defects in the neurocranium and prdm1a and prdm5 double mutants also show more severe phenotypes. Overall, our data reveal that prdm3, 5, and 16 are involved in the zebrafish craniofacial development and that prdm1a may interact with prdm3, 5, and 16 in the formation of the craniofacial skeleton in zebrafish. Copyright © 2012 Wiley Periodicals, Inc.

  4. Redundant Roles of PRDM Family Members in Zebrafish Craniofacial Development

    PubMed Central

    Ding, Hai-Lei; Clouthier, David E.; Artinger, Kristin B.

    2014-01-01

    Background PRDM proteins are evolutionary conserved Zn-Finger transcription factors that share a characteristic protein domain organization. Previous studies have shown that prdm1a is required for the specification and differentiation of neural crest cells in the zebrafish. Results Here we examine other members of this family, specifically prdm3, 5, and 16, in the differentiation of the zebrafish craniofacial skeleton. prdm3 and prdm16 are strongly expressed in the pharyngeal arches, while prdm5 is expressed specifically in the area of the forming neurocranium. Knockdown of prdm3 and prdm16 results in a reduction in the neural crest markers dlx2a and barx1 and defects in both the viscerocranium and the neurocranium. The knockdown of prdm3 and prdm16 in combination is additive in the neurocranium, but not in the viscerocranium. Injection of sub-optimal doses of prdm1a with prdm3 or prdm16 Morpholinos together leads to more severe phenotypes in the viscerocranium and neurocranium. prdm5 mutants have defects in the neurocranium and prdm1a and prdm5 double mutants also show more severe phenotypes. Conclusions Overall, our data reveal that prdm3, 5, and 16 are involved in the zebrafish craniofacial development and that prdm1a may interact with prdm3, 5, and 16 in the formation of the craniofacial skeleton in zebrafish. PMID:23109401

  5. Zinc and cadmium accumulation in single zebrafish ( Danio rerio) embryos — A total reflection X-ray fluorescence spectrometry application

    NASA Astrophysics Data System (ADS)

    Mages, Margarete; Bandow, Nicole; Küster, Eberhard; Brack, Werner; von Tümpling, Wolf

    2008-12-01

    Trace metals such as Cadmium (Cd) and Zinc (Zn) are known to exhibit adverse effects on many aquatic organisms including early life stages of fish. In contact with contaminated sediment, fish eggs and embryos may be exposed to metals via the water phase as well as via direct contact with contaminated particles. This may result in body burdens that are difficult to predict and may vary according to individual micro scale exposure conditions. The highly sensitive total reflection X-ray fluorescence spectrometry (TXRF) may provide a tool to analyse individual embryos for internal contaminant concentrations and thus helps to develop a better understanding of dose-response relationships. To test this hypothesis, embryos of Danio rerio were exposed to Cd and Zn spiked sediment in different treatments applying an ion exchange resin for modification of bioavailable concentrations. The TXRF analysis indicated individual embryos with dramatically enhanced exposure compared to other individuals despite uniform exposure conditions on a macro scale. Ion exchanger reduced embryo Zn concentrations to values close to control value with a comparably low standard deviation. Cadmium concentrations in embryos were in the range of 4000 to 7000 µg/g with a median of 5740 µg/g. A commercial ion exchanger reduced individual body burdens by a factor 50 to 100. Individual peak body burdens of up to 3160 µg/g were accompanied by reduced weight of the fish eggs due to early death i.e. coagulation. The investigation of exposure and effects on an individual-based scale may significantly help to reduce uncertainty and inconsistencies occurring in conventional analysis of pooled fish embryo samples.

  6. Isolation of a small molecule with anti-MRSA activity from a mangrove symbiont Streptomyces sp. PVRK-1 and its biomedical studies in Zebrafish embryos

    PubMed Central

    Kannan, Rajaretinam Rajesh; Iniyan, Appadurai Muthamil; Prakash, Vincent Samuel Gnana

    2011-01-01

    Objective The aim of the present study was to isolate the anti-MRSA (Methicillin Resistant Staphylococcus aureus) molecule from the Mangrove symbiont Streptomyces and its biomedical studies in Zebrafish embryos. Methods MRSA was isolated from the pus samples of Colachal hospitals and confirmed by amplification of mecA gene. Anti-MRSA molecule producing strain was identified by 16s rRNA gene sequencing. Anti-MRSA compound production was optimized by Solid State Fermentation (SSF) and the purification of the active molecule was carried out by TLC and RP-HPLC. The inhibitory concentration and LC50 were calculated using Statistical software SPSS. The Biomedical studies including the cardiac assay and organ toxicity assessment were carried out in Zebrafish. Results The bioactive anti-MRSA small molecule A2 was purified by TLC with Rf value of 0.37 with 1.389 retention time at RP-HPLC. The Inhibitory Concentration of the purified molecule A2 was 30 µg/mL but, the inhibitory concentration of the MRSA in the infected embryo was 32-34 µg/mL for TLC purified molecule A2 with LC50 mean value was 61.504 µg/mL. Zebrafish toxicity was assessed in 48-60 µg/mL by observing the physiological deformities and the heart beat rates (HBR) of embryos for anti MRSA molecule showed the mean of 41.33-41.67 HBR/15 seconds for 40 µg/mL and control was 42.33-42.67 for 15 seconds which significantly showed that the anti-MRSA molecule A2 did not affected the HBR. Conclusions Anti-MRSA molecule from Streptomyces sp PVRK-1 was isolated and biomedical studies in Zebrafish model assessed that the molecule was non toxic at the minimal inhibitory concentration of MRSA. PMID:23569790

  7. Isolation of a small molecule with anti-MRSA activity from a mangrove symbiont Streptomyces sp. PVRK-1 and its biomedical studies in Zebrafish embryos.

