Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments.

Science.gov (United States)

Felker, A; Mosimann, C

2016-01-01

Spatiotemporal transgene regulation by transgenic DNA recombinases is a central tool for reverse genetics in multicellular organisms, with excellent applications for misexpression and lineage tracing experiments. One of the most widespread technologies for this purpose is Cre recombinase-controlled lox site recombination that is attracting increasing interest in the zebrafish field. Tol2-mediated zebrafish transgenesis provides a stable platform to integrate lox cassette transgenes, while the amenability of the zebrafish embryo to drug treatments makes the model an ideal candidate for tamoxifen-inducible CreERT2 experiments. In addition, advanced transgenesis technologies such as phiC31 or CRISPR-Cas9-based knock-ins are even further promoting zebrafish transgenesis for Cre/lox applications. In this chapter, we will first introduce the basics of Cre/lox methodology, CreERT2 regulation by tamoxifen, as well as the utility of Tol2 and other contemporary transgenesis techniques for Cre/lox experiments. We will then outline in detail practical experimental steps for efficient transgenesis toward the creation of single-insertion transgenes and will introduce protocols for 4-hydroxytamoxifen-mediated CreERT2 induction to perform spatiotemporal lox transgene regulation experiments in zebrafish embryos. Last, we will discuss advanced experimental applications of Cre/lox beyond traditional lineage tracing approaches. Copyright © 2016 Elsevier Inc. All rights reserved.

Zebrafish CaV2.1 Calcium Channels Are Tailored for Fast Synchronous Neuromuscular Transmission

Science.gov (United States)

Naranjo, David; Wen, Hua; Brehm, Paul

2015-01-01

The CaV2.2 (N-type) and CaV2.1 (P/Q-type) voltage-dependent calcium channels are prevalent throughout the nervous system where they mediate synaptic transmission, but the basis for the selective presence at individual synapses still remains an open question. The CaV2.1 channels have been proposed to respond more effectively to brief action potentials (APs), an idea supported by computational modeling. However, the side-by-side comparison of CaV2.1 and CaV2.2 kinetics in intact neurons failed to reveal differences. As an alternative means for direct functional comparison we expressed zebrafish CaV2.1 and CaV2.2 α-subunits, along with their accessory subunits, in HEK293 cells. HEK cells lack calcium currents, thereby circumventing the need for pharmacological inhibition of mixed calcium channel isoforms present in neurons. HEK cells also have a simplified morphology compared to neurons, which improves voltage control. Our measurements revealed faster kinetics and shallower voltage-dependence of activation and deactivation for CaV2.1. Additionally, recordings of calcium current in response to a command waveform based on the motorneuron AP show, directly, more effective activation of CaV2.1. Analysis of calcium currents associated with the AP waveform indicate an approximately fourfold greater open probability (PO) for CaV2.1. The efficient activation of CaV2.1 channels during APs may contribute to the highly reliable transmission at zebrafish neuromuscular junctions. PMID:25650925

Characterization of Na+ and Ca2+ channels in zebrafish dorsal root ganglion neurons.

Directory of Open Access Journals (Sweden)

Yu-Jin Won

Full Text Available BACKGROUND: Dorsal root ganglia (DRG somata from rodents have provided an excellent model system to study ion channel properties and modulation using electrophysiological investigation. As in other vertebrates, zebrafish (Danio rerio DRG are organized segmentally and possess peripheral axons that bifurcate into each body segment. However, the electrical properties of zebrafish DRG sensory neurons, as compared with their mammalian counterparts, are relatively unexplored because a preparation suitable for electrophysiological studies has not been available. METHODOLOGY/PRINCIPAL FINDINGS: We show enzymatically dissociated DRG neurons from juvenile zebrafish expressing Isl2b-promoter driven EGFP were easily identified with fluorescence microscopy and amenable to conventional whole-cell patch-clamp studies. Two kinetically distinct TTX-sensitive Na(+ currents (rapidly- and slowly-inactivating were discovered. Rapidly-inactivating I(Na were preferentially expressed in relatively large neurons, while slowly-inactivating I(Na was more prevalent in smaller DRG neurons. RT-PCR analysis suggests zscn1aa/ab, zscn8aa/ab, zscn4ab and zscn5Laa are possible candidates for these I(Na components. Voltage-gated Ca(2+ currents (I(Ca were primarily (87% comprised of a high-voltage activated component arising from ω-conotoxin GVIA-sensitive Ca(V2.2 (N-type Ca(2+ channels. A few DRG neurons (8% displayed a miniscule low-voltage-activated component. I(Ca in zebrafish DRG neurons were modulated by neurotransmitters via either voltage-dependent or -independent G-protein signaling pathway with large cell-to-cell response variability. CONCLUSIONS/SIGNIFICANCE: Our present results indicate that, as in higher vertebrates, zebrafish DRG neurons are heterogeneous being composed of functionally distinct subpopulations that may correlate with different sensory modalities. These findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and

High magnetic field induced otolith fusion in the zebrafish larvae.

Science.gov (United States)

Pais-Roldán, Patricia; Singh, Ajeet Pratap; Schulz, Hildegard; Yu, Xin

2016-04-11

Magnetoreception in animals illustrates the interaction of biological systems with the geomagnetic field (geoMF). However, there are few studies that identified the impact of high magnetic field (MF) exposure from Magnetic Resonance Imaging (MRI) scanners (>100,000 times of geoMF) on specific biological targets. Here, we investigated the effects of a 14 Tesla MRI scanner on zebrafish larvae. All zebrafish larvae aligned parallel to the B0 field, i.e. the static MF, in the MRI scanner. The two otoliths (ear stones) in the otic vesicles of zebrafish larvae older than 24 hours post fertilization (hpf) fused together after the high MF exposure as short as 2 hours, yielding a single-otolith phenotype with aberrant swimming behavior. The otolith fusion was blocked in zebrafish larvae under anesthesia or embedded in agarose. Hair cells may play an important role on the MF-induced otolith fusion. This work provided direct evidence to show that high MF interacts with the otic vesicle of zebrafish larvae and causes otolith fusion in an "all-or-none" manner. The MF-induced otolith fusion may facilitate the searching for MF sensors using genetically amenable vertebrate animal models, such as zebrafish.

Injury-induced ctgfa directs glial bridging and spinal cord regeneration in zebrafish

Science.gov (United States)

Mokalled, Mayssa H.; Patra, Chinmoy; Dickson, Amy L.; Endo, Toyokazu; Stainier, Didier Y. R.; Poss, Kenneth D.

2016-01-01

Unlike mammals, zebrafish efficiently regenerate functional nervous system tissue after major spinal cord injury. Whereas glial scarring presents a roadblock for mammalian spinal cord repair, glial cells in zebrafish form a bridge across severed spinal cord tissue and facilitate regeneration, a relatively unexplored process. Here, we performed a genome-wide profiling screen for secreted factors that are upregulated during zebrafish spinal cord regeneration. We find that connective tissue growth factor a (ctgfa) is induced in and around glial cells that participate in initial bridging events. Mutations in ctgfa disrupt spinal cord repair, while transgenic ctgfa overexpression and local human CTGF recombinant protein delivery accelerate bridging and functional regeneration. Our study reveals that CTGF is necessary and sufficient to stimulate glial bridging and natural spinal cord regeneration. PMID:27811277

Acquisition of glial cells missing 2 enhancers contributes to a diversity of ionocytes in zebrafish.

Directory of Open Access Journals (Sweden)

Takanori Shono

Full Text Available Glial cells missing 2 (gcm2 encoding a GCM-motif transcription factor is expressed in the parathyroid in amniotes. In contrast, gcm2 is expressed in pharyngeal pouches (a homologous site of the parathyroid, gills, and H(+-ATPase-rich cells (HRCs, a subset of ionocytes on the skin surface of the teleost fish zebrafish. Ionocytes are specialized cells that are involved in osmotic homeostasis in aquatic vertebrates. Here, we showed that gcm2 is essential for the development of HRCs and Na(+-Cl(- co-transporter-rich cells (NCCCs, another subset of ionocytes in zebrafish. We also identified gcm2 enhancer regions that control gcm2 expression in ionocytes of zebrafish. Comparisons of the gcm2 locus with its neighboring regions revealed no conserved elements between zebrafish and tetrapods. Furthermore, We observed gcm2 expression patterns in embryos of the teleost fishes Medaka (Oryzias latipes and fugu (Fugu niphobles, the extant primitive ray-finned fishes Polypterus (Polypterus senegalus and sturgeon (a hybrid of Huso huso × Acipenser ruhenus, and the amphibian Xenopus (Xenopus laevis. Although gcm2-expressing cells were observed on the skin surface of Medaka and fugu, they were not found in Polypterus, sturgeon, or Xenopus. Our results suggest that an acquisition of enhancers for the expression of gcm2 contributes to a diversity of ionocytes in zebrafish during evolution.

Zebrafish swimming in the flow: a particle image velocimetry study

Directory of Open Access Journals (Sweden)

Violet Mwaffo

2017-11-01

Full Text Available Zebrafish is emerging as a species of choice for the study of a number of biomechanics problems, including balance development, schooling, and neuromuscular transmission. The precise quantification of the flow physics around swimming zebrafish is critical toward a mechanistic understanding of the complex swimming style of this fresh-water species. Although previous studies have elucidated the vortical structures in the wake of zebrafish swimming in placid water, the flow physics of zebrafish swimming against a water current remains unexplored. In an effort to illuminate zebrafish swimming in a dynamic environment reminiscent of its natural habitat, we experimentally investigated the locomotion and hydrodynamics of a single zebrafish swimming in a miniature water tunnel using particle image velocimetry. Our results on zebrafish locomotion detail the role of flow speed on tail beat undulations, heading direction, and swimming speed. Our findings on zebrafish hydrodynamics offer a precise quantification of vortex shedding during zebrafish swimming and demonstrate that locomotory patterns play a central role on the flow physics. This knowledge may help clarify the evolutionary advantage of burst and cruise swimming movements in zebrafish.

Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a-/- mutants

LENUS (Irish Health Repository)

Sapetto-Rebow, Beata

2011-11-23

Abstract Background Genetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse Top2a knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish top2a mutants arises from maternal expression of top2a or compensation from its top2b paralogue. Results Here, we describe bloody minded (blm), a novel mutant of zebrafish top2a. In contrast to mouse Top2a nulls, zebrafish top2a mutants survive to larval stages (4-5 day post fertilization). Developmental analyses demonstrate abundant expression of maternal top2a but not top2b. Inhibition or poisoning of maternal topoisomerase II delays embryonic development by extending the cell cycle M-phase. Zygotic top2a and top2b are co-expressed in the zebrafish CNS, but endogenous or ectopic top2b RNA appear unable to prevent the blm phenotype. Conclusions We conclude that maternal top2a enables zebrafish development before the mid-zygotic transition (MZT) and that zebrafish top2a and top2b are not functionally redundant during development after activation of the zygotic genome.

Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a-/- mutants

Directory of Open Access Journals (Sweden)

Sapetto-Rebow Beata

2011-11-01

Full Text Available Abstract Background Genetic alterations in human topoisomerase II alpha (TOP2A are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse Top2a knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish top2a mutants arises from maternal expression of top2a or compensation from its top2b paralogue. Results Here, we describe bloody minded (blm, a novel mutant of zebrafish top2a. In contrast to mouse Top2a nulls, zebrafish top2a mutants survive to larval stages (4-5 day post fertilization. Developmental analyses demonstrate abundant expression of maternal top2a but not top2b. Inhibition or poisoning of maternal topoisomerase II delays embryonic development by extending the cell cycle M-phase. Zygotic top2a and top2b are co-expressed in the zebrafish CNS, but endogenous or ectopic top2b RNA appear unable to prevent the blm phenotype. Conclusions We conclude that maternal top2a enables zebrafish development before the mid-zygotic transition (MZT and that zebrafish top2a and top2b are not functionally redundant during development after activation of the zygotic genome.

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

Science.gov (United States)

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

2009-01-01

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

Expanded progenitor populations, vitreo-retinal abnormalities, and Müller glial reactivity in the zebrafish leprechaun/patched2 retina

Directory of Open Access Journals (Sweden)

Bibliowicz Jonathan

2009-10-01

Full Text Available Abstract Background The roles of the Hedgehog (Hh pathway in controlling vertebrate retinal development have been studied extensively; however, species- and context-dependent findings have provided differing conclusions. Hh signaling has been shown to control both population size and cell cycle kinetics of proliferating retinal progenitors, and to modulate differentiation within the retina by regulating the timing of cell cycle exit. While cell cycle exit has in turn been shown to control cell fate decisions within the retina, a direct role for the Hh pathway in retinal cell fate decisions has yet to be established in vivo. Results To gain further insight into Hh pathway function in the retina, we have analyzed retinal development in leprechaun/patched2 mutant zebrafish. While lep/ptc2 mutants possessed more cells in their retinas, all cell types, except for Müller glia, were present at identical ratios as those observed in wild-type siblings. lep/ptc2 mutants possessed a localized upregulation of GFAP, a marker for 'reactive' glia, as well as morphological abnormalities at the vitreo-retinal interface, where Müller glial endfeet terminate. In addition, analysis of the over-proliferation phenotype at the ciliary marginal zone (CMZ revealed that the number of proliferating progenitors, but not the rate of proliferation, was increased in lep/ptc2 mutants. Conclusion Our results indicate that Patched2-dependent Hh signaling does not likely play an integral role in neuronal cell fate decisions in the zebrafish retina. ptc2 deficiency in zebrafish results in defects at the vitreo-retinal interface and Müller glial reactivity. These phenotypes are similar to the ocular abnormalities observed in human patients suffering from Basal Cell Naevus Syndrome (BCNS, a disorder that has been linked to mutations in the human PTCH gene (the orthologue of the zebrafish ptc2, and point to the utility of the lep/ptc2 mutant line as a model for the study of BCNS

Zebrafish brd2a and brd2b are paralogous members of the bromodomain-ET (BET family of transcriptional coregulators that show structural and expression divergence

Directory of Open Access Journals (Sweden)

Bee Katharine J

2008-04-01

Full Text Available Abstract Background Brd2 belongs to the bromodomain-extraterminal domain (BET family of transcriptional co-regulators, and functions as a pivotal histone-directed recruitment scaffold in chromatin modification complexes affecting signal-dependent transcription. Brd2 facilitates expression of genes promoting proliferation and is implicated in apoptosis and in egg maturation and meiotic competence in mammals; it is also a susceptibility gene for juvenile myoclonic epilepsy (JME in humans. The brd2 ortholog in Drosophila is a maternal effect, embryonic lethal gene that regulates several homeotic loci, including Ultrabithorax. Despite its importance, there are few systematic studies of Brd2 developmental expression in any organism. To help elucidate both conserved and novel gene functions, we cloned and characterized expression of brd2 cDNAs in zebrafish, a vertebrate system useful for genetic analysis of development and disease, and for study of the evolution of gene families and functional diversity in chordates. Results We identify cDNAs representing two paralogous brd2 loci in zebrafish, brd2a on chromosome 19 and brd2b on chromosome 16. By sequence similarity, syntenic and phylogenetic analyses, we present evidence for structural divergence of brd2 after gene duplication in fishes. brd2 paralogs show potential for modular domain combinations, and exhibit distinct RNA expression patterns throughout development. RNA in situ hybridizations in oocytes and embryos implicate brd2a and brd2b as maternal effect genes involved in egg polarity and egg to embryo transition, and as zygotic genes important for development of the vertebrate nervous system and for morphogenesis and differentiation of the digestive tract. Patterns of brd2 developmental expression in zebrafish are consistent with its proposed role in Homeobox gene regulation. Conclusion Expression profiles of zebrafish brd2 paralogs support a role in vertebrate developmental patterning and

Zebrafish have an ethanol-inducible hepatic 4-nitrophenol hydroxylase that is not CYP2E1-like.

Science.gov (United States)

Hartman, Jessica H; Kozal, Jordan S; Di Giulio, Richard T; Meyer, Joel N

2017-09-01

Zebrafish are an attractive model organism for toxicology; however, an important consideration in translating between species is xenobiotic metabolism/bioactivation. CYP2E1 metabolizes small hydrophobic molecules, e.g. ethanol, cigarette smoke, and diesel exhaust components. CYP2E1 is thought to only be conserved in mammals, but recent reports identified homologous zebrafish cytochrome P450s. Herein, ex vivo biochemical measurements show that unlike mammals, zebrafish possess a low-affinity 4-nitrophenol hydroxylase (K m ∼0.6 mM) in hepatic microsomes and mitochondria that is inducible only 1.5- to 2-fold by ethanol and is insensitive to 4-methylpyrazole inhibition. In closing, we suggest creating improved models to study CYP2E1 in zebrafish. Copyright © 2017 Elsevier B.V. All rights reserved.

Gonadotropin-releasing hormone 2 suppresses food intake in the zebrafish, Danio rerio

Directory of Open Access Journals (Sweden)

Ryo eNishiguchi

2012-10-01

Full Text Available Gonadotropin-releasing hormone (GnRH is an evolutionarily conserved neuropeptide with 10 amino acid residues, of which several structural variants exist. A molecular form known as GnRH2 ([His5 Trp7 Tyr8]GnRH, also known as chicken GnRH II is widely distributed in vertebrates except for rodents, and has recently been implicated in the regulation of feeding behavior in goldfish. However, the influence of GnRH2 on feeding behavior in other fish has not yet been studied. In the present study, therefore, we investigated the role of GnRH2 in the regulation of feeding behavior in a zebrafish model, and examined its involvement in food intake after intracerebroventricular (ICV administration. ICV injection of GnRH2 at 0.1 and 1 pmol/g body weight (BW induced a marked decrease of food consumption in a dose-dependent manner during 30 min after feeding. Cumulative food intake was significantly decreased by ICV injection of GnRH2 at 1 pmol/g BW during the 30-min post-treatment observation period. The anorexigenic action of GnRH2 was completely blocked by treatment with the GnRH type I receptor antagonist Antide at 50 pmol/g BW. We also examined the effect of feeding condition on the expression level of the GnRH2 transcript in the hypothalamus. Levels of GnRH2 mRNA obtained from fish that had been provided excess food for 7 days were higher than those in fish that had been fed normally. These results suggest that, in zebrafish, GnRH2 acts as an anorexigenic factor, as is the case in goldfish.

Direction selectivity in the larval zebrafish tectum is mediated by asymmetric inhibition

Directory of Open Access Journals (Sweden)

Abhinav eGrama

2012-09-01

Full Text Available The extraction of the direction of motion is an important computation performed by many sensory systems and in particular, the mechanism by which direction selective ganglion cells (DS-RGCs in the retina acquire their selective properties, has been studied extensively. However, whether DS-RGCs simply relay this information to downstream areas or whether additional and potentially de-novo processing occurs in these recipient structures is a matter of great interest. Neurons in the larval zebrafish tectum, the largest retino-recipent area in this animal, show direction selective responses to moving visual stimuli but how these properties are acquired is still unknown. In order to study this, we first used two-photon calcium imaging to classify the population responses of tectal cells to bars moving at different speeds and in different directions. Subsequently, we performed in-vivo whole cell electrophysiology on these direction selective tectal neurons and we found that their inhibitory inputs were strongly biased towards the null direction of motion, whereas the excitatory inputs showed little selectivity. In addition, we found that excitatory currents evoked by a stimulus moving in the preferred direction occurred before the inhibitory currents whereas a stimulus moving in the null direction evoked currents in the reverse temporal order. The membrane potential modulations resulting from these currents were enhanced by the spike generation mechanism to generate amplified direction selectivity in the spike output. Thus our results implicate a local inhibitory circuit in generating direction selectivity in tectal neurons.

Genomic Organization of Zebrafish microRNAs

Directory of Open Access Journals (Sweden)

Paydar Ima

2008-05-01

Full Text Available Abstract Background microRNAs (miRNAs are small (~22 nt non-coding RNAs that regulate cell movement, specification, and development. Expression of miRNAs is highly regulated, both spatially and temporally. Based on direct cloning, sequence conservation, and predicted secondary structures, a large number of miRNAs have been identified in higher eukaryotic genomes but whether these RNAs are simply a subset of a much larger number of noncoding RNA families is unknown. This is especially true in zebrafish where genome sequencing and annotation is not yet complete. Results We analyzed the zebrafish genome to identify the number and location of proven and predicted miRNAs resulting in the identification of 35 new miRNAs. We then grouped all 415 zebrafish miRNAs into families based on seed sequence identity as a means to identify possible functional redundancy. Based on genomic location and expression analysis, we also identified those miRNAs that are likely to be encoded as part of polycistronic transcripts. Lastly, as a resource, we compiled existing zebrafish miRNA expression data and, where possible, listed all experimentally proven mRNA targets. Conclusion Current analysis indicates the zebrafish genome encodes 415 miRNAs which can be grouped into 44 families. The largest of these families (the miR-430 family contains 72 members largely clustered in two main locations along chromosome 4. Thus far, most zebrafish miRNAs exhibit tissue specific patterns of expression.

Characterization of sleep in zebrafish and insomnia in hypocretin receptor mutants.

Directory of Open Access Journals (Sweden)

Tohei Yokogawa

2007-10-01

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

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Science.gov (United States)

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

Inward rectifier potassium current (I K1) and Kir2 composition of the zebrafish (Danio rerio) heart.

Science.gov (United States)

Hassinen, Minna; Haverinen, Jaakko; Hardy, Matt E; Shiels, Holly A; Vornanen, Matti

2015-12-01

Electrophysiological properties and molecular background of the zebrafish (Danio rerio) cardiac inward rectifier current (IK1) were examined. Ventricular myocytes of zebrafish have a robust (-6.7 ± 1.2 pA pF(-1) at -120 mV) strongly rectifying and Ba(2+)-sensitive (IC50 = 3.8 μM) IK1. Transcripts of six Kir2 channels (drKir2.1a, drKir2.1b, drKir2.2a, drKir2.2b, drKir2.3, and drKir2.4) were expressed in the zebrafish heart. drKir2.4 and drKir2.2a were the dominant isoforms in both the ventricle (92.9 ± 1.5 and 6.3 ± 1.5%) and the atrium (28.9 ± 2.9 and 64.7 ± 3.0%). The remaining four channels comprised together less than 1 and 7 % of the total transcripts in ventricle and atrium, respectively. The four main gene products (drKir2.1a, drKir2.2a, drKir2.2b, drKir2.4) were cloned, sequenced, and expressed in HEK cells for electrophysiological characterization. drKir2.1a was the most weakly rectifying (passed more outward current) and drKir2.2b the most strongly rectifying (passed less outward current) channel, whilst drKir2.2a and drKir2.4 were intermediate between the two. In regard to sensitivity to Ba(2+) block, drKir2.4 was the most sensitive (IC50 = 1.8 μM) and drKir2.1a the least sensitive channel (IC50 = 132 μM). These findings indicate that the Kir2 isoform composition of the zebrafish heart markedly differs from that of mammalian hearts. Furthermore orthologous Kir2 channels (Kir2.1 and Kir2.4) of zebrafish and mammals show striking differences in Ba(2+)-sensitivity. Structural and functional differences needs to be taken into account when zebrafish is used as a model for human cardiac electrophysiology, cardiac diseases, and in screening cardioactive substances.

A novel role for MAPKAPK2 in morphogenesis during zebrafish development.

Directory of Open Access Journals (Sweden)

Beth A Holloway

2009-03-01

Full Text Available One of the earliest morphogenetic processes in the development of many animals is epiboly. In the zebrafish, epiboly ensues when the animally localized blastoderm cells spread, thin over, and enclose the vegetally localized yolk. Only a few factors are known to function in this fundamental process. We identified a maternal-effect mutant, betty boop (bbp, which displays a novel defect in epiboly, wherein the blastoderm margin constricts dramatically, precisely when half of the yolk cell is covered by the blastoderm, causing the yolk cell to burst. Whole-blastoderm transplants and mRNA microinjection rescue demonstrate that Bbp functions in the yolk cell to regulate epiboly. We positionally cloned the maternal-effect bbp mutant gene and identified it as the zebrafish homolog of the serine-threonine kinase Mitogen Activated Protein Kinase Activated Protein Kinase 2, or MAPKAPK2, which was not previously known to function in embryonic development. We show that the regulation of MAPKAPK2 is conserved and p38 MAP kinase functions upstream of MAPKAPK2 in regulating epiboly in the zebrafish embryo. Dramatic alterations in calcium dynamics, together with the massive marginal constrictive force observed in bbp mutants, indicate precocious constriction of an F-actin network within the yolk cell, which first forms at 50% epiboly and regulates epiboly progression. We show that MAPKAPK2 activity and its regulator p38 MAPK function in the yolk cell to regulate the process of epiboly, identifying a new pathway regulating this cell movement process. We postulate that a p38 MAPKAPK2 kinase cascade modulates the activity of F-actin at the yolk cell margin circumference allowing the gradual closure of the blastopore as epiboly progresses.

Object recognition memory in zebrafish.

Science.gov (United States)

May, Zacnicte; Morrill, Adam; Holcombe, Adam; Johnston, Travis; Gallup, Joshua; Fouad, Karim; Schalomon, Melike; Hamilton, Trevor James

2016-01-01

The novel object recognition, or novel-object preference (NOP) test is employed to assess recognition memory in a variety of organisms. The subject is exposed to two identical objects, then after a delay, it is placed back in the original environment containing one of the original objects and a novel object. If the subject spends more time exploring one object, this can be interpreted as memory retention. To date, this test has not been fully explored in zebrafish (Danio rerio). Zebrafish possess recognition memory for simple 2- and 3-dimensional geometrical shapes, yet it is unknown if this translates to complex 3-dimensional objects. In this study we evaluated recognition memory in zebrafish using complex objects of different sizes. Contrary to rodents, zebrafish preferentially explored familiar over novel objects. Familiarity preference disappeared after delays of 5 mins. Leopard danios, another strain of D. rerio, also preferred the familiar object after a 1 min delay. Object preference could be re-established in zebra danios by administration of nicotine tartrate salt (50mg/L) prior to stimuli presentation, suggesting a memory-enhancing effect of nicotine. Additionally, exploration biases were present only when the objects were of intermediate size (2 × 5 cm). Our results demonstrate zebra and leopard danios have recognition memory, and that low nicotine doses can improve this memory type in zebra danios. However, exploration biases, from which memory is inferred, depend on object size. These findings suggest zebrafish ecology might influence object preference, as zebrafish neophobia could reflect natural anti-predatory behaviour. Copyright © 2015 Elsevier B.V. All rights reserved.

Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia

Directory of Open Access Journals (Sweden)

Chao Liu

2015-08-01

Full Text Available Apolipoprotein C-II (APOC2 is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases.

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

Science.gov (United States)

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

2016-04-01

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

The endocannabinoid gene faah2a modulates stress-associated behavior in zebrafish.

Directory of Open Access Journals (Sweden)

Randall G Krug

Full Text Available The ability to orchestrate appropriate physiological and behavioral responses to stress is important for survival, and is often dysfunctional in neuropsychiatric disorders that account for leading causes of global disability burden. Numerous studies have shown that the endocannabinoid neurotransmitter system is able to regulate stress responses and could serve as a therapeutic target for the management of these disorders. We used quantitative reverse transcriptase-polymerase chain reactions to show that genes encoding enzymes that synthesize (abhd4, gde1, napepld, enzymes that degrade (faah, faah2a, faah2b, and receptors that bind (cnr1, cnr2, gpr55-like endocannabinoids are expressed in zebrafish (Danio rerio. These genes are conserved in many other vertebrates, including humans, but fatty acid amide hydrolase 2 has been lost in mice and rats. We engineered transcription activator-like effector nucleases to create zebrafish with mutations in cnr1 and faah2a to test the role of these genes in modulating stress-associated behavior. We showed that disruption of cnr1 potentiated locomotor responses to hyperosmotic stress. The increased response to stress was consistent with rodent literature and served to validate the use of zebrafish in this field. Moreover, we showed for the first time that disruption of faah2a attenuated the locomotor responses to hyperosmotic stress. This later finding suggests that FAAH2 may be an important mediator of stress responses in non-rodent vertebrates. Accordingly, FAAH and FAAH2 modulators could provide distinct therapeutic options for stress-aggravated disorders.

Social dominance modulates eavesdropping in zebrafish

Science.gov (United States)

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

2015-01-01

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

Zebrafish models for the functional genomics of neurogenetic disorders.

Science.gov (United States)

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

2011-03-01

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

Als2 mRNA splicing variants detected in KO mice rescue severe motor dysfunction phenotype in Als2 knock-down zebrafish.

Science.gov (United States)

Gros-Louis, Francois; Kriz, Jasna; Kabashi, Edor; McDearmid, Jonathan; Millecamps, Stéphanie; Urushitani, Makoto; Lin, Li; Dion, Patrick; Zhu, Qinzhang; Drapeau, Pierre; Julien, Jean-Pierre; Rouleau, Guy A

2008-09-01

Recessive ALS2 mutations are linked to three related but slightly different neurodegenerative disorders: amyotrophic lateral sclerosis, hereditary spastic paraplegia and primary lateral sclerosis. To investigate the function of the ALS2 encoded protein, we generated Als2 knock-out (KO) mice and zAls2 knock-down zebrafish. The Als2(-/-) mice lacking exon 2 and part of exon 3 developed mild signs of neurodegeneration compatible with axonal transport deficiency. In contrast, zAls2 knock-down zebrafish had severe developmental abnormalities, swimming deficits and motor neuron perturbation. We identified, by RT-PCR, northern and western blotting novel Als2 transcripts in mouse central nervous system. These Als2 transcripts were present in Als2 null mice as well as in wild-type littermates and some rescued the zebrafish phenotype. Thus, we speculate that the newly identified Als2 mRNA species prevent the Als2 KO mice from developing severe neurodegenerative disease and might also regulate the severity of the motor neurons phenotype observed in ALS2 patients.

Evaluating human cancer cell metastasis in zebrafish

International Nuclear Information System (INIS)

Teng, Yong; Xie, Xiayang; Walker, Steven; White, David T; Mumm, Jeff S; Cowell, John K

2013-01-01

In vivo metastasis assays have traditionally been performed in mice, but the process is inefficient and costly. However, since zebrafish do not develop an adaptive immune system until 14 days post-fertilization, human cancer cells can survive and metastasize when transplanted into zebrafish larvae. Despite isolated reports, there has been no systematic evaluation of the robustness of this system to date. Individual cell lines were stained with CM-Dil and injected into the perivitelline space of 2-day old zebrafish larvae. After 2-4 days fish were imaged using confocal microscopy and the number of metastatic cells was determined using Fiji software. To determine whether zebrafish can faithfully report metastatic potential in human cancer cells, we injected a series of cells with different metastatic potential into the perivitelline space of 2 day old embryos. Using cells from breast, prostate, colon and pancreas we demonstrated that the degree of cell metastasis in fish is proportional to their invasion potential in vitro. Highly metastatic cells such as MDA231, DU145, SW620 and ASPC-1 are seen in the vasculature and throughout the body of the fish after only 24–48 hours. Importantly, cells that are not invasive in vitro such as T47D, LNCaP and HT29 do not metastasize in fish. Inactivation of JAK1/2 in fibrosarcoma cells leads to loss of invasion in vitro and metastasis in vivo, and in zebrafish these cells show limited spread throughout the zebrafish body compared with the highly metastatic parental cells. Further, knockdown of WASF3 in DU145 cells which leads to loss of invasion in vitro and metastasis in vivo also results in suppression of metastasis in zebrafish. In a cancer progression model involving normal MCF10A breast epithelial cells, the degree of invasion/metastasis in vitro and in mice is mirrored in zebrafish. Using a modified version of Fiji software, it is possible to quantify individual metastatic cells in the transparent larvae to correlate with

Role of Nrf2 antioxidant defense in mitigating cadmium-induced oxidative stress in the olfactory system of zebrafish

Energy Technology Data Exchange (ETDEWEB)

Wang, Lu; Gallagher, Evan P., E-mail: evang3@uw.edu

2013-01-15

Exposure to trace metals can disrupt olfactory function in fish leading to a loss of behaviors critical to survival. Cadmium (Cd) is an olfactory toxicant that elicits cellular oxidative stress as a mechanism of toxicity while also inducing protective cellular antioxidant genes via activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway. However, the molecular mechanisms of Cd-induced olfactory injury have not been characterized. In the present study, we investigated the role of the Nrf2-mediated antioxidant defense pathway in protecting against Cd-induced olfactory injury in zebrafish. A dose-dependent induction of Nrf2-regulated antioxidant genes associated with cellular responses to oxidative stress was observed in the olfactory system of adult zebrafish following 24 h Cd exposure. Zebrafish larvae exposed to Cd for 3 h showed increased glutathione S-transferase pi (gst pi), glutamate–cysteine ligase catalytic subunit (gclc), heme oxygenase 1 (hmox1) and peroxiredoxin 1 (prdx1) mRNA levels indicative of Nrf2 activation, and which were blocked by morpholino-mediated Nrf2 knockdown. The inhibition of antioxidant gene induction in Cd-exposed Nrf2 morphants was associated with disruption of olfactory driven behaviors, increased cell death and loss of olfactory sensory neurons (OSNs). Nrf2 morphants also exhibited a downregulation of OSN-specific genes after Cd exposure. Pre-incubation of embryos with sulforaphane (SFN) partially protected against Cd-induced olfactory tissue damage. Collectively, our results indicate that oxidative stress is an important mechanism of Cd-mediated injury in the zebrafish olfactory system. Moreover, the Nrf2 pathway plays a protective role against cellular oxidative damage and is important in maintaining zebrafish olfactory function. -- Highlights: ► Oxidative stress is an important mechanism of Cd-mediated olfactory injury. ► Cd induces antioxidant gene expression in the zebrafish olfactory system. ► The

Whole-body cortisol response of zebrafish to acute net handling stress

Science.gov (United States)

Ramsay, J.M.; Feist, G.W.; Varga, Z.M.; Westerfield, M.; Kent, M.L.; Schreck, C.B.

2009-01-01

Zebrafish, Danio rerio, are frequently handled during husbandry and experimental procedures in the laboratory, yet little is known about the physiological responses to such stressors. We measured the whole-body cortisol levels of adult zebrafish subjected to net stress and air exposure at intervals over a 24 h period; cortisol recovered to near control levels by about 1 h post-net-stress (PNS). We then measured cortisol at frequent intervals over a 1 h period. Cortisol levels were more than 2-fold higher in net stressed fish at 3 min PNS and continued to increase peaking at 15 min PNS, when cortisol levels were 6-fold greater than the control cortisol. Mean cortisol declined from 15 to 60 min PNS, and at 60 min, net-stressed cortisol was similar to control cortisol. Because the age of fish differed between studies, we examined resting cortisol levels of fish of different ages (3, 7, 13, and 19 months). The resting cortisol values among tanks with the same age fish differed significantly but there was no clear effect of age. Our study is the first to report the response and recovery of cortisol after net handling for laboratory-reared zebrafish. ?? 2009 Elsevier B.V.

Targeted knock-in of CreER T2 in zebrafish using CRISPR/Cas9.

Science.gov (United States)

Kesavan, Gokul; Hammer, Juliane; Hans, Stefan; Brand, Michael

2018-04-01

New genome-editing approaches, such as the CRISPR/Cas system, have opened up great opportunities to insert or delete genes at targeted loci and have revolutionized genetics in model organisms like the zebrafish. The Cre-loxp recombination system is widely used to activate or inactivate genes with high spatial and temporal specificity. Using a CRISPR/Cas9-mediated knock-in strategy, we inserted a zebrafish codon-optimized CreER T2 transgene at the otx2 gene locus to generate a conditional Cre-driver line. We chose otx2 as it is a patterning gene of the anterior neural plate that is expressed during early development. By knocking in CreER T2 upstream of the endogenous ATG of otx2, we utilized this gene's native promoter and enhancer elements to perfectly match CreER T2 and endogenous otx2 expression patterns. Next, by combining this novel driver line with a Cre-dependent reporter line, we show that only in the presence of tamoxifen can efficient Cre-loxp-mediated recombination be achieved in the anterior neural plate-derived tissues like the telencephalon, the eye and the optic tectum. Our results imply that the otx2:CreER T2 transgenic fish will be a valuable tool for lineage tracing and conditional mutant studies in larval and adult zebrafish.

DNA methylation regulates gabrb2 mRNA expression: developmental variations and disruptions in l-methionine-induced zebrafish with schizophrenia-like symptoms.

Science.gov (United States)

Wang, L; Jiang, W; Lin, Q; Zhang, Y; Zhao, C

2016-11-01

Single nucleotide polymorphisms (SNPs) in the human type A gamma-aminobutyric acid (GABA) receptor β 2 subunit gene (GABRB2) have been associated with schizophrenia and quantitatively correlated with mRNA expression in the postmortem brain tissue of patients with schizophrenia. l-Methionine (MET) administration has been reported to cause a recrudescence of psychotic symptoms in patients with schizophrenia, and similar symptoms have been generated in MET-induced mice. In this study, a zebrafish animal model was used to evaluate the relationship between the gabrb2 mRNA expression and its promoter DNA methylation in developmental and MET-induced schizophrenia-like zebrafish. The results indicated developmental increases in global DNA methylation and decreases in gabrb2 promoter methylation in zebrafish. A significant increase in gabrb2 mRNA levels was observed after GABA was synthesized. Additionally, the MET-triggered schizophrenia-like symptoms in adult zebrafish, involving social withdrawal and cognitive dysfunction analyzed with social interaction and T-maze behavioral tests, were accompanied by significantly increased DNA methylation levels in the global genome and the gabrb2 promoter. Furthermore, the significant correlation between gabrb2 mRNA expression and gabrb2 promoter methylation observed in the developmental stages became non-significant in MET-triggered adult zebrafish. These findings demonstrate that gabrb2 mRNA expression is associated with DNA methylation varies by developmental stage and show that these epigenetic association mechanisms are disrupted in MET-triggered adult zebrafish with schizophrenia-like symptoms. In conclusion, these results provide plausible epigenetic evidence of the GABA A receptor β 2 subunit involvement in the schizophrenia-like behaviors and demonstrate the potential use of zebrafish models in neuropsychiatric research. © 2016 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.

Aqueous Hg(2+) associates with TiO2 nanoparticles according to particle size, changes particle agglomeration, and becomes less bioavailable to zebrafish.

Science.gov (United States)

Boran, Halis; Boyle, David; Altinok, Ilhan; Patsiou, Danae; Henry, Theodore B

2016-05-01

Engineered nanoparticles (NPs) have unique physicochemistry and potential to interact with other substances in the aqueous phase. Here, gene [metallothionein 2 (mt2)] expression changes in larval zebrafish were used to evaluate the association between aqueous Hg(2+) and TiO2 (NPs and bulk particle size control) to investigate the relationship between changes in Hg(2+) behavior and TiO2 size. During 24h exposures, TiO2 agglomerates increased in size and in the presence of 25μg Hg(2+)/L, greater increases in size were observed. The concentration of Hg(2+) in suspension also decreased in the presence of TiO2-NPs. Mercury increased expression of mt2 in larval zebrafish, but this response was lessened when zebrafish were exposed to Hg(2+) in the presence of TiO2-NPs, and which suggests that TiO2-NPs alter the bioavailability of Hg(2+) to zebrafish larvae. This ameliorative effect of TiO2 was also likely due to surface binding of Hg(2+) because a greater decrease in mt2 expression was observed in the presence of 1mg/L TiO2-NPs than 1mg/L TiO2-bulk. In conclusion, the results show that Hg(2+) will associate with TiO2-NPs, TiO2-NPs that have associated Hg(2+) will settle out of the aqueous phase more rapidly, and agglomerates will deliver associated Hg(2+) to sediment surfaces. Copyright © 2016 Elsevier B.V. All rights reserved.

Redundant roles of PRDM family members in zebrafish craniofacial development.

Science.gov (United States)

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.

Report of Workshop on Euthanasia for Zebrafish-A Matter of Welfare and Science.

Science.gov (United States)

Köhler, Almut; Collymore, Chereen; Finger-Baier, Karin; Geisler, Robert; Kaufmann, Larissa; Pounder, Kieran C; Schulte-Merker, Stefan; Valentim, Ana; Varga, Zoltan M; Weiss, Jürgen; Strähle, Uwe

2017-12-01

The increasing importance of zebrafish as a biomedical model organism is reflected by the steadily growing number of publications and laboratories working with this species. Regulatory recommendations for euthanasia as issued in Directive 2010/63/EU are, however, based on experience with fish species used for food production and do not take the small size and specific physiology of zebrafish into account. Consequently, the currently recommended methods of euthanasia in the Directive 2010/63/EU are either not applicable or may interfere with research goals. An international workshop was held in Karlsruhe, Germany, March 9, 2017, to discuss and propose alternative methods for euthanasia of zebrafish. The aim was to identify methods that adequately address the physiology of zebrafish and its use as a biomedical research model, follow the principles of the 3Rs (Replacement, Reduction, and Refinement) in animal experimentation and consider animal welfare during anesthesia and euthanasia. The results of the workshop are summarized here in the form of a white paper.

Subdivisions of the adult zebrafish pallium based on molecular marker analysis [version 2; referees: 2 approved, 1 approved with reservations

Directory of Open Access Journals (Sweden)

Julia Ganz

2015-11-01

Full Text Available Background: The telencephalon shows a remarkable structural diversity among vertebrates. In particular, the everted telencephalon of ray-finned fishes has a markedly different morphology compared to the evaginated telencephalon of all other vertebrates. This difference in development has hampered the comparison between different areas of the pallium of ray-finned fishes and the pallial nuclei of all other vertebrates. Various models of homology between pallial subdivisions in ray-finned fishes and the pallial nuclei in tetrapods have been proposed based on connectional, neurochemical, gene expression and functional data. However, no consensus has been reached so far. In recent years, the analysis of conserved developmental marker genes has assisted the identification of homologies for different parts of the telencephalon among several tetrapod species. Results: We have investigated the gene expression pattern of conserved marker genes in the adult zebrafish (Danio rerio pallium to identify pallial subdivisions and their homology to pallial nuclei in tetrapods. Combinatorial expression analysis of ascl1a, eomesa, emx1, emx2, emx3, and Prox1 identifies four main divisions in the adult zebrafish pallium. Within these subdivisions, we propose that Dm is homologous to the pallial amygdala in tetrapods and that the dorsal subdivision of Dl is homologous to part of the hippocampal formation in mouse. We have complemented this analysis be examining the gene expression of emx1, emx2 and emx3 in the zebrafish larval brain. Conclusions: Based on our gene expression data, we propose a new model of subdivisions in the adult zebrafish pallium and their putative homologies to pallial nuclei in tetrapods. Pallial nuclei control sensory, motor, and cognitive functions, like memory, learning and emotion. The identification of pallial subdivisions in the adult zebrafish and their homologies to pallial nuclei in tetrapods will contribute to the use of the zebrafish

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

Science.gov (United States)

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

2014-09-07

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

Mercury-induced epigenetic transgenerational inheritance of abnormal neurobehavior is correlated with sperm epimutations in zebrafish.

Directory of Open Access Journals (Sweden)

Michael J Carvan

Full Text Available Methylmercury (MeHg is a ubiquitous environmental neurotoxicant, with human exposures predominantly resulting from fish consumption. Developmental exposure of zebrafish to MeHg is known to alter their neurobehavior. The current study investigated the direct exposure and transgenerational effects of MeHg, at tissue doses similar to those detected in exposed human populations, on sperm epimutations (i.e., differential DNA methylation regions [DMRs] and neurobehavior (i.e., visual startle and spontaneous locomotion in zebrafish, an established human health model. F0 generation embryos were exposed to MeHg (0, 1, 3, 10, 30, and 100 nM for 24 hours ex vivo. F0 generation control and MeHg-exposed lineages were reared to adults and bred to yield the F1 generation, which was subsequently bred to the F2 generation. Direct exposure (F0 generation and transgenerational actions (F2 generation were then evaluated. Hyperactivity and visual deficit were observed in the unexposed descendants (F2 generation of the MeHg-exposed lineage compared to control. An increase in F2 generation sperm epimutations was observed relative to the F0 generation. Investigation of the DMRs in the F2 generation MeHg-exposed lineage sperm revealed associated genes in the neuroactive ligand-receptor interaction and actin-cytoskeleton pathways being effected, which correlate to the observed neurobehavioral phenotypes. Developmental MeHg-induced epigenetic transgenerational inheritance of abnormal neurobehavior is correlated with sperm epimutations in F2 generation adult zebrafish. Therefore, mercury can promote the epigenetic transgenerational inheritance of disease in zebrafish, which significantly impacts its environmental health considerations in all species including humans.

klf2ash317 Mutant Zebrafish Do Not Recapitulate Morpholino-Induced Vascular and Haematopoietic Phenotypes.

Directory of Open Access Journals (Sweden)

Peter Novodvorsky

Full Text Available The zinc-finger transcription factor Krϋppel-like factor 2 (KLF2 transduces blood flow into molecular signals responsible for a wide range of responses within the vasculature. KLF2 maintains a healthy, quiescent endothelial phenotype. Previous studies report a range of phenotypes following morpholino antisense oligonucleotide-induced klf2a knockdown in zebrafish. Targeted genome editing is an increasingly applied method for functional assessment of candidate genes. We therefore generated a stable klf2a mutant zebrafish and characterised its cardiovascular and haematopoietic development.Using Transcription Activator-Like Effector Nucleases (TALEN we generated a klf2a mutant (klf2ash317 with a 14bp deletion leading to a premature stop codon in exon 2. Western blotting confirmed loss of wild type Klf2a protein and the presence of a truncated protein in klf2ash317 mutants. Homozygous klf2ash317 mutants exhibit no defects in vascular patterning, survive to adulthood and are fertile, without displaying previously described morphant phenotypes such as high-output cardiac failure, reduced haematopoetic stem cell (HSC development or impaired formation of the 5th accessory aortic arch. Homozygous klf2ash317 mutation did not reduce angiogenesis in zebrafish with homozygous mutations in von Hippel Lindau (vhl, a form of angiogenesis that is dependent on blood flow. We examined expression of three klf family members in wildtype and klf2ash317 zebrafish. We detected vascular expression of klf2b (but not klf4a or biklf/klf4b/klf17 in wildtypes but found no differences in expression that might account for the lack of phenotype in klf2ash317 mutants. klf2b morpholino knockdown did not affect heart rate or impair formation of the 5th accessory aortic arch in either wildtypes or klf2ash317 mutants.The klf2ash317 mutation produces a truncated Klf2a protein but, unlike morpholino induced klf2a knockdown, does not affect cardiovascular development.

Cell migration during heart regeneration in zebrafish.

Science.gov (United States)

Tahara, Naoyuki; Brush, Michael; Kawakami, Yasuhiko

2016-07-01

Zebrafish possess the remarkable ability to regenerate injured hearts as adults, which contrasts the very limited ability in mammals. Although very limited, mammalian hearts do in fact have measurable levels of cardiomyocyte regeneration. Therefore, elucidating mechanisms of zebrafish heart regeneration would provide information of naturally occurring regeneration to potentially apply to mammalian studies, in addition to addressing this biologically interesting phenomenon in itself. Studies over the past 13 years have identified processes and mechanisms of heart regeneration in zebrafish. After heart injury, pre-existing cardiomyocytes dedifferentiate, enter the cell cycle, and repair the injured myocardium. This process requires interaction with epicardial cells, endocardial cells, and vascular endothelial cells. Epicardial cells envelope the heart, while endocardial cells make up the inner lining of the heart. They provide paracrine signals to cardiomyocytes to regenerate the injured myocardium, which is vascularized during heart regeneration. In addition, accumulating results suggest that local migration of these major cardiac cell types have roles in heart regeneration. In this review, we summarize the characteristics of various heart injury methods used in the research community and regeneration of the major cardiac cell types. Then, we discuss local migration of these cardiac cell types and immune cells during heart regeneration. Developmental Dynamics 245:774-787, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

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

Science.gov (United States)

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

2018-01-01

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

Global gene expression profile induced by the UV-filter 2-ethyl-hexyl-4-trimethoxycinnamate (EHMC) in zebrafish (Danio rerio)

International Nuclear Information System (INIS)

Zucchi, Sara; Oggier, Daniela M.; Fent, Karl

2011-01-01

Residues of the UV-filter 2-ethyl-hexyl-4-trimethoxycinnamate (EHMC) are ubiquitously found in aquatic biota but potential adverse effects in fish are fairly unknown. To identify molecular effects and modes of action of EHMC we applied a gene expression profiling in zebrafish using whole genome microarrays. Transcriptome analysis and validation of targeted genes were performed after 14 days of exposure of male zebrafish. Concentrations of 2.2 μg/L and 890 μg/L EHMC lead to alteration of 1096 and 1137 transcripts, respectively, belonging to many pathways. Genes involved in lipid metabolism and estrogenic pathway (vtg1), lipid biosynthesis (ptgds), vitamin A metabolic process (rbp2a), DNA damage and apoptosis (gadd45b), and regulation of cell growth (igfbp1a) were investigated by qRT-PCR analysis in whole body, liver, brain and testis. The analysis showed tissue-specific gene profiles and revealed that EHMC slightly affects the transcription of genes involved in hormonal pathways including vtg1, esr1, esr2b, ar, cyp19b and hsd17β3. - Highlights: → The UV-filter EHMC accumulates in biota and shows expressional changes in fish. → Molecular effects of EHMC are demonstrated by microarrays and qRT-PCR in zebrafish. → Expressional changes in zebrafish occur at environmentally relevant concentrations. → The expressional changes point to interference of EHMC with the sex hormone system. → Additionally, many pathways are affected demonstrating multiple activities of EHMC. - Gene expression changes by 2-ethyl-hexyl-4-trimethoxycinnamate in zebrafish.

Global gene expression profile induced by the UV-filter 2-ethyl-hexyl-4-trimethoxycinnamate (EHMC) in zebrafish (Danio rerio)

Energy Technology Data Exchange (ETDEWEB)

Zucchi, Sara [University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Gruendensrasse 40, CH-4132 Muttenz (Switzerland); Oggier, Daniela M. [University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Gruendensrasse 40, CH-4132 Muttenz (Switzerland); University of Zuerich, Institute of Plant Biology, Division of Limnology, 8802 Kilchberg (Switzerland); Fent, Karl, E-mail: karl.fent@fhnw.ch [University of Applied Sciences Northwestern Switzerland, School of Life Sciences, Gruendensrasse 40, CH-4132 Muttenz (Switzerland); Swiss Federal Institute of Technology Zuerich (ETH Zuerich), Department of Environmental Sciences, 8092 Zuerich (Switzerland)

2011-10-15

Residues of the UV-filter 2-ethyl-hexyl-4-trimethoxycinnamate (EHMC) are ubiquitously found in aquatic biota but potential adverse effects in fish are fairly unknown. To identify molecular effects and modes of action of EHMC we applied a gene expression profiling in zebrafish using whole genome microarrays. Transcriptome analysis and validation of targeted genes were performed after 14 days of exposure of male zebrafish. Concentrations of 2.2 {mu}g/L and 890 {mu}g/L EHMC lead to alteration of 1096 and 1137 transcripts, respectively, belonging to many pathways. Genes involved in lipid metabolism and estrogenic pathway (vtg1), lipid biosynthesis (ptgds), vitamin A metabolic process (rbp2a), DNA damage and apoptosis (gadd45b), and regulation of cell growth (igfbp1a) were investigated by qRT-PCR analysis in whole body, liver, brain and testis. The analysis showed tissue-specific gene profiles and revealed that EHMC slightly affects the transcription of genes involved in hormonal pathways including vtg1, esr1, esr2b, ar, cyp19b and hsd17{beta}3. - Highlights: > The UV-filter EHMC accumulates in biota and shows expressional changes in fish. > Molecular effects of EHMC are demonstrated by microarrays and qRT-PCR in zebrafish. > Expressional changes in zebrafish occur at environmentally relevant concentrations. > The expressional changes point to interference of EHMC with the sex hormone system. > Additionally, many pathways are affected demonstrating multiple activities of EHMC. - Gene expression changes by 2-ethyl-hexyl-4-trimethoxycinnamate in zebrafish.

A variant of fibroblast growth factor receptor 2 (Fgfr2 regulates left-right asymmetry in zebrafish.

Directory of Open Access Journals (Sweden)

Da-Wei Liu

Full Text Available Many organs in vertebrates are left-right asymmetrical located. For example, liver is at the right side and stomach is at the left side in human. Fibroblast growth factor (Fgf signaling is important for left-right asymmetry. To investigate the roles of Fgfr2 signaling in zebrafish left-right asymmetry, we used splicing blocking morpholinos to specifically block the splicing of fgfr2b and fgfr2c variants, respectively. We found that the relative position of the liver and the pancreas were disrupted in fgfr2c morphants. Furthermore, the left-right asymmetry of the heart became random. Expression pattern of the laterality controlling genes, spaw and pitx2c, also became random in the morphants. Furthermore, lefty1 was not expressed in the posterior notochord, indicating that the molecular midline barrier had been disrupted. It was also not expressed in the brain diencephalon. Kupffer's vesicle (KV size became smaller in fgfr2c morphants. Furthermore, KV cilia were shorter in fgfr2c morphants. We conclude that the fgfr2c isoform plays an important role in the left-right asymmetry during zebrafish development.

A variant of fibroblast growth factor receptor 2 (Fgfr2) regulates left-right asymmetry in zebrafish.

Science.gov (United States)

Liu, Da-Wei; Hsu, Chia-Hao; Tsai, Su-Mei; Hsiao, Chung-Der; Wang, Wen-Pin

2011-01-01

Many organs in vertebrates are left-right asymmetrical located. For example, liver is at the right side and stomach is at the left side in human. Fibroblast growth factor (Fgf) signaling is important for left-right asymmetry. To investigate the roles of Fgfr2 signaling in zebrafish left-right asymmetry, we used splicing blocking morpholinos to specifically block the splicing of fgfr2b and fgfr2c variants, respectively. We found that the relative position of the liver and the pancreas were disrupted in fgfr2c morphants. Furthermore, the left-right asymmetry of the heart became random. Expression pattern of the laterality controlling genes, spaw and pitx2c, also became random in the morphants. Furthermore, lefty1 was not expressed in the posterior notochord, indicating that the molecular midline barrier had been disrupted. It was also not expressed in the brain diencephalon. Kupffer's vesicle (KV) size became smaller in fgfr2c morphants. Furthermore, KV cilia were shorter in fgfr2c morphants. We conclude that the fgfr2c isoform plays an important role in the left-right asymmetry during zebrafish development.

Fish can show emotional fever: stress-induced hyperthermia in zebrafish.

Science.gov (United States)

Rey, Sonia; Huntingford, Felicity A; Boltaña, Sebastian; Vargas, Reynaldo; Knowles, Toby G; Mackenzie, Simon

2015-11-22

Whether fishes are sentient beings remains an unresolved and controversial question. Among characteristics thought to reflect a low level of sentience in fishes is an inability to show stress-induced hyperthermia (SIH), a transient rise in body temperature shown in response to a variety of stressors. This is a real fever response, so is often referred to as 'emotional fever'. It has been suggested that the capacity for emotional fever evolved only in amniotes (mammals, birds and reptiles), in association with the evolution of consciousness in these groups. According to this view, lack of emotional fever in fishes reflects a lack of consciousness. We report here on a study in which six zebrafish groups with access to a temperature gradient were either left as undisturbed controls or subjected to a short period of confinement. The results were striking: compared to controls, stressed zebrafish spent significantly more time at higher temperatures, achieving an estimated rise in body temperature of about 2-4°C. Thus, zebrafish clearly have the capacity to show emotional fever. While the link between emotion and consciousness is still debated, this finding removes a key argument for lack of consciousness in fishes. © 2015 The Authors.

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

Science.gov (United States)

Postlethwait, J H

2006-01-01

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

In silico and in situ characterization of the zebrafish (Danio rerio gnrh3 (sGnRH gene

Directory of Open Access Journals (Sweden)

Husebye Harald

2002-08-01

Full Text Available Abstract Background Gonadotropin releasing hormone (GnRH is responsible for stimulation of gonadotropic hormone (GtH in the hypothalamus-pituitary-gonadal axis (HPG. The regulatory mechanisms responsible for brain specificity make the promoter attractive for in silico analysis and reporter gene studies in zebrafish (Danio rerio. Results We have characterized a zebrafish [Trp7, Leu8] or salmon (s GnRH variant, gnrh3. The gene includes a 1.6 Kb upstream regulatory region and displays the conserved structure of 4 exons and 3 introns, as seen in other species. An in silico defined enhancer at -976 in the zebrafish promoter, containing adjacent binding sites for Oct-1, CREB and Sp1, was predicted in 2 mammalian and 5 teleost GnRH promoters. Reporter gene studies confirmed the importance of this enhancer for cell specific expression in zebrafish. Interestingly the promoter of human GnRH-I, known as mammalian GnRH (mGnRH, was shown capable of driving cell specific reporter gene expression in transgenic zebrafish. Conclusions The characterized zebrafish Gnrh3 decapeptide exhibits complete homology to the Atlantic salmon (Salmo salar GnRH-III variant. In silico analysis of mammalian and teleost GnRH promoters revealed a conserved enhancer possessing binding sites for Oct-1, CREB and Sp1. Transgenic and transient reporter gene expression in zebrafish larvae, confirmed the importance of the in silico defined zebrafish enhancer at -976. The capability of the human GnRH-I promoter of directing cell specific reporter gene expression in zebrafish supports orthology between GnRH-I and GnRH-III.

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

Energy Technology Data Exchange (ETDEWEB)

Schmidt, Hayden R. [Department of Biology, Whittier College, Whittier, CA 90608 (United States); Radić, Zoran; Taylor, Palmer [Department of Pharmacology, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, CA 92093-0650 (United States); Fradinger, Erica A., E-mail: efrading@whittier.edu [Department of Biology, Whittier College, Whittier, CA 90608 (United States)

2015-04-15

The zebrafish is rapidly becoming an important model system for screening of new therapeutics. Here we evaluated the zebrafish as a potential pharmacological model for screening novel oxime antidotes to organophosphate (OP)-inhibited acetylcholinesterase (AChE). The k{sub i} values determined for chlorpyrifos oxon (CPO) and dichlorvos (DDVP) showed that CPO was a more potent inhibitor of both human and zebrafish AChE, but overall zebrafish AChE was less sensitive to OP inhibition. In contrast, aldoxime antidotes, the quaternary ammonium 2-PAM and tertiary amine RS-194B, showed generally similar overall reactivation kinetics, k{sub r}, in both zebrafish and human AChE. However, differences between the K{sub ox} and k{sub 2} constants suggest that zebrafish AChE associates more tightly with oximes, but has a slower maximal reactivation rate than human AChE. Homology modeling suggests that these kinetic differences result from divergences in the amino acids lining the entrance to the active site gorge. Although 2-PAM had the more favorable in vitro reactivation kinetics, RS-194B was more effective antidote in vivo. In intact zebrafish embryos, antidotal treatment with RS-194B rescued embryos from OP toxicity, whereas 2-PAM had no effect. Dechorionation of the embryos prior to antidotal treatment allowed both 2-PAM and RS-194B to rescue zebrafish embryos from OP toxicity. Interestingly, RS-194B and 2-PAM alone increased cholinergic motor activity in dechorionated embryos possibly due to the reversible inhibition kinetics, K{sub i} and αK{sub i}, of the oximes. Together these results demonstrate that the zebrafish at various developmental stages provides an excellent model for investigating membrane penetrant antidotes to OP exposure. - Highlights: • Zebrafish AChE shares significant structural similarities with human AChE. • OP-inhibited zebrafish and human AChE exhibit similar reactivation kinetics. • The zebrafish chorion is permeable to BBB penetrant and not

Influence of magnetic field on zebrafish activity and orientation in a plus maze.

Science.gov (United States)

Osipova, Elena A; Pavlova, Vera V; Nepomnyashchikh, Valentin A; Krylov, Viacheslav V

2016-01-01

We describe an impact of the geomagnetic field (GMF) and its modification on zebrafish's orientation and locomotor activity in a plus maze with four arms oriented to the north, east, south and west. Zebrafish's directional preferences were bimodal in GMF: they visited two arms oriented in opposed directions (east-west) most frequently. This bimodal preference remained stable for same individuals across experiments divided by several days. When the horizontal GMF component was turned 90° clockwise, the preference accordingly shifted by 90° to arms oriented to the north and south. Other modifications of GMF (reversal of both vertical and horizontal GMF components; reversal of vertical component only; and reversal of horizontal component only) did not exert any discernible effect on the orientation of zebrafish. The 90° turn of horizontal component also resulted in a significant increase of fish's locomotor activity in comparison with the natural GMF. This increase became even more pronounced when the horizontal component was repeatedly turned by 90° and back with 1min interval between turns. Our results show that GMF and its variations should be taken into account when interpreting zebrafish's directional preferences and locomotor activity in mazes and other experimental devices. Copyright © 2015. Published by Elsevier B.V.

Differential Toxicity of mDia Formin-Directed Functional Agonists and Antagonists in Developing Zebrafish

Directory of Open Access Journals (Sweden)

Hunter LeCorgne

2018-04-01

Full Text Available The mammalian Diaphanous-related (mDia formins are cytoskeletal regulators that assemble and, in some cases, bundle filamentous actin (F-actin, as well as stabilize microtubules. The development of small molecule antagonists and agonists that interrogate mDia formin function has allowed us to investigate the roles of formins in disease states. A small molecule inhibitor of FH2 domain (SMIFH2 inhibits mDia-dependent actin dynamics and abrogates tumor cell migration and cell division in vitro and ex vivo tissue explants. mDia formin activation with small molecule intramimics IMM01/02 and mDia2-DAD peptides inhibited glioblastoma motility and invasion in vitro and ex vivo rat brain slices. However, SMIFH2, IMMs, and mDia2 DAD efficacy in vivo remains largely unexplored and potential toxicity across a range of developmental phenotypes has not been thoroughly characterized. In this study, we performed an in vivo screen of early life-stage toxicity in Danio rerio zebrafish embryos 2 days post-fertilization (dpf in response to SMIFH2, IMM01/02, and mDia2 DAD. SMIFH2 at concentrations ≥5–10 μM induced significant defects in developing zebrafish, including shorter body lengths, tail curvature and defective tail cellularity, craniofacial malformations, pericardial edema, absent and/or compromised vasculature function and flow, depressed heart rates and increased mortality. Conversely, IMM and mDia2 DAD peptides were minimally toxic at concentrations up to 10–20 and 50 μM, respectively. SMIFH2's therapeutic potential may therefore be limited by its substantial in vivo toxicity at functional concentrations. mDia formin agonism with IMMs and mDia2 DADs may therefore be a more effective and less toxic anti-invasive therapeutic approach.

spv locus aggravates Salmonella infection of zebrafish adult by inducing Th1/Th2 shift to Th2 polarization.

Science.gov (United States)

Wu, Shu-Yan; Wang, Li-Dan; Xu, Guang-Mei; Yang, Si-di; Deng, Qi-Feng; Li, Yuan-Yuan; Huang, Rui

2017-08-01

Salmonella enterica serovar typhimurium (S. typhimurium) are facultative intracellular enteric pathogens causing disease with a broad range of hosts. It was known that Th1-type cytokines such as IFN-γ, IL-12, and TNF-α etc. could induce protective immunity against intracellular pathogens, while Th2-type cytokines such as IL-4, IL-10, and IL-13 etc. are proved to help pathogens survive inside hosts and cause severe infection. One of the critical virulence factor attributes to the pathogenesis of S. typhimurium is Salmonella plasmid virulence genes (spv). Until now, the interaction between spv locus and the predictable generation of Th1 or Th2 immune responses to Salmonella has not been identified. In this study, zebrafish adults were employed to explore the effect of spv locus on Salmonella pathogenesis as well as host adaptive immune responses especially shift of Th1/Th2 balance. The pathological changes of intestines and livers in zebrafish were observed by hematoxylin-eosin (HE) staining and electron microscopy. Levels of the transcription factors of Th1 (Tbx21) and Th2 (GATA3) were measured by real-time quantitative PCR (RT-qPCR). Expression of cytokines were determined by using RT-qPCR and ELISA, respectively. Results showed that spv operon aggravates damage of zebrafish. Furthermore, it demonstrated that spv locus could inhibit the transcription of tbx21 gene and suppress the expression of cytokines IFN-γ, IL-12 and TNF-α. On the contrary, the transcription of gata3 gene could be promoted and the expression of cytokines IL-4, IL-10 and IL-13 were enhanced by spv locus. Taken together, our data revealed that spv locus could aggravate Salmonella infection of zebrafish adult by inducing an imbalance of Th1/Th2 immune response and resulting in a detrimental Th2 bias of host. Copyright © 2017. Published by Elsevier Ltd.

Pharmacological Modulation of Hemodynamics in Adult Zebrafish In Vivo.

Directory of Open Access Journals (Sweden)

Daniel Brönnimann

Full Text Available Hemodynamic parameters in zebrafish receive increasing attention because of their important role in cardiovascular processes such as atherosclerosis, hematopoiesis, sprouting and intussusceptive angiogenesis. To study underlying mechanisms, the precise modulation of parameters like blood flow velocity or shear stress is centrally important. Questions related to blood flow have been addressed in the past in either embryonic or ex vivo-zebrafish models but little information is available for adult animals. Here we describe a pharmacological approach to modulate cardiac and hemodynamic parameters in adult zebrafish in vivo.Adult zebrafish were paralyzed and orally perfused with salt water. The drugs isoprenaline and sodium nitroprusside were directly applied with the perfusate, thus closely resembling the preferred method for drug delivery in zebrafish, namely within the water. Drug effects on the heart and on blood flow in the submental vein were studied using electrocardiograms, in vivo-microscopy and mathematical flow simulations.Under control conditions, heart rate, blood flow velocity and shear stress varied less than ± 5%. Maximal chronotropic effects of isoprenaline were achieved at a concentration of 50 μmol/L, where it increased the heart rate by 22.6 ± 1.3% (n = 4; p < 0.0001. Blood flow velocity and shear stress in the submental vein were not significantly increased. Sodium nitroprusside at 1 mmol/L did not alter the heart rate but increased blood flow velocity by 110.46 ± 19.64% (p = 0.01 and shear stress by 117.96 ± 23.65% (n = 9; p = 0.03.In this study, we demonstrate that cardiac and hemodynamic parameters in adult zebrafish can be efficiently modulated by isoprenaline and sodium nitroprusside. Together with the suitability of the zebrafish for in vivo-microscopy and genetic modifications, the methodology described permits studying biological processes that are dependent on hemodynamic alterations.

Inheritance patterns of morphological laterality in mouth opening of zebrafish, Danio rerio.

Science.gov (United States)

Hata, Hiroki; Hori, Michio

2012-01-01

The inheritance patterns of asymmetry in mouth opening in zebrafish were investigated using crossing experiments. Zebrafish exhibit asymmetric laterality in mouth opening, with each individual having either a leftward (righty) or rightward (lefty) bias. All righty incrosses produced only righty F(1), whereas all lefty incrosses resulted in an F(1) L:R ratio of 2:1. All test crosses between lefty and righty individuals resulted in an F(1) L:R=1:1. These results were consistent with the hereditary pattern for Japanese medaka, three Tanganyikan cichlids, and a Japanese riverine goby. The pattern suggests a one-locus two-allele Mendelian model of inheritance, with the lefty allele being dominant over righty and the dominant homozygote being lethal. To determine the reason for the absence of lefty homozygotes, the survival rates of the offspring were examined according to developmental stage. Survival did not differ among combinations of parent laterality. Thus the mechanism underlying the lethality of the dominant homozygote remains unclear. This study showed that the mouth-opening laterality of zebrafish is genetically determined and that the direction follows a Mendelian inheritance pattern that is shared among cypriniform zebrafish, beloniform medaka, perciform cichlids, and a goby, suggesting a common genetic background in mouth-opening laterality among these species.