    PubMed

    Kannan, Rajaretinam Rajesh; Iniyan, Appadurai Muthamil; Prakash, Vincent Samuel Gnana

    2011-10-01

    The aim of the present study was to isolate the anti-MRSA (Methicillin Resistant Staphylococcus aureus) molecule from the Mangrove symbiont Streptomyces and its biomedical studies in Zebrafish embryos. MRSA was isolated from the pus samples of Colachal hospitals and confirmed by amplification of mecA gene. Anti-MRSA molecule producing strain was identified by 16s rRNA gene sequencing. Anti-MRSA compound production was optimized by Solid State Fermentation (SSF) and the purification of the active molecule was carried out by TLC and RP-HPLC. The inhibitory concentration and LC50 were calculated using Statistical software SPSS. The Biomedical studies including the cardiac assay and organ toxicity assessment were carried out in Zebrafish. The bioactive anti-MRSA small molecule A2 was purified by TLC with Rf value of 0.37 with 1.389 retention time at RP-HPLC. The Inhibitory Concentration of the purified molecule A2 was 30 µg/mL but, the inhibitory concentration of the MRSA in the infected embryo was 32-34 µg/mL for TLC purified molecule A2 with LC50 mean value was 61.504 µg/mL. Zebrafish toxicity was assessed in 48-60 µg/mL by observing the physiological deformities and the heart beat rates (HBR) of embryos for anti MRSA molecule showed the mean of 41.33-41.67 HBR/15 seconds for 40 µg/mL and control was 42.33-42.67 for 15 seconds which significantly showed that the anti-MRSA molecule A2 did not affected the HBR. Anti-MRSA molecule from Streptomyces sp PVRK-1 was isolated and biomedical studies in Zebrafish model assessed that the molecule was non toxic at the minimal inhibitory concentration of MRSA.

  8. Early zebrafish development: It’s in the maternal genes

    PubMed Central

    Abrams, Elliott W.; Mullins, Mary C.

    2009-01-01

    Summary The earliest stages of embryonic development in all animals examined rely on maternal gene products that are generated during oogenesis and supplied to the egg. The period of maternal control of embryonic development varies among animals according to the onset of zygotic transcription and the persistence of maternal gene products. This maternal regulation has been little studied in vertebrates, due to the difficulty in manipulating maternal gene function and lack of basic molecular information. However, recent maternal-effect screens in the zebrafish have generated more than 40 unique mutants that are providing new molecular entry points to the maternal control of early vertebrate development. Here we discuss recent studies of 12 zebrafish mutant genes that illuminate the maternal molecular controls on embryonic development, including advances in the regulation of animal-vegetal polarity, egg activation, cleavage development, body plan formation, tissue morphogenesis, microRNA function and germ cell development. PMID:19608405

  9. TRNA mutations that affect decoding fidelity deregulate development and the proteostasis network in zebrafish

    PubMed Central

    Reverendo, Marisa; Soares, Ana R; Pereira, Patrícia M; Carreto, Laura; Ferreira, Violeta; Gatti, Evelina; Pierre, Philippe; Moura, Gabriela R; Santos, Manuel A

    2014-01-01

    Mutations in genes that encode tRNAs, aminoacyl-tRNA syntheases, tRNA modifying enzymes and other tRNA interacting partners are associated with neuropathies, cancer, type-II diabetes and hearing loss, but how these mutations cause disease is unclear. We have hypothesized that levels of tRNA decoding error (mistranslation) that do not fully impair embryonic development can accelerate cell degeneration through proteome instability and saturation of the proteostasis network. To test this hypothesis we have induced mistranslation in zebrafish embryos using mutant tRNAs that misincorporate Serine (Ser) at various non-cognate codon sites. Embryo viability was affected and malformations were observed, but a significant proportion of embryos survived by activating the unfolded protein response (UPR), the ubiquitin proteasome pathway (UPP) and downregulating protein biosynthesis. Accumulation of reactive oxygen species (ROS), mitochondrial and nuclear DNA damage and disruption of the mitochondrial network, were also observed, suggesting that mistranslation had a strong negative impact on protein synthesis rate, ER and mitochondrial homeostasis. We postulate that mistranslation promotes gradual cellular degeneration and disease through protein aggregation, mitochondrial dysfunction and genome instability. PMID:25483040

  10. rbm47, a novel RNA binding protein, regulates zebrafish head development.

    PubMed

    Guan, Rui; El-Rass, Suzan; Spillane, David; Lam, Simon; Wang, Yuodong; Wu, Jing; Chen, Zhuchu; Wang, Anan; Jia, Zhengping; Keating, Armand; Hu, Jim; Wen, Xiao-Yan

    2013-12-01

    Vertebrate trunk induction requires inhibition of bone morphogenetic protein (BMP) signaling, whereas vertebrate head induction requires concerted inhibition of both Wnt and BMP signaling. RNA binding proteins play diverse roles in embryonic development and their roles in vertebrate head development remain to be elucidated. We first characterized the human RBM47 as an RNA binding protein that specifically binds RNA but not single-stranded DNA. Next, we knocked down rbm47 gene function in zebrafish using morpholinos targeting the start codon and exon-1/intron-1 splice junction. Down-regulation of rbm47 resulted in headless and small head phenotypes, which can be rescued by a wnt8a blocking morpholino. To further reveal the mechanism of rbm47's role in head development, microarrays were performed to screen genes differentially expressed in normal and knockdown embryos. epcam and a2ml were identified as the most significantly up- and down-regulated genes, respectively. The microarrays also confirmed up-regulation of several genes involved in head development, including gsk3a, otx2, and chordin, which are important regulators of Wnt signaling. Altogether, our findings reveal that Rbm47 is a novel RNA-binding protein critical for head formation and embryonic patterning during zebrafish embryogenesis which may act through a Wnt8a signaling pathway. Copyright © 2013 Wiley Periodicals, Inc.

  11. Metabolism of clofibric acid in zebrafish embryos (Danio rerio) as determined by liquid chromatography-high resolution-mass spectrometry.

    PubMed

    Brox, Stephan; Seiwert, Bettina; Haase, Nora; Küster, Eberhard; Reemtsma, Thorsten

    2016-01-01

    The zebrafish embryo (ZFE) is increasingly used in ecotoxicology research but detailed knowledge of its metabolic potential is still limited. This study focuses on the xenobiotic metabolism of ZFE at different life-stages using the pharmaceutical compound clofibric acid as study compound. Liquid chromatography with quadrupole-time-of-flight mass spectrometry (LC-QToF-MS) is used to detect and to identify the transformation products (TPs). In screening experiments, a total of 18 TPs was detected and structure proposals were elaborated for 17 TPs, formed by phase I and phase II metabolism. Biotransformation of clofibric acid by the ZFE involves conjugation with sulfate or glucuronic acid, and, reported here for the first time, with carnitine, taurine, and aminomethanesulfonic acid. Further yet unknown cyclization products were identified using non-target screening that may represent a new detoxification pathway. Sulfate containing TPs occurred already after 3h of exposure (7hpf), and from 48h of exposure (52hpf) onwards, all TPs were detected. The detection of these TPs indicates the activity of phase I and phase II enzymes already at early life-stages. Additionally, the excretion of one TP into the exposure medium was observed. The results of this study outline the high metabolic potential of the ZFE with respect to the transformation of xenobiotics. Similarities but also differences to other test systems were observed. Biotransformation of test chemicals in toxicity testing with ZFE may therefore need further consideration. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Sodium selenite/selenium nanoparticles (SeNPs) protect cardiomyoblasts and zebrafish embryos against ethanol induced oxidative stress.

    PubMed

    Kalishwaralal, Kalimuthu; Jeyabharathi, Subhaschandrabose; Sundar, Krishnan; Muthukumaran, Azhaguchamy

    2015-10-01

    Alcoholic cardiomyopathy is the damage caused to the heart muscles due to high level of alcohol consumption resulting in enlargement and inflammation of the heart. Selenium is an important trace element that is beneficial to human health. Selenium protects the cells by preventing the formation of free radicals in the body. In the present study, protein mediated synthesis of SeNPs was investigated. Two different sizes of SeNPs were synthesized using BSA and keratin. The synthesized SeNPs were characterized by scanning electron microscopy (SEM) with elemental composition analysis Energy Dispersive X-ray spectroscopy(EDX) and X-ray diffraction (XRD). This study demonstrates the in vitro and in vivo antioxidative effects of sodium selenite and SeNPs. Further selenium and SeNPs were evaluated for their ability to protect against 1% ethanol induced oxidative stress in H9C2 cell line. The selenium and SeNPs were found to reduce the 1% ethanol-induced oxidative damage through scavenging intracellular reactive oxygen species. The selenium and SeNPs could also prevent pericardial edema induced ethanol treatment and reduced apoptosis and cell death in zebrafish embryos. The results indicate that selenium and SeNPs could potentially be used as an additive in alcoholic beverage industry to control the cardiomyopathy. Copyright © 2015 Elsevier GmbH. All rights reserved.

  13. Dmrt1 is necessary for male sexual development in zebrafish

    PubMed Central

    Webster, Kaitlyn A.; Schach, Ursula; Ordaz, Angel; Steinfeld, Jocelyn S.; Draper, Bruce W.; Siegfried, Kellee R.

    2018-01-01

    The dmrt1 (doublesex and mab-3 related transcription factor 1) gene is a key regulator of sex determination and/or gonadal sex differentiation across metazoan animals. This is unusual given that sex determination genes are typically not well conserved. The mechanisms by which zebrafish sex is determined have remained elusive due to the lack of sex chromosomes and the complex polygenic nature of sex determination in domesticated strains. To investigate the role of dmrt1 in zebrafish sex determination and gonad development, we isolated mutations disrupting this gene. We found that the majority of dmrt1 mutant fish develop as fertile females suggesting a complete male-to-female sex reversal in mutant animals that would have otherwise developed as males. A small percentage of mutant animals became males, but were sterile and displayed testicular dysgenesis. Therefore zebrafish dmrt1 functions in male sex determination and testis development. Mutant males had aberrant gonadal development at the onset of gonadal sex-differentiation, displaying reduced oocyte apoptosis followed by development of intersex gonads and failed testis morphogenesis and spermatogenesis. By contrast, female ovaries developed normally. We found that Dmrt1 is necessary for normal transcriptional regulation of the amh (anti-Müllerian hormone) and foxl2 (forkhead box L2) genes, which are thought to be important for male or female sexual development respectively. Interestingly, we identified one dmrt1 mutant allele that cooperates with a linked segregation distorter locus to generate an apparent XY sex determination mechanism. We conclude that dmrt1 is dispensable for ovary development but necessary for testis development in zebrafish, and that dmrt1 promotes male development by transcriptionally regulating male and female genes as has been described in other animals. Furthermore, the strong sex-ratio bias caused by dmrt1 reduction-of-function points to potential mechanisms through which sex

  14. Assessing embryo development using swept source optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Caujolle, S.; Cernat, R.; Silvestri, G.; Marques, M. J.; Bradu, A.; Feuchter, T.; Robinson, G.; Griffin, D.; Podoleanu, A.

    2018-03-01

    A detailed assessment of embryo development would assist biologists with selecting the most suitable embryos for transfer leading to higher pregnancy rates. Currently, only low resolution microscopy is employed to perform this assessment. Although this method delivers some information on the embryo surface morphology, no specific details are shown related to its inner structure. Using a Master-Slave Swept-Source Optical Coherence Tomography (SS-OCT), images of bovine embryos from day 7 after fertilization were collected from different depths. The dynamic changes inside the embryos were examined, in detail and in real-time from several depths. To prove our ability to characterize the morphology, a single embryo was imaged over 26 hours. The embryo was deprived of its life support environment, leading to its death. Over this period, clear morphological changes were observed.