Zebrafish-Based Discovery of Antiseizure Compounds from the Red Sea: Pseurotin A2 and Azaspirofuran A.

Science.gov (United States)

Copmans, Daniëlle; Rateb, Mostafa; Tabudravu, Jioji N; Pérez-Bonilla, Mercedes; Dirkx, Nina; Vallorani, Riccardo; Diaz, Caridad; Pérez Del Palacio, José; Smith, Alan J; Ebel, Rainer; Reyes, Fernando; Jaspars, Marcel; de Witte, Peter A M

2018-04-19

In search for novel antiseizure drugs (ASDs), the European FP7-funded PharmaSea project used zebrafish embryos and larvae as a drug discovery platform to screen marine natural products to identify promising antiseizure hits in vivo for further development. Within the framework of this project, seven known heterospirocyclic γ-lactams, namely, pseurotin A, pseurotin A 2 , pseurotin F1, 11- O-methylpseurotin A, pseurotin D, azaspirofuran A, and azaspirofuran B, were isolated from the bioactive marine fungus Aspergillus fumigatus, and their antiseizure activity was evaluated in the larval zebrafish pentylenetetrazole (PTZ) seizure model. Pseurotin A 2 and azaspirofuran A were identified as antiseizure hits, while their close chemical analogues were inactive. Besides, electrophysiological analysis from the zebrafish midbrain demonstrated that pseurotin A 2 and azaspirofuran A also ameliorate PTZ-induced epileptiform discharges. Next, to determine whether these findings translate to mammalians, both compounds were analyzed in the mouse 6 Hz (44 mA) psychomotor seizure model. They lowered the seizure duration dose-dependently, thereby confirming their antiseizure properties and suggesting activity against drug-resistant seizures. Finally, in a thorough ADMET assessment, pseurotin A 2 and azaspirofuran A were found to be drug-like. Based on the prominent antiseizure activity in both species and the drug-likeness, we propose pseurotin A 2 and azaspirofuran A as lead compounds that are worth further investigation for the treatment of epileptic seizures. This study not only provides the first evidence of antiseizure activity of pseurotins and azaspirofurans, but also demonstrates the value of the zebrafish model in (marine) natural product drug discovery in general, and for ASD discovery in particular.

Molecular genetics of pituitary development in zebrafish.

Science.gov (United States)

Pogoda, Hans-Martin; Hammerschmidt, Matthias

2007-08-01

The pituitary gland of vertebrates consists of two major parts, the neurohypophysis (NH) and the adenohypophysis (AH). As a central part of the hypothalamo-hypophyseal system (HHS), it constitutes a functional link between the nervous and the endocrine system to regulate basic body functions, such as growth, metabolism and reproduction. The development of the AH has been intensively studied in mouse, serving as a model for organogenesis and differential cell specification. However, given that the AH is a relatively recent evolutionary advance of the chordate phylum, it is also interesting to understand its development in lower chordate systems. In recent years, the zebrafish has emerged as a powerful lower vertebrate system for developmental studies, being amenable for large-scale genetic approaches, embryological manipulations, and in vivo imaging. Here, we present an overview of current knowledge of the mechanisms and genetic control of pituitary formation during zebrafish development. First, we describe the components of the zebrafish HHS, and the different pituitary cell types and hormones, followed by a description of the different steps of normal pituitary development. The central part of the review deals with the genes found to be essential for zebrafish AH development, accompanied by a description of the corresponding mutant phenotypes. Finally, we discuss future directions, with particular focus on evolutionary aspects, and some novel functional aspects with growing medical and social relevance.

Dithiocarbamates are teratogenic to developing zebrafish through inhibition of lysyl oxidase activity

International Nuclear Information System (INIS)

Boxtel, Antonius L. van; Kamstra, Jorke H.; Fluitsma, Donna M.; Legler, Juliette

2010-01-01

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 effects 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.

Establishing zebrafish as a novel exercise model: swimming economy, swimming-enhanced growth and muscle growth marker gene expression.

Directory of Open Access Journals (Sweden)

Arjan P Palstra

Full Text Available BACKGROUND: Zebrafish has been largely accepted as a vertebrate multidisciplinary model but its usefulness as a model for exercise physiology has been hampered by the scarce knowledge on its swimming economy, optimal swimming speeds and cost of transport. Therefore, we have performed individual and group-wise swimming experiments to quantify swimming economy and to demonstrate the exercise effects on growth in adult zebrafish. METHODOLOGY/PRINCIPAL FINDINGS: Individual zebrafish (n = 10 were able to swim at a critical swimming speed (U(crit of 0.548±0.007 m s(-1 or 18.0 standard body lengths (BL s(-1. The optimal swimming speed (U(opt at which energetic efficiency is highest was 0.396±0.019 m s(-1 (13.0 BL s(-1 corresponding to 72.26±0.29% of U(crit. The cost of transport at optimal swimming speed (COT(opt was 25.23±4.03 µmol g(-1 m(-1. A group-wise experiment was conducted with zebrafish (n = 83 swimming at U(opt for 6 h day(-1 for 5 days week(-1 for 4 weeks vs. zebrafish (n = 84 that rested during this period. Swimming zebrafish increased their total body length by 5.6% and body weight by 41.1% as compared to resting fish. For the first time, a highly significant exercise-induced growth is demonstrated in adult zebrafish. Expression analysis of a set of muscle growth marker genes revealed clear regulatory roles in relation to swimming-enhanced growth for genes such as growth hormone receptor b (ghrb, insulin-like growth factor 1 receptor a (igf1ra, troponin C (stnnc, slow myosin heavy chain 1 (smyhc1, troponin I2 (tnni2, myosin heavy polypeptide 2 (myhz2 and myostatin (mstnb. CONCLUSIONS/SIGNIFICANCE: From the results of our study we can conclude that zebrafish can be used as an exercise model for enhanced growth, with implications in basic, biomedical and applied sciences, such as aquaculture.

Silver nanoparticles enhance wound healing in zebrafish (Danio rerio).

Science.gov (United States)

Seo, Seung Beom; Dananjaya, S H S; Nikapitiya, Chamilani; Park, Bae Keun; Gooneratne, Ravi; Kim, Tae-Yoon; Lee, Jehee; Kim, Cheol-Hee; De Zoysa, Mahanama

2017-09-01

Silver nanoparticles (AgNPs) were successfully synthesized by a chemical reduction method, physico-chemically characterized and their effect on wound-healing activity in zebrafish was investigated. The prepared AgNPs were circular-shaped, water soluble with average diameter and zeta potential of 72.66 nm and -0.45 mv, respectively. Following the creation of a laser skin wound on zebrafish, the effect of AgNPs on wound-healing activity was tested by two methods, direct skin application (2 μg/wound) and immersion in a solution of AgNPs and water (50 μg/L). The zebrafish were followed for 20 days post-wounding (dpw) by visual observation of wound size, calculating wound healing percentage (WHP), and histological examination. Visually, both direct skin application and immersion AgNPs treatments displayed clear and faster wound closure at 5, 10 and 20 dpw compared to the controls, which was confirmed by 5 dpw histology data. At 5 dpw, WHP was highest in the AgNPs immersion group (36.6%) > AgNPs direct application group (23.7%) > controls (18.2%), showing that WHP was most effective in fish immersed in AgNPs solution. In general, exposure to AgNPs induced gene expression of selected wound-healing-related genes, namely, transforming growth factor (TGF-β), matrix metalloproteinase (MMP) -9 and -13, pro-inflammatory cytokines (IL-1β and TNF-α) and antioxidant enzymes (superoxide dismutase and catalase), which observed differentiation at 12 and 24 h against the control; but the results were not consistently significant, and many either reached basal levels or were down regulated at 5 dpw in the wounded muscle. These results suggest that AgNPs are effective in acceleration of wound healing and altered the expression of some wound-healing-related genes. However, the detailed mechanism of enhanced wound healing remains to be investigated in fish. Copyright © 2017 Elsevier Ltd. All rights reserved.

Expression of sall4 in taste buds of zebrafish.

Science.gov (United States)

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

2013-07-01

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

Development of the zebrafish myoseptum with emphasis on the myotendinous junction.

Science.gov (United States)

Charvet, Benjamin; Malbouyres, Marilyne; Pagnon-Minot, Aurélie; Ruggiero, Florence; Le Guellec, Dominique

2011-12-01

Zebrafish myosepta connect two adjacent muscle cells and transmit muscular forces to axial structures during swimming via the myotendinous junction (MTJ). The MTJ establishes transmembrane linkages system consisting of extracellular matrix molecules (ECM) surrounding the basement membrane, cytoskeletal elements anchored to sarcolema, and all intermediate proteins that link ECM to actin filaments. Using a series of zebrafish specimens aged between 24 h post-fertilization and 2 years old, the present paper describes at the transmission electron microscope level the development of extracellular and intracellular elements of the MTJ. The transverse myoseptum development starts during the segmentation period by deposition of sparse and loosely organized collagen fibrils. During the hatching period, a link between actin filaments and sarcolemma is established. The basal lamina underlining sarcolemma is well differentiated. Later, collagen fibrils display an orthogonal orientation and fibroblast-like cells invade the myoseptal stroma. A dense network of collagen fibrils is progressively formed that both anchor myoseptal fibroblasts and sarcolemmal basement membrane. The differentiation of a functional MTJ is achieved when sarcolemma interacts with both cytoskeletal filaments and extracellular components. This solid structural link between contractile apparatus and ECM leads to sarcolemma deformations resulting in the formation of regular invaginations, and allows force transmission during muscle contraction. This paper presents the first ultrastructural atlas of the zebrafish MTJ development, which represents an useful tool to analyse the mechanisms of the myotendinous system formation and their disruption in muscle disorders.

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

Directory of Open Access Journals (Sweden)

Yann Gibert

2011-01-01

Full Text Available 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.

Examination of a Palatogenic Gene Program in Zebrafish

Science.gov (United States)

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

2011-01-01

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

The inflammatory bowel disease (IBD susceptibility genes NOD1 and NOD2 have conserved anti-bacterial roles in zebrafish

Directory of Open Access Journals (Sweden)

Stefan H. Oehlers

2011-11-01

Inflammatory bowel disease (IBD, in the form of Crohn’s disease (CD or ulcerative colitis (UC, is a debilitating chronic immune disorder of the intestine. A complex etiology resulting from dysfunctional interactions between the intestinal immune system and its microflora, influenced by host genetic susceptibility, makes disease modeling challenging. Mutations in NOD2 have the highest disease-specific risk association for CD, and a related gene, NOD1, is associated with UC. NOD1 and NOD2 encode intracellular bacterial sensor proteins acting as innate immune triggers, and represent promising therapeutic targets. The zebrafish has the potential to aid in modeling genetic and environmental aspects of IBD pathogenesis. Here, we report the characterization of the Nod signaling components in the zebrafish larval intestine. The nod1 and nod2 genes are expressed in intestinal epithelial cells and neutrophils together with the Nod signaling pathway genes ripk2, a20, aamp, cd147, centaurin b1, erbin and grim-19. Using a zebrafish embryo Salmonella infection model, morpholino-mediated depletion of Nod1 or Nod2 reduced the ability of embryos to control systemic infection. Depletion of Nod1 or Nod2 decreased expression of dual oxidase in the intestinal epithelium and impaired the ability of larvae to reduce intracellular bacterial burden. This work highlights the potential use of zebrafish larvae in the study of components of IBD pathogenesis.

Image-Based Measurement of H2O2 Reaction-Diffusion in Wounded Zebrafish Larvae.

Science.gov (United States)

Jelcic, Mark; Enyedi, Balázs; Xavier, João B; Niethammer, Philipp

2017-05-09

Epithelial injury induces rapid recruitment of antimicrobial leukocytes to the wound site. In zebrafish larvae, activation of the epithelial NADPH oxidase Duox at the wound margin is required early during this response. Before injury, leukocytes are near the vascular region, that is, ∼100-300 μm away from the injury site. How Duox establishes long-range signaling to leukocytes is unclear. We conceived that extracellular hydrogen peroxide (H 2 O 2 ) generated by Duox diffuses through the tissue to directly regulate chemotactic signaling in these cells. But before it can oxidize cellular proteins, H 2 O 2 must get past the antioxidant barriers that protect the cellular proteome. To test whether, or on which length scales this occurs during physiological wound signaling, we developed a computational method based on reaction-diffusion principles that infers H 2 O 2 degradation rates from intravital H 2 O 2 -biosensor imaging data. Our results indicate that at high tissue H 2 O 2 levels the peroxiredoxin-thioredoxin antioxidant chain becomes overwhelmed, and H 2 O 2 degradation stalls or ceases. Although the wound H 2 O 2 gradient reaches deep into the tissue, it likely overcomes antioxidant barriers only within ∼30 μm of the wound margin. Thus, Duox-mediated long-range signaling may require other spatial relay mechanisms besides extracellular H 2 O 2 diffusion. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Evaluation of visible implant elastomer tags in zebrafish (Danio rerio

Directory of Open Access Journals (Sweden)

Claudia Hohn

2013-11-01

The use of the visible implant elastomer (VIE tagging system in zebrafish (Danio rerio was examined. Two tag orientations (horizontal and vertical at the dorsal fin base were tested for tag retention, tag fragmentation and whether VIE tags affected growth and survival of juvenile zebrafish (1–4 month post hatch. Six tag locations (abdomen, anal fin base, caudal peduncle, dorsal fin base, pectoral fin base, isthmus and 5 tag colors (yellow, red, pink, orange, blue were evaluated for ease of VIE tag application and tag visibility in adult zebrafish. Long-term retention (1 year and multiple tagging sites (right and left of dorsal fin and pectoral fin base were examined in adult zebrafish. Lastly, survival of recombination activation gene 1−/− (rag1−/− zebrafish was evaluated after VIE tagging. The best tag location was the dorsal fin base, and the most visible tag color was pink. Growth rate of juvenile zebrafish was not affected by VIE tagging. Horizontal tagging is recommended in early stages of fish growth (1–2 months post hatch. VIE tags were retained for 1 year and tagging did not interfere with long-term growth and survival. There was no mortality associated with VIE tagging in rag1−/− zebrafish. The VIE tagging system is highly suitable for small-sized zebrafish. When familiar with the procedure, 120 adult zebrafish can be tagged in one hour. It does not increase mortality in adult zebrafish or interfere with growth in juvenile or adult zebrafish.

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

Energy Technology Data Exchange (ETDEWEB)

Massarsky, Andrey, E-mail: andrey.massarsky@duke.edu [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Bone, Audrey J. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Dong, Wu [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); School of Animal Science and Technology, Inner Mongolia Provincial Key Laboratory for Toxicants and Animal Disease, Inner Mongolia University for the Nationalities, Tongliao, Inner Mongolia 028000 (China); Hinton, David E. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States); Prasad, G.L. [RAI Services Company, Winston-Salem, NC 27101 (United States); Di Giulio, Richard T. [Nicholas School of the Environment, Duke University, Durham, NC 27708 (United States)

2016-10-15

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) 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) pathways. �

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

International Nuclear Information System (INIS)

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

2009-01-01

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

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

Science.gov (United States)

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.

Zebrafish: an animal model for research in veterinary medicine.

Science.gov (United States)

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

2015-01-01

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

HCV IRES-mediated core expression in zebrafish.

Directory of Open Access Journals (Sweden)

Ye Zhao

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

Characterization of brn1.2 and corticotropin-releasing hormone genes in zebrafish

OpenAIRE

Chandrasekar, Gayathri

2007-01-01

The zebrafish (Danio rerio), a tropical fresh water fish originally found in the rivers of India and Bangladesh has become a popular vertebrate model system over the last decade. The rapid sequencing of the zebrafish genome together with the latest advances in forward and reverse genetics has made this model organism more fascinating as it can be used to decipher the genetic mechanisms involved in the vertebrate development. Corticotropin-releasing hormone (CRH) regulates t...

Microcystin-LR exposure induces developmental neurotoxicity in zebrafish embryo

International Nuclear Information System (INIS)

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

2016-01-01

Microcystin-LR (MCLR) is a commonly acting potent hepatotoxin and has been pointed out of potentially causing developmental neurotoxicity, but the exact mechanism is little known. In this study, zebrafish embryos were exposed to 0, 0.8, 1.6 or 3.2 mg/L MCLR for 120 h. MCLR exposure through submersion caused serious hatching delay and body length decrease. The content of MCLR in zebrafish larvae was analyzed and the results demonstrated that MCLR can accumulate in zebrafish larvae. The locomotor speed of zebrafish larvae was decreased. Furthermore, the dopamine and acetylcholine (ACh) content were detected to be significantly decreased in MCLR exposure groups. And the acetylcholinesterase (AChE) activity was significantly increased after exposure to 1.6 and 3.2 mg/L MCLR. The transcription pattern of manf, chrnα7 and ache gene was consistent with the change of the dopamine content, ACh content and AChE activity. Gene expression involved in the development of neurons was also measured. α1-tubulin and shha gene expression were down-regulated, whereas mbp and gap43 gene expression were observed to be significantly up-regulated upon exposure to MCLR. The above results indicated that MCLR-induced developmental toxicity might attribute to the disorder of cholinergic system, dopaminergic signaling, and the development of neurons. - Highlights: • MCLR accumulation induces developmental neurotoxicity in zebrafish embryo. • The decrease of dopamine levels might be associated with the MCLR-induced developmental neurotoxicity in zebrafish larvae. • The alternation of cholinergic system might contribute to the change of neurobehavior in zebrafish larvae exposure with MCLR. - MCLR accumulation induces developmental neurotoxicity by affecting cholinergic system, dopaminergic signaling, and the development of neurons in zebrafish embryo.

Monitoring Toxic Ionic Liquids in Zebrafish ( Danio rerio) with Desorption Electrospray Ionization Mass Spectrometry Imaging (DESI-MSI)

Science.gov (United States)

Perez, Consuelo J.; Tata, Alessandra; de Campos, Michel L.; Peng, Chun; Ifa, Demian R.

2017-06-01

Ambient mass spectrometry imaging has become an increasingly powerful technique for the direct analysis of biological tissues in the open environment with minimal sample preparation and fast analysis times. In this study, we introduce desorption electrospray ionization mass spectrometry imaging (DESI-MSI) as a novel, rapid, and sensitive approach to localize the accumulation of a mildly toxic ionic liquid (IL), AMMOENG 130 in zebrafish ( Danio rerio). The work demonstrates that DESI-MSI has the potential to rapidly monitor the accumulation of IL pollutants in aquatic organisms. AMMOENG 130 is a quaternary ammonium-based IL reported to be broadly used as a surfactant in commercialized detergents. It is known to exhibit acute toxicity to zebrafish causing extensive damage to gill secondary lamellae and increasing membrane permeability. Zebrafish were exposed to the IL in a static 96-h exposure study in concentrations near the LC50 of 1.25, 2.5, and 5.0 mg/L. DESI-MS analysis of zebrafish gills demonstrated the appearance of a dealkylated AMMOENG 130 metabolite in the lowest concentration of exposure identified by a high resolution hybrid LTQ-Orbitrap mass spectrometer as the trimethylstearylammonium ion, [C21H46N]+. With DESI-MSI, the accumulation of AMMOENG 130 and its dealkylated metabolite in zebrafish tissue was found in the nervous and respiratory systems. AMMOENG 130 and the metabolite were capable of penetrating the blood brain barrier of the fish with significant accumulation in the brain. Hence, we report for the first time the simultaneous characterization, distribution, and metabolism of a toxic IL in whole body zebrafish analyzed by DESI-MSI. This ambient mass spectrometry imaging technique shows great promise for the direct analysis of biological tissues to qualitatively monitor foreign, toxic, and persistent compounds in aquatic organisms from the environment. [Figure not available: see fulltext.

Identification of estrogen target genes during zebrafish embryonic development through transcriptomic analysis.

Directory of Open Access Journals (Sweden)

Ruixin Hao

Full Text Available 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 fertilization (dpf, harvested at 1, 2, 3 and 4 dpf, and subjected to RNA extraction for transcriptome analysis using microarrays. Differentially expressed genes by E2-treatment were analyzed with hierarchical clustering followed by biological process and tissue enrichment analysis. Markedly distinct sets of genes were up and down-regulated by E2 at the four different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by E2 were relatively similar throughout zebrafish development. According to knowledge-based tissue enrichment, estrogen responsive genes were clustered mainly in the liver, pancreas and brain. This was in line with the developmental dynamics of estrogen-target tissues that were visualized using transgenic zebrafish containing estrogen responsive elements driving the expression of GFP (Tg(5xERE:GFP. Finally, the identified embryonic estrogen-responsive genes were compared to already published estrogen-responsive genes identified in male adult zebrafish (Gene Expression Omnibus database. The expressions of a few genes were co-regulated by E2 in both embryonic and adult zebrafish. These could potentially be used as estrogenic biomarkers for exposure to estrogens or estrogenic endocrine disruptors in zebrafish. In conclusion, our data suggests that estrogen effects on early embryonic zebrafish development are stage- and tissue- specific.

Changes in Neurotransmitter Profiles during Early Zebrafish (Danio rerio) Development and after Pesticide Exposure.

Science.gov (United States)

Tufi, Sara; Leonards, Pim; Lamoree, Marja; de Boer, Jacob; Legler, Juliette; Legradi, Jessica

2016-03-15

During early development, neurotransmitters are important stimulants for the development of the central nervous system. Although the development of different neuronal cell types during early zebrafish (Danio rerio) development is well-studied, little is known of the levels of neurotransmitters, their precursors and metabolites during development, and how these levels are affected by exposure to environmental contaminants. A method based on hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry has been applied for the first time to zebrafish embryos and larvae to study five neurotransmitter systems in parallel, including the dopaminergic-andrenergic, glutaminergic-GABAnergic, serotoninergic, histaminergic, and cholinergic systems. Our method enables the quantification of neurotransmitters and their precursors and metabolites in whole zebrafish from the period of zygote to free-swimming larvae 6 days postfertilization (dpf). We observed a developmental stage-dependent pattern with clear differences between the first 2 days of development and the following days. Whereas the neurotransmitter levels steadily increased, the precursors showed a peak at 3 dpf. After exposure to several pesticides, significant differences in concentrations of neurotransmitters and precursors were observed. Our study revealed new insights about neurotransmitter systems during early zebrafish development and showed the usefulness of our approach for environmental neurotoxicity studies.

Developmental effects of aerosols and coal burning particles in zebrafish embryos

International Nuclear Information System (INIS)

Olivares, Alba; Drooge, Barend L. van; Casado, Marta; Prats, Eva; Serra, Montserrat; Ven, Leo T. van der; Kamstra, Jorke H.; Hamers, Timo; Hermsen, Sanne; Grimalt, Joan O.; Piña, Benjamin

2013-01-01

Embryo toxicity of particles generated by combustion processes is of special concern for human health. A significant part of these toxic effects is linked to the binding of some pollutants (like polycyclic aromatic hydrocarbons or PAHs) to the Aryl hydrocarbon Receptor (AhR) and the activation of target genes, like the cytochrome P4501A. This activity was analyzed for ambient air and coal-combustion particle extracts in zebrafish embryos (the cyp1aDarT assay) and in two single-cell bioassays: the yeast-based YCM-RYA and the DR-luc (rat cells) assay. Observed AhR ligand activity of samples generally correlated to the predicted toxic effect according to their PAH composition, except for one of the coal combustion samples with an anomalously high activity in the cyp1aDarT assay. This sample induced deformities in zebrafish embryos. We concluded that the combination of morphological and molecular assays may detect embryonic toxic effects that cannot be predicted from chemical analyses or single-cell bioassays. -- Highlights: ► Samples from air particulated matter and coal waste gob showed embryo toxicity in zebrafish. ► PAHs composition of samples does not adequately predict the toxic effects in zebrafish. ► Active coal waste gob samples show maximal AhR-ligand activity and induce deformations in zebrafish embryos. -- Aerosols and coal burning particles showed a strong developmental toxicity in zebrafish, in a degree that cannot be directly predicted from chemical analyses or single-cell bioassays

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

Science.gov (United States)

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

2018-02-01

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

TOXICITY EVALUATION OF NEW ENGINEERED NANOMATERIALS IN ZEBRAFISH

Directory of Open Access Journals (Sweden)

Maria Violetta Brundo

2016-04-01

Full Text Available The effect of the nanoparticles on the marine organisms, depends on their size, chemical composition, surface structure, solubility and shape.In order to take advantage from their activity, preserving the surrounding environment from a possible pollution, we are trying to trap the nanoparticles into new nanomaterials. The nanomaterials tested were synthesized proposing a ground-breaking approach by an upside-down vision of the Au/TiO2nano-system to avoid the release of nanoparticles. The system was synthesized by wrapping Au nanoparticles with a thin layer of TiO2. The non-toxicity of the nano-system was established by testing the effect of the material on zebrafish larvae. Danio rerio o zebrafish was considered a excellent model for the environmental biomonitoring of aquatic environments and the Zebrafish Embryo Toxicity Test is considered an alternative method of animal test. For this reason zebrafish larvae were exposed to different concentrations of nanoparticles of TiO2 and Au and new nanomaterials. As biomarkers of exposure, we evaluated the expression of metallothioneins by immunohistochemistry analysis and western blotting analysis also. The results obtained by toxicity test showed that neither mortality as well as sublethal effects were induced by the different nanomaterials and nanoparticles tested. Only zebrafish larvae exposed to free Au nanoparticles showed a different response to anti-MT antibody. In fact, the immunolocalization analysis highlighted an increase of the metallothioneins synthesis.

pitx2 Deficiency results in abnormal ocular and craniofacial development in zebrafish.

Directory of Open Access Journals (Sweden)

Yi Liu

Full Text Available 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 pitx2(ex4/5 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 pitx2(ex4/5 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 pitx2(ex4/5 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.

Hypothalamic Projections to the Optic Tectum in Larval Zebrafish

Science.gov (United States)

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

2018-01-01

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

Hypothalamic Projections to the Optic Tectum in Larval Zebrafish

Directory of Open Access Journals (Sweden)

Lucy A. Heap

2018-01-01

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

Zebrafish brain mapping--standardized spaces, length scales, and the power of N and n.

Science.gov (United States)

Hunter, Paul R; Hendry, Aenea C; Lowe, Andrew S

2015-06-01

Mapping anatomical and functional parameters of the zebrafish brain is moving apace. Research communities undertaking such studies are becoming ever larger and more diverse. The unique features, tools, and technologies associated with zebrafish are propelling them as the 21st century model organism for brain mapping. Uniquely positioned as a vertebrate model system, the zebrafish enables imaging of anatomy and function at different length scales from intraneuronal compartments to sparsely distributed whole brain patterns. With a variety of diverse and established statistical modeling and analytic methods available from the wider brain mapping communities, the richness of zebrafish neuroimaging data is being realized. The statistical power of population observations (N) within and across many samples (n) projected onto a standardized space will provide vast databases for data-driven biological approaches. This article reviews key brain mapping initiatives at different levels of scale that highlight the potential of zebrafish brain mapping. By way of introduction to the next wave of brain mappers, an accessible introduction to the key concepts and caveats associated with neuroimaging are outlined and discussed. © 2014 Wiley Periodicals, Inc.

A zebrafish model of inflammatory lymphangiogenesis

Directory of Open Access Journals (Sweden)

Kazuhide S. Okuda

2015-10-01

Full Text Available Inflammatory bowel disease (IBD is a disabling chronic inflammatory disease of the gastrointestinal tract. IBD patients have increased intestinal lymphatic vessel density and recent studies have shown that this may contribute to the resolution of IBD. However, the molecular mechanisms involved in IBD-associated lymphangiogenesis are still unclear. In this study, we established a novel inflammatory lymphangiogenesis model in zebrafish larvae involving colitogenic challenge stimulated by exposure to 2,4,6-trinitrobenzenesulfonic acid (TNBS or dextran sodium sulphate (DSS. Treatment with either TNBS or DSS resulted in vascular endothelial growth factor receptor (Vegfr-dependent lymphangiogenesis in the zebrafish intestine. Reduction of intestinal inflammation by the administration of the IBD therapeutic, 5-aminosalicylic acid, reduced intestinal lymphatic expansion. Zebrafish macrophages express vascular growth factors vegfaa, vegfc and vegfd and chemical ablation of these cells inhibits intestinal lymphatic expansion, suggesting that the recruitment of macrophages to the intestine upon colitogenic challenge is required for intestinal inflammatory lymphangiogenesis. Importantly, this study highlights the potential of zebrafish as an inflammatory lymphangiogenesis model that can be used to investigate the role and mechanism of lymphangiogenesis in inflammatory diseases such as IBD.

Expression profiles for six zebrafish genes during gonadal sex differentiation

DEFF Research Database (Denmark)

Jørgensen, Anne; Morthorst, Jane Ebsen; Andersen, Ole

2008-01-01

BACKGROUND: The mechanism of sex determination in zebrafish is largely unknown and neither sex chromosomes nor a sex-determining gene have been identified. This indicates that sex determination in zebrafish is mediated by genetic signals from autosomal genes. The aim of this study was to determine...... the precise timing of expression of six genes previously suggested to be associated with sex differentiation in zebrafish. The current study investigates the expression of all six genes in the same individual fish with extensive sampling dates during sex determination and -differentiation. RESULTS......: In the present study, we have used quantitative real-time PCR to investigate the expression of ar, sox9a, dmrt1, fig alpha, cyp19a1a and cyp19a1b during the expected sex determination and gonadal sex differentiation period. The expression of the genes expected to be high in males (ar, sox9a and dmrt1a) and high...

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

Science.gov (United States)

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.

Skin too thin? The developing utility of zebrafish skin (neuro)pharmacology for CNS drug discovery research.

Science.gov (United States)

Nguyen, Michael; Poudel, Manoj K; Stewart, Adam Michael; Kalueff, Allan V

2013-09-01

Skin coloration can be affected by many genetic, environmental and pharmacological factors. Zebrafish (Danio rerio) are a useful and versatile model organism in biomedical research due to their genetic tractability, physiological homology to mammals, low cost, reproducibility and high throughput. Zebrafish coloration is mediated by chromatophores - the skin color pigment cells largely controlled by endocrine and neural mechanisms. The characteristic darkening of zebrafish skin is caused by the dispersion (and paling - by aggregation) of melanosomes (pigment-containing organelles), which show high homology to mammalian structures. Various pharmacological agents potently affect zebrafish coloration - the phenotype that often accompanies behavioral effects of the drugs, and may be used for drug discovery. Although zebrafish behavior and skin responses are usually not directly related, they share common regulatory (neural, endocrine) mechanisms, and therefore may be assessed in parallel during psychotropic drug screening. For example, some psychoactive drugs can potently affect zebrafish skin coloration. Can we use this knowledge to refine phenotype-driven psychotropic drug discovery? Here, we present current models using zebrafish skin coloration assays, and discuss how these models may be applied to enhance in vivo CNS drug discovery. Copyright © 2013 Elsevier Inc. All rights reserved.

Daily shoaling patterns in the zebrafish Danio rerio

Directory of Open Access Journals (Sweden)

Timothy PACIOREK, Scott McROBERT

2013-12-01

Full Text Available Shoaling intensity in zebrafish Danio rerio is believed to vary throughout subjective day and night hours. This experiment examines long term variations in shoaling behavior. Adult zebrafish Danio rerio were maintained under a 12:12 LD cycle (with dim red light serving as reduced visibility during subjective dark hours, and their shoaling behavior was monitored every hours for a three-day period of time. Our results show that zebrafish perform shoaling behavior throughout subjective day and under reduced visibility conditions, although mean shoaling times during the light phase were significantly higher than mean shoaling times during the dark phase. However, on the 3rd day of the experiment, mean shoaling times during the subjective night had increased and mean shoaling times during the subjective day had decreased. This shift in intensity was not seen on the first two days of the study, and may represent the influence of experience on the behavior of the test fish. We believe this study shows that shoaling behavior changes with light/dark cycles and that fish shoal even during reduced visibility conditions [Current Zoology 59 (6:754–760, 2013].

Immunostaining of dissected zebrafish embryonic heart.

Science.gov (United States)

Yang, Jingchun; Xu, Xiaolei

2012-01-10

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects. The expression of any gene can be manipulated via morpholino technology or RNA injection. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis. Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals. Copyright © 2012 Journal of Visualized Experiments

Prolonged hypoxia increases survival even in Zebrafish (Danio rerio showing cardiac arrhythmia.

Directory of Open Access Journals (Sweden)

Renate Kopp

Full Text Available Tolerance towards hypoxia is highly pronounced in zebrafish. In this study even beneficial effects of hypoxia, specifically enhanced survival of zebrafish larvae, could be demonstrated. This effect was actually more pronounced in breakdance mutants, which phenotypically show cardiac arrhythmia. Breakdance mutants (bre are characterized by chronically reduced cardiac output. Despite an about 50% heart rate reduction, they become adults, but survival rate significantly drops to 40%. Normoxic bre animals demonstrate increased hypoxia inducible factor 1 a (Hif-1α expression, which indicates an activated hypoxic signaling pathway. Consequently, cardiovascular acclimation, like cardiac hypertrophy and increased erythrocyte concentration, occurs. Thus, it was hypothesized, that under hypoxic conditions survival might be even more reduced. When bre mutants were exposed to hypoxic conditions, they surprisingly showed higher survival rates than under normoxic conditions and even reached wildtype values. In hypoxic wildtype zebrafish, survival yet exceeded normoxic control values. To specify physiological acclimation, cardiovascular and metabolic parameters were measured before hypoxia started (3 dpf, when the first differences in survival rate occurred (7 dpf and when survival rate plateaued (15 dpf. Hypoxic animals expectedly demonstrated Hif-1α accumulation and consequently enhanced convective oxygen carrying capacity. Moreover, bre animals showed a significantly enhanced heart rate under hypoxic conditions, which reached normoxic wildtype values. This improvement in convective oxygen transport ensured a sufficient oxygen and nutrient supply and was also reflected in the significantly higher mitochondrial activity. The highly optimized energy metabolism observed in hypoxic zebrafish larvae might be decisive for periods of higher energy demand due to organ development, growth and increased activity. However, hypoxia increased survival only during a

Colonizing the embryonic zebrafish gut with anaerobic bacteria derived from the human gastrointestinal tract.