  15. Nestin is essential for zebrafish brain and eye development through control of progenitor cell apoptosis.

    PubMed

    Chen, Hua-Ling; Yuh, Chiou-Hwa; Wu, Kenneth K

    2010-02-19

    Nestin is expressed in neural progenitor cells (NPC) of developing brain. Despite its wide use as an NPC marker, the function of nestin in embryo development is unclear. As nestin is conserved in zebrafish and its predicted sequence is clustered with the mammalian nestin orthologue, we used zebrafish as a model to investigate its role in embryogenesis. Injection of nestin morpholino (MO) into fertilized eggs induced time- and dose-dependent brain and eye developmental defects. Nestin morphants exhibited characteristic morphological changes including small head, small eyes and hydrocephalus. Histological examinations show reduced hind- and mid-brain size, dilated ventricle, poorly organized retina and underdeveloped lens. Injection of control nestin MO did not induce brain or eye changes. Nestin MO injection reduced expression of ascl1b (achaete-scute complex-like 1b), a marker of NPCs, without affecting its distribution. Nestin MO did not influence Elavl3/4 (Embryonic lethal, abnormal vision, Drosophila-like 3/4) (a neuronal marker), or otx2 (a midbrain neuronal marker), but severely perturbed cranial motor nerve development and axon distribution. To determine whether the developmental defects are due to excessive NPC apoptosis and/or reduced NPC proliferation, we analyzed apoptosis by TUNEL assay and acridine orange staining and proliferation by BrdU incorporation, pcna and mcm5 expressions. Excessive apoptosis was noted in hindbrain and midbrain cells. Apoptotic signals were colocalized with ascl1b. Proliferation markers were not significantly altered by nestin MO. These results suggest that nestin is essential for zebrafish brain and eye development probably through control of progenitor cell apoptosis.

  16. Identification of the Zinc Finger Protein ZRANB2 as a Novel Maternal Lipopolysaccharide-binding Protein That Protects Embryos of Zebrafish against Gram-negative Bacterial Infections*

    PubMed Central

    Wang, Xia; Du, Xiaoyuan; Li, Hongyan; Zhang, Shicui

    2016-01-01

    Zinc finger ZRANB2 proteins are widespread in animals, but their functions and mechanisms remain poorly defined. Here we clearly demonstrate that ZRANB2 is a newly identified LPS-binding protein present abundantly in the eggs/embryos of zebrafish. We also show that recombinant ZRANB2 (rZRANB2) acts as a pattern recognition receptor capable of identifying the bacterial signature molecule LPS as well as binding the Gram-negative bacteria Escherichia coli, Vibrio anguilarum, and Aeromonas hydrophila and functions as an antibacterial effector molecule capable of directly killing the bacteria. Furthermore, we reveal that N-terminal residues 11–37 consisting of the first ZnF_RBZ domain are indispensable for ZRANB2 antimicrobial activity. Importantly, microinjection of rZRANB2 into early embryos significantly enhanced the resistance of the embryos against pathogenic A. hydrophila challenge, and this enhanced bacterial resistance was markedly reduced by co-injection of anti-ZRANB2 antibody. Moreover, precipitation of ZRANB2 in the embryo extracts by preincubation with anti-ZRANB2 antibody caused a marked decrease in the antibacterial activity of the extracts against the bacteria tested. In addition, the N-terminal peptide Z1/37 or Z11/37 with in vitro antibacterial activity also promoted the resistance of embryos against A. hydrophila, but the peptide Z38/198 without in vitro antibacterial activity did not. Collectively, these results indicate that ZRANB2 is a maternal LPS-binding protein that can protect the early embryos of zebrafish against pathogenic attacks, a novel role ever assigned to ZRANB2 proteins. This work also provides new insights into the immunological function of the zinc finger proteins that are widely distributed in various animals. PMID:26740623

  17. GLUT2-mediated glucose uptake and availability are required for embryonic brain development in zebrafish.

    PubMed

    Marín-Juez, Rubén; Rovira, Mireia; Crespo, Diego; van der Vaart, Michiel; Spaink, Herman P; Planas, Josep V

    2015-01-01

    Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 (glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing.

  18. GLUT2-mediated glucose uptake and availability are required for embryonic brain development in zebrafish

    PubMed Central

    Marín-Juez, Rubén; Rovira, Mireia; Crespo, Diego; van der Vaart, Michiel; Spaink, Herman P; Planas, Josep V

    2015-01-01

    Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 (glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing. PMID:25294126

  19. Ethanol affects the development of sensory hair cells in larval zebrafish (Danio rerio).

    PubMed

    Uribe, Phillip M; Asuncion, James D; Matsui, Jonathan I

    2013-01-01

    Children born to mothers with substantial alcohol consumption during pregnancy can present a number of morphological, cognitive, and sensory abnormalities, including hearing deficits, collectively known as fetal alcohol syndrome (FAS). The goal of this study was to determine if the zebrafish lateral line could be used to study sensory hair cell abnormalities caused by exposure to ethanol during embryogenesis. Some lateral line sensory hair cells are present at 2 days post-fertilization (dpf) and are functional by 5 dpf. Zebrafish embryos were raised in fish water supplemented with varying concentrations of ethanol (0.75%-1.75% by volume) from 2 dpf through 5 dpf. Ethanol treatment during development resulted in many physical abnormalities characteristic of FAS in humans. Also, the number of sensory hair cells decreased as the concentration of ethanol increased in a dose-dependent manner. The dye FM 1-43FX was used to detect the presence of functional mechanotransduction channels. The percentage of FM 1-43-labeled hair cells decreased as the concentration of ethanol increased. Methanol treatment did not affect the development of hair cells. The cell cycle markers proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) demonstrated that ethanol reduced the number of sensory hair cells, as a consequence of decreased cellular proliferation. There was also a significant increase in the rate of apoptosis, as determined by TUNEL-labeling, in neuromasts following ethanol treatment during larval development. Therefore, zebrafish are a useful animal model to study the effects of hair cell developmental disorders associated with FAS.