Science.gov (United States)

Toh, Michael C; Goodyear, Mara; Daigneault, Michelle; Allen-Vercoe, Emma; Van Raay, Terence J

2013-06-01

The zebrafish has become increasingly popular for microbiological research. It has been used as an infection model for a variety of pathogens, and is also emerging as a tool for studying interactions between a host and its resident microbial communities. The mouse microbiota has been transplanted into the zebrafish gut, but to our knowledge, there has been no attempt to introduce a bacterial community derived from the human gut. We explored two methods for colonizing the developing gut of 5-day-old germ-free zebrafish larvae with a defined anaerobic microbial community derived from a single human fecal sample. Both environmental exposure (static immersion) and direct microinjection into the gut resulted in the establishment of two species-Lactobacillus paracasei and Eubacterium limosum-from a community of 30 strains consisting of 22 anaerobic species. Of particular interest is E. limosum, which, as a strict anaerobe, represents a group of bacteria which until now have not been shown to colonize the developing zebrafish gut. Our success here indicates that further investigation of zebrafish as a tool for studying human gut microbial communities is warranted.

An α-smooth muscle actin (acta2/αsma zebrafish transgenic line marking vascular mural cells and visceral smooth muscle cells.

Directory of Open Access Journals (Sweden)

Thomas R Whitesell

Full Text Available Mural cells of the vascular system include vascular smooth muscle cells (SMCs and pericytes whose role is to stabilize and/or provide contractility to blood vessels. One of the earliest markers of mural cell development in vertebrates is α smooth muscle actin (acta2; αsma, which is expressed by pericytes and SMCs. In vivo models of vascular mural cell development in zebrafish are currently lacking, therefore we developed two transgenic zebrafish lines driving expression of GFP or mCherry in acta2-expressing cells. These transgenic fish were used to trace the live development of mural cells in embryonic and larval transgenic zebrafish. acta2:EGFP transgenic animals show expression that largely mirrors native acta2 expression, with early pan-muscle expression starting at 24 hpf in the heart muscle, followed by skeletal and visceral muscle. At 3.5 dpf, expression in the bulbus arteriosus and ventral aorta marks the first expression in vascular smooth muscle. Over the next 10 days of development, the number of acta2:EGFP positive cells and the number of types of blood vessels associated with mural cells increases. Interestingly, the mural cells are not motile and remain in the same position once they express the acta2:EGFP transgene. Taken together, our data suggests that zebrafish mural cells develop relatively late, and have little mobility once they associate with vessels.

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

Science.gov (United States)

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

2018-01-01

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

Oral exposure of adult zebrafish (Danio rerio) to 2,4,6-tribromophenol affects reproduction

DEFF Research Database (Denmark)

Halden, Anna Norman; Nyholm, Jenny Rattfelt; Andersson, Patrik L

2010-01-01

The bromophenol 2,4,6-tribromophenol (TBP) is widely used as an industrial chemical, formed by degradation of tetrabromobisphenol-A, and it occurs naturally in marine organisms. Concentrations of TBP in fish have been related to intake via feed, but little is known about effects on fish health...... after oral exposure. In this study, we exposed adult male and female zebrafish (Danio rerio) to TBP via feed in nominal concentrations of 33, 330, and 3300 mu g/g feed (or control feed) for 6 weeks to assess the effects of TBP on reproductive output, gonad morphology, circulatory vitellogenin levels......, and early embryo development. The aim was also to investigate the extent to which TBP was metabolised to 2,4,6-tribromoanisole (IBA) in dietary exposed zebrafish, and the amounts of TBP and TBA found in offspring. After 6 weeks of exposure, we found about 3% of the daily dose of TBP in adult fish...

Ethanol Exposure Causes Muscle Degeneration in Zebrafish

Directory of Open Access Journals (Sweden)

Elizabeth C. Coffey

2018-03-01

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

Genetic determinants of hyaloid and retinal vasculature in zebrafish

Directory of Open Access Journals (Sweden)

Hyde David R

2007-10-01

Full Text Available Abstract Background The retinal vasculature is a capillary network of blood vessels that nourishes the inner retina of most mammals. Developmental abnormalities or microvascular complications in the retinal vasculature result in severe human eye diseases that lead to blindness. To exploit the advantages of zebrafish for genetic, developmental and pharmacological studies of retinal vasculature, we characterised the intraocular vasculature in zebrafish. Results We show a detailed morphological and developmental analysis of the retinal blood supply in zebrafish. Similar to the transient hyaloid vasculature in mammalian embryos, vessels are first found attached to the zebrafish lens at 2.5 days post fertilisation. These vessels progressively lose contact with the lens and by 30 days post fertilisation adhere to the inner limiting membrane of the juvenile retina. Ultrastructure analysis shows these vessels to exhibit distinctive hallmarks of mammalian retinal vasculature. For example, smooth muscle actin-expressing pericytes are ensheathed by the basal lamina of the blood vessel, and vesicle vacuolar organelles (VVO, subcellular mediators of vessel-retinal nourishment, are present. Finally, we identify 9 genes with cell membrane, extracellular matrix and unknown identity that are necessary for zebrafish hyaloid and retinal vasculature development. Conclusion Zebrafish have a retinal blood supply with a characteristic developmental and adult morphology. Abnormalities of these intraocular vessels are easily observed, enabling application of genetic and chemical approaches in zebrafish to identify molecular regulators of hyaloid and retinal vasculature in development and disease.

Toxicity assessment of zebrafish following exposure to CdTe QDs

Energy Technology Data Exchange (ETDEWEB)

Zhang, Wei, E-mail: wzhang@ecust.edu.cn [State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, East China University of Science and Technology, Shanghai 200237 (China); Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237 (China); School of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai 200237 (China); Lin, Kuangfei, E-mail: kflin@ecust.edu.cn [State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, East China University of Science and Technology, Shanghai 200237 (China); Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237 (China); School of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai 200237 (China); Miao, Youna [State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, East China University of Science and Technology, Shanghai 200237 (China); Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai 200237 (China); School of Resource and Environmental Engineering, East China University of Science and Technology, Shanghai 200237 (China); Dong, Qiaoxiang; Huang, Changjiang; Wang, Huili [Zhejiang Provincial Key Lab for Technology and Application of Model Organisms, Wenzhou Medical College, Wenzhou 325035 (China); Guo, Meijin [State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237 (China); Cui, Xinhong [Shanghai Institute of Landscape Gardening, Shanghai 200233 (China)

2012-04-30

Highlights: Black-Right-Pointing-Pointer The LC{sub 50} of TGA-CdTe for zebrafish at 120 hpf was 185.9 nM. Black-Right-Pointing-Pointer Zebrafish exposed to TGA-CdTe resulted in lower hatch rate and more malformation. Black-Right-Pointing-Pointer Body length and heart beat of zebrafish declined after exposure to TGA-CdTe. Black-Right-Pointing-Pointer Larvae exposure to TGA-CdTe elicited a higher basal swimming rate. Black-Right-Pointing-Pointer Abnormal vascular of FLI-1 transgenic zebrafish larvae exposed to TGA-CdTe occurred. - Abstract: CdTe quantum dots (QDs) are nanocrystals of unique composition and properties that have found many new commercial applications; therefore, their potential toxicity to aquatic organisms has become a hot research topic. The lab study was performed to determine the developmental and behavioral toxicities to zebrafish under continuous exposure to low concentrations of CdTe QDs (1-400 nM) coated with thioglycolic acid (TGA). The results show: (1) the 120 h LC{sub 50} of 185.9 nM, (2) the lower hatch rate and body length, more malformations, and less heart beat and swimming speed of the exposed zebrafish, (3) the brief burst and a higher basal swimming rate of the exposed zebrafish larvae during a rapid transition from light-to-dark, and (4) the vascular hyperplasia, vascular bifurcation, vascular crossing and turbulence of the exposed FLI-1 transgenic zebrafish larvae.

GLUT2-mediated glucose uptake and availability are required for embryonic brain development in zebrafish.

Science.gov (United States)

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.

Roles of brca2 (fancd1 in oocyte nuclear architecture, gametogenesis, gonad tumors, and genome stability in zebrafish.

Directory of Open Access Journals (Sweden)

Adriana Rodríguez-Marí

2011-03-01

Full Text Available Mild mutations in BRCA2 (FANCD1 cause Fanconi anemia (FA when homozygous, while severe mutations cause common cancers including breast, ovarian, and prostate cancers when heterozygous. Here we report a zebrafish brca2 insertional mutant that shares phenotypes with human patients and identifies a novel brca2 function in oogenesis. Experiments showed that mutant embryos and mutant cells in culture experienced genome instability, as do cells in FA patients. In wild-type zebrafish, meiotic cells expressed brca2; and, unexpectedly, transcripts in oocytes localized asymmetrically to the animal pole. In juvenile brca2 mutants, oocytes failed to progress through meiosis, leading to female-to-male sex reversal. Adult mutants became sterile males due to the meiotic arrest of spermatocytes, which then died by apoptosis, followed by neoplastic proliferation of gonad somatic cells that was similar to neoplasia observed in ageing dead end (dnd-knockdown males, which lack germ cells. The construction of animals doubly mutant for brca2 and the apoptotic gene tp53 (p53 rescued brca2-dependent sex reversal. Double mutants developed oocytes and became sterile females that produced only aberrant embryos and showed elevated risk for invasive ovarian tumors. Oocytes in double-mutant females showed normal localization of brca2 and pou5f1 transcripts to the animal pole and vasa transcripts to the vegetal pole, but had a polarized rather than symmetrical nucleus with the distribution of nucleoli and chromosomes to opposite nuclear poles; this result revealed a novel role for Brca2 in establishing or maintaining oocyte nuclear architecture. Mutating tp53 did not rescue the infertility phenotype in brca2 mutant males, suggesting that brca2 plays an essential role in zebrafish spermatogenesis. Overall, this work verified zebrafish as a model for the role of Brca2 in human disease and uncovered a novel function of Brca2 in vertebrate oocyte nuclear architecture.

Influence of carbon nanotube length on toxicity to zebrafish embryos

Directory of Open Access Journals (Sweden)

Cheng J

2012-07-01

Full Text Available Jinping Cheng,1,2 Shuk Han Cheng11Department of Biology and Chemistry, City University of Hong Kong, Hong Kong; 2State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai, ChinaAbstract: There is currently a large difference of opinion in nanotoxicology studies of nanomaterials. There is concern about why some studies have indicated that there is strong toxicity, while others have not. In this study, the length of carbon nanotubes greatly affected their toxicity in zebrafish embryos. Multiwalled carbon nanotubes (MWCNTs were sonicated in a nitric acid solution for 24 hours and 48 hours. The modified MWCNTs were tested in early developing zebrafish embryo. MWCNTs prepared with the longer sonication time resulted in severe developmental toxicity; however, the shorter sonication time did not induce any obvious toxicity in the tested developing zebrafish embryos. The cellular and molecular changes of the affected zebrafish embryos were studied and the observed phenotypes scored. This study suggests that length plays an important role in the in vivo toxicity of functionalized CNTs. This study will help in furthering the understanding on current differences in toxicity studies of nanomaterials.Keywords: length, carbon nanotubes, sonication, developmental toxicity, zebrafish

Somatic mutagenesis with a Sleeping Beauty transposon system leads to solid tumor formation in zebrafish.

Directory of Open Access Journals (Sweden)

Maura McGrail

2011-04-01

Full Text Available Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700-6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers.

Circadian clocks, rhythmic synaptic plasticity and the sleep-wake cycle in zebrafish.

Science.gov (United States)

Elbaz, Idan; Foulkes, Nicholas S; Gothilf, Yoav; Appelbaum, Lior

2013-01-01

The circadian clock and homeostatic processes are fundamental mechanisms that regulate sleep. Surprisingly, despite decades of research, we still do not know why we sleep. Intriguing hypotheses suggest that sleep regulates synaptic plasticity and consequently has a beneficial role in learning and memory. However, direct evidence is still limited and the molecular regulatory mechanisms remain unclear. The zebrafish provides a powerful vertebrate model system that enables simple genetic manipulation, imaging of neuronal circuits and synapses in living animals, and the monitoring of behavioral performance during day and night. Thus, the zebrafish has become an attractive model to study circadian and homeostatic processes that regulate sleep. Zebrafish clock- and sleep-related genes have been cloned, neuronal circuits that exhibit circadian rhythms of activity and synaptic plasticity have been studied, and rhythmic behavioral outputs have been characterized. Integration of this data could lead to a better understanding of sleep regulation. Here, we review the progress of circadian clock and sleep studies in zebrafish with special emphasis on the genetic and neuroendocrine mechanisms that regulate rhythms of melatonin secretion, structural synaptic plasticity, locomotor activity and sleep.

Directional cell migration establishes the axes of planar polarity in the posterior lateral-line organ of the zebrafish.

Science.gov (United States)

López-Schier, Hernán; Starr, Catherine J; Kappler, James A; Kollmar, Richard; Hudspeth, A J

2004-09-01

The proper orientation of mechanosensory hair cells along the lateral-line organ of a fish or amphibian is essential for the animal's ability to sense directional water movements. Within the sensory epithelium, hair cells are polarized in a stereotyped manner, but the mechanisms that control their alignment relative to the body axes are unknown. We have found, however, that neuromasts can be oriented either parallel or perpendicular to the anteroposterior body axis. By characterizing the strauss mutant zebrafish line and by tracking labeled cells, we have demonstrated that neuromasts of these two orientations originate from, respectively, the first and second primordia. Furthermore, altering the migratory pathway of a primordium reorients a neuromast's axis of planar polarity. We propose that the global orientation of hair cells relative to the body axes is established through an interaction between directional movement by primordial cells and the timing of neuromast maturation.

Identification and characterization of zebrafish thrombocytes.

Science.gov (United States)

Jagadeeswaran, P; Sheehan, J P; Craig, F E; Troyer, D

1999-12-01

To analyse primary haemostasis in the zebrafish we have identified and characterized the zebrafish thrombocyte by morphologic, immunologic and functional approaches. Novel methods were developed for harvesting zebrafish blood with preservation of thrombocytes, and assaying whole blood adhesion/aggregation responses in microtitre plates. Light and electron microscopy of the thrombocyte illustrated morphological characteristics including the formation of aggregates, pseudopodia, and surface-connected vesicles analagous to the platelet canalicular system. Immunostaining with polyclonal antisera versus human platelet glycoproteins demonstrated the presence of glycoprotein Ib and IIb/IIIa-like complexes on the thrombocyte surface. Whole blood assays for adhesion/aggregation and ATP release showed ristocetin-induced adhesion without ATP release, and platelet agonist (collagen, arachidonic acid) induced aggregation with ATP release. Blood harvested from zebrafish treated with aspirin demonstrated inhibition of arachidonic acid induced aggregation and agonist induced ATP release, consistent with at least partial dependence on an intact cyclo oxygenase pathway. The combined morphologic immunologic and functional evidence suggest that the zebrafish thrombocyte is the haemostatic homologue of the mammalian platelet. Conservation of major haemostatic pathways involved in platelet function and coagulation suggests that the zebrafish is a relevant model for mammalian haemostasis and thrombosis.

Effect of sublethal concentrations of zinc on reproduction in the zebrafish, Brachydanio rerio Hamilton--Buchanan

Energy Technology Data Exchange (ETDEWEB)

Speranza, A.W.; Seeley, R.J.; Seeley, V.A.; Perlmutter, A.

1977-03-01

Adult zebrafish, when held in water containing a threshold concentration (55 ppM) of zinc for a 9-day period in which the gametes were maturing, showed a delay in spawning. When spawning did occur, the experimental pairs of fish produced an average of 165 eggs of which only 21.1 percent were viable. In contrast, control pairs of fish produced an average of 434 eggs of which 90.2 percent were viable. In addition, survival of the eggs to hatching was significantly lower than in controls, the survival rate for the experimentals being 0.9 percent, that of the controls, 63.3 percent. The adverse effects on zebrafish of exposure to zinc can be reversed by returning the fish to a zinc-free environment.

The hazard assessment of nanostructured CeO{sub 2}-based mixed oxides on the zebrafish Danio rerio under environmentally relevant UV-A exposure

Energy Technology Data Exchange (ETDEWEB)

Jemec, Anita, E-mail: anita.jemec@bf.uni-lj.si [National Institute of Chemistry, Laboratory for Environmental Sciences and Engineering, Hajdrihova 19, SI-1001 Ljubljana (Slovenia); University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, SI-1000 Ljubljana (Slovenia); Djinović, Petar; Črnivec, Ilja Gasan Osojnik; Pintar, Albin [National Institute of Chemistry, Laboratory for Environmental Sciences and Engineering, Hajdrihova 19, SI-1001 Ljubljana (Slovenia)

2015-02-15

The effect of nanomaterials on biota under realistic environmental conditions is an important question. However, there is still a lack of knowledge on how different illumination conditions alter the toxicity of some photocatalytic nanomaterials. We have investigated how environmentally relevant UV-A exposure (intensity 8.50 ± 0.61 W/m{sup 2}, exposure dose 9.0 J/cm{sup 2}) affected the toxicity of cerium oxide (CeO{sub 2})-based nanostructured materials to the early-life stages of zebrafish Danio rerio. Pure cerium oxide (CeO{sub 2}), copper–cerium (CuO–CeO{sub 2}) (with a nominal 10, 15 and 20 mol.% CuO content), cerium–zirconium (CeO{sub 2}–ZrO{sub 2}) and nickel and cobalt (Ni–Co) deposited over CeO{sub 2}–ZrO{sub 2} were tested. It was found that under both illumination regimes, none of the tested materials affected the normal development or induced mortality of zebrafish early-life stages up to 100 mg/L. Only in the case of CuO–CeO{sub 2}, the growth of larvae was decreased (96 h LOEC values for CuCe10, CuCe15 and CuCe20 were 50, 50 and 10 mg/L, respectively). To conclude, CeO{sub 2}-based nanostructured materials are not severely toxic to zebrafish and environmentally relevant UV-A exposure does not enhance their toxicity. - Highlights: • CeO{sub 2}–ZrO{sub 2} nanomaterials and pure CeO{sub 2} (up to 100 mg/L) were not harmful to zebrafish. • Only CuO modified CeO{sub 2} affected the growth of zebrafish larvae. • UV-A radiation did not enhance the toxicity of tested nanomaterials.

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

Science.gov (United States)

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

2014-07-01

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

Effects of specific and prolonged expression of zebrafish growth factors, Fgf2 and Lif in primordial germ cells in vivo

Energy Technology Data Exchange (ETDEWEB)

Wong, Ten-Tsao, E-mail: wong20@purdue.edu [Department of Animal Sciences, Purdue University, 901 W. State Street, West Lafayette, IN 47907 (United States); Collodi, Paul [Department of Animal Sciences, Purdue University, 901 W. State Street, West Lafayette, IN 47907 (United States)

2013-01-04

Highlights: Black-Right-Pointing-Pointer We discovered that nanos3 3 Prime UTR prolonged PGC-specific protein expression up to 26 days. Black-Right-Pointing-Pointer Expression of Fgf2 in PGCs significantly increased PGC number at later developmental stages. Black-Right-Pointing-Pointer Expression of Lif in PGCs resulted in a significant disruption of PGC migration. Black-Right-Pointing-Pointer Lif illicited its effect on PGC migration through Lif receptor a. Black-Right-Pointing-Pointer Our approach could be used to achieve prolonged PGC-specific expression of other proteins. -- Abstract: Primordial germ cells (PGCs), specified early in development, proliferate and migrate to the developing gonad before sexual differentiation occurs in the embryo and eventually give rise to spermatogonia or oogonia. In this study, we discovered that nanos3 3 Prime UTR, a common method used to label PGCs, not only directed PGC-specific expression of DsRed but also prolonged this expression up to 26 days post fertilization (dpf) when DsRed-nanos3 3 Prime UTR hybrid mRNAs were introduced into 1- to 2-cell-stage embryos. As such, we employed this knowledge to express zebrafish leukemia inhibitory factor (Lif), basic fibroblast growth factor (Fgf2) and bone morphogenetic protein 4 (Bmp4) in the PGCs and evaluate their effects on PGC development in vivo for over a period of 3 weeks. The results show that expression of Fgf2 significantly increased PGC number at 14- and 21-dpf while Bmp4 resulted in severe ventralization and death of the embryos by 3 days. Expression of Lif resulted in a significant disruption of PGC migration. Mopholino knockdown experiments indicated that Lif illicited its effect on PGC migration through Lif receptor a (Lifra) but not Lifrb. The general approach described in this study could be used to achieve prolonged PGC-specific expression of other proteins to investigate their roles in germ cell and gonad development. The results also indicate that zebrafish PGCs

Culturable gut microbiota diversity in zebrafish.

Science.gov (United States)

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

2012-03-01

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

Human amyloidogenic light chain proteins result in cardiac dysfunction, cell death, and early mortality in zebrafish.

Science.gov (United States)

Mishra, Shikha; Guan, Jian; Plovie, Eva; Seldin, David C; Connors, Lawreen H; Merlini, Giampaolo; Falk, Rodney H; MacRae, Calum A; Liao, Ronglih

2013-07-01

Systemic amyloid light-chain (AL) amyloidosis is associated with rapidly progressive and fatal cardiomyopathy resulting from the direct cardiotoxic effects of circulating AL light chain (AL-LC) proteins and the indirect effects of AL fibril tissue infiltration. Cardiac amyloidosis is resistant to standard heart failure therapies, and, to date, there are limited treatment options for these patients. The mechanisms underlying the development of cardiac amyloidosis and AL-LC cardiotoxicity are largely unknown, and their study has been limited by the lack of a suitable in vivo model system. Here, we establish an in vivo zebrafish model of human AL-LC-induced cardiotoxicity. AL-LC isolated from AL cardiomyopathy patients or control nonamyloidogenic LC protein isolated from multiple myeloma patients (Con-LC) was directly injected into the circulation of zebrafish at 48 h postfertilization. AL-LC injection resulted in impaired cardiac function, pericardial edema, and increased cell death relative to Con-LC, culminating in compromised survival with 100% mortality within 2 wk, independent of AL fibril deposition. Prior work has implicated noncanonical p38 MAPK activation in the pathogenesis of AL-LC-induced cardiotoxicity, and p38 MAPK inhibition via SB-203580 rescued AL-LC-induced cardiac dysfunction and cell death and attenuated mortality in zebrafish. This in vivo zebrafish model of AL-LC cardiotoxicity demonstrates that antagonism of p38 MAPK within the AL-LC cardiotoxic signaling response may serve to improve cardiac function and mortality in AL cardiomyopathy. Furthermore, this in vivo model system will allow for further study of the molecular underpinnings of AL cardiotoxicity and identification of novel therapeutic strategies.

Swimming Effects on Developing Zebrafish

NARCIS (Netherlands)

Kranenbarg, S.; Pelster, B.

2013-01-01

Zebrafish represent an important vertebrate model species in developmental biology. This chapter reviews the effects of exercise on the development of the musculoskeletal system, the cardiovascular system, metabolic capacities of developing zebrafish, and regulation of these processes on the gene

Laser capture microdissection of gonads from juvenile zebrafish

DEFF Research Database (Denmark)

Jørgensen, Anne; Nielsen, John; Morthorst, Jane Ebsen

2009-01-01

was adjusted and optimised to isolate juvenile zebrafish gonads. Results: The juvenile zebrafish gonad is not morphologically distinguishable when using dehydrated cryosections on membrane slides and a specific staining method is necessary to identify the gonads. The protocol setup in this study allows......Background: Investigating gonadal gene expression is important in attempting to elucidate the molecular mechanism of sex determination and differentiation in the model species zebrafish. However, the small size of juvenile zebrafish and correspondingly their gonads complicates this type...... of investigation. Furthermore, the lack of a genetic sex marker in juvenile zebrafish prevents pooling gonads from several individuals. The aim of this study was to establish a method to isolate the gonads from individual juvenile zebrafish allowing future investigations of gonadal gene expression during sex...

The cytochrome P450 2AA gene cluster in zebrafish (Danio rerio): Expression of CYP2AA1 and CYP2AA2 and response to phenobarbital-type inducers

Energy Technology Data Exchange (ETDEWEB)

Kubota, Akira [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Bainy, Afonso C.D. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Departamento de Bioquímica, CCB, Universidade Federal de Santa Catarina, Florianopolis, SC 88040-900 (Brazil); Woodin, Bruce R.; Goldstone, Jared V. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Stegeman, John J., E-mail: jstegeman@whoi.edu [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States)

2013-10-01

The cytochrome P450 (CYP) 2 gene family is the largest and most diverse CYP gene family in vertebrates. In zebrafish, we have identified 10 genes in a new subfamily, CYP2AA, which does not show orthology to any human or other mammalian CYP genes. Here we report evolutionary and structural relationships of the 10 CYP2AA genes and expression of the first two genes, CYP2AA1 and CYP2AA2. Parsimony reconstruction of the tandem duplication pattern for the CYP2AA cluster suggests that CYP2AA1, CYP2AA2 and CYP2AA3 likely arose in the earlier duplication events and thus are most diverged in function from the other CYP2AAs. On the other hand, CYP2AA8 and CYP2AA9 are genes that arose in the latest duplication event, implying functional similarity between these two CYPs. A molecular model of CYP2AA1 showing the sequence conservation across the CYP2AA cluster reveals that the regions with the highest variability within the cluster map onto CYP2AA1 near the substrate access channels, suggesting differing substrate specificities. Zebrafish CYP2AA1 transcript was expressed predominantly in the intestine, while CYP2AA2 was most highly expressed in the kidney, suggesting differing roles in physiology. In the liver CYP2AA2 expression but not that of CYP2AA1, was increased by 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and, to a lesser extent, by phenobarbital (PB). In contrast, pregnenolone 16α-carbonitrile (PCN) increased CYP2AA1 expression, but not CYP2AA2 in the liver. The results identify a CYP2 subfamily in zebrafish that includes genes apparently induced by PB-type chemicals and PXR agonists, the first concrete in vivo evidence for a PB-type response in fish. - Highlights: • A tandemly duplicated cluster of ten CYP2AA genes was described in zebrafish. • Parsimony and duplication analyses suggest pathways to CYP2AA diversity. • Homology models reveal amino acid positions possibly related to functional diversity. • The CYP2AA locus does not share synteny with

The cytochrome P450 2AA gene cluster in zebrafish (Danio rerio): Expression of CYP2AA1 and CYP2AA2 and response to phenobarbital-type inducers

International Nuclear Information System (INIS)

Kubota, Akira; Bainy, Afonso C.D.; Woodin, Bruce R.; Goldstone, Jared V.; Stegeman, John J.

2013-01-01

The cytochrome P450 (CYP) 2 gene family is the largest and most diverse CYP gene family in vertebrates. In zebrafish, we have identified 10 genes in a new subfamily, CYP2AA, which does not show orthology to any human or other mammalian CYP genes. Here we report evolutionary and structural relationships of the 10 CYP2AA genes and expression of the first two genes, CYP2AA1 and CYP2AA2. Parsimony reconstruction of the tandem duplication pattern for the CYP2AA cluster suggests that CYP2AA1, CYP2AA2 and CYP2AA3 likely arose in the earlier duplication events and thus are most diverged in function from the other CYP2AAs. On the other hand, CYP2AA8 and CYP2AA9 are genes that arose in the latest duplication event, implying functional similarity between these two CYPs. A molecular model of CYP2AA1 showing the sequence conservation across the CYP2AA cluster reveals that the regions with the highest variability within the cluster map onto CYP2AA1 near the substrate access channels, suggesting differing substrate specificities. Zebrafish CYP2AA1 transcript was expressed predominantly in the intestine, while CYP2AA2 was most highly expressed in the kidney, suggesting differing roles in physiology. In the liver CYP2AA2 expression but not that of CYP2AA1, was increased by 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and, to a lesser extent, by phenobarbital (PB). In contrast, pregnenolone 16α-carbonitrile (PCN) increased CYP2AA1 expression, but not CYP2AA2 in the liver. The results identify a CYP2 subfamily in zebrafish that includes genes apparently induced by PB-type chemicals and PXR agonists, the first concrete in vivo evidence for a PB-type response in fish. - Highlights: • A tandemly duplicated cluster of ten CYP2AA genes was described in zebrafish. • Parsimony and duplication analyses suggest pathways to CYP2AA diversity. • Homology models reveal amino acid positions possibly related to functional diversity. • The CYP2AA locus does not share synteny with

Heart-specific expression of laminopathic mutations in transgenic zebrafish.

Science.gov (United States)

Verma, Ajay D; Parnaik, Veena K

2017-07-01

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

New tides: using zebrafish to study renal regeneration.

Science.gov (United States)

McCampbell, Kristen K; Wingert, Rebecca A

2014-02-01

Over the past several decades, the zebrafish has become one of the major vertebrate model organisms used in biomedical research. In this arena, the zebrafish has emerged as an applicable system for the study of kidney diseases and renal regeneration. The relevance of the zebrafish model for nephrology research has been increasingly appreciated as the understanding of zebrafish kidney structure, ontogeny, and the response to damage has steadily expanded. Recent studies have documented the amazing regenerative characteristics of the zebrafish kidney, which include the ability to replace epithelial populations after acute injury and to grow new renal functional units, termed nephrons. Here we discuss how nephron composition is conserved between zebrafish and mammals, and highlight how recent findings from zebrafish studies utilizing transgenic technologies and chemical genetics can complement traditional murine approaches in the effort to dissect how the kidney responds to acute damage and identify therapeutics that enhance human renal regeneration. Copyright © 2014 Mosby, Inc. All rights reserved.

Reproductive impairment in the zebrafish, Danio rerio, upon chronic exposure to 1,2,3-trichlorobenzene.

NARCIS (Netherlands)

Roex, E.W.M.; Giovannangelo, M.E.C.A.; van Gestel, C.A.M.

2001-01-01

Most organic pollutants are supposed to act via the mechanism of nonpolar narcosis upon acute exposure. Because the chronic effects of these compounds are still relatively unknown, in this study a chronic toxicity experiment was performed with zebrafish, Danio rerio, exposed to 1, 2,

Graph theoretical model of a sensorimotor connectome in zebrafish.

Science.gov (United States)

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

2012-01-01

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

Sensory hair cell regeneration in the zebrafish lateral line.

Science.gov (United States)

Lush, Mark E; Piotrowski, Tatjana

2014-10-01

Damage or destruction of sensory hair cells in the inner ear leads to hearing or balance deficits that can be debilitating, especially in older adults. Unfortunately, the damage is permanent, as regeneration of the inner ear sensory epithelia does not occur in mammals. Zebrafish and other non-mammalian vertebrates have the remarkable ability to regenerate sensory hair cells and understanding the molecular and cellular basis for this regenerative ability will hopefully aid us in designing therapies to induce regeneration in mammals. Zebrafish not only possess hair cells in the ear but also in the sensory lateral line system. Hair cells in both organs are functionally analogous to hair cells in the inner ear of mammals. The lateral line is a mechanosensory system found in most aquatic vertebrates that detects water motion and aids in predator avoidance, prey capture, schooling, and mating. Although hair cell regeneration occurs in both the ear and lateral line, most research to date has focused on the lateral line due to its relatively simple structure and accessibility. Here we review the recent discoveries made during the characterization of hair cell regeneration in zebrafish. Copyright © 2014 Wiley Periodicals, Inc.

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

Science.gov (United States)

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.

The importance of Zebrafish in biomedical research.

Science.gov (United States)

Tavares, Bárbara; Santos Lopes, Susana

2013-01-01

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

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

Science.gov (United States)

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

2017-11-01

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

Cyp1a reporter zebrafish reveals target tissues for dioxin

Energy Technology Data Exchange (ETDEWEB)

Kim, Kun-Hee [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Park, Hye-Jeong [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); Kim, Jin Hee [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Kim, Suhyun [Graduate School of Medicine, Korea University, Ansan (Korea, Republic of); Williams, Darren R. [New Drug Targets Laboratory, School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju (Korea, Republic of); Kim, Myeong-Kyu [Department of Neurology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Jung, Young Do [Department of Biochemistry, Chonnam National University Medical School, Gwangju (Korea, Republic of); Teraoka, Hiroki [School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu (Japan); Park, Hae-Chul [Graduate School of Medicine, Korea University, Ansan (Korea, Republic of); Choy, Hyon E., E-mail: hyonchoy@chonnam.ac.kr [Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Shin, Boo Ahn, E-mail: bashin@chonnam.ac.kr [Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Choi, Seok-Yong, E-mail: zebrafish@chonnam.ac.kr [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); School of Biological Sciences and Technology, Chonnam National University, Gwangju (Korea, Republic of)

2013-06-15

Highlights: •2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic anthropogenic substance ever identified. •Transgenic cyp1a reporter zebrafish reveals target tissues for TCDD. •The retinal bipolar cells, otic vesicle, lateral line, pancreas, cloaca and pectoral fin bud are novel targets in zebrafish for TCDD. •Our findings will further understanding of human health risks by TCDD. -- Abstract: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the unintentional byproduct of various industrial processes, is classified as human carcinogen and could disrupt reproductive, developmental and endocrine systems. Induction of cyp1a1 is used as an indicator of TCDD exposure. We sought to determine tissues that are vulnerable to TCDD toxicity using a transgenic zebrafish (Danio rerio) model. We inserted a nuclear enhanced green fluorescent protein gene (EGFP) into the start codon of a zebrafish cyp1a gene in a fosmid clone using DNA recombineering. The resulting recombineered fosmid was then used to generate cyp1a reporter zebrafish, embryos of which were exposed to TCDD. Expression pattern of EGFP in the reporter zebrafish mirrored that of endogenous cyp1a mRNA. In addition, exposure of the embryos to TCDD at as low as 10 pM for 72 h, which does not elicit morphological abnormalities of embryos, markedly increased GFP expression. Furthermore, the reporter embryos responded to other AhR ligands as well. Exposure of the embryos to TCDD revealed previously reported (the cardiovascular system, liver, pancreas, kidney, swim bladder and skin) and unreported target tissues (retinal bipolar cells, otic vesicle, lateral line, cloaca and pectoral fin bud) for TCDD. Transgenic cyp1a reporter zebrafish we have developed can further understanding of ecotoxicological relevance and human health risks by TCDD. In addition, they could be used to identify agonists of AhR and antidotes to TCDD toxicity.