  20. Biomedical properties of a series of ruthenium-N-heterocyclic carbene complexes based on oxidant activity in vitro and assessment in vivo of biosafety in zebrafish embryos.

    PubMed

    Alfaro, Juan M; Prades, Amparo; del Carmen Ramos, María; Peris, Eduardo; Ripoll-Gómez, Jorge; Poyatos, Macarena; Burgos, Javier S

    2010-03-01

    N-Heterocyclic carbene (NHC) ligands have attracted great interest over the last decade for their use in the design of homogenous catalysts. NHC-based metal complexes have interesting potential biomedical applications, such as in antimicrobial and cancer therapy, which are beginning to be explored more fully. We have studied here the oxidant activities of a series of Ru(II) complexes in vitro and zebrafish (Danio rerio) have been used as a model in vivo to investigate and characterize the toxicity of some of these compounds. Dual behavior was observed for the NHC-based complexes as they behaved as antioxidants at low concentrations but showed pro-oxidant capacity at higher concentrations. Zebrafish embryos were exposed to Ru(II) complexes under several different conditions (0 or 24 h postfertilization, with or without the chorion) and various parameters, such as viability, edema, heart rate, blood coagulation, pigmentation, scoliosis, malformation, and hatching, were tested. In general, zebrafish embryos were not harmed by exposure to Ru(II) complexes whatever the experimental conditions. Several toxicity profiles were observed depending upon the chemical structure of the compound in question. Their characteristics as pro-oxidant and/or antioxidant agents together with their biosafety may point to their having biomedical applications as antitumoral or neuroprotective drugs.

  1. Blocking VEGF signaling delays development of replacement teeth in zebrafish.

    PubMed

    Crucke, J; Huysseune, A

    2015-01-01

    The dentition in zebrafish is extremely and richly vascularized, but the function of the vasculature, in view of the continuous replacement of the teeth, remains elusive. Through application of SU5416, a vascular endothelial growth factor receptor inhibitor, we studied the role of the blood vessels in the dentition of the zebrafish. We were unable to show an effect on the development of first-generation teeth as well as first tooth replacement. However, in juvenile fish, a delay was observed in the developmental state of the replacement tooth compared with what was expected based on the maturation state of the functional tooth. Furthermore, we observed a difference between treated and nontreated fish in the distance between blood vessels and developing replacement teeth. In conclusion, our results provide support for a nutritive, rather than an inductive, function of the vasculature in the process of tooth development and replacement. © International & American Associations for Dental Research 2014.

  2. Malformation of certain brain blood vessels caused by TCDD activation of Ahr2/Arnt1 signaling in developing zebrafish.

    PubMed

    Teraoka, Hiroki; Ogawa, Akira; Kubota, Akira; Stegeman, John J; Peterson, Richard E; Hiraga, Takeo

    2010-08-15

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes various signs of toxicity in early life stages of vertebrates through activation of the aryl hydrocarbon receptor (AHR). The AHR also plays important roles in normal development in mice, and AHR(-/-) mice show abnormal development of vascular structures in various blood vessels. Our previous studies revealed that Ahr type 2 (Ahr2) activation by TCDD and beta-naphthoflavone (BNF) caused a significant decrease in blood flow in the dorsal midbrain of zebrafish embryos. Here we report effects of TCDD exposure on the morphology of some blood vessels in the head of developing zebrafish. TCDD caused concentration-dependent anatomical rearrangements in the shape of the prosencephalic artery in zebrafish larvae. In contrast, no major vascular defects were recognized in the trunk and tail regions following exposure to TCDD at least at the concentrations used. Essentially, the same observations were also confirmed in BNF-exposed larvae. Knock-down of either Ahr2 or Ahr nuclear translocator type 1 (Arnt1) by morpholino oligonucleotides (MOs) protected larvae against abnormal shape of the prosencephalic artery caused by TCDD and BNF. On the other hand, knock-down of Ahr2 or Arnt1 in vehicle-exposed zebrafish larvae had no clear effect on morphology of the prosencephalic artery or trunk vessels. Ascorbic acid, an antioxidant, protected against the TCDD-induced decrease in blood flow through the prosencephalic artery, but not the abnormal morphological changes in the shape of this artery. These results indicate that activation of Ahr2/Arnt1 pathway by TCDD and BNF affects the shape of certain blood vessels in the brain of developing zebrafish. (c) 2010 Elsevier B.V. All rights reserved.

  3. N-acetylcysteine prevents ketamine-induced adverse effects on development, heart rate and monoaminergic neurons in zebrafish.

    PubMed

    Robinson, Bonnie; Dumas, Melanie; Gu, Qiang; Kanungo, Jyotshna

    2018-06-08

    N-acetylcysteine, a precursor molecule of glutathione, is an antioxidant. Ketamine, a pediatric anesthetic, has been implicated in cardiotoxicity and neurotoxicity including modulation of monoaminergic systems in mammals and zebrafish. Here, we show that N-acetylcysteine prevents ketamine's adverse effects on development and monoaminergic neurons in zebrafish embryos. The effects of ketamine and N-acetylcysteine alone or in combination were measured on the heart rate, body length, brain serotonergic neurons and tyrosine hydroxylase-immunoreactive (TH-IR) neurons. In the absence of N-acetylcysteine, a concentration of ketamine that produces an internal embryo exposure level comparable to human anesthetic plasma concentrations significantly reduced heart rate and body length and those effects were prevented by N-acetylcysteine co-treatment. Ketamine also reduced the areas occupied by serotonergic neurons in the brain, whereas N-acetylcysteine co-exposure counteracted this effect. TH-IR neurons in the embryo brain and TH-IR cells in the trunk were significantly reduced with ketamine treatment, but not in the presence of N-acetylcysteine. In our continued search for compounds that can prevent ketamine toxicity, this study using specific endpoints of developmental toxicity, cardiotoxicity and neurotoxicity, demonstrates protective effects of N-acetylcysteine against ketamine's adverse effects. This is the first study that shows the protective effects of N-acetylcysteine on ketamine-induced developmental defects of monoaminergic neurons as observed in a whole organism. Published by Elsevier B.V.

  4. Comparison of molecular marker expression in early zebrafish brain development following chronic ethanol or morpholino treatment.

    PubMed

    Zhang, Chengjin; Boa-Amponsem, Oswald; Cole, Gregory J

    2017-08-01

    This study was undertaken to ascertain whether defined markers of early zebrafish brain development are affected by chronic ethanol exposure or morpholino knockdown of agrin, sonic hedgehog, retinoic acid, and fibroblast growth factors, four signaling molecules that are suggested to be ethanol sensitive. Zebrafish embryos were exposed to 2% ethanol from 6 to 24 hpf or injected with agrin, shha, aldh1a3, or fgf8a morpholinos. In situ hybridization was employed to analyze otx2, pax6a, epha4a, krx20, pax2a, fgf8a, wnt1, and eng2b expression during early brain development. Our results showed that pax6a mRNA expression was decreased in eye, forebrain, and hindbrain of both chronic ethanol exposed and select MO treatments. Epha4a expression in rhombomere R1 boundary was decreased in chronic ethanol exposure and aldh1a3 morphants, lost in fgf8a morphants, but largely unaffected in agrin and shha morphants. Ectopic pax6a and epha4a expression in midbrain was only found in fgf8a morphants. These results suggest that while chronic ethanol induces obvious morphological change in brain architecture, many molecular markers of these brain structures are relatively unaffected by ethanol exposure.

  5. pitx2 Deficiency Results in Abnormal Ocular and Craniofacial Development in Zebrafish

    PubMed Central

    Liu, Yi; Semina, Elena V.

    2012-01-01

    Human PITX2 mutations are associated with Axenfeld-Rieger syndrome, an autosomal-dominant developmental disorder that involves ocular anterior segment defects, dental hypoplasia, craniofacial dysmorphism and umbilical abnormalities. Characterization of the PITX2 pathway and identification of the mechanisms underlying the anomalies associated with PITX2 deficiency is important for better understanding of normal development and disease; studies of pitx2 function in animal models can facilitate these analyses. A knockdown of pitx2 in zebrafish was generated using a morpholino that targeted all known alternative transcripts of the pitx2 gene; morphant embryos generated with the pitx2ex4/5 splicing-blocking oligomer produced abnormal transcripts predicted to encode truncated pitx2 proteins lacking the third (recognition) helix of the DNA-binding homeodomain. The morphological phenotype of pitx2ex4/5 morphants included small head and eyes, jaw abnormalities and pericardial edema; lethality was observed at ∼6–8-dpf. Cartilage staining revealed a reduction in size and an abnormal shape/position of the elements of the mandibular and hyoid pharyngeal arches; the ceratobranchial arches were also decreased in size. Histological and marker analyses of the misshapen eyes of the pitx2ex4/5 morphants identified anterior segment dysgenesis and disordered hyaloid vasculature. In summary, we demonstrate that pitx2 is essential for proper eye and craniofacial development in zebrafish and, therefore, that PITX2/pitx2 function is conserved in vertebrates. PMID:22303467

  6. A critical period for functional vestibular development in zebrafish

    NASA Technical Reports Server (NTRS)

    Moorman, Stephen J.; Cordova, Rodolfo; Davies, Sarah A.

    2002-01-01

    We have determined a critical period for vestibular development in zebrafish by using a bioreactor designed by NASA to simulate microgravity for cells in culture. A critical period is defined as the briefest period of time during development when stimulus deprivation results in long lasting or permanent sensory deficits. Zebrafish eggs were collected within 3 hours of being laid and fertilized. In experiment 1, eggs were placed in the bioreactor at 3, 24, 30, 36, 48, or 72 hours postfertilization (hPF) and maintained in the bioreactor until 96 hPF. In experiment 2, eggs were placed in the bioreactor immediately after they were collected and maintained in the bioreactor until 24, 36, 48, 60, 66, 72, or 96 hPF. Beginning at 96 hPF, all larvae had their vestibulo-ocular reflexes (VOR) evaluated once each day for 5 days. Only larvae that hatched from eggs that were placed in the bioreactor before 30 hPF in experiment 1 or removed from the bioreactor later than 66 hPF in experiment 2 had VOR deficits that persisted for at least 5 days. These data suggest a critical period for vestibular development in the zebrafish that begins before 30 hPF and ends after 66 hPF. To confirm this, zebrafish eggs were placed in the bioreactor at 24 hPF and removed at 72 hPF. VORs were evaluated in these larvae once each day for 5 days beginning at 96 hPF. These larvae had VOR deficits that persisted for at least 5 days. In addition, larvae that had been maintained in the bioreactor from 24 to 66 hPF or from 30 to 72 hPF, had only temporary VOR deficits. In a final experiment, zebrafish eggs were placed in the bioreactor at 3 hPF and removed at 96 hPF but the bioreactor was turned off from 24 hPF to 72 hPF. These larvae had normal VORs when they were removed from the bioreactor at 96 hPF. Taken as a whole, these data support the idea that there is a critical period for functional maturation of the zebrafish vestibular system. The developmental period identified includes the timeframe

  7. Transcriptomic and phenotypic profiling in developing zebrafish exposed to thyroid hormone receptor agonists

    SciT

    Haggard, Derik E.; Noyes, Pamela D.; Waters, Katrina M.

    There is a need to develop novel, high-throughput screening and prioritization methods to identify chemicals with adverse estrogen, androgen, and thyroid activity to protect human health and the environment and is of interest to the Endocrine Disruptor Screening Program. The current aim is to explore the utility of zebrafish as a testing paradigm to classify endocrine activity using phenotypically anchored transcriptome profiling. Transcriptome analysis was conducted on embryos exposed to 25 estrogen-, androgen-, or thyroid-active chemicals at a concentration that elicited adverse malformations or mortality at 120 hours post-fertilization in 80% of the animals exposed. Analysis of the top 1000more » significant differentially expressed transcripts across all treatments identified a unique transcriptional and phenotypic profile for thyroid hormone receptor agonists, which can be used as a biomarker screen for potential thyroid hormone agonists.« less

  8. 4D blood flow mapping using SPIM-microPIV in the developing zebrafish heart

    NASA Astrophysics Data System (ADS)

    Zickus, Vytautas; Taylor, Jonathan M.