The primary role of zebrafish nanog is in extra-embryonic tissue.

Science.gov (United States)

Gagnon, James A; Obbad, Kamal; Schier, Alexander F

2018-01-09

The role of the zebrafish transcription factor Nanog has been controversial. It has been suggested that Nanog is primarily required for the proper formation of the extra-embryonic yolk syncytial layer (YSL) and only indirectly regulates gene expression in embryonic cells. In an alternative scenario, Nanog has been proposed to directly regulate transcription in embryonic cells during zygotic genome activation. To clarify the roles of Nanog, we performed a detailed analysis of zebrafish nanog mutants. Whereas zygotic nanog mutants survive to adulthood, maternal-zygotic (MZ nanog ) and maternal mutants exhibit developmental arrest at the blastula stage. In the absence of Nanog, YSL formation and epiboly are abnormal, embryonic tissue detaches from the yolk, and the expression of dozens of YSL and embryonic genes is reduced. Epiboly defects can be rescued by generating chimeric embryos of MZ nanog embryonic tissue with wild-type vegetal tissue that includes the YSL and yolk cell. Notably, cells lacking Nanog readily respond to Nodal signals and when transplanted into wild-type hosts proliferate and contribute to embryonic tissues and adult organs from all germ layers. These results indicate that zebrafish Nanog is necessary for proper YSL development but is not directly required for embryonic cell differentiation. © 2018. Published by The Company of Biologists Ltd.

Zebrafish neurobehavioral phenomics for aquatic neuropharmacology and toxicology research.

Science.gov (United States)

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

2016-01-01

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

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

Science.gov (United States)

McGinnis, Courtney L; Crivello, Joseph F

2011-05-01

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

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

Science.gov (United States)

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

2016-10-07

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

Episodic-like memory in zebrafish.

Science.gov (United States)

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

2016-11-01

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

Implications of exposure to dextran-coated and uncoated iron oxide nanoparticles to developmental toxicity in zebrafish

Science.gov (United States)

de Oliveira, Giovanna Medeiros Tavares; de Oliveira, Elisa Magno Nunes; Pereira, Talita Carneiro Brandão; Papaléo, Ricardo Meurer; Bogo, Maurício Reis

2017-12-01

Iron oxide nanoparticles (IONPS) have been widely investigated as a platform for a new class of multifunctional theranostic agents. They are considered biocompatible, and some formulations are already available in the market for clinical use. However, contradictory results regarding toxicity of IONPs raise a concern about the potential harm of these nanoparticles. Changes in the nanoparticle (NP) physicochemical properties or exposure media can significantly alter their behavior and, as a consequence, their toxic effects. Here, behavior and two-step RT-qPCR were employed to access the potential toxicological effects of dextran-coated IONPs (CLIO-NH2) and uncoated IONPs (UCIO) in zebrafish larvae. Animals were exposed for 7 days to NP solutions ranging from 0.1-100 μg/mL directly mixed to the system water. UCIO showed high decantation and instability in solution, altering zebrafish mortality but showing no alterations in behavior and molecular expression analysis. CLIO-NH2 exposure did not cause significant mortality or changes in hatching rate of zebrafish larvae; however, behavior and expression profiles of the group exposed to lower concentration (1 μg/mL) presented a tendency to decrease the locomotor activity and apoptotic pathway activation.

Circadian clocks, rhythmic synaptic plasticity and the sleep-wake cycle in zebrafish

Directory of Open Access Journals (Sweden)

Idan eElbaz

2013-02-01

Full Text Available The circadian clock and homeostatic processes are fundamental mechanisms that regulate sleep. Surprisingly, despite decades of research, we still do not know why we sleep. Intriguing hypotheses suggest that sleep regulates synaptic plasticity and consequently has a beneficial role in learning and memory. However, direct evidence is still limited and the molecular regulatory mechanisms remain unclear. The zebrafish provides a powerful vertebrate model system that enables simple genetic manipulation, imaging of neuronal circuits and synapses in living animals, and the monitoring of behavioral performance during day and night. Thus, the zebrafish has become an attractive model to study circadian and homeostatic processes that regulate sleep. Zebrafish clock- and sleep-related genes have been cloned, neuronal circuits that exhibit circadian rhythms of activity and synaptic plasticity have been studied, and rhythmic behavioral outputs have been characterized. Integration of this data could lead to a better understanding of sleep regulation. Here, we review the progress of circadian clock and sleep studies in zebrafish with special emphasis on the genetic and neuroendocrine mechanisms that regulate rhythms of melatonin secretion, structural synaptic plasticity, locomotor activity and sleep.

Graph theoretical model of a sensorimotor connectome in zebrafish.

Directory of Open Access Journals (Sweden)

Michael Stobb

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

Defective glycinergic synaptic transmission in zebrafish motility mutants

Directory of Open Access Journals (Sweden)

Hiromi Hirata

2010-01-01

Full Text Available Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR β subunit genes. These mutants exhibit a loss of glycinergic synaptic transmission due to a lack of synaptic aggregation of GlyRs. Due to the consequent loss of reciprocal inhibition of motor circuits between the two sides of the spinal cord, motor neurons activate simultaneously on both sides resulting in bilateral contraction of axial muscles of beo mutants, eliciting the so-called ‘accordion’ phenotype. Similar defects in GlyR subunit genes have been observed in several mammals and are the basis for human hyperekplexia/startle disease. By contrast, zebrafish shocked (sho mutants have a defect in slc6a9, encoding GlyT1, a glycine transporter that is expressed by astroglial cells surrounding the glycinergic synapse in the hindbrain and spinal cord. GlyT1 mediates rapid uptake of glycine from the synaptic cleft, terminating synaptic transmission. In zebrafish sho mutants, there appears to be elevated extracellular glycine resulting in persistent inhibition of postsynaptic neurons and subsequent reduced motility, causing the ‘twitch once’ phenotype. We review current knowledge regarding zebrafish ‘accordion’ and ‘twitch once’ mutants, including beo and sho, and report the identification of a new α2 subunit that revises the phylogeny of zebrafish GlyRs.

Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

Science.gov (United States)

Hirata, Hiromi; Carta, Eloisa; Yamanaka, Iori; Harvey, Robert J.; Kuwada, John Y.

2009-01-01

Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo) mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR) β subunit genes. These mutants exhibit a loss of glycinergic synaptic transmission due to a lack of synaptic aggregation of GlyRs. Due to the consequent loss of reciprocal inhibition of motor circuits between the two sides of the spinal cord, motor neurons activate simultaneously on both sides resulting in bilateral contraction of axial muscles of beo mutants, eliciting the so-called ‘accordion’ phenotype. Similar defects in GlyR subunit genes have been observed in several mammals and are the basis for human hyperekplexia/startle disease. By contrast, zebrafish shocked (sho) mutants have a defect in slc6a9, encoding GlyT1, a glycine transporter that is expressed by astroglial cells surrounding the glycinergic synapse in the hindbrain and spinal cord. GlyT1 mediates rapid uptake of glycine from the synaptic cleft, terminating synaptic transmission. In zebrafish sho mutants, there appears to be elevated extracellular glycine resulting in persistent inhibition of postsynaptic neurons and subsequent reduced motility, causing the ‘twitch-once’ phenotype. We review current knowledge regarding zebrafish ‘accordion’ and ‘twitch-once’ mutants, including beo and sho, and report the identification of a new α2 subunit that revises the phylogeny of zebrafish GlyRs. PMID:20161699

Fog1 is required for cardiac looping in zebrafish

OpenAIRE

Walton, R. Zaak; Bruce, Ashley E.E.; Olivey, Harold E.; Najib, Khalid; Johnson, Vanitha; Earley, Judy U.; Ho, Robert K.; Svensson, Eric C.

2006-01-01

To further our understanding of FOG gene function during cardiac development, we utilized zebrafish to examine FOG’s role in the early steps of heart morphogenesis. We identified fragments of three fog genes in the zebrafish genomic database and isolated full-length coding sequences for each of these genes by using a combination of RT-PCR and 5′-RACE. One gene was similar to murine FOG-1 (fog1), while the remaining two were similar to murine FOG-2 (fog2a and fog2b). All Fog proteins were able...

The role of Nrf1 and Nrf2 in the regulation of glutathione and redox dynamics in the developing zebrafish embryo

Directory of Open Access Journals (Sweden)

Karilyn E. Sant

2017-10-01

Full Text Available Redox signaling is important for embryogenesis, guiding pathways that govern processes crucial for embryo patterning, including cell polarization, proliferation, and apoptosis. Exposure to pro-oxidants during this period can be deleterious, resulting in altered physiology, teratogenesis, later-life diseases, or lethality. We previously reported that the glutathione antioxidant defense system becomes increasingly robust, including a doubling of total glutathione and dynamic shifts in the glutathione redox potential at specific stages during embryonic development in the zebrafish, Danio rerio. However, the mechanisms underlying these changes are unclear, as is the effectiveness of the glutathione system in ameliorating oxidative insults to the embryo at different stages. Here, we examine how the glutathione system responds to the model pro-oxidants tert-butylhydroperoxide and tert-butylhydroquinone at different developmental stages, and the role of Nuclear factor erythroid 2-related factor (Nrf proteins in regulating developmental glutathione redox status. Embryos became increasingly sensitive to pro-oxidants after 72 h post-fertilization (hpf, after which the duration of the recovery period for the glutathione redox potential was increased. To determine whether the doubling of glutathione or the dynamic changes in glutathione redox potential are mediated by zebrafish paralogs of Nrf transcription factors, morpholino oligonucleotides were used to knock down translation of Nrf1 and Nrf2 (nrf1a, nrf1b, nrf2a, nrf2b. Knockdown of Nrf1a or Nrf1b perturbed glutathione redox state until 72 hpf. Knockdown of Nrf2 paralogs also perturbed glutathione redox state but did not significantly affect the response of glutathione to pro-oxidants. Nrf1b morphants had decreased gene expression of glutathione synthesis enzymes, while hsp70 increased in Nrf2b morphants. This work demonstrates that despite having a more robust glutathione system, embryos become more

The Zebrafish Model Organism Database (ZFIN)

Data.gov (United States)

U.S. Department of Health & Human Services — ZFIN serves as the zebrafish model organism database. It aims to: a) be the community database resource for the laboratory use of zebrafish, b) develop and support...

Developmental nephrotoxicity of aristolochic acid in a zebrafish model

International Nuclear Information System (INIS)

Ding, Yu-Ju; Chen, Yau-Hung

2012-01-01

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

Developmental nephrotoxicity of aristolochic acid in a zebrafish model

Energy Technology Data Exchange (ETDEWEB)

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

2012-05-15

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

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

Energy Technology Data Exchange (ETDEWEB)

Bourassa, Daisy; Gleber, Sophie-Charlotte; Vogt, Stefan; Shin, Chong Hyun; Fahrni, Christoph J.

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 as 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.

Involvement of purinergic system in inflammation and toxicity induced by copper in zebrafish larvae

Energy Technology Data Exchange (ETDEWEB)

Leite, Carlos Eduardo, E-mail: carlos.leite@pucrs.br [Instituto de Toxicologia e Farmacologia, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, CEP 90619-900 (Brazil); Programa de Pós-Graduação em Medicina: Ciências Médicas, Universidade Federal do Rio Grande do Sul, Porto Alegre, CEP 90035-003 (Brazil); Maboni, Lucas de Oliveira [Instituto de Toxicologia e Farmacologia, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, CEP 90619-900 (Brazil); Faculdade de Biociências, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, CEP 90619-900 (Brazil); Cruz, Fernanda Fernandes [Instituto de Toxicologia e Farmacologia, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, CEP 90619-900 (Brazil); Faculdade de Farmácia, Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, CEP 90619-900 (Brazil); Rosemberg, Denis Broock [Programa de Pós-graduação em Ciências Ambientais, Universidade Comunitária da Região de Chapecó, Chapecó, CEP 89809-000 (Brazil); and others

2013-11-01

The use of zebrafish (Danio rerio) is increasing as an intermediate preclinical model, to prioritize drug candidates for mammalian testing. As the immune system of the zebrafish is quite similar to that of mammals, models of inflammation are being developed for the screening of new drugs. The characterization of these models is crucial for studies that seek for mechanisms of action and specific pharmacological targets. It is well known that copper is a metal that induces damage and cell migration to hair cells of lateral line of zebrafish. Extracellular nucleotides/nucleosides, as ATP and adenosine (ADO), act as endogenous signaling molecules during tissue damage by exerting effects on inflammatory and immune responses. The present study aimed to characterize the inflammatory status, and to investigate the involvement of the purinergic system in copper-induced inflammation in zebrafish larvae. Fishes of 7 days post-fertilization were exposed to 10 μM of copper for a period of 24 h. The grade of oxidative stress, inflammatory status, copper uptake, the activity and the gene expression of the enzymes responsible for controlling the levels of nucleotides and adenosine were evaluated. Due to the copper accumulation in zebrafish larvae tissues, the damage and oxidative stress were exacerbated over time, resulting in an inflammatory process involving IL-1β, TNF-α, COX-2 and PGE{sub 2}. Within the purinergic system, the mechanisms that control the ADO levels were the most involved, mainly the reactions performed by the isoenzyme ADA 2. In conclusion, our data shed new lights on the mechanisms related to copper-induced inflammation in zebrafish larvae. - Graphical abstract: This scheme provides a chronological proposition for the biochemical events induced by copper in zebrafish larvae. The dashed line shows the absorption of copper over the exposure time. After 1 h of exposure to copper, the release of PGE{sub 2} occurs, followed by an increase of MPO (as a consequence

Involvement of purinergic system in inflammation and toxicity induced by copper in zebrafish larvae

International Nuclear Information System (INIS)

Leite, Carlos Eduardo; Maboni, Lucas de Oliveira; Cruz, Fernanda Fernandes; Rosemberg, Denis Broock

2013-01-01

The use of zebrafish (Danio rerio) is increasing as an intermediate preclinical model, to prioritize drug candidates for mammalian testing. As the immune system of the zebrafish is quite similar to that of mammals, models of inflammation are being developed for the screening of new drugs. The characterization of these models is crucial for studies that seek for mechanisms of action and specific pharmacological targets. It is well known that copper is a metal that induces damage and cell migration to hair cells of lateral line of zebrafish. Extracellular nucleotides/nucleosides, as ATP and adenosine (ADO), act as endogenous signaling molecules during tissue damage by exerting effects on inflammatory and immune responses. The present study aimed to characterize the inflammatory status, and to investigate the involvement of the purinergic system in copper-induced inflammation in zebrafish larvae. Fishes of 7 days post-fertilization were exposed to 10 μM of copper for a period of 24 h. The grade of oxidative stress, inflammatory status, copper uptake, the activity and the gene expression of the enzymes responsible for controlling the levels of nucleotides and adenosine were evaluated. Due to the copper accumulation in zebrafish larvae tissues, the damage and oxidative stress were exacerbated over time, resulting in an inflammatory process involving IL-1β, TNF-α, COX-2 and PGE 2 . Within the purinergic system, the mechanisms that control the ADO levels were the most involved, mainly the reactions performed by the isoenzyme ADA 2. In conclusion, our data shed new lights on the mechanisms related to copper-induced inflammation in zebrafish larvae. - Graphical abstract: This scheme provides a chronological proposition for the biochemical events induced by copper in zebrafish larvae. The dashed line shows the absorption of copper over the exposure time. After 1 h of exposure to copper, the release of PGE 2 occurs, followed by an increase of MPO (as a consequence of

Large-scale assessment of the zebrafish embryo as a possible predictive model in toxicity testing.

Directory of Open Access Journals (Sweden)

Shaukat Ali

Full Text Available BACKGROUND: In the drug discovery pipeline, safety pharmacology is a major issue. The zebrafish has been proposed as a model that can bridge the gap in this field between cell assays (which are cost-effective, but low in data content and rodent assays (which are high in data content, but less cost-efficient. However, zebrafish assays are only likely to be useful if they can be shown to have high predictive power. We examined this issue by assaying 60 water-soluble compounds representing a range of chemical classes and toxicological mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: Over 20,000 wild-type zebrafish embryos (including controls were cultured individually in defined buffer in 96-well plates. Embryos were exposed for a 96 hour period starting at 24 hours post fertilization. A logarithmic concentration series was used for range-finding, followed by a narrower geometric series for LC(50 determination. Zebrafish embryo LC(50 (log mmol/L, and published data on rodent LD(50 (log mmol/kg, were found to be strongly correlated (using Kendall's rank correlation tau and Pearson's product-moment correlation. The slope of the regression line for the full set of compounds was 0.73403. However, we found that the slope was strongly influenced by compound class. Thus, while most compounds had a similar toxicity level in both species, some compounds were markedly more toxic in zebrafish than in rodents, or vice versa. CONCLUSIONS: For the substances examined here, in aggregate, the zebrafish embryo model has good predictivity for toxicity in rodents. However, the correlation between zebrafish and rodent toxicity varies considerably between individual compounds and compound class. We discuss the strengths and limitations of the zebrafish model in light of these findings.

Zebrafish hoxd4a acts upstream of meis1.1 to direct vasculogenesis, angiogenesis and hematopoiesis.

Directory of Open Access Journals (Sweden)

Aseervatham Anusha Amali

Full Text Available Mice lacking the 4th-group paralog Hoxd4 display malformations of the anterior vertebral column, but are viable and fertile. Here, we report that zebrafish embryos having decreased function of the orthologous hoxd4a gene manifest striking perturbations in vasculogenesis, angiogenesis and primitive and definitive hematopoiesis. These defects are preceded by reduced expression of the hemangioblast markers scl1, lmo2 and fli1 within the posterior lateral plate mesoderm (PLM at 13 hours post fertilization (hpf. Epistasis analysis revealed that hoxd4a acts upstream of meis1.1 but downstream of cdx4 as early as the shield stage in ventral-most mesoderm fated to give rise to hemangioblasts, leading us to propose that loss of hoxd4a function disrupts hemangioblast specification. These findings place hoxd4a high in a genetic hierarchy directing hemangioblast formation downstream of cdx1/cdx4 and upstream of meis1.1. An additional consequence of impaired hoxd4a and meis1.1 expression is the deregulation of multiple Hox genes implicated in vasculogenesis and hematopoiesis which may further contribute to the defects described here. Our results add to evidence implicating key roles for Hox genes in their initial phase of expression early in gastrulation.

A novel zinc finger protein 219-like (ZNF219L) is involved in the regulation of collagen type 2 alpha 1a (col2a1a) gene expression in zebrafish notochord.

Science.gov (United States)

Lien, Huang-Wei; Yang, Chung-Hsiang; Cheng, Chia-Hsiung; Hung, Chin-Chun; Liao, Wei-Hao; Hwang, Pung-Pung; Han, Yu-San; Huang, Chang-Jen

2013-01-01

The notochord is required for body plan patterning in vertebrates, and defects in notochord development during embryogenesis can lead to diseases affecting the adult. It is therefore important to elucidate the gene regulatory mechanism underlying notochord formation. In this study, we cloned the zebrafish zinc finger 219-like (ZNF219L) based on mammalian ZNF219, which contains nine C2H2-type zinc finger domains. Through whole-mount in situ hybridization, we found that znf219L mRNA is mainly expressed in the zebrafish midbrain-hindbrain boundary, hindbrain, and notochord during development. The znf219L morpholino knockdown caused partial abnormal notochord phenotype and reduced expression of endogenous col2a1a in the notochord specifically. In addition, ZNF219L could recognize binding sites with GGGGG motifs and trigger augmented activity of the col2a1a promoter in a luciferase assay. Furthermore, in vitro binding experiments revealed that ZNF219L recognizes the GGGGG motifs in the promoter region of the zebrafish col2a1a gene through its sixth and ninth zinc finger domains. Taken together, our results reveal that ZNF219L is involved in regulating the expression of col2a1a in zebrafish notochord specifically.

Egfl6 is involved in zebrafish notochord development.

Science.gov (United States)

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.

Macrophage–Microbe Interactions: Lessons from the Zebrafish Model

Directory of Open Access Journals (Sweden)

Nagisa Yoshida

2017-12-01

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

Mechanisms of toxicity of di(2-ethylhexyl) phthalate on the reproductive health of male zebrafish

Energy Technology Data Exchange (ETDEWEB)

Uren-Webster, Tamsyn M.; Lewis, Ceri; Filby, Amy L.; Paull, Gregory C. [Hatherly Laboratories, School of Biosciences, University of Exeter, Prince of Wales Road, Exeter, Devon EX4 4PS (United Kingdom); Santos, Eduarda M., E-mail: e.santos@exeter.ac.uk [Hatherly Laboratories, School of Biosciences, University of Exeter, Prince of Wales Road, Exeter, Devon EX4 4PS (United Kingdom)

2010-09-01

Phthalates are ubiquitous in the aquatic environment and are known to adversely affect male reproductive health in mammals through interactions with multiple receptor systems. However, little is known about the risks they pose to fish. This project investigated the effects of di(2-ethylhexyl) phthalate (DEHP), the most commonly used phthalate, on the reproductive health of male zebrafish (Danio rerio). Males were treated with 0.5, 50 and 5000 mg DEHP kg{sup -1} (body weight) for a period of 10 days via intraperitoneal injection. The effects of the exposure were assessed by analysing fertilisation success, testis histology, sperm DNA integrity and transcript profiles of the liver and testis. A significant increase in the hepatosomatic index and levels of hepatic vitellogenin transcript were observed following exposure to 5000 mg DEHP kg{sup -1}. Exposure to 5000 mg DEHP kg{sup -1} also resulted in a reduction in fertilisation success of oocytes spawned by untreated females. However, survival and development of the resulting embryos were unaffected by all treatments, and no evidence of DEHP-induced sperm DNA damage was observed. Exposure to 50 and 5000 mg DEHP kg{sup -1} caused alterations in the proportion of germ cells at specific stages of spermatogenesis in the testis, including a reduction in the proportion of spermatozoa and an increase in the proportion of spermatocytes, suggesting that DEHP may inhibit the progression of meiosis. In parallel, exposure to 5000 mg DEHP kg{sup -1} increased the levels of two peroxisome proliferator-activated receptor (PPAR) responsive genes (acyl-coenzyme A oxidase 1 (acox1) and enoyl-coenzyme A, hydratase/3-hydroxyacyl coenzyme A dehydrogenase (ehhadh). These data demonstrated that exposure to high concentrations of DEHP disrupts spermatogenesis in adult zebrafish with a consequent decrease in their ability to fertilise oocytes spawned by untreated females. Furthermore, our data suggest that the adverse effects caused by

Two zebrafish G2A homologs activate multiple intracellular signaling pathways in acidic environment

Energy Technology Data Exchange (ETDEWEB)

Ichijo, Yuta; Mochimaru, Yuta [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Azuma, Morio [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190-Gofuku, Toyama 930-8555 (Japan); Satou, Kazuhiro; Negishi, Jun [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Nakakura, Takashi [Department of Anatomy, Graduate School of Medicine, Teikyo University, 2-11-1 Itabashi-Ku, Tokyo 173-8605 (Japan); Oshima, Natsuki [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Mogi, Chihiro; Sato, Koichi [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Matsuda, Kouhei [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190-Gofuku, Toyama 930-8555 (Japan); Okajima, Fumikazu [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Tomura, Hideaki, E-mail: tomurah@meiji.ac.jp [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan)

2016-01-01

Human G2A is activated by various stimuli such as lysophosphatidylcholine (LPC), 9-hydroxyoctadecadienoic acid (9-HODE), and protons. The receptor is coupled to multiple intracellular signaling pathways, including the G{sub s}-protein/cAMP/CRE, G{sub 12/13}-protein/Rho/SRE, and G{sub q}-protein/phospholipase C/NFAT pathways. In the present study, we examined whether zebrafish G2A homologs (zG2A-a and zG2A-b) could respond to these stimuli and activate multiple intracellular signaling pathways. We also examined whether histidine residue and basic amino acid residue in the N-terminus of the homologs also play roles similar to those played by human G2A residues if the homologs sense protons. We found that the zG2A-a showed the high CRE, SRE, and NFAT activities, however, zG2A-b showed only the high SRE activity under a pH of 8.0. Extracellular acidification from pH 7.4 to 6.3 ameliorated these activities in zG2A-a-expressing cells. On the other hand, acidification ameliorated the SRE activity but not the CRE and NFAT activities in zG2A-b-expressing cells. LPC or 9-HODE did not modify any activity of either homolog. The substitution of histidine residue at the 174{sup th} position from the N-terminus of zG2A-a to asparagine residue attenuated proton-induced CRE and NFAT activities but not SRE activity. The substitution of arginine residue at the 32nd position from the N-terminus of zG2A-a to the alanine residue also attenuated its high and the proton-induced CRE and NFAT activities. On the contrary, the substitution did not attenuate SRE activity. The substitution of the arginine residue at the 10th position from the N-terminus of zG2A-b to the alanine residue also did not attenuate its high or the proton-induced SRE activity. These results indicate that zebrafish G2A homologs were activated by protons but not by LPC and 9-HODE, and the activation mechanisms of the homologs were similar to those of human G2A. - Highlights: • Zebrafish two G2A homologs are proton

FishNet: an online database of zebrafish anatomy

Directory of Open Access Journals (Sweden)

Gibson Abigail J

2007-08-01

Full Text Available Abstract Background Over the last two decades, zebrafish have been established as a genetically versatile model system for investigating many different aspects of vertebrate developmental biology. With the credentials of zebrafish as a developmental model now well recognized, the emerging new opportunity is the wider application of zebrafish biology to aspects of human disease modelling. This rapidly increasing use of zebrafish as a model for human disease has necessarily generated interest in the anatomy of later developmental phases such as the larval, juvenile, and adult stages, during which many of the key aspects of organ morphogenesis and maturation take place. Anatomical resources and references that encompass these stages are non-existent in zebrafish and there is therefore an urgent need to understand how different organ systems and anatomical structures develop throughout the life of the fish. Results To overcome this deficit we have utilized the technique of optical projection tomography to produce three-dimensional (3D models of larval fish. In order to view and display these models we have created FishNet http://www.fishnet.org.au, an interactive reference of zebrafish anatomy spanning the range of zebrafish development from 24 h until adulthood. Conclusion FishNet contains more than 36 000 images of larval zebrafish, with more than 1 500 of these being annotated. The 3D models can be manipulated on screen or virtually sectioned. This resource represents the first complete embryo to adult atlas for any species in 3D.

Assessment of locomotion behavior in adult Zebrafish after acute exposure to different pharmacological reference compounds

Directory of Open Access Journals (Sweden)

Pankaj Gupta

2014-01-01

Full Text Available Objectives: The objective of the present study was to assess locomotor behavior of adult zebrafish after acute exposure to different pharmacological reference compounds. Materials and Methods: Adult zebrafish of 4-5-months-old were exposed to different concentrations of known reference compounds for 15 min. The test was conducted separately for each drug concentration as well as control. Locomotor activity parameters viz. distance travelled, speed, total mobile time, and total immobile time were recorded for each animal during the exposure period. Results: Out of 11 compounds tested, nine compounds showed decrease in locomotor behavior with significant changes in distance travelled, speed, total mobile time, and total immobile time. Caffeine exhibited biphasic response in locomotion behavior, while scopolamine failed to induce any significant changes. Conclusion: In view of the above findings, these results suggested that exposure of adult zebrafish with different known compounds produce the expected changes in the locomotion behavior; therefore, adult zebrafish can be used an alternative approach for the assessment of new chemical entities for their effect on locomotor behavior.

Zebrafish: A Versatile Animal Model for Fertility Research

Directory of Open Access Journals (Sweden)

Jing Ying Hoo

2016-01-01

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

Mutations in zebrafish pitx2 model congenital malformations in Axenfeld-Rieger syndrome but do not disrupt left-right placement of visceral organs.

Science.gov (United States)

Ji, Yongchang; Buel, Sharleen M; Amack, Jeffrey D

2016-08-01

Pitx2 is a conserved homeodomain transcription factor that has multiple functions during embryonic development. Mutations in human PITX2 cause autosomal dominant Axenfeld-Rieger syndrome (ARS), characterized by congenital eye and tooth malformations. Pitx2(-/-) knockout mouse models recapitulate aspects of ARS, but are embryonic lethal. To date, ARS treatments remain limited to managing individual symptoms due to an incomplete understanding of PITX2 function. In addition to regulating eye and tooth development, Pitx2 is a target of a conserved Nodal (TGFβ) signaling pathway that mediates left-right (LR) asymmetry of visceral organs. Based on its highly conserved asymmetric expression domain, the Nodal-Pitx2 axis has long been considered a common denominator of LR development in vertebrate embryos. However, functions of Pitx2 during asymmetric organ morphogenesis are not well understood. To gain new insight into Pitx2 function we used genome editing to create mutations in the zebrafish pitx2 gene. Mutations in the pitx2 homeodomain caused phenotypes reminiscent of ARS, including aberrant development of the cornea and anterior chamber of the eye and reduced or absent teeth. Intriguingly, LR asymmetric looping of the heart and gut was normal in pitx2 mutants. These results suggest conserved roles for Pitx2 in eye and tooth development and indicate Pitx2 is not required for asymmetric looping of zebrafish visceral organs. This work establishes zebrafish pitx2 mutants as a new animal model for investigating mechanisms underlying congenital malformations in ARS and high-throughput drug screening for ARS therapeutics. Additionally, pitx2 mutants present a unique opportunity to identify new genes involved in vertebrate LR patterning. We show Nodal signaling-independent of Pitx2-controls asymmetric expression of the fatty acid elongase elovl6 in zebrafish, pointing to a potential novel pathway during LR organogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

Quo natas, Danio?—Recent Progress in Modeling Cancer in Zebrafish

Directory of Open Access Journals (Sweden)

Stefanie Kirchberger

2017-08-01

Full Text Available Over the last decade, zebrafish has proven to be a powerful model in cancer research. Zebrafish form tumors that histologically and genetically resemble human cancers. The live imaging and cost-effective compound screening possible with zebrafish especially complement classic mouse cancer models. Here, we report recent progress in the field, including genetically engineered zebrafish cancer models, xenotransplantation of human cancer cells into zebrafish, promising approaches toward live investigation of the tumor microenvironment, and identification of therapeutic strategies by performing compound screens on zebrafish cancer models. Given the recent advances in genome editing, personalized zebrafish cancer models are now a realistic possibility. In addition, ongoing automation will soon allow high-throughput compound screening using zebrafish cancer models to be part of preclinical precision medicine approaches.

Biotransformation in the zebrafish embryo -temporal gene transcription changes of cytochrome P450 enzymes and internal exposure dynamics of the AhR binding xenobiotic benz[a]anthracene.