    2018-02-01

    Fluid-structure interaction in the developing heart is an active area of research in developmental biology. However, investigation of heart dynamics is mostly limited to computational uid dynamics simulations using heart wall structure information only, or single plane blood ow information - so there is a need for 3D + time resolved data to fully understand cardiac function. We present an imaging platform combining selective plane illumination microscopy (SPIM) with micro particle image velocimetry (μPIV) to enable 3D-resolved flow mapping in a microscopic environment, free from many of the sources of error and bias present in traditional epi uorescence-based μPIV systems. By using our new system in conjunction with optical heart beat synchronization, we demonstrate the ability obtain non-invasive 3D + time resolved blood flow measurements in the heart of a living zebrafish embryo.

  9. Abnormal cerebellar development and ataxia in CARP VIII morphant zebrafish.

    PubMed

    Aspatwar, Ashok; Tolvanen, Martti E E; Jokitalo, Eija; Parikka, Mataleena; Ortutay, Csaba; Harjula, Sanna-Kaisa E; Rämet, Mika; Vihinen, Mauno; Parkkila, Seppo

    2013-02-01

    Congenital ataxia and mental retardation are mainly caused by variations in the genes that affect brain development. Recent reports have shown that mutations in the CA8 gene are associated with mental retardation and ataxia in humans and ataxia in mice. The gene product, carbonic anhydrase-related protein VIII (CARP VIII), is predominantly present in cerebellar Purkinje cells, where it interacts with the inositol 1,4,5-trisphosphate receptor type 1, a calcium channel. In this study, we investigated the effects of the loss of function of CARP VIII during embryonic development in zebrafish using antisense morpholino oligonucleotides against the CA8 gene. Knockdown of CA8 in zebrafish larvae resulted in a curved body axis, pericardial edema and abnormal movement patterns. Histologic examination revealed gross morphologic defects in the cerebellar region and in the muscle. Electron microscopy studies showed increased neuronal cell death in developing larvae injected with CA8 antisense morpholinos. These data suggest a pivotal role for CARP VIII during embryonic development. Furthermore, suppression of CA8 expression leads to defects in motor and coordination functions, mimicking the ataxic human phenotype. This work reveals an evolutionarily conserved function of CARP VIII in brain development and introduces a novel zebrafish model in which to investigate the mechanisms of CARP VIII-related ataxia and mental retardation in humans.

  10. Maternal thyroid hormones are essential for neural development in zebrafish.

    PubMed

    Campinho, Marco A; Saraiva, João; Florindo, Claudia; Power, Deborah M

    2014-07-01

    Teleost eggs contain an abundant store of maternal thyroid hormones (THs), and early in zebrafish embryonic development, all the genes necessary for TH signaling are expressed. Nonetheless the function of THs in embryonic development remains elusive. To test the hypothesis that THs are fundamental for zebrafish embryonic development, an monocarboxilic transporter 8 (Mct8) knockdown strategy was deployed to prevent maternal TH uptake. Absence of maternal THs did not affect early specification of the neural epithelia but profoundly modified later dorsal specification of the brain and spinal cord as well as specific neuron differentiation. Maternal THs acted upstream of pax2a, pax7, and pax8 genes but downstream of shha and fgf8a signaling. The lack of inhibitory spinal cord interneurons and increased motoneurons in the mct8 morphants is consistent with their stiff axial body and impaired mobility. The mct8 mutations are associated with X-linked mental retardation in humans, and the cellular and molecular consequences of MCT8 knockdown during embryonic development in zebrafish provides new insight into the potential role of THs in this condition.

  11. Maternal Thyroid Hormones Are Essential for Neural Development in Zebrafish

    PubMed Central

    Saraiva, João; Florindo, Claudia; Power, Deborah M.

    2014-01-01

    Teleost eggs contain an abundant store of maternal thyroid hormones (THs), and early in zebrafish embryonic development, all the genes necessary for TH signaling are expressed. Nonetheless the function of THs in embryonic development remains elusive. To test the hypothesis that THs are fundamental for zebrafish embryonic development, an monocarboxilic transporter 8 (Mct8) knockdown strategy was deployed to prevent maternal TH uptake. Absence of maternal THs did not affect early specification of the neural epithelia but profoundly modified later dorsal specification of the brain and spinal cord as well as specific neuron differentiation. Maternal THs acted upstream of pax2a, pax7, and pax8 genes but downstream of shha and fgf8a signaling. The lack of inhibitory spinal cord interneurons and increased motoneurons in the mct8 morphants is consistent with their stiff axial body and impaired mobility. The mct8 mutations are associated with X-linked mental retardation in humans, and the cellular and molecular consequences of MCT8 knockdown during embryonic development in zebrafish provides new insight into the potential role of THs in this condition. PMID:24877564

  12. Development of a new clinically applicable device for embryo evaluation which measures embryo oxygen consumption.

    PubMed

    Kurosawa, Hiroki; Utsunomiya, Hiroki; Shiga, Naomi; Takahashi, Aiko; Ihara, Motomasa; Ishibashi, Masumi; Nishimoto, Mitsuo; Watanabe, Zen; Abe, Hiroyuki; Kumagai, Jin; Terada, Yukihiro; Igarashi, Hideki; Takahashi, Toshifumi; Fukui, Atsushi; Suganuma, Ryota; Tachibana, Masahito; Yaegashi, Nobuo

    2016-10-01

    Does a new system-the chip-sensing embryo respiration monitoring system (CERMs)-enable evaluation of embryo viability for potential application in a clinical IVF setting? The system enabled the oxygen consumption rate of spheroids, bovine embryos and frozen-thawed human embryos to be measured, and this rate corresponded to the developmental potential of embryos. To date, no reliable and clinically suitable objective evaluation methods for embryos are available, which circumvent the differences in inter-observer subjective view. Existing systems such as the scanning electrochemical microscopy (SECM) technique, which enables the measurement of oxygen consumption rate in embryos, need improvement in usability before they can be applied to a clinical setting. This is a prospective original research study. The feasibility of measuring the oxygen consumption rate was assessed using CERMs for 9 spheroids, 9 bovine embryos and 30 redundant frozen-thawed human embryos. The endpoints for the study were whether CERMs could detect a dissolved oxygen gradient with high sensitivity, had comparable accuracy to the SECM measuring system with improved usability, and could predict the development of an embryo to a blastocyst by measuring the oxygen consumption rate. The relationship between the oxygen consumption rate and standard morphological evaluation was also examined. We developed a new CERMs, which enables the oxygen consumption rate to be measured automatically using an electrochemical method. The device was initially used for measuring a dissolved oxygen concentration gradient in order to calculate oxygen consumption rate using nine spheroids. Next, we evaluated data correlation between the CERMs and the SECM measuring systems using nine bovine embryos. Finally, the oxygen consumption rates of 30 human embryos, which were frozen-thawed on 2nd day after fertilization, were measured by CERMs at 6, 24, 48, 72 and 96 h after thawing with standard morphological evaluation

  13. OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing.

    PubMed

    Busquet, François; Strecker, Ruben; Rawlings, Jane M; Belanger, Scott E; Braunbeck, Thomas; Carr, Gregory J; Cenijn, Peter; Fochtman, Przemyslaw; Gourmelon, Anne; Hübler, Nicole; Kleensang, André; Knöbel, Melanie; Kussatz, Carola; Legler, Juliette; Lillicrap, Adam; Martínez-Jerónimo, Fernando; Polleichtner, Christian; Rzodeczko, Helena; Salinas, Edward; Schneider, Katharina E; Scholz, Stefan; van den Brandhof, Evert-Jan; van der Ven, Leo T M; Walter-Rohde, Susanne; Weigt, Stefan; Witters, Hilda; Halder, Marlies

    2014-08-01

    The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV<30%) for most chemicals and laboratories. The reproducibility was lower (CV>30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  14. In vivo toxic effects of 4-methoxy-5-hydroxy-canthin-6-one in zebrafish embryos via copper dyshomeostasis and oxidative stress.

    PubMed

    Gong, Guiyi; Jiang, Lingling; Lin, Qinghua; Liu, Wenyuan; He, Ming-Fang; Zhang, Jie; Feng, Feng; Qu, Wei; Xie, Ning

    2018-01-01

    Dysfunction of copper homeostasis can lead to a host of disorders, which might be toxic sometimes. 4-Methoxy-5-hydroxy-canthin-6-one (CAN) is one of the major constituents from Picrasma quassioides and responsible for its therapeutic effects. In this work, we evaluated the toxic effect of CAN (7.5μM) on zebrafish embryos. CAN treatment decreased survival, delayed hatching time and induced malformations (loss of pigmentation, pericardial edema, as well as hematologic and neurologic abnormalities). Besides, exogenous copper supplementation rescued the pigmentation and cardiovascular defects in CAN-treated embryos. Further spectroscopic studies revealed a copper-chelating activity of CAN. Then its regulation on the expressions of copper homeostasis related genes also be analyzed. In addition, CAN lowered the total activity of SOD, elevated the ROS production and altered the oxidative related genes transcriptions, which led to oxidative stress. In conclusion, we demonstrated that CAN (7.5μM) might exert its toxic effects in zebrafish embryos by causing copper dyshomeostasis and oxidative stress. It will give insight into the risk assessment and prevention of CAN-mediated toxicity. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. RhoA/ROCK pathway activity is essential for the correct localization of the germ plasm mRNAs in zebrafish embryos.

    PubMed

    Miranda-Rodríguez, Jerónimo Roberto; Salas-Vidal, Enrique; Lomelí, Hilda; Zurita, Mario; Schnabel, Denhi

    2017-01-01

    Zebrafish germ plasm is composed of mRNAs such as vasa and nanos and of proteins such as Bucky ball, all of which localize symmetrically in four aggregates at the distal region of the first two cleavage furrows. The coordination of actin microfilaments, microtubules and kinesin is essential for the correct localization of the germ plasm. Rho-GTPases, through their effectors, coordinate cytoskeletal dynamics. We address the participation of RhoA and its effector ROCK in germ plasm localization during the transition from two- to eight-cell embryos. We found that active RhoA is enriched along the cleavage furrow during the first two division cycles, whereas ROCK localizes at the distal region of the cleavage furrows in a similar pattern as the germ plasm mRNAs. Specific inhibition of RhoA and ROCK affected microtubules organization at the cleavage furrow; these caused the incorrect localization of the germ plasm mRNAs. The incorrect localization of the germ plasm led to a dramatic change in the number of germ cells during the blastula and 24hpf embryo stages without affecting any other developmental processes. We demonstrate that the Rho/ROCK pathway is intimately related to the determination of germ cells in zebrafish embryos. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Cadmium affects muscle type development and axon growth in zebrafish embryonic somitogenesis.

    PubMed

    Hen Chow, Elly Suk; Cheng, Shuk Han

    2003-05-01

    We have previously reported that exposure to cadmium during zebrafish embryonic development caused morphological malformations of organs and ectopic expression of genes involved in regulating developmental process. One of the most common developmental defects observed was altered axial curvature resulting from defects in the myotomes of the somites. In this study, we investigated the mechanisms of cadmium-induced toxicity in zebrafish somitogenesis. We showed that the critical period of exposure was the gastrulation period, which actually preceded the formation of the first morpholo