Science.gov (United States)

Kühnert, Agnes; Vogs, Carolina; Seiwert, Bettina; Aulhorn, Silke; Altenburger, Rolf; Hollert, Henner; Küster, Eberhard; Busch, Wibke

2017-11-01

Not much is known about the biotransformation capability of zebrafish (Danio rerio) embryos. For understanding possible toxicity differences to adult fish, it might be crucial to understand the biotransformation of chemicals in zebrafish embryos i.e. as part of toxicokinetics. The biotransformation capabilities were analysed for two different stages of zebrafish embryos in conjunction with the internal concentrations of a xenobiotic. Zebrafish embryos of the late cleavage/early blastula period (2-26 hpf) and the early pharyngula period (26-50 hpf) were exposed for 24 h to the AhR binding compound benz[a]anthracene (BaA). Time dependent changes in cyp transcription (cyp1a, cyp1b1, cyp1c1 and cyp1c2) as well as concentration & time-dependent courses of BaA in the fish embryo and the exposure medium were analysed. Additionally, the CYP mediated formation of biotransformation products was investigated. We found correlations between transcriptional responses and the internal concentration for both exposure types. These correlations were depending on the start of the exposure i.e. the age of the exposed embryo. While no significant induction of the examined gene transcripts was observed in the first 12 h of exposure beginning in the blastula period a correlation was apparent when exposure started later i.e. in the pharyngula period. A significant induction of cyp1a was detected already after 1.5 h of BaA exposure. Gene transcripts for cyp1b1, cyp1c1 and cyp1c2 showed expressions distinctly different from cyp1a and were, in general, less inducible by BaA in both exposure windows. The toxicokinetic analysis showed that the biotransformation capability was fivefold higher in the older fish embryos. Biotransformation products of phase I reactions were found between 32 hpf and 50 hpf and were tentatively identified as benz[a]anthracene-phenol and benz[a]anthracene-dihydrodiol-epoxide. In conclusion, not only duration but also onset of exposure in relation to the

Arsenic transport by zebrafish aquaglyceroporins

Directory of Open Access Journals (Sweden)

Landfear Scott M

2009-11-01

Full Text Available Abstract Background Arsenic is one of the most ubiquitous toxins and endangers the health of tens of millions of humans worldwide. It is a mainly a water-borne contaminant. Inorganic trivalent arsenic (AsIII is one of the major species that exists environmentally. The transport of AsIII has been studied in microbes, plants and mammals. Members of the aquaglyceroporin family have been shown to actively conduct AsIII and its organic metabolite, monomethylarsenite (MAsIII. However, the transport of AsIII and MAsIII in in any fish species has not been characterized. Results In this study, five members of the aquaglyceroporin family from zebrafish (Danio rerio were cloned, and their ability to transport water, glycerol, and trivalent arsenicals (AsIII and MAsIII and antimonite (SbIII was investigated. Genes for at least seven aquaglyceroporins have been annotated in the zebrafish genome project. Here, five genes which are close homologues to human AQP3, AQP9 and AQP10 were cloned from a zebrafish cDNA preparation. These genes were named aqp3, aqp3l, aqp9a, aqp9b and aqp10 according to their similarities to the corresponding human AQPs. Expression of aqp9a, aqp9b, aqp3, aqp3l and aqp10 in multiple zebrafish organs were examined by RT-PCR. Our results demonstrated that these aquaglyceroporins exhibited different tissue expression. They are all detected in more than one tissue. The ability of these five aquaglyceroporins to transport water, glycerol and the metalloids arsenic and antimony was examined following expression in oocytes from Xenopus leavis. Each of these channels showed substantial glycerol transport at equivalent rates. These aquaglyceroporins also facilitate uptake of inorganic AsIII, MAsIII and SbIII. Arsenic accumulation in fish larvae and in different tissues from adult zebrafish was studied following short-term arsenic exposure. The results showed that liver is the major organ of arsenic accumulation; other tissues such as gill, eye

A Student Team in a University of Michigan Biomedical Engineering Design Course Constructs a Microfluidic Bioreactor for Studies of Zebrafish Development

Science.gov (United States)

Shen, Yu-chi; Li, David; Al-Shoaibi, Ali; Bersano-Begey, Tom; Chen, Hao; Ali, Shahid; Flak, Betsy; Perrin, Catherine; Winslow, Max; Shah, Harsh; Ramamurthy, Poornapriya; Schmedlen, Rachael H.; Takayama, Shuichi

2009-01-01

Abstract The zebrafish is a valuable model for teaching developmental, molecular, and cell biology; aquatic sciences; comparative anatomy; physiology; and genetics. Here we demonstrate that zebrafish provide an excellent model system to teach engineering principles. A seven-member undergraduate team in a biomedical engineering class designed, built, and tested a zebrafish microfluidic bioreactor applying microfluidics, an emerging engineering technology, to study zebrafish development. During the semester, students learned engineering and biology experimental design, chip microfabrication, mathematical modeling, zebrafish husbandry, principles of developmental biology, fluid dynamics, microscopy, and basic molecular biology theory and techniques. The team worked to maximize each person's contribution and presented weekly written and oral reports. Two postdoctoral fellows, a graduate student, and three faculty instructors coordinated and directed the team in an optimal blending of engineering, molecular, and developmental biology skill sets. The students presented two posters, including one at the Zebrafish meetings in Madison, Wisconsin (June 2008). PMID:19292670

Noninvasive technique for measurement of heartbeat regularity in zebrafish (Danio rerio embryos

Directory of Open Access Journals (Sweden)

Cheng Shuk

2009-02-01

Full Text Available Abstract Background Zebrafish (Danio rerio, due to its optical accessibility and similarity to human, has emerged as model organism for cardiac research. Although various methods have been developed to assess cardiac functions in zebrafish embryos, there lacks a method to assess heartbeat regularity in blood vessels. Heartbeat regularity is an important parameter for cardiac function and is associated with cardiotoxicity in human being. Using stereomicroscope and digital video camera, we have developed a simple, noninvasive method to measure the heart rate and heartbeat regularity in peripheral blood vessels. Anesthetized embryos were mounted laterally in agarose on a slide and the caudal blood circulation of zebrafish embryo was video-recorded under stereomicroscope and the data was analyzed by custom-made software. The heart rate was determined by digital motion analysis and power spectral analysis through extraction of frequency characteristics of the cardiac rhythm. The heartbeat regularity, defined as the rhythmicity index, was determined by short-time Fourier Transform analysis. Results The heart rate measured by this noninvasive method in zebrafish embryos at 52 hour post-fertilization was similar to that determined by direct visual counting of ventricle beating (p > 0.05. In addition, the method was validated by a known cardiotoxic drug, terfenadine, which affects heartbeat regularity in humans and induces bradycardia and atrioventricular blockage in zebrafish. A significant decrease in heart rate was found by our method in treated embryos (p p Conclusion The data support and validate this rapid, simple, noninvasive method, which includes video image analysis and frequency analysis. This method is capable of measuring the heart rate and heartbeat regularity simultaneously via the analysis of caudal blood flow in zebrafish embryos. With the advantages of rapid sample preparation procedures, automatic image analysis and data analysis, this

Normal anatomy and histology of the adult zebrafish.

Science.gov (United States)

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

2011-08-01

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

Rac1-PAK2 pathway is essential for zebrafish heart regeneration

Energy Technology Data Exchange (ETDEWEB)

Peng, Xiangwen [State Key Laboratory of Genetic Engineering, Department of Genetics, School of Life Sciences, Fudan University, Shanghai 201203 (China); He, Quanze [Center for Reproduction and Genetics, Suzhou Municipal Hospital, Jiangsu 215002 (China); Li, Guobao; Ma, Jinmin [State Key Laboratory of Genetic Engineering, Department of Genetics, School of Life Sciences, Fudan University, Shanghai 201203 (China); Zhong, Tao P., E-mail: taozhongfudan@yahoo.com [State Key Laboratory of Genetic Engineering, Department of Genetics, School of Life Sciences, Fudan University, Shanghai 201203 (China); Department of Medicine, Vanderbilt University School of Medicine, TN 37232 (United States)

2016-04-15

P-21 activated kinases, or PAKs, are serine–threonine kinases that play important roles in diverse heart functions include heart development, cardiovascular development and function in a range of models; however, the mechanisms by which PAKs mediate heart regeneration are unknown. Here, we demonstrate that PAK2 and PAK4 expression is induced in cardiomyocytes and vessels, respectively, following zebrafish heart injury. Inhibition of PAK2 and PAK4 using a specific small molecule inhibitor impedes cardiomyocyte proliferation/dedifferentiation and cardiovascular regeneration, respectively. Cdc42 is specifically expressed in the ventricle and may function upstream of PAK2 but not PAK4 under normal conditions and that cardiomyocyte proliferentation during heart regeneration relies on Rac1-mediated activation of Pak2. Our results indicate that PAKs play a key role in heart regeneration.

Rac1-PAK2 pathway is essential for zebrafish heart regeneration

International Nuclear Information System (INIS)

Peng, Xiangwen; He, Quanze; Li, Guobao; Ma, Jinmin; Zhong, Tao P.

2016-01-01

P-21 activated kinases, or PAKs, are serine–threonine kinases that play important roles in diverse heart functions include heart development, cardiovascular development and function in a range of models; however, the mechanisms by which PAKs mediate heart regeneration are unknown. Here, we demonstrate that PAK2 and PAK4 expression is induced in cardiomyocytes and vessels, respectively, following zebrafish heart injury. Inhibition of PAK2 and PAK4 using a specific small molecule inhibitor impedes cardiomyocyte proliferation/dedifferentiation and cardiovascular regeneration, respectively. Cdc42 is specifically expressed in the ventricle and may function upstream of PAK2 but not PAK4 under normal conditions and that cardiomyocyte proliferentation during heart regeneration relies on Rac1-mediated activation of Pak2. Our results indicate that PAKs play a key role in heart regeneration.

Endocrine disruption of courtship behaviour and reproduction in zebrafish (Danio rerio)

DEFF Research Database (Denmark)

Broch-Lips, Mia Gina Gruwier

2011-01-01

of the reversibility of hormonally induced shifts in sex ratio of zebrafish. In the first part of this study zebrafish were exposed to three different environmentally relevant concentrations of the synthetic oestrogen17α-ethinylestradiol (EE2) from egg stage to sexual maturity. Secondary sexual characteristics...... as fertilizing the spawned eggs. It was further demonstrated that the exposure to TB led to irreversible masculinisation of zebrafish which is in contrast with the partial reversibility of oestrogen induced sex change. During my investigations leading to this thesis it became apparent that sexual behaviour...... courtship behaviour have only been scarcely investigated. The aim of this project was to learn more about the effects of EDCS on the courtship behaviour and reproduction in zebrafish as well as investigating the reversibility of observed effects. I furthermore observed some interesting aspects...

Embryological exposure to valproic acid induces social interaction deficits in zebrafish (Danio rerio): A developmental behavior analysis.

Science.gov (United States)

Zimmermann, Fernanda Francine; Gaspary, Karina Vidarte; Leite, Carlos Eduardo; De Paula Cognato, Giana; Bonan, Carla Denise

2015-01-01

Changes in social behavior are associated with brain disorders, including mood disorders, stress, schizophrenia, Alzheimer's disease, and autism spectrum disorders (ASD). Autism is a complex neurodevelopmental disorder characterized by deficits in social interaction, impaired communication, anxiety, hyperactivity, and the presence of restricted interests. Zebrafish is one of the most social vertebrates used as a model in biomedical research, contributing to an understanding of the mechanisms that underlie social behavior. Valproic acid (VPA) is used as an anti-epileptic drug and mood stabilizer; however, prenatal VPA exposure in humans has been associated with an increased incidence of autism and it can also affect fetal brain development. Therefore, we conducted a behavioral screening at different periods of zebrafish development at 6, 30, 70, and 120dpf (days postfertilization) after VPA exposure in the early development stage to investigate social behavior, locomotion, aggression, and anxiety. VPA (48μM) exposure during the first 48hpf (hours postfertilization) did not promote changes on survival, morphology, and hatching rate at 24hpf, 48hpf, and 72hpf. The behavioral patterns suggest that VPA exposure induces changes in locomotor activity and anxiety at different developmental periods in zebrafish. Furthermore, a social interaction deficit is present at 70dpf and 120dpf. VPA exposure did not affect aggression in the adult stage at 70dpf and 120dpf. This is the first study that demonstrated zebrafish exposed to VPA during the first 48h of development exhibit deficits in social interaction, anxiety, and hyperactivity at different developmental periods. Copyright © 2015 Elsevier Inc. All rights reserved.

Neurotransmitter-Regulated Regeneration in the Zebrafish Retina

Directory of Open Access Journals (Sweden)

Mahesh B. Rao

2017-04-01

Full Text Available Summary: Current efforts to repair damaged or diseased mammalian retinas are inefficient and largely incapable of fully restoring vision. Conversely, the zebrafish retina is capable of spontaneous regeneration upon damage using Müller glia (MG-derived progenitors. Understanding how zebrafish MG initiate regeneration may help develop new treatments that prompt mammalian retinas to regenerate. We show that inhibition of γ-aminobutyric acid (GABA signaling facilitates initiation of MG proliferation. GABA levels decrease following damage, and MG are positioned to detect decreased ambient levels and undergo dedifferentiation. Using pharmacological and genetic approaches, we demonstrate that GABAA receptor inhibition stimulates regeneration in undamaged retinas while activation inhibits regeneration in damaged retinas. : Unlike mammals, zebrafish regenerate following retina damage from a resident adult stem cell (Müller glia. Dissecting the mechanisms that zebrafish use could lead to new therapeutic targets to treat retinal diseases. Patton and colleagues have discovered a mechanism by which decreased GABA levels are sensed by Müller glia to initiate a regenerative response. Keywords: zebrafish, retina, regeneration, Müller glia, GABA

Reprimo tissue-specific expression pattern is conserved between zebrafish and human.

Directory of Open Access Journals (Sweden)

Ricardo J Figueroa

Full Text Available Reprimo (RPRM, a member of the RPRM gene family, is a tumor-suppressor gene involved in the regulation of the p53-mediated cell cycle arrest at G2/M. RPRM has been associated with malignant tumor progression and proposed as a potential biomarker for early cancer detection. However, the expression and role of RPRM, as well as its family, are poorly understood and their physiology is as yet unstudied. In this scenario, a model system like the zebrafish could serve to dissect the role of the RPRM family members in vivo. Phylogenetic analysis reveals that RPRM and RPRML have been differentially retained by most species throughout vertebrate evolution, yet RPRM3 has been retained only in a small group of distantly related species, including zebrafish. Herein, we characterized the spatiotemporal expression of RPRM (present in zebrafish as an infraclass duplication rprma/rprmb, RPRML and RPRM3 in the zebrafish. By whole-mount in situ hybridization (WISH and fluorescent in situ hybridization (FISH, we demonstrate that rprm (rprma/rprmb and rprml show a similar spatiotemporal expression profile during zebrafish development. At early developmental stages rprmb is expressed in somites. After one day post-fertilization, rprm (rprma/rprmb and rprml are expressed in the notochord, brain, blood vessels and digestive tube. On the other hand, rprm3 shows the most unique expression profile, being expressed only in the central nervous system (CNS. We assessed the expression patterns of RPRM gene transcripts in adult zebrafish and human RPRM protein product in tissue samples by RT-qPCR and immunohistochemistry (IHC staining, respectively. Strikingly, tissue-specific expression patterns of the RPRM transcripts and protein are conserved between zebrafish and humans. We propose the zebrafish as a powerful tool to elucidate the both physiological and pathological roles of the RPRM gene family.

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

Science.gov (United States)

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

2018-05-01

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

Oxidative stress and DNA damage induced by imidacloprid in zebrafish (Danio rerio).

Science.gov (United States)

Ge, Weili; Yan, Saihong; Wang, Jinhua; Zhu, Lusheng; Chen, Aimei; Wang, Jun

2015-02-18

Imidacloprid is a neonicotinoid insecticide that can have negative effects on nontarget animals. The present study was conducted to assess the toxicity of various imidacloprid doses (0.3, 1.25, and 5 mg/mL) on zebrafish sampled after 7, 14, 21, and 28 days of exposure. The levels of catalase (CAT), superoxide dismutase (SOD), reactive oxygen species (ROS), glutathione-S-transferase (GST), and malondialdehyde (MDA) and the extent of DNA damage were measured to evaluate the toxicity of imidacloprid on zebrafish. SOD and GST activities were noticeably increased during early exposure but were inhibited toward the end of the exposure period. In addition, the CAT levels decreased to the control level following their elevation during early exposure. High concentrations of imidacloprid (1.25 and 5 mg/L) induced excessive ROS production and markedly increased MDA content on the 21st day of exposure. DNA damage was dose- and time-dependent. In conclusion, the present study showed that imidacloprid can induce oxidative stress and DNA damage in zebrafish.

Variations in dysfunction of sister chromatid cohesion in esco2 mutant zebrafish reflect the phenotypic diversity of Roberts syndrome

Directory of Open Access Journals (Sweden)

Stefanie M. Percival

2015-08-01

Full Text Available Mutations in ESCO2, one of two establishment of cohesion factors necessary for proper sister chromatid cohesion (SCC, cause a spectrum of developmental defects in the autosomal-recessive disorder Roberts syndrome (RBS, warranting in vivo analysis of the consequence of cohesion dysfunction. Through a genetic screen in zebrafish targeting embryonic-lethal mutants that have increased genomic instability, we have identified an esco2 mutant zebrafish. Utilizing the natural transparency of zebrafish embryos, we have developed a novel technique to observe chromosome dynamics within a single cell during mitosis in a live vertebrate embryo. Within esco2 mutant embryos, we observed premature chromatid separation, a unique chromosome scattering, prolonged mitotic delay, and genomic instability in the form of anaphase bridges and micronuclei formation. Cytogenetic studies indicated complete chromatid separation and high levels of aneuploidy within mutant embryos. Amongst aneuploid spreads, we predominantly observed decreases in chromosome number, suggesting that either cells with micronuclei or micronuclei themselves are eliminated. We also demonstrated that the genomic instability leads to p53-dependent neural tube apoptosis. Surprisingly, although many cells required Esco2 to establish cohesion, 10-20% of cells had only weakened cohesion in the absence of Esco2, suggesting that compensatory cohesion mechanisms exist in these cells that undergo a normal mitotic division. These studies provide a unique in vivo vertebrate view of the mitotic defects and consequences of cohesion establishment loss, and they provide a compensation-based model to explain the RBS phenotypes.

Zebrafish kidney phagocytes utilize macropinocytosis and Ca+-dependent endocytic mechanisms.

Directory of Open Access Journals (Sweden)

Claudia Hohn

Full Text Available BACKGROUND: The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including active endocytosis by macrophages and granulocytes. Endocytosis can be used as a reliable measure of selective and non-selective mechanisms of antigen uptake in the early phase of an immune response. Numerous assays have been developed to measure this response in a variety of mammalian and fish species. The small size of the zebrafish has prevented the large-scale collection of monocytes/macrophages and granulocytes for these endocytic assays. METHODOLOGY/PRINCIPAL FINDINGS: Pooled zebrafish kidney hematopoietic tissues were used as a source of phagocytic cells for flow-cytometry based endocytic assays. FITC-Dextran, Lucifer Yellow and FITC-Edwardsiella ictaluri were used to evaluate selective and non-selective mechanisms of uptake in zebrafish phagocytes. CONCLUSIONS/SIGNIFICANCE: Zebrafish kidney phagocytes characterized as monocytes/macrophages, neutrophils and lymphocytes utilize macropinocytosis and Ca(2+-dependant endocytosis mechanisms of antigen uptake. These cells do not appear to utilize a mannose receptor. Heat-killed Edwardsiella ictaluri induces cytoskeletal interactions for internalization in zebrafish kidney monocytes/macrophages and granulocytes. The proposed method is easy to implement and should prove especially useful in immunological, toxicological and epidemiological research.

Functional Analysis of Nuclear Estrogen Receptors in Zebrafish Reproduction by Genome Editing Approach.

Science.gov (United States)

Lu, Huijie; Cui, Yong; Jiang, Liwen; Ge, Wei

2017-07-01

Estrogens signal through both nuclear and membrane receptors with most reported effects being mediated via the nuclear estrogen receptors (nERs). Although much work has been reported on nERs in the zebrafish, there is a lack of direct genetic evidence for their functional roles and importance in reproduction. To address this issue, we undertook this study to disrupt all three nERs in the zebrafish, namely esr1 (ERα), esr2a (ERβII), and esr2b (ERβI), by the genome-editing technology clustered regularly interspaced short palindromic repeats and its associated nuclease (CRISPR/Cas9). Using this loss-of-function genetic approach, we successfully created three mutant zebrafish lines with each nER knocked out. In addition, we also generated all possible double and triple knockouts of the three nERs. The phenotypes of these mutants in reproduction were analyzed in all single, double, and triple nER knockouts in both females and males. Surprisingly, all three single nER mutant fish lines display normal reproductive development and function in both females and males, suggesting functional redundancy among these nERs. Further analysis of double and triple knockouts showed that nERs, especially Esr2a and Esr2b, were essential for female reproduction, and loss of these two nERs led to an arrest of folliculogenesis at previtellogenic stage II followed by sex reversal from female to male. In addition, the current study also revealed a unique role for Esr2a in follicle cell proliferation and transdifferentiation, follicle growth, and chorion formation. Taken together, this study provides the most comprehensive genetic analysis for differential functions of esr1, esr2a, and esr2b in fish reproduction. Copyright © 2017 Endocrine Society.

Biosecurity and Health Monitoring at the Zebrafish International Resource Center.

Science.gov (United States)

Murray, Katrina N; Varga, Zoltán M; Kent, Michael L

2016-07-01

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

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

Energy Technology Data Exchange (ETDEWEB)

Zeng, Cheng [College of Fisheries, Huazhong Agricultural University, Wuhan 430070 (China); Sun, Hong [Hubei Maternal and Child Health Hospital, Wuhan 430070 (China); Xie, Ping [Donghu Experimental Station of Lake Ecosystems, State Key Laboratory for Freshwater Ecology and Biotechnology of China, Institute of Hydrobiology, The Chinese Academy of Sciences, Wuhan 430072 (China); Wang, Jianghua; Zhang, Guirong; Chen, Nan [College of Fisheries, Huazhong Agricultural University, Wuhan 430070 (China); Yan, Wei, E-mail: Yanwei75126@163.com [Institute of Agricultural Quality Standards and Testing Technology, Hubei Academy of Agricultural Sciences, Wuhan 430064 (China); Li, Guangyu, E-mail: ligy2001@163.com [College of Fisheries, Huazhong Agricultural University, Wuhan 430070 (China); Freshwater Aquaculture Collaborative Innovation Center of Hubei Province, Wuhan 430070 (China)

2014-04-01

Highlights: • MCLR-induced apoptosis in the heart of developing embryos leads to the growth delay in zebrafish. • MCLR-triggered apoptosis might be induced by ROS. • P53–Bax–Bcl-2 and caspase-dependent apoptotic pathway contribute greatly to MCLR-induced apoptosis. Abstract: 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.

Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond.

Science.gov (United States)

Kalueff, Allan V; Gebhardt, Michael; Stewart, Adam Michael; Cachat, Jonathan M; Brimmer, Mallorie; Chawla, Jonathan S; Craddock, Cassandra; Kyzar, Evan J; Roth, Andrew; Landsman, Samuel; Gaikwad, Siddharth; Robinson, Kyle; Baatrup, Erik; Tierney, Keith; Shamchuk, Angela; Norton, William; Miller, Noam; Nicolson, Teresa; Braubach, Oliver; Gilman, Charles P; Pittman, Julian; Rosemberg, Denis B; Gerlai, Robert; Echevarria, David; Lamb, Elisabeth; Neuhauss, Stephan C F; Weng, Wei; Bally-Cuif, Laure; Schneider, Henning

2013-03-01

Zebrafish (Danio rerio) are rapidly gaining popularity in translational neuroscience and behavioral research. Physiological similarity to mammals, ease of genetic manipulations, sensitivity to pharmacological and genetic factors, robust behavior, low cost, and potential for high-throughput screening contribute to the growing utility of zebrafish models in this field. Understanding zebrafish behavioral phenotypes provides important insights into neural pathways, physiological biomarkers, and genetic underpinnings of normal and pathological brain function. Novel zebrafish paradigms continue to appear with an encouraging pace, thus necessitating a consistent terminology and improved understanding of the behavioral repertoire. What can zebrafish 'do', and how does their altered brain function translate into behavioral actions? To help address these questions, we have developed a detailed catalog of zebrafish behaviors (Zebrafish Behavior Catalog, ZBC) that covers both larval and adult models. Representing a beginning of creating a more comprehensive ethogram of zebrafish behavior, this effort will improve interpretation of published findings, foster cross-species behavioral modeling, and encourage new groups to apply zebrafish neurobehavioral paradigms in their research. In addition, this glossary creates a framework for developing a zebrafish neurobehavioral ontology, ultimately to become part of a unified animal neurobehavioral ontology, which collectively will contribute to better integration of biological data within and across species.

Application of embryonic and adult zebrafish for nanotoxicity assessment.

Science.gov (United States)

Wang, Jiangxin; Zhu, Xiaoshan; Chen, Yongsheng; Chang, Yung

2012-01-01

As an emerging model for toxicological studies, zebrafish has been explored for nanotoxicity assessment. In addition to endpoint examination of embryo/fish mortality and/or developmental disorders, molecular analyses of differential gene expression have also been employed to evaluate toxic effects associated with the exposure to nanomaterials. Here, we describe zebrafish-based assays, including both embryo and adult, for evaluation of nanotoxicity caused by metal oxide nanoparticles (NPs), in particular, zinc oxide (ZnO) and titanium oxide (TiO(2)) nanoparticles.

The role of Sox6 in zebrafish muscle fiber type specification.

Science.gov (United States)

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

2015-01-01

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

Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish

International Nuclear Information System (INIS)

Jönsson, Maria E.; Kubota, Akira; Timme-Laragy, Alicia R.; Woodin, Bruce; Stegeman, John J.

2012-01-01

The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P450 1 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on β-catenin dependent transcription, histological effects, and Ahr2 dependence of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24 h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC 50 values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2 nM PCB126 approximately 30% of eleutheroembryos failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells. -- Highlights: ► PCB126 caused cellular changes in the developing swim bladder. ► Swim bladder inflation was not related to expression of CYP1 or cox-2. �

Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish

Energy Technology Data Exchange (ETDEWEB)

Jönsson, Maria E., E-mail: maria.jonsson@ebc.uu.se [Dept. of Environmental Toxicology, Evolutionary Biology, Centre, Uppsala University, Norbyvägen 18A, 752 36 Uppsala (Sweden); Biology Department, Redfield 3-42 MS 32, Woods Hole Oceanographic Institution, Woods Hole, MA, 02543 (United States); Kubota, Akira, E-mail: akubota@whoi.edu [Biology Department, Redfield 3-42 MS 32, Woods Hole Oceanographic Institution, Woods Hole, MA, 02543 (United States); Timme-Laragy, Alicia R., E-mail: atimmelaragy@whoi.edu [Biology Department, Redfield 3-42 MS 32, Woods Hole Oceanographic Institution, Woods Hole, MA, 02543 (United States); Division of Environmental Health, Department of Public Health, School of Public Health and Health Sciences, University of Massachusetts, Amherst, MA, 01003 (United States); Woodin, Bruce, E-mail: bwoodin@whoi.edu [Biology Department, Redfield 3-42 MS 32, Woods Hole Oceanographic Institution, Woods Hole, MA, 02543 (United States); Stegeman, John J., E-mail: jstegeman@whoi.edu [Biology Department, Redfield 3-42 MS 32, Woods Hole Oceanographic Institution, Woods Hole, MA, 02543 (United States)

2012-12-01

The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P450 1 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on β-catenin dependent transcription, histological effects, and Ahr2 dependence of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24 h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC{sub 50} values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2 nM PCB126 approximately 30% of eleutheroembryos failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells. -- Highlights: ► PCB126 caused cellular changes in the developing swim bladder. ► Swim bladder inflation was not related to expression of CYP1 or cox

Deletion of Pr130 Interrupts Cardiac Development in Zebrafish

Directory of Open Access Journals (Sweden)

Jie Yang

2016-11-01

Full Text Available Protein phosphatase 2 regulatory subunit B, alpha (PPP2R3A, a regulatory subunit of protein phosphatase 2A (PP2A, is a major serine/threonine phosphatase that regulates crucial function in development and growth. Previous research has implied that PPP2R3A was involved in heart failure, and PR130, the largest transcription of PPP2R3A, functioning in the calcium release of sarcoplasmic reticulum (SR, plays an important role in the excitation-contraction (EC coupling. To obtain a better understanding of PR130 functions in myocardium and cardiac development, two pr130-deletion zebrafish lines were generated using clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated proteins (Cas system. Pr130-knockout zebrafish exhibited cardiac looping defects and decreased cardiac function (decreased fractional area and fractional shortening. Hematoxylin and eosin (H&E staining demonstrated reduced cardiomyocytes. Subsequent transmission electron microscopy revealed that the bright and dark bands were narrowed and blurred, the Z- and M-lines were fogged, and the gaps between longitudinal myocardial fibers were increased. Additionally, increased apoptosis was observed in cardiomyocyte in pr130-knockout zebrafish compared to wild-type (WT. Taken together, our results suggest that pr130 is required for normal myocardium formation and efficient cardiac contractile function.

Kctd10 regulates heart morphogenesis by repressing the transcriptional activity of Tbx5a in zebrafish

Science.gov (United States)

Tong, Xiangjun; Zu, Yao; Li, Zengpeng; Li, Wenyuan; Ying, Lingxiao; Yang, Jing; Wang, Xin; He, Shuonan; Liu, Da; Zhu, Zuoyan; Chen, Jianming; Lin, Shuo; Zhang, Bo

2014-01-01

The T-box transcription factor Tbx5 (Tbx5a in zebrafish) plays a crucial role in the formation of cardiac chambers in a dose-dependent manner. Its deregulation leads to congenital heart disease. However, little is known regarding its regulation. Here we isolate a zebrafish mutant with heart malformations, called 34c. The affected gene is identified as kctd10, a member of the potassium channel tetramerization domain (KCTD)-containing family. In the mutant, the expressions of the atrioventricular canal marker genes, such as tbx2b, hyaluronan synthase 2 (has2), notch1b and bmp4, are changed. The knockdown of tbx5 rescues the ectopic expression of has2, and knockdown of either tbx5a or has2 alleviates the heart defects. We show that Kctd10 directly binds to Tbx5 to repress its transcriptional activity. Our results reveal a new essential factor for cardiac development and suggest that KCTD10 could be considered as a new causative gene of congenital heart disease.

Regulation of embryonic hematopoiesis by a cytokine-inducible SH2 domain homolog in zebrafish.

Science.gov (United States)

Lewis, Rowena S; Noor, Suzita M; Fraser, Fiona W; Sertori, Robert; Liongue, Clifford; Ward, Alister C

2014-06-15

Cytokine-inducible SH2 domain-containing protein (CISH), a member of the suppressor of cytokine signaling family of negative feedback regulators, is induced by cytokines that activate STAT5 and can inhibit STAT5 signaling in vitro. However, demonstration of a definitive in vivo role for CISH during development has remained elusive. This study employed expression analysis and morpholino-mediated knockdown in zebrafish in concert with bioinformatics and biochemical approaches to investigate CISH function. Two zebrafish CISH paralogs were identified, cish.a and cish.b, with high overall conservation (43-46% identity) with their mammalian counterparts. The cish.a gene was maternally derived, with transcripts present throughout embryogenesis, and increasing at 4-5 d after fertilization, whereas cish.b expression commenced at 8 h after fertilization. Expression of cish.a was regulated by the JAK2/STAT5 pathway via conserved tetrameric STAT5 binding sites (TTCN3GAA) in its promoter. Injection of morpholinos targeting cish.a, but not cish.b or control morpholinos, resulted in enhanced embryonic erythropoiesis, myelopoiesis, and lymphopoiesis, including a 2- 3-fold increase in erythrocytic markers. This occurred concomitantly with increased activation of STAT5. This study indicates that CISH functions as a conserved in vivo target and regulator of STAT5 in the control of embryonic hematopoiesis. Copyright © 2014 by The American Association of Immunologists, Inc.

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

Science.gov (United States)

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

2016-04-01

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

The removal process of 2,2-dichloroacetamide (DCAcAm), a new disinfection by-product, in drinking water treatment process and its toxicity on zebrafish.

Science.gov (United States)

Lin, Tao; Zhou, Dongju; Yu, Shilin; Chen, Wei

2016-09-01

The removal process of 2,2-dichloroacetamide (DCAcAm), a new disinfection by-product (DBP) in conventional drinking water treatment plant (C-DWTP) and advanced DWTP (ADWTP) was studied with newly maximum formation potential (MFP) process. It was demonstrated that the advanced treatment displayed greater removal efficiency towards DCAcAm formation potential (MFP) than the conventional treatment. The hydrophilic natural organic matter and natural organic matter with molecular weight 10 kDa leaded to more DCAcAm formation, and the aromatic protein was inferred as one part of DCAcAm precursor. DCAcAm was found to cause delayed development and malformation to zebrafish embryos at embryonic growth stage. Compared with heart toxicity, it caused a significant neuron toxicity. It also could cause the acute DNA damage to adult zebrafish, which should be extremely cautioned. Copyright © 2016 Elsevier Ltd. All rights reserved.

Zebrafish models in neuropsychopharmacology and CNS drug discovery.

Science.gov (United States)

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

2017-07-01

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

Chronic bisphenol A exposure alters behaviors of zebrafish (Danio rerio)

International Nuclear Information System (INIS)

Wang, Ju; Wang, Xia; Xiong, Can; Liu, Jian; Hu, Bing; Zheng, Lei

2015-01-01

The adult zebrafish (Danio rerio) were exposed to treated-effluent concentration of bisphenol A (BPA) or 17β-estradiol (E2) for 6 months to evaluate their effects on behavioral characteristics: motor behavior, aggression, group preference, novel tank test and light/dark preference. E2 exposure evidently dampened fish locomotor activity, while BPA exposure had no marked effect. Interestingly, BPA-exposed fish reduced their aggressive behavior compared with control or E2. Both BPA and E2 exposure induced a significant decrease in group preference, as well as a weaker adaptability to new environment, exhibiting lower latency to reach the top, more entries to the top, longer time spent in the top, fewer frequent freezing, and fewer erratic movements. Furthermore, the circadian rhythmicity of light/dark preference was altered by either BPA or E2 exposure. Our results suggest that chronic exposure of treated-effluent concentration BPA or E2 induced various behavioral anomalies in adult fish and enhanced ecological risk to wildlife. - Highlights: • BPA exposure induces various adult behavioral anomalies. • BPA exposure decreases social interaction and environmental adaptation of zebrafish. • BPA exposure increases ecological risk to wildlife. - Chronic bisphenol A exposure alters zebrafish behaviors.

Developmental toxicity of CdTe QDs in zebrafish embryos and larvae

International Nuclear Information System (INIS)

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

2013-01-01

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

Developmental toxicity of CdTe QDs in zebrafish embryos and larvae

Science.gov (United States)

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.

Developmental toxicity of CdTe QDs in zebrafish embryos and larvae

Energy Technology Data Exchange (ETDEWEB)

Duan, Junchao; Yu, Yongbo [School of Public Health, Capital Medical University (China); Li, Yang [School of Public Health, Jilin University (China); Yu, Yang; Li, Yanbo; Huang, Peili; Zhou, Xianqing [School of Public Health, Capital Medical University (China); Peng, Shuangqing, E-mail: pengsq@hotmail.com [Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Evaluation and Research Centre for Toxicology (China); Sun, Zhiwei, E-mail: zwsun@ccmu.edu.cn [School of Public Health, Capital Medical University (China)

2013-07-15

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.

Detection of vitellogenin incorporation into zebrafish oocytes by FITC fluorescence

Directory of Open Access Journals (Sweden)

Yokoi Hayato

2011-04-01

Full Text Available Abstract Background Large volumes of lymph can be collected from the eye-sacs of bubble-eye goldfish. We attempted to induce vitellogenin (Vtg in the eye-sac lymph of bubble-eye goldfish and develop a method for visualizing Vtg incorporation by zebrafish oocytes using FITC-labeling. Methods Estrogen efficiently induced Vtg in the eye-sac lymph of goldfish. After FITC-labeled Vtg was prepared, it was injected into mature female zebrafish. Results Incorporation of FITC-labeled Vtg by zebrafish oocytes was detected in in vivo and in vitro experiments. The embryos obtained from zebrafish females injected with FITC-labeled Vtg emitted FITC fluorescence from the yolk sac and developed normally. Conclusion This method for achieving Vtg incorporation by zebrafish oocytes could be useful in experiments related to the development and endocrinology of zebrafish oocytes.

Definition of the zebrafish genome using flow cytometry and cytogenetic mapping

Directory of Open Access Journals (Sweden)

Zhou Yi

2007-06-01

Full Text Available Abstract Background The zebrafish (Danio rerio is an important vertebrate model organism system for biomedical research. The syntenic conservation between the zebrafish and human genome allows one to investigate the function of human genes using the zebrafish model. To facilitate analysis of the zebrafish genome, genetic maps have been constructed and sequence annotation of a reference zebrafish genome is ongoing. However, the duplicative nature of teleost genomes, including the zebrafish, complicates accurate assembly and annotation of a representative genome sequence. Cytogenetic approaches provide "anchors" that can be integrated with accumulating genomic data. Results Here, we cytogenetically define the zebrafish genome by first estimating the size of each linkage group (LG chromosome using flow cytometry, followed by the cytogenetic mapping of 575 bacterial artificial chromosome (BAC clones onto metaphase chromosomes. Of the 575 BAC clones, 544 clones localized to apparently unique chromosomal locations. 93.8% of these clones were assigned to a specific LG chromosome location using fluorescence in situ hybridization (FISH and compared to the LG chromosome assignment reported in the zebrafish genome databases. Thirty-one BAC clones localized to multiple chromosomal locations in several different hybridization patterns. From these data, a refined second generation probe panel for each LG chromosome was also constructed. Conclusion The chromosomal mapping of the 575 large-insert DNA clones allows for these clones to be integrated into existing zebrafish mapping data. An accurately annotated zebrafish reference genome serves as a valuable resource for investigating the molecular basis of human diseases using zebrafish mutant models.

Biotransformation of ginsenosides F4 and Rg6 in zebrafish.

Science.gov (United States)

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

2017-03-28

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

Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas.

Science.gov (United States)

Won, Minho; Ro, Hyunju; Dawid, Igor B

2015-10-06

The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use.

Effects of 2,2',4,4'-tetrabromodiphenyl ether on neurobehavior and memory change and bcl-2, c-fos, grin1b and lingo1b gene expression in male zebrafish (Danio rerio).

Science.gov (United States)

Zheng, Shukai; Liu, Caixia; Huang, Yanhong; Bao, Mian; Huang, Yuanni; Wu, Kusheng

2017-10-15

Polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants in various environmental matrices and organisms and pose a threat to neural systems of organisms. However, though quite a few studies have explored the effect of PBDEs on neural behaviors such as learning and memory abilities in animals, their mechanisms are less known. We used the zebrafish model to evaluate neurotoxicity of PBDEs and observe changes in behavior and related gene expression. In behavioral testing, 50 zebrafish were divided into five groups treated with different concentrations of BDE-47. T-maze exploration was used for learning and memory testing, which was recorded by camera every 7days. After 21days, all fish were killed, and the gene expression of c-fos, bcl-2, lingo1b and grin1b in brain tissue was analyzed by RT-qPCR. The behavioral changes (latency to leave the start zone, reach the reward zone, and stay in the reward zone; accuracy in choosing the right maze arm, accumulation of freezing bouts, etc.) were related to BDE-47 concentration and had a time-effect relation with increasing exposure days, especially with 500μg/L BDE-47. BDE-47 elevated brain bcl-2, grin1b and lingo1b expression. The expression of c-fos showed an increase with 50 and 100μg/L BDE-47 exposure. The PBDE BDE-47 had a negative impact on the neurobehaviors of zebrafish and affected the expression of c-fos, bcl-2, lingo1b and grin1b in zebrafish brain tissue. Copyright © 2017 Elsevier Inc. All rights reserved.

Biosecurity and Health Monitoring at the Zebrafish International Resource Center

OpenAIRE

Murray, Katrina N.; Varga, Zolt?n M.; Kent, Michael L.

2016-01-01

The Zebrafish International Resource Center (ZIRC) is a repository and distribution center for mutant, transgenic, and wild-type zebrafish. In recent years annual imports of new zebrafish lines to ZIRC have increased tremendously. In addition, after 15 years of research, we have identified some of the most virulent pathogens affecting zebrafish that should be avoided in large production facilities, such as ZIRC. Therefore, while importing a high volume of new lines we prioritize safeguarding ...

Zebrafish foxo3b negatively regulates canonical Wnt signaling to affect early embryogenesis.

Directory of Open Access Journals (Sweden)

Xun-wei Xie

Full Text Available FOXO genes are involved in many aspects of development and vascular homeostasis by regulating cell apoptosis, proliferation, and the control of oxidative stress. In addition, FOXO genes have been showed to inhibit Wnt/β-catenin signaling by competing with T cell factor to bind to β-catenin. However, how important of this inhibition in vivo, particularly in embryogenesis is still unknown. To demonstrate the roles of FOXO genes in embryogenesis will help us to further understand their relevant physiological functions. Zebrafish foxo3b gene, an orthologue of mammalian FOXO3, was expressed maternally and distributed ubiquitously during early embryogenesis and later restricted to brain. After morpholino-mediated knockdown of foxo3b, the zebrafish embryos exhibited defects in axis and neuroectoderm formation, suggesting its critical role in early embryogenesis. The embryo-developmental marker gene staining at different stages, phenotype analysis and rescue assays revealed that foxo3b acted its role through negatively regulating both maternal and zygotic Wnt/β-catenin signaling. Moreover, we found that foxo3b could interact with zebrafish β-catenin1 and β-catenin2 to suppress their transactivation in vitro and in vivo, further confirming its role relevant to the inhibition of Wnt/β-catenin signaling. Taken together, we revealed that foxo3b played a very important role in embryogenesis and negatively regulated maternal and zygotic Wnt/β-catenin signaling by directly interacting with both β-catenin1 and β-catenin2. Our studies provide an in vivo model for illustrating function of FOXO transcription factors in embryogenesis.

The Complete and Updated "Rotifer Polyculture Method" for Rearing First Feeding Zebrafish

Science.gov (United States)

Lawrence, Christian; Best, Jason; Cockington, Jason; Henry, Eric C.; Hurley, Shane; James, Althea; Lapointe, Christopher; Maloney, Kara; Sanders, Erik

2016-01-01

The zebrafish (Danio rerio) is a model organism of increasing importance in many fields of science. One of the most demanding technical aspects of culture of this species in the laboratory is rearing first-feeding larvae to the juvenile stage with high rates of growth and survival. The central management challenge of this developmental period revolves around delivering highly nutritious feed items to the fish on a nearly continuous basis without compromising water quality. Because larval zebrafish are well-adapted to feed on small zooplankton in the water column, live prey items such as brachionid rotifers, Artemia, and Paramecium are widely recognized as the feeds of choice, at least until the fish reach the juvenile stage and are able to efficiently feed on processed diets. This protocol describes a method whereby newly hatched zebrafish larvae are cultured together with live saltwater rotifers (Brachionus plicatilis) in the same system. This polyculture approach provides fish with an "on-demand", nutrient-rich live food source without producing chemical waste at levels that would otherwise limit performance. Importantly, because the system harnesses both the natural high productivity of the rotifers and the behavioral preferences of the fish, the labor involved with maintenance is low. The following protocol details an updated, step-by-step procedure that incorporates rotifer production (scalable to any desired level) for use in a polyculture of zebrafish larvae and rotifers that promotes maximal performance during the first 5 days of exogenous feeding. PMID:26863035

Exposure to tributyltin induces endoplasmic reticulum stress and the unfolded protein response in zebrafish.

Science.gov (United States)

Komoike, Yuta; Matsuoka, Masato

2013-10-15

Tributyltin (TBT) is a major marine contaminant and causes endocrine disruption, hepatotoxicity, immunotoxicity, and neurotoxicity. However, the molecular mechanisms underlying the toxicity of TBT have not been fully elucidated. We examined whether exposure to TBT induces the endoplasmic reticulum (ER) stress response in zebrafish, a model organism. Zebrafish-derived BRF41 fibroblast cells were exposed to 0.5 or 1 μM TBT for 0.5-16 h and subsequently lysed and immunoblotted to detect ER stress-related proteins. Zebrafish embryos, grown until 32 h post fertilization (hpf), were exposed to 1 μM TBT for 16 h and used in whole mount in situ hybridization and immunohistochemistry to visualize the expression of ER chaperones and an ER stress-related apoptosis factor. Exposure of the BRF41 cells to TBT caused phosphorylation of the zebrafish homolog of protein kinase RNA-activated-like ER kinase (PERK), eukaryotic translation initiation factor 2 alpha (eIF2α), and inositol-requiring enzyme 1 (IRE1), characteristic splicing of X-box binding protein 1 (XBP1) mRNA, and enhanced expression of activating transcription factor 4 (ATF4) protein. In TBT-exposed zebrafish embryos, ectopic expression of the gene encoding zebrafish homolog of the 78 kDa glucose-regulating protein (GRP78) and gene encoding CCAAT/enhancer-binding protein homologous protein (CHOP) was detected in the precursors of the neuromast, which is a sensory organ for detecting water flow and vibration. Our in vitro and in vivo studies revealed that exposure of zebrafish to TBT induces the ER stress response via activation of both the PERK-eIF2α and IRE1-XBP1 pathways of the unfolded protein response (UPR) in an organ-specific manner. Copyright © 2013 Elsevier B.V. All rights reserved.

Effects of titanium dioxide nanoparticles exposure on parkinsonism in zebrafish larvae and PC12.

Science.gov (United States)

Hu, Qinglian; Guo, Fengliang; Zhao, Fenghui; Fu, Zhengwei

2017-04-01

Nanomaterials hold significant potential for industrial and biomedical application these years. Therefore, the relationship between nanoparticles and neurodegenerative disease is of enormous interest. In this contribution, zebrafish embryos and PC12 cell lines were selected for studying neurotoxicity of titanium dioxide nanoparticles (TiO 2 NPs). After exposure of different concentrations of TiO 2 NPs to embryos from fertilization to 96 hpf, the hatching time of zebrafish was decreased, accompanied by an increase in malformation rate. However, no significant increases in mortality relative to control were observed. These results indicated that TiO 2 NPs exposure hold a risk for premature of zebrafish embryos, but not fatal. The further investigation confirmed that TiO 2 NPs could accumulate in the brain of zebrafish larvae, resulting in reactive oxygen species (ROS) generation and cell death of hypothalamus. Meanwhile, q-PCR analysis showed that TiO 2 NPs exposure increased the pink1, parkin, α-syn and uchl1 gene expression, which are related with the formation of Lewy bodies. We also observed loss of dopaminergic neurons in zebrafish and in vitro. These remarkable hallmarks are all linked to these Parkinson's disease (PD) symptoms. Our results indicate that TiO 2 NPs exposure induces neurotoxicity in vivo and in vitro, which poses a significant risk factor for the development of PD. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Science.gov (United States)

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

2013-01-01

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

Developmental Toxicity of Zinc Oxide Nanoparticles to Zebrafish (Danio rerio: A Transcriptomic Analysis.

Directory of Open Access Journals (Sweden)

Jin Soo Choi

Full Text Available Zinc oxide nanoparticles (ZnO NPs are being utilized in an increasing number of fields and commercial applications. While their general toxicity and associated oxidative stress have been extensively studied, the toxicological pathways that they induce in developmental stages are still largely unknown. In this study, the developmental toxicity of ZnO NPs to embryonic/larval zebrafish was investigated. The transcriptional expression profiles induced by ZnO NPs were also investigated to ascertain novel genomic responses related to their specific toxicity pathway. Zebrafish embryos were exposed to 0.01, 0.1, 1, and 10 mg/L ZnO NPs for 96 h post-fertilization. The toxicity of ZnO NPs, based on their Zn concentration, was quite similar to that in embryonic/larval zebrafish exposed to corresponding ZnSO4 concentrations. Pericardial edema and yolk-sac edema were the principal malformations induced by ZnO NPs. Gene-expression profiling using microarrays demonstrated 689 genes that were differentially regulated (fold change >1.5 following exposure to ZnO NPs (498 upregulated, 191 downregulated. Several genes that were differentially regulated following ZnO NP exposure shared similar biological pathways with those observed with ZnSO4 exposure, but six genes (aicda, cyb5d1, edar, intl2, ogfrl2 and tnfsf13b associated with inflammation and the immune system responded specifically to ZnO NPs (either in the opposite direction or were unchanged in ZnSO4 exposure. Real-time reverse-transcription quantitative polymerase chain reaction confirmed that the responses of these genes to ZnO NPs were significantly different from their response to ZnSO4 exposure. ZnO NPs may affect genes related to inflammation and the immune system, resulting in yolk-sac edema and pericardia edema in embryonic/larval developmental stages. These results will assist in elucidating the mechanisms of toxicity of ZnO NPs during development of zebrafish.

Short-term memory in zebrafish (Danio rerio).

Science.gov (United States)

Jia, Jason; Fernandes, Yohaan; Gerlai, Robert

2014-08-15

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

Characterization of behavioral and endocrine effects of LSD on zebrafish.

Science.gov (United States)

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

2010-12-25

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

Kaempferol Identified by Zebrafish Assay and Fine Fractionations Strategy from Dysosma versipellis Inhibits Angiogenesis through VEGF and FGF Pathways

Science.gov (United States)

Liang, Fang; Han, Yuxiang; Gao, Hao; Xin, Shengchang; Chen, Shaodan; Wang, Nan; Qin, Wei; Zhong, Hanbing; Lin, Shuo; Yao, Xinsheng; Li, Song

2015-01-01

Natural products are a rich resource for the discovery of therapeutic substances. By directly using 504 fine fractions from isolated traditional Chinese medicine plants, we performed a transgenic zebrafish based screen for anti-angiogenesis substances. One fraction, DYVE-D3, was found to inhibit the growth of intersegmental vessels in the zebrafish vasculature. Bioassay-guided isolation of DYVE-D3 indicates that the flavonoid kaempferol was the active substance. Kaempferol also inhibited the proliferation and migration of HUVECs in vitro. Furthermore, we found that kaempferol suppressed angiogenesis through inhibiting VEGFR2 expression, which can be enhanced by FGF inhibition. In summary, this study shows that the construction of fine fraction libraries allows efficient identification of active substances from natural products. PMID:26446489

Modafinil decreases anxiety-like behaviour in zebrafish

Directory of Open Access Journals (Sweden)

Adrian Johnson

2017-02-01

Full Text Available Modafinil (2-((diphenylmethylsulfinylacetamide, a selective dopamine and norepinephrine transporter inhibitor, is most commonly prescribed for narcolepsy but has gained recent interest for treating a variety of disorders. Zebrafish (Danio rerio are becoming a model of choice for pharmacological and behavioural research. To investigate the behavioural effects of modafinil on anxiety, we administered doses of 0, 2, 20, and 200 mg/L for 30 minutes then tested zebrafish in the novel approach test. In this test, the fish was placed into a circular arena with a novel object in the center and motion-tracking software was used to quantify the time the fish spent in the outer area of the arena (thigmotaxis zone, middle third of the arena (transition zone and center of the arena, as well as total distance traveled, immobility and meandering. Modafinil caused a decrease in time spent in the thigmotaxis zone and increased time spent in the transition zone across all doses. Modafinil did not significantly alter the time spent in the center zone (near the novel object, the distance moved, meandering, or the duration of time spent immobile. We also validated this test as a measure of anxiety with the administration of ethanol (1% which decreased time spent in the thigmotaxis zone and increased time spent in the transition zone. These results suggest that modafinil decreases anxiety-like behaviour in zebrafish.

Characterization of the Zebrafish Homolog of Zipper Interacting Protein Kinase

Directory of Open Access Journals (Sweden)

Brandon W. Carr

2014-06-01

Full Text Available Zipper-interacting protein kinase (ZIPK is a conserved vertebrate-specific regulator of actomyosin contractility in smooth muscle and non-muscle cells. Murine ZIPK has undergone an unusual divergence in sequence and regulation compared to other ZIPK orthologs. In humans, subcellular localization is controlled by phosphorylation of threonines 299 and 300. In contrast, ZIPK subcellular localization in mouse and rat is controlled by interaction with PAR-4. We carried out a comparative biochemical characterization of the regulation of the zebrafish ortholog of ZIPK. Like the human orthologs zebrafish ZIPK undergoes nucleocytoplasmic-shuttling and is abundant in the cytoplasm, unlike the primarily nuclear rat ZIPK. Rat ZIPK, but not human or zebrafish ZIPK, interacts with zebrafish PAR-4. Mutation of the conserved residues required for activation of the mammalian orthologs abrogated activity of the zebrafish ZIPK. In contrast to the human ortholog, mutation of threonine 299 and 300 in the zebrafish ZIPK has no effect on the activity or subcellular localization. Thus, we found that zebrafish ZIPK functions in a manner most similar to the human ZIPK and quite distinct from murine orthologs, yet the regulation of subcellular localization is not conserved.

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

Science.gov (United States)

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.

Ahr2-dependance of PCB126 effects on the swimbladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish

Science.gov (United States)

Jönsson, Maria E.; Kubota, Akira; Timme-Laragy, Alicia; Woodin, Bruce; Stegeman, John J.

2012-01-01

The teleost swimbladder is assumed a homolog of the tetrapod lung. Both swimbladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR1) agonists; in zebrafish (Danio rerio) the swimbladder fails to inflate with exposure to 3,3’,4,4’,5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P4501 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swimbladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on β-catenin dependent transcription, histological effects, and Ahr2 dependance of the effect of PCB126 on swimbladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24 h and then held in clean water until day 4, a normal time for swimbladder inflation. The effects of PCB126 were concentration-dependent with EC50 values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swimbladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swimbladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2 nM PCB126 approximately 30% of eleutheroembryos2 failed to inflate the swimbladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swimbladder. Our results indicate that PCB126 blocks swimbladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swimbladder cells. PMID:23036320

Distribution and chronotropic effects of serotonin in the zebrafish heart.

Science.gov (United States)

Stoyek, Matthew R; Jonz, Michael G; Smith, Frank M; Croll, Roger P

2017-09-01

Several lines of evidence suggest that serotonin (5-HT) has a regulatory role in cardiovascular function from embryogenesis through adulthood. However, the reported actions of 5-HT are often contradictory and include bradycardia or tachycardia, hypotension or hypertension, and vasodilation or vasoconstriction. Clarifying such cardiac effects requires further research and may benefit from utilizing a model simpler than the mammalian hearts traditionally used in these studies. In the present study, we describe the cardiac distribution and chronotropic responses of 5-HT in the zebrafish heart. A combined anatomical, electrophysiological, and pharmacological approach was used to investigate the involvement of 5-HT pathways, and to compare neural and direct myocardial pathways of biological action. Immunohistochemical methods revealed 5-HT in endocardial cells, glial-like cells, and intracardiac neurons in the atrium. Electrocardiogram (ECG) recordings combined with the administration of pharmacological agents demonstrated that 5-HT acted predominantly through direct myocardial pathways resulting in a reduction of heart rate. Overall, the results of this study contribute significant advances in the establishment of the zebrafish as a new model for studies of the role of 5-HT in autonomic cardiac control. Copyright © 2017 Elsevier B.V. All rights reserved.

Assessment of thyroid endocrine system impairment and oxidative stress mediated by cobalt ferrite (CoFe2 O4 ) nanoparticles in zebrafish larvae.

Science.gov (United States)

Ahmad, Farooq; Liu, Xiaoyi; Zhou, Ying; Yao, Hongzhou; Zhao, Fangfang; Ling, Zhaoxing; Xu, Chao

2016-12-01

Fascinating super paramagnetic uniqueness of iron oxide particles at nano-scale level make them extremely useful in the state of the art therapies, equipments, and techniques. Cobalt ferrite (CoFe 2 O 4 ) magnetic nanoparticles (MNPs) are extensively used in nano-based medicine and electronics, results in extensive discharge and accumulation into the environment. However, very limited information is available for their endocrine disrupting potential in aquatic organisms. In this study, the thyroid endocrine disrupting ability of CoFe 2 O 4 NPs in Zebrafish larvae for 168-h post fertilization (hpf) was evaluated. The results showed the elevated amounts of T4 and T3 hormones by malformation of hypothalamus pituitary axis in zebrafish larvae. These elevated levels of whole body THs leads to delayed hatching, head and eye malformation, arrested development, and alterations in metabolism. The influence of THs disruption on ROS production and change in activities of catalase (CAT), mu-glutathione s-transferase (mu-GST), and acid phosphatase (AP) were also studied. The production of significantly higher amounts of in vivo generation of ROS leads to membrane damage and oxidative stress. Presences of NPs and NPs agglomerates/aggregates were also the contributing factors in mechanical damaging the membranes and physiological structure of thyroid axis. The increased activities of CAT, mu-GST, and AP confirmed the increased oxidative stress, possible DNA, and metabolic alterations, respectively. The excessive production of in vivo ROS leads to severe apoptosis in head, eye, and heart region confirming that malformation leads to malfunctioning of hypothalamus pituitary axis. ROS-induced oxidative DNA damage by formation of 8-OHdG DNA adducts elaborates the genotoxicity potential of CoFe 2 O 4 NPs. This study will help us to better understand the risk and assessment of endocrine disrupting potential of nanoparticles. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 2068

Chronic perfluorooctane sulfonate (PFOS) exposure induces hepatic steatosis in zebrafish

International Nuclear Information System (INIS)

Cheng, Jiangfei; Lv, Suping; Nie, Shangfei; Liu, Jing; Tong, Shoufang; Kang, Ning; Xiao, Yanyan; Dong, Qiaoxiang; Huang, Changjiang; Yang, Dongren

2016-01-01

Highlights: • PFOS chronic exposure induces sex-dependent hepatic steotosis in zebrafish. • PFOS interferes with β-oxidation, lipid synthesis, and lipid hepatic export process. • Zebrafish could be used as an alternative model for PFOS chronic toxicity screening. - Abstract: Perfluorooctane sulfonate (PFOS), one persistent organic pollutant, has been widely detected in the environment, wildlife and human. Currently few studies have documented the effects of chronic PFOS exposure on lipid metabolism, especially in aquatic organisms. The underlying mechanisms of hepatotoxicity induced by chronic PFOS exposure are still largely unknown. The present study defined the effects of chronic exposure to low level of PFOS on lipid metabolism using zebrafish as a model system. Our findings revealed a severe hepatic steatosis in the liver of males treated with 0.5 μM PFOS as evidenced by hepatosomatic index, histological assessment and liver lipid profiles. Quantitative PCR assay further indicated that PFOS significantly increase the transcriptional expression of nuclear receptors (nr1h3, rara, rxrgb, nr1l2) and the genes associated with fatty acid oxidation (acox1, acadm, cpt1a). In addition, chronic PFOS exposure significantly decreased liver ATP content and serum level of VLDL/LDL lipoprotein in males. Taken together, these findings suggest that chronic PFOS exposure induces hepatic steatosis in zebrafish via disturbing lipid biosynthesis, fatty acid β-oxidation and excretion of VLDL/LDL lipoprotein, and also demonstrate the validity of using zebrafish as an alternative model for PFOS chronic toxicity screening.

Chronic perfluorooctane sulfonate (PFOS) exposure induces hepatic steatosis in zebrafish

Energy Technology Data Exchange (ETDEWEB)

Cheng, Jiangfei; Lv, Suping; Nie, Shangfei; Liu, Jing; Tong, Shoufang; Kang, Ning; Xiao, Yanyan; Dong, Qiaoxiang [Zhejiang Provincial Key Laboratory for Technology and Application of Model Organisms (China); Institute of Environmental Safety and Human Health, Wenzhou Medical University, Wenzhou, 325035 (China); Huang, Changjiang, E-mail: cjhuang5711@163.com [Zhejiang Provincial Key Laboratory for Technology and Application of Model Organisms (China); Institute of Environmental Safety and Human Health, Wenzhou Medical University, Wenzhou, 325035 (China); Yang, Dongren, E-mail: yangdongren@yahoo.com [Zhejiang Provincial Key Laboratory for Technology and Application of Model Organisms (China); Institute of Environmental Safety and Human Health, Wenzhou Medical University, Wenzhou, 325035 (China)

2016-07-15

Highlights: • PFOS chronic exposure induces sex-dependent hepatic steotosis in zebrafish. • PFOS interferes with β-oxidation, lipid synthesis, and lipid hepatic export process. • Zebrafish could be used as an alternative model for PFOS chronic toxicity screening. - Abstract: Perfluorooctane sulfonate (PFOS), one persistent organic pollutant, has been widely detected in the environment, wildlife and human. Currently few studies have documented the effects of chronic PFOS exposure on lipid metabolism, especially in aquatic organisms. The underlying mechanisms of hepatotoxicity induced by chronic PFOS exposure are still largely unknown. The present study defined the effects of chronic exposure to low level of PFOS on lipid metabolism using zebrafish as a model system. Our findings revealed a severe hepatic steatosis in the liver of males treated with 0.5 μM PFOS as evidenced by hepatosomatic index, histological assessment and liver lipid profiles. Quantitative PCR assay further indicated that PFOS significantly increase the transcriptional expression of nuclear receptors (nr1h3, rara, rxrgb, nr1l2) and the genes associated with fatty acid oxidation (acox1, acadm, cpt1a). In addition, chronic PFOS exposure significantly decreased liver ATP content and serum level of VLDL/LDL lipoprotein in males. Taken together, these findings suggest that chronic PFOS exposure induces hepatic steatosis in zebrafish via disturbing lipid biosynthesis, fatty acid β-oxidation and excretion of VLDL/LDL lipoprotein, and also demonstrate the validity of using zebrafish as an alternative model for PFOS chronic toxicity screening.

Silver nanoparticles induce endoplasmatic reticulum stress response in zebrafish

Energy Technology Data Exchange (ETDEWEB)

Christen, Verena [University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz (Switzerland); Capelle, Martinus [Crucell, P.O. Box 2048, NL-2301 Leiden (Netherlands); Fent, Karl, E-mail: karl.fent@fhnw.ch [University of Applied Sciences and Arts Northwestern Switzerland, School of Life Sciences, Gründenstrasse 40, CH-4132 Muttenz (Switzerland); Swiss Federal Institute of Technology Zürich, Department of Environmental Systems Science, CH-8092 Zürich (Switzerland)

2013-10-15

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

Silver nanoparticles induce endoplasmatic reticulum stress response in zebrafish

International Nuclear Information System (INIS)

Christen, Verena; Capelle, Martinus; Fent, Karl

2013-01-01

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

Indomethacin treatment prior to pentylenetetrazole-induced seizures downregulates the expression of il1b and cox2 and decreases seizure-like behavior in zebrafish larvae.

Science.gov (United States)

Barbalho, Patrícia Gonçalves; Lopes-Cendes, Iscia; Maurer-Morelli, Claudia Vianna

2016-03-09

It has been demonstrated that the zebrafish model of pentylenetetrazole (PTZ)-evoked seizures and the well-established rodent models of epilepsy are similar pertaining to behavior, electrographic features, and c-fos expression. Although this zebrafish model is suitable for studying seizures, to date, inflammatory response after seizures has not been investigated using this model. Because a relationship between epilepsy and inflammation has been established, in the present study we investigated the transcript levels of the proinflammatory cytokines interleukin-1 beta (il1b) and cyclooxygenase-2 (cox2a and cox2b) after PTZ-induced seizures in the brain of zebrafish 7 days post fertilization. Furthermore, we exposed the fish to the nonsteroidal anti-inflammatory drug indomethacin prior to PTZ, and we measured its effect on seizure latency, number of seizure behaviors, and mRNA expression of il1b, cox2b, and c-fos. We used quantitative real-time PCR to assess the mRNA expression of il1b, cox2a, cox2b, and c-fos, and visual inspection was used to monitor seizure latency and the number of seizure-like behaviors. We found a short-term upregulation of il1b, and we revealed that cox2b, but not cox2a, was induced after seizures. Indomethacin treatment prior to PTZ-induced seizures downregulated the mRNA expression of il1b, cox2b, and c-fos. Moreover, we observed that in larvae exposed to indomethacin, seizure latency increased and the number of seizure-like behaviors decreased. This is the first study showing that il1b and cox-2 transcripts are upregulated following PTZ-induced seizures in zebrafish. In addition, we demonstrated the anticonvulsant effect of indomethacin based on (1) the inhibition of PTZ-induced c-fos transcription, (2) increase in seizure latency, and (3) decrease in the number of seizure-like behaviors. Furthermore, anti-inflammatory effect of indomethacin is clearly demonstrated by the downregulation of the mRNA expression of il1b and cox2b. Our results

Role of zebrafish cytochrome P450 CYP1C genes in the reduced mesencephalic vein blood flow caused by activation of AHR2

International Nuclear Information System (INIS)

Kubota, Akira; Stegeman, John J.; Woodin, Bruce R.; Iwanaga, Toshihiko; Harano, Ryo; Peterson, Richard E.; Hiraga, Takeo; Teraoka, Hiroki

2011-01-01

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). We previously reported a sensitive and useful endpoint of TCDD developmental toxicity in zebrafish, namely a decrease in blood flow in the dorsal midbrain, but downstream genes involved in the effect are not known. The present study addressed the role of zebrafish cytochrome P450 1C (CYP1C) genes in association with a decrease in mesencephalic vein (MsV) blood flow. The CYP1C subfamily was recently discovered in fish and includes the paralogues CYP1C1 and CYP1C2, both of which are induced via AHR2 in zebrafish embryos. We used morpholino antisense oligonucleotides (MO or morpholino) to block initiation of translation of the target genes. TCDD-induced mRNA expression of CYP1Cs and a decrease in MsV blood flow were both blocked by gene knockdown of AHR2. Gene knockdown of CYP1C1 by two different morpholinos and CYP1C2 by two different morpholinos, but not by their 5 nucleotide-mismatch controls, was effective in blocking reduced MsV blood flow caused by TCDD. The same CYP1C-MOs prevented reduction of blood flow in the MsV caused by β-naphthoflavone (BNF), representing another class of AHR agonists. Whole-mount in situ hybridization revealed that mRNA expression of CYP1C1 and CYP1C2 was induced by TCDD most strongly in branchiogenic primordia and pectoral fin buds. In situ hybridization using head transverse sections showed that TCDD increased the expression of both CYP1Cs in endothelial cells of blood vessels, including the MsV. These results indicate a potential role of CYP1C1 and CYP1C2 in the local circulation failure induced by AHR2 activation in the dorsal midbrain of the zebrafish embryo. - Research Highlights: → We examine the roles of zebrafish CYP1C1 and CYP1C2 in TCDD developmental toxicity. → TCDD induces mRNA expression of both CYP1Cs in the mesencephalic vein. → Knockdown of each

Zebrafish syntenic relationship to human/mouse genomes revealed by radiation hybrid mapping

International Nuclear Information System (INIS)

Samonte, Irene E.

2007-01-01

Zebrafish (Danio rerio) is an excellent model system for vertebrate developmental analysis and a new model for human disorders. In this study, however, zebrafish was used to determine its syntenic relationship to human/mouse genomes using the zebrafish-hamster radiation hybrid panel. The focus was on genes residing on chromosomes 6 and 17 of human and mouse, respectively, and some other genes of either immunologic or evolutionary importance. Gene sequences of interest and zebrafish expressed sequence tags deposited in the GenBank were used in identifying zebrafish homologs. Polymerase chain reaction (PCR) amplification, cloning and subcloning, sequencing, and phylogenetic analysis were done to confirm the homology of the candidate genes in zebrafish. The promising markers were then tested in the 94 zebrafish-hamster radiation hybrid panel cell lines and submitted for logarithm of the odds (LOD) score analysis to position genes on the zebrafish map. A total of 19 loci were successfully mapped to zebrafish linkage groups 1, 14, 15, 19, and 20. Four of these loci were positioned in linkage group 20, whereas, 3 more loci were added in linkage group 19, thus increasing to 34 loci the number of human genes syntenic to the group. With the sequencing of the zebrafish genome, about 20 more MHC genes were reported linked on the same group. (Author)

Interordinal chimera formation between medaka and zebrafish for analyzing stem cell differentiation.

Science.gov (United States)

Hong, Ni; Chen, Songlin; Ge, Ruowen; Song, Jianxing; Yi, Meisheng; Hong, Yunhan

2012-08-10

Chimera formation is a standard test for pluripotency of stem cells in vivo. Interspecific chimera formation between distantly related organisms offers also an attractive approach for propagating endangered species. Parameters influencing interspecies chimera formation have remained poorly elucidated. Here, we report interordinal chimera formation between medaka and zebrafish, which separated ∼320 million years ago and exhibit a more than 2-fold difference in developmental speed. We show that, on transplantation into zebrafish blastulae, both noncultivated blastomeres and long-term cultivated embryonic stem (ES) cells of medaka adopted the zebrafish developmental program and differentiated into physiologically functional cell types including pigment cells, blood cells, and cardiomyocytes. We also show that medaka ES cells express differentiation gene markers during chimeric embryogenesis. Therefore, the evolutionary distance and different embryogenesis speeds do not produce donor-host incompatibility to compromise chimera formation between medaka and zebrafish, and molecular markers are valuable for analyzing lineage commitment and cell differentiation in interspecific chimeric embryos.

Relationships among msx gene structure and function in zebrafish and other vertebrates.

Science.gov (United States)

Ekker, M; Akimenko, M A; Allende, M L; Smith, R; Drouin, G; Langille, R M; Weinberg, E S; Westerfield, M

1997-10-01

The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.

Fluoride caused thyroid endocrine disruption in male zebrafish (Danio rerio).

Science.gov (United States)

Jianjie, Chen; Wenjuan, Xue; Jinling, Cao; Jie, Song; Ruhui, Jia; Meiyan, Li

2016-02-01

Excessive fluoride in natural water ecosystem has the potential to detrimentally affect thyroid endocrine system, but little is known of such effects or underlying mechanisms in fish. In the present study, we evaluated the effects of fluoride on growth performance, thyroid histopathology, thyroid hormone levels, and gene expressions in the HPT axis in male zebrafish (Danio rerio) exposed to different determined concentrations of 0.1, 0.9, 2.0 and 4.1 M of fluoride to investigate the effects of fluoride on thyroid endocrine system and the potential toxic mechanisms caused by fluoride. The results indicated that the growth of the male zebrafish used in the experiments was significantly inhibited, the thyroid microtrastructure was changed, and the levels of T3 and T4 were disturbed in fluoride-exposed male fish. In addition, the expressional profiles of genes in HPT axis displayed alteration. The expressions of all studied genes were significantly increased in all fluoride-exposed male fish after exposure for 45 days. The transcriptional levels of corticotrophin-releasing hormone (CRH), thyroid-stimulating hormone (TSH), thyroglobulin (TG), sodium iodide symporter (NIS), iodothyronine I (DIO1), and thyroid hormone receptor alpha (TRα) were also elevated in all fluoride-exposed male fish after 90 days of exposure, while the inconsistent expressions were found in the mRNA of iodothyronineⅡ (DIO2), UDP glucuronosyltransferase 1 family a, b (UGT1ab), transthyretin (TTR), and thyroid hormone receptor beta (TRβ). These results demonstrated that fluoride could notably inhibit the growth of zebrafish, and significantly affect thyroid endocrine system by changing the microtrastructure of thyroid, altering thyroid hormone levels and endocrine-related gene expressions in male zebrafish. All above indicated that fluoride could pose a great threat to thyroid endocrine system, thus detrimentally affected the normal function of thyroid of male zebrafish. Copyright © 2015

Myomaker mediates fusion of fast myocytes in zebrafish embryos

Energy Technology Data Exchange (ETDEWEB)

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 were unable to swim, even under effect of a tactile stimulation. Confocal observations showed a marked phenotype characterized by the persistence of mononucleated muscle cells in the fast myotome at developmental stages where these cells normally fuse to form multinucleated myotubes. This indicates that myomaker is essential for myocyte fusion in zebrafish. Thus, there is an evolutionary conservation of myomaker expression and function among Teleostomi.

Transcriptome analysis of zebrafish embryogenesis using microarrays.

Directory of Open Access Journals (Sweden)

Sinnakaruppan Mathavan

2005-08-01

Full Text Available Zebrafish (Danio rerio is a well-recognized model for the study of vertebrate developmental genetics, yet at the same time little is known about the transcriptional events that underlie zebrafish embryogenesis. Here we have employed microarray analysis to study the temporal activity of developmentally regulated genes during zebrafish embryogenesis. Transcriptome analysis at 12 different embryonic time points covering five different developmental stages (maternal, blastula, gastrula, segmentation, and pharyngula revealed a highly dynamic transcriptional profile. Hierarchical clustering, stage-specific clustering, and algorithms to detect onset and peak of gene expression revealed clearly demarcated transcript clusters with maximum gene activity at distinct developmental stages as well as co-regulated expression of gene groups involved in dedicated functions such as organogenesis. Our study also revealed a previously unidentified cohort of genes that are transcribed prior to the mid-blastula transition, a time point earlier than when the zygotic genome was traditionally thought to become active. Here we provide, for the first time to our knowledge, a comprehensive list of developmentally regulated zebrafish genes and their expression profiles during embryogenesis, including novel information on the temporal expression of several thousand previously uncharacterized genes. The expression data generated from this study are accessible to all interested scientists from our institute resource database (http://giscompute.gis.a-star.edu.sg/~govind/zebrafish/data_download.html.

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

Science.gov (United States)

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

2015-04-01

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

The toxicity of a new disinfection by-product, 2,2-dichloroacetamide (DCAcAm), on adult zebrafish (Danio rerio) and its occurrence in the chlorinated drinking water.

Science.gov (United States)

Yu, Shilin; Lin, Tao; Chen, Wei; Tao, Hui

2015-11-01

The detection method of 2,2-dichloroacetamide (DCAcAm), a new disinfection by-product (DBP) in chlorinated drinking water, was established using a gas chromatograph coupled with a micro-electron capture detector. The chlorinated water samples were taken from ten drinking water treatment plants around Yangtze River or Taihu Lake in China. The concentration of DCAcAm was detected ranging from 0.5 to 1.8μg/L in the waterworks around Yangtze River, and 1.5-2.6μg/L around Taihu Lake. The toxicity of DCAcAm on adult zebrafish was assessed by investigating the metabolism damage with multiple metabolic biomarkers and the accumulation capability with bio-concentration factor. The results showed that DCAcAm could cause the acute metabolism damage and was easily accumulated in zebrafish, and should be extremely cautioned. Copyright © 2015 Elsevier Ltd. All rights reserved.

Zebrafish Axenic Larvae Colonization with Human Intestinal Microbiota.

Science.gov (United States)

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

2018-04-01

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

Zebrafish Database: Customizable, Free, and Open-Source Solution for Facility Management.

Science.gov (United States)

Yakulov, Toma Antonov; Walz, Gerd

2015-12-01

Zebrafish Database is a web-based customizable database solution, which can be easily adapted to serve both single laboratories and facilities housing thousands of zebrafish lines. The database allows the users to keep track of details regarding the various genomic features, zebrafish lines, zebrafish batches, and their respective locations. Advanced search and reporting options are available. Unique features are the ability to upload files and images that are associated with the respective records and an integrated calendar component that supports multiple calendars and categories. Built on the basis of the Joomla content management system, the Zebrafish Database is easily extendable without the need for advanced programming skills.

Method for somatic cell nuclear transfer in zebrafish.

Science.gov (United States)

Siripattarapravat, Kannika; Cibelli, Jose B

2011-01-01

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

Disruption of apoptosis pathways involved in zebrafish gonad differentiation by 17α-ethinylestradiol and fadrozole exposures

International Nuclear Information System (INIS)

Luzio, Ana; Matos, Manuela; Santos, Dércia; Fontaínhas-Fernandes, António A.; Monteiro, Sandra M.

2016-01-01

Highlights: • Apoptosis in females is avoided by anti-apoptotic pathways and in males is essential to the “juvenile ovary” failure. • BIRC5 is central to the regulation of zebrafish spermatogenesis. • EE2 did not change sex ratios, but Fadrozole induced masculinization with a significant increase in male proportion. • The few females identified after exposure to Fadrozole may have avoided sex reversal by increasing anti-apoptotic proteins. • EE2 increased the pro-apoptotic genes/proteins in males, promoting gonad differentiation. - Abstract: Zebrafish (Danio rerio) sex determination seems to involve genetic factors (GSD) but also environmental factors (ESD), such as endocrine disrupting chemicals (EDCs) that are known to mimic endogenous hormones and disrupt gonad differentiation. Apoptosis has also been proposed to play a crucial role in zebrafish gonad differentiation. Nevertheless, the interactions between EDCs and apoptosis have received little attention. Thus, this study aimed to assess if and which apoptotic pathways are involved in zebrafish gonad differentiation and how EDCs may interfere with this process. With these purposes, zebrafish were exposed to 17α-ethinylestradiol (EE_2, 4 ng/L) and fadrozole (Fad, 50 μg/L) from 2 h to 35 days post-fertilization (dpf). Afterwards, a gene expression analysis by qRT-PCR and a stereological analysis, based on systematic sampling and protein immunohistochemistry, were performed. The death receptors (FAS; TRADD), anti-apoptotic (BCL-2; MDM2), pro-apoptotic (CASP-2 and −6) and cell proliferation (BIRC5/survivin; JUN) genes and proteins were evaluated. In general, apoptosis was inhibited in females through the involvement of anti-apoptotic pathways, while in males apoptosis seemed to be crucial to the failure of the “juvenile ovary” development and the induction of testes transformation. The JUN protein was shown to be necessary in juvenile ovaries, while the BIRC5 protein seemed to be involved in

Disruption of apoptosis pathways involved in zebrafish gonad differentiation by 17α-ethinylestradiol and fadrozole exposures

Energy Technology Data Exchange (ETDEWEB)

Luzio, Ana, E-mail: aluzio@utad.pt [Centre for the Research and Technology of Agro-Environmental and Biological Sciences, CITAB, Departamento de Biologia e Ambiente (DeBA), University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801 (Portugal); Life Sciences and Environment School, University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801 (Portugal); Matos, Manuela [University of Lisbon, Faculty of Sciences, BioISI– Biosystems & Integrative Sciences Institute, Campo Grande, 1749-016 Lisbon (Portugal); Department of Genetics and Biotechnology, Life Sciences and Environment School (ECVA), University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801 (Portugal); Santos, Dércia [Life Sciences and Environment School, University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801 (Portugal); Fontaínhas-Fernandes, António A.; Monteiro, Sandra M. [Centre for the Research and Technology of Agro-Environmental and Biological Sciences, CITAB, Departamento de Biologia e Ambiente (DeBA), University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801 (Portugal); Life Sciences and Environment School, University of Trás-os-Montes and Alto Douro, UTAD, Quinta de Prados, Vila Real, 5000-801 (Portugal); and others

2016-08-15

Highlights: • Apoptosis in females is avoided by anti-apoptotic pathways and in males is essential to the “juvenile ovary” failure. • BIRC5 is central to the regulation of zebrafish spermatogenesis. • EE2 did not change sex ratios, but Fadrozole induced masculinization with a significant increase in male proportion. • The few females identified after exposure to Fadrozole may have avoided sex reversal by increasing anti-apoptotic proteins. • EE2 increased the pro-apoptotic genes/proteins in males, promoting gonad differentiation. - Abstract: Zebrafish (Danio rerio) sex determination seems to involve genetic factors (GSD) but also environmental factors (ESD), such as endocrine disrupting chemicals (EDCs) that are known to mimic endogenous hormones and disrupt gonad differentiation. Apoptosis has also been proposed to play a crucial role in zebrafish gonad differentiation. Nevertheless, the interactions between EDCs and apoptosis have received little attention. Thus, this study aimed to assess if and which apoptotic pathways are involved in zebrafish gonad differentiation and how EDCs may interfere with this process. With these purposes, zebrafish were exposed to 17α-ethinylestradiol (EE{sub 2}, 4 ng/L) and fadrozole (Fad, 50 μg/L) from 2 h to 35 days post-fertilization (dpf). Afterwards, a gene expression analysis by qRT-PCR and a stereological analysis, based on systematic sampling and protein immunohistochemistry, were performed. The death receptors (FAS; TRADD), anti-apoptotic (BCL-2; MDM2), pro-apoptotic (CASP-2 and −6) and cell proliferation (BIRC5/survivin; JUN) genes and proteins were evaluated. In general, apoptosis was inhibited in females through the involvement of anti-apoptotic pathways, while in males apoptosis seemed to be crucial to the failure of the “juvenile ovary” development and the induction of testes transformation. The JUN protein was shown to be necessary in juvenile ovaries, while the BIRC5 protein seemed to be involved

Evolution of the osteoblast: skeletogenesis in gar and zebrafish

Directory of Open Access Journals (Sweden)

Eames B Frank

2012-03-01

Full Text Available Abstract Background Although the vertebrate skeleton arose in the sea 500 million years ago, our understanding of the molecular fingerprints of chondrocytes and osteoblasts may be biased because it is informed mainly by research on land animals. In fact, the molecular fingerprint of teleost osteoblasts differs in key ways from that of tetrapods, but we do not know the origin of these novel gene functions. They either arose as neofunctionalization events after the teleost genome duplication (TGD, or they represent preserved ancestral functions that pre-date the TGD. Here, we provide evolutionary perspective to the molecular fingerprints of skeletal cells and assess the role of genome duplication in generating novel gene functions. We compared the molecular fingerprints of skeletogenic cells in two ray-finned fish: zebrafish (Danio rerio--a teleost--and the spotted gar (Lepisosteus oculatus--a "living fossil" representative of a lineage that diverged from the teleost lineage prior to the TGD (i.e., the teleost sister group. We analyzed developing embryos for expression of the structural collagen genes col1a2, col2a1, col10a1, and col11a2 in well-formed cartilage and bone, and studied expression of skeletal regulators, including the transcription factor genes sox9 and runx2, during mesenchymal condensation. Results Results provided no evidence for the evolution of novel functions among gene duplicates in zebrafish compared to the gar outgroup, but our findings shed light on the evolution of the osteoblast. Zebrafish and gar chondrocytes both expressed col10a1 as they matured, but both species' osteoblasts also expressed col10a1, which tetrapod osteoblasts do not express. This novel finding, along with sox9 and col2a1 expression in developing osteoblasts of both zebrafish and gar, demonstrates that osteoblasts of both a teleost and a basally diverging ray-fin fish express components of the supposed chondrocyte molecular fingerprint. Conclusions Our

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

Science.gov (United States)

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

2016-01-01

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

Effects of Short-Term Exposure to Sublethal Concentrations of Silver Nanoparticles on Histopathology and Electron Microscope Ultrastructure of Zebrafish (Danio Rerio Gills

Directory of Open Access Journals (Sweden)

Borhan Mansouri

2015-11-01

Full Text Available Background: The increasing use of nanomaterials and nanoproducts has increased the possibility of contamination of the environment, which may have adverse effects on different organisms. The aim of this study was to evaluate the effects of silver nanoparticles on histopathology and gill ultrastructure of zebrafish (Danio rerio under laboratory conditions. Methods: Zebrafish were exposed to four concentrations of silver nanoparticles (0.0015, 0.00375, 0.0075, and 0.015 mg/l for a period of 4 days. Gill ultrastructure and histopathological changes were studied using scanning electron microscope and haematoxylin - eosin staining. Results: Exposure to silver nanoparticles significantly (P < 0.001 increased the diameter of gill filaments and secondary lamellae, while silver nanoparticles significantly reduced the length of the secondary gills in zebrafish. Moreover, other changes such as vacuolization, dilated and clubbed tips, hyperplasia, edema, fusion, swelling of mucocytes, hypertrophy, and necrosis were observed. The effects of silver nanoparticles in zebrafish gills were dose dependent. Conclusion: Based on the adverse effects of AgNPs on zebrafish gills, silver nanoparticle solutions can be hazardous pollutants for the environment.

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

Energy Technology Data Exchange (ETDEWEB)

Bouwmeester, Manon C.; Ruiter, Sander; Lommelaars, Tobias; Sippel, Josefine; Hodemaekers, Hennie M. [Center for Health Protection, National Institute for Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven (Netherlands); Brandhof, Evert-Jan van den [Center for Environmental Quality, National Institute for Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven (Netherlands); Pennings, Jeroen L.A. [Center for Health Protection, National Institute for Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven (Netherlands); Kamstra, Jorke H. [Institute for Environmental Studies (IVM), VU University, De Boelelaan 1085, 1081 HV Amsterdam (Netherlands); Jelinek, Jaroslav [Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA (United States); Issa, Jean-Pierre J. [Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA (United States); Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Legler, Juliette [Institute for Environmental Studies (IVM), VU University, De Boelelaan 1085, 1081 HV Amsterdam (Netherlands); Ven, Leo T.M. van der, E-mail: leo.van.der.ven@rivm.nl [Center for Health Protection, National Institute for Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven (Netherlands)

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

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

International Nuclear Information System (INIS)

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

2016-01-01

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

Selectivity of natural, synthetic and environmental estrogens for zebrafish estrogen receptors

Energy Technology Data Exchange (ETDEWEB)

Pinto, Caroline [Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5056 (United States); Grimaldi, Marina; Boulahtouf, Abdelhay [Institut de Recherche en Cancérologie de Montpellier, Institut National de la Santé de la Recherche Médicale U896, Institut Régional de Cancérologie de Montpellier, Université Montpellier 1, 34298 Montpellier (France); Pakdel, Farzad [Institut de Recherche sur la Santé, Environnement et Travail (IRSET), INSERM U1085, Université de Rennes 1, Rennes (France); Brion, François; Aït-Aïssa, Sélim [Unité Écotoxicologie In Vitro et In Vivo, INERIS, Parc ALATA, 60550 Verneuil-en-Halatte (France); Cavaillès, Vincent [Institut de Recherche en Cancérologie de Montpellier, Institut National de la Santé de la Recherche Médicale U896, Institut Régional de Cancérologie de Montpellier, Université Montpellier 1, 34298 Montpellier (France); Bourguet, William [U1054, Centre de Biochimie Structurale, CNRS UMR5048, Université Montpellier 1 et 2, 34290 Montpellier (France); Gustafsson, Jan-Ake [Center for Nuclear Receptors and Cell Signaling, Department of Biology and Biochemistry, University of Houston, Houston, TX 77204-5056 (United States); Department of Biosciences and Nutrition, Karolinska Institutet, 14183 Huddinge (Sweden); and others

2014-10-01

Zebrafish, Danio rerio, is increasingly used as an animal model to study the effects of pharmaceuticals and environmental estrogens. As most of these estrogens have only been tested on human estrogen receptors (ERs), it is necessary to measure their effects on zebrafish ERs. In humans there are two distinct nuclear ERs (hERα and hERβ), whereas the zebrafish genome encodes three ERs, zfERα and two zfERβs (zfERβ1 and zfERβ2). In this study, we established HeLa-based reporter cell lines stably expressing each of the three zfERs. We first reported that estrogens more efficiently activate the zfERs at 28 °C as compared to 37 °C, thus reflecting the physiological temperature of zebrafish in wildlife. We then showed significant differences in the ability of agonist and antagonist estrogens to modulate activation of the three zfER isotypes in comparison to hERs. Environmental compounds (bisphenol A, alkylphenols, mycoestrogens) which are hER panagonists and hERβ selective agonists displayed greater potency for zfERα as compared to zfERβs. Among hERα selective synthetic agonists, PPT did not activate zfERα while 16α-LE2 was the most zfERα selective compound. Altogether, these results confirm that all hER ligands control in a similar manner the transcriptional activity of zfERs although significant differences in selectivity were observed among subtypes. The zfER subtype selective ligands that we identified thus represent new valuable tools to dissect the physiological roles of the different zfERs. Finally, our work also points out that care has to be taken in transposing the results obtained using the zebrafish as a model for human physiopathology. - Highlights: • Zebrafish is increasingly used to study the effects of estrogens. • We assessed the activity of pharmaceutical and environmental estrogens on zfERs. • Environmental estrogens displayed greater potency for zfERα compared to zfERβs. • hERβ selective agonists displayed greater potency for zf

Collagenolytic Activity Is Associated with Scar Resolution in Zebrafish Hearts after Cryoinjury

Science.gov (United States)

Gamba, Laurent; Amin-Javaheri, Armaan; Kim, Jieun; Warburton, David; Lien, Ching-Ling

2017-01-01

Myocardial infarction is the major cause of cardiac injury in western countries and can result in a massive loss of heart cells, leading eventually to heart failure. A fibrotic collagen-rich scar may prevent ventricular wall rupture, but also may result in heart failure because of its stiffness. In zebrafish, cardiac cryoinjury triggers a fibrotic response and scarring. Unlike with mammals, zebrafish heart has the striking ability to regenerate and to resolve the scar. Thus, understanding the mechanisms of scar resolution in zebrafish heart might facilitate the design of new therapeutic approaches to improve the recovery of patients. To visualize the collagenolytic activity within the zebrafish heart following cryoinjury, we used an in situ collagen zymography assay. We detected expression of mmp2 and mmp14a and these matrix metalloproteinases might contribute to the collagenase activity. Collagenolytic activity was present in the wound area, but decreased as the myocardium regenerated. Comparison with neonatal mouse hearts that failed to regenerate after transmural cryoinjury revealed a similar collagenolytic activity in the scar. These findings suggest that collagenolytic activity may be key to how the zebrafish heart resolves its scar; however, it is not sufficient in mouse hearts that lack efficient myocardial regeneration. PMID:29367534

Zebrafish as a Model System for Investigating the Compensatory Regulation of Ionic Balance during Metabolic Acidosis

Directory of Open Access Journals (Sweden)

Lletta Lewis

2018-04-01

Full Text Available Zebrafish (Danio rerio have become an important model for integrative physiological research. Zebrafish inhabit a hypo-osmotic environment; to maintain ionic and acid-base homeostasis, they must actively take up ions and secrete acid to the water. The gills in the adult and the skin at larval stage are the primary sites of ionic regulation in zebrafish. The uptake of ions in zebrafish is mediated by specific ion transporting cells termed ionocytes. Similarly, in mammals, ion reabsorption and acid excretion occur in specific cell types in the terminal region of the renal tubules (distal convoluted tubule and collecting duct. Previous studies have suggested that functional regulation of several ion transporters/channels in the zebrafish ionocytes resembles that in the mammalian renal cells. Additionally, several mechanisms involved in regulating the epithelial ion transport during metabolic acidosis are found to be similar between zebrafish and mammals. In this article, we systemically review the similarities and differences in ionic regulation between zebrafish and mammals during metabolic acidosis. We summarize the available information on the regulation of epithelial ion transporters during acidosis, with a focus on epithelial Na+, Cl− and Ca2+ transporters in zebrafish ionocytes and mammalian renal cells. We also discuss the neuroendocrine responses to acid exposure, and their potential role in ionic compensation. Finally, we identify several knowledge gaps that would benefit from further study.

Metal uptake and acute toxicity in zebrafish: Common mechanisms across multiple metals

Energy Technology Data Exchange (ETDEWEB)

Alsop, Derek, E-mail: alsopde@mcmaster.ca [Department of Biology, McMaster University, 1280 Main St. W., Hamilton, ON L8S 4K1 (Canada); Wood, Chris M. [Department of Biology, McMaster University, 1280 Main St. W., Hamilton, ON L8S 4K1 (Canada)

2011-10-15

All metals tested reduced calcium uptake in zebrafish larvae. However, it was whole body sodium loss that was functionally related to toxicity. The zebrafish larvae acute toxicity assay save time, space and resources. - Abstract: Zebrafish larvae (Danio rerio) were used to examine the mechanisms of action and acute toxicities of metals. Larvae had similar physiological responses and sensitivities to waterborne metals as adults. While cadmium and zinc have previously been shown to reduce Ca{sup 2+} uptake, copper and nickel also decreased Ca{sup 2+} uptake, suggesting that the epithelial transport of all these metals is through Ca{sup 2+} pathways. However, exposure to cadmium, copper or nickel for up to 48 h had little or no effect on total whole body Ca{sup 2+} levels, indicating that the reduction of Ca{sup 2+} uptake is not the acute toxic mechanism of these metals. Instead, mortalities were effectively related to whole body Na{sup +}, which decreased up to 39% after 48 h exposures to different metals around their respective 96 h LC50s. Decreases in whole body K{sup +} were also observed, although they were not as pronounced or frequent as Na{sup +} losses. None of the metals tested inhibited Na{sup +} uptake in zebrafish (Na{sup +} uptake was in fact increased with exposure) and the observed losses of Na{sup +}, K{sup +}, Ca{sup 2+} and Mg{sup 2+} were proportional to the ionic gradients between the plasma and water, indicating diffusive ion loss with metal exposure. This study has shown that there is a common pathway for metal uptake and a common mechanism of acute toxicity across groups of metals in zebrafish. The disruption of ion uptake accompanying metal exposure does not appear to be responsible for the acute toxicity of metals, as has been previously suggested, but rather the toxicity is instead due to total ion loss (predominantly Na{sup +}).

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

Directory of Open Access Journals (Sweden)

Moayad Saad

2018-02-01

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

The role of Fanconi anemia/BRCA genes in zebrafish sex determination.

Science.gov (United States)

Rodríguez-Marí, Adriana; Postlethwait, John H

2011-01-01

Fanconi anemia (FA) is a human disease of bone marrow failure, leukemia, squamous cell carcinoma, and developmental anomalies, including hypogonadism and infertility. Bone marrow transplants improve hematopoietic phenotypes but do not prevent other cancers. FA arises from mutation in any of the 15 FANC genes that cooperate to repair double stranded DNA breaks by homologous recombination. Zebrafish has a single ortholog of each human FANC gene and unexpectedly, mutations in at least two of them (fancl and fancd1(brca2)) lead to female-to-male sex reversal. Investigations show that, as in human, zebrafish fanc genes are required for genome stability and for suppressing apoptosis in tissue culture cells, in embryos treated with DNA damaging agents, and in meiotic germ cells. The sex reversal phenotype requires the action of Tp53 (p53), an activator of apoptosis. These results suggest that in normal sex determination, zebrafish oocytes passing through meiosis signal the gonadal soma to maintain expression of aromatase, an enzyme that converts androgen to estrogen, thereby feminizing the gonad and the individual. According to this model, normal male and female zebrafish differ in genetic factors that control the strength of the late meiotic oocyte-derived signal, probably by regulating the number of meiotic oocytes, which environmental factors can also alter. Transcripts from fancd1(brca2) localize at the animal pole of the zebrafish oocyte cytoplasm and are required for normal oocyte nuclear architecture, for normal embryonic development, and for preventing ovarian tumors. Embryonic DNA repair and sex reversal phenotypes provide assays for the screening of small molecule libraries for therapeutic substances for FA. Copyright © 2011 Elsevier Inc. All rights reserved.

A review of monoaminergic neuropsychopharmacology in zebrafish.

Science.gov (United States)

Maximino, Caio; Herculano, Anderson Manoel

2010-12-01

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

Hypoxia-induced retinopathy model in adult zebrafish

DEFF Research Database (Denmark)

Cao, Ziquan; Jensen, Lasse D.; Rouhi, Pegah

2010-01-01

Hypoxia-induced vascular responses, including angiogenesis, vascular remodeling and vascular leakage, significantly contribute to the onset, development and progression of retinopathy. However, until recently there were no appropriate animal disease models recapitulating adult retinopathy available....... In this article, we describe protocols that create hypoxia-induced retinopathy in adult zebrafish. Adult fli1: EGFP zebrafish are placed in hypoxic water for 3-10 d and retinal neovascularization is analyzed using confocal microscopy. It usually takes 11 d to obtain conclusive results using the hypoxia......-induced retinopathy model in adult zebrafish. This model provides a unique opportunity to study kinetically the development of retinopathy in adult animals using noninvasive protocols and to assess therapeutic efficacy of orally active antiangiogenic drugs....

Thyroid endocrine disruption in zebrafish larvae after exposure to mono-(2-ethylhexyl phthalate (MEHP.

Directory of Open Access Journals (Sweden)

Wenhui Zhai

Full Text Available Phthalates are extensively used as plasticizers in a variety of daily-life products, resulting in widespread distribution in aquatic environments. However, limited information is available on the endocrine disrupting effects of phthalates in aquatic organisms. The aim of the present study was to examine whether exposure to mono-(2-ethylhexyl phthalate (MEHP, the hydrolytic metabolite of di-(2-ethylhexyl phthalate (DEHP disrupts thyroid endocrine system in fish. In this study, zebrafish (Danio rerio embryos were exposed to different concentrations of MEHP (1.6, 8, 40, and 200 μg/L from 2 h post-fertilization (hpf to 168 hpf. The whole-body content of thyroid hormone and transcription of genes involved in the hypothalamic-pituitary-thyroid (HPT axis were examined. Treatment with MEHP significantly decreased whole-body T4 contents and increased whole-body T3 contents, indicating thyroid endocrine disruption. The upregulation of genes related to thyroid hormone metabolism (Dio2 and UGT1ab might be responsible for decreased T4 contents. Elevated gene transcription of Dio1 was also observed in this study, which might assist to degrade increased T3 contents. Exposure to MEHP also significantly induced transcription of genes involved in thyroid development (Nkx2.1 and Pax8 and thyroid hormone synthesis (TSHβ, NIS and TG. However, the genes encoding proteins involved in TH transport (transthyretin, TTR was transcriptionally significantly down-regulated after exposure to MEHP. Overall, these results demonstrate that acute exposure to MEHP alters whole-body contents of thyroid hormones in zebrafish embryos/larvae and changes the transcription of genes involved in the HPT axis, thus exerting thyroid endocrine toxicity.

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

Directory of Open Access Journals (Sweden)

Hanna Luukinen

2018-01-01

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

Short-term exposure of arsenite disrupted thyroid endocrine system and altered gene transcription in the HPT axis in zebrafish.

Science.gov (United States)

Sun, Hong-Jie; Li, Hong-Bo; Xiang, Ping; Zhang, Xiaowei; Ma, Lena Q

2015-10-01

Arsenic (As) pollution in aquatic environment may adversely impact fish health by disrupting their thyroid hormone homeostasis. In this study, we explored the effect of short-term exposure of arsenite (AsIII) on thyroid endocrine system in zebrafish. We measured As concentrations, As speciation, and thyroid hormone thyroxine levels in whole zebrafish, oxidative stress (H2O2) and damage (MDA) in the liver, and gene transcription in hypothalamic-pituitary-thyroid (HPT) axis in the brain and liver tissues of zebrafish after exposing to different AsIII concentrations for 48 h. Result indicated that exposure to AsIII increased inorganic As in zebrafish to 0.46-0.72 mg kg(-1), induced oxidative stress with H2O2 being increased by 1.4-2.5 times and caused oxidative damage with MDA being augmented by 1.6 times. AsIII exposure increased thyroxine levels by 1.3-1.4 times and modulated gene transcription in HPT axis. Our study showed AsIII caused oxidative damage, affected thyroid endocrine system and altered gene transcription in HPT axis in zebrafish. Published by Elsevier Ltd.

3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy

Directory of Open Access Journals (Sweden)

Juneyong Eum

2016-11-01

Full Text Available Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis.

Functional inhibition of UQCRB suppresses angiogenesis in zebrafish

Energy Technology Data Exchange (ETDEWEB)

Cho, Yoon Sun; Jung, Hye Jin [Chemical Genomics National Research Laboratory, Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of); Seok, Seung Hyeok [Department of Microbiology and Immunology, Institute for Experimental Animals, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Payumo, Alexander Y.; Chen, James K. [Department of Chemical and Systems Biology, Stanford University School of Medicine, Stanford, CA 94305 (United States); Kwon, Ho Jeong, E-mail: kwonhj@yonsei.ac.kr [Chemical Genomics National Research Laboratory, Department of Biotechnology, Translational Research Center for Protein Function Control, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749 (Korea, Republic of)

2013-04-19

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

Phenotype classification of zebrafish embryos by supervised learning.

Directory of Open Access Journals (Sweden)

Nathalie Jeanray

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

Functional inhibition of UQCRB suppresses angiogenesis in zebrafish

International Nuclear Information System (INIS)

Cho, Yoon Sun; Jung, Hye Jin; Seok, Seung Hyeok; Payumo, Alexander Y.; Chen, James K.; Kwon, Ho Jeong

2013-01-01

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

Social learning of an associative foraging task in zebrafish

Science.gov (United States)

Zala, Sarah M.; Määttänen, Ilmari

2013-05-01

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

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

Science.gov (United States)

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

2016-06-01

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 only 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. © 2015 Wiley Periodicals, Inc.

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

Science.gov (United States)

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.

The neural basis of visual behaviors in the larval zebrafish.

Science.gov (United States)

Portugues, Ruben; Engert, Florian

2009-12-01

We review visually guided behaviors in larval zebrafish and summarise what is known about the neural processing that results in these behaviors, paying particular attention to the progress made in the last 2 years. Using the examples of the optokinetic reflex, the optomotor response, prey tracking and the visual startle response, we illustrate how the larval zebrafish presents us with a very promising model vertebrate system that allows neurocientists to integrate functional and behavioral studies and from which we can expect illuminating insights in the near future. Copyright 2009 Elsevier Ltd. All rights reserved.

Uranium-induced sensory alterations in the zebrafish Danio rerio

Energy Technology Data Exchange (ETDEWEB)

Faucher, K., E-mail: kfaucher@hotmail.fr [Laboratoire d' ecotoxicologie des radionucleides (LECO), Institut de Radioprotection et Surete Nucleaire, Centre de Cadarache, Batiment 186, BP3, 13115 Saint Paul lez Durance (France); Floriani, M.; Gilbin, R.; Adam-Guillermin, C. [Laboratoire d' ecotoxicologie des radionucleides (LECO), Institut de Radioprotection et Surete Nucleaire, Centre de Cadarache, Batiment 186, BP3, 13115 Saint Paul lez Durance (France)

2012-11-15

The effect of chronic exposure to uranium ions (UO{sub 2}{sup 2+}) on sensory tissues including the olfactory and lateral line systems was investigated in zebrafish (Danio rerio) using scanning electron microscopy. The aim of this study was to determine whether exposure to uranium damaged sensory tissues in fish. The fish were exposed to uranium at the concentration of 250 {mu}g l{sup -1} for 10 days followed by a depuration period of 23 days. Measurements of uranium uptake in different fish organs: olfactory rosettes and bulbs, brain, skin, and muscles, were also determined by ICP-AES and ICP-MS during the entire experimental period. The results showed that uranium displayed a strong affinity for sensory structures in direct contact with the surrounding medium, such as the olfactory and lateral line systems distributed on the skin. A decreasing gradient of uranium concentration was found: olfactory rosettes > olfactory bulbs > skin > muscles > brain. At the end of the experiment, uranium was present in non-negligible quantities in sensory tissues. In parallel, fish exposed to uranium showed severe sensory tissue alterations at the level of the olfactory and lateral line systems. In both sensory systems, the gross morphology was altered and the sensory hair cells were significantly damaged very early after the initiation of exposure (from the 3rd day). At the end of the experiment, after 23 days of depuration, the lateral line system still displayed slight tissue alterations, but approximately 80% of the neuromasts in this system had regenerated. In contrast, the olfactory system took more time to recover, as more than half of the olfactory rosettes observed remained destroyed at the end of the experiment. This study showed, for the first time, that uranium is able to damage fish sensory tissues to such an extent that tissue regeneration is delayed.

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

Energy Technology Data Exchange (ETDEWEB)

Berger, Joachim, E-mail: Joachim.Berger@Monash.edu [Australian Regenerative Medicine Institute, EMBL Australia, Monash University, Clayton (Australia); Sztal, Tamar; Currie, Peter D. [Australian Regenerative Medicine Institute, EMBL Australia, Monash University, Clayton (Australia)

2012-07-13

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

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

International Nuclear Information System (INIS)

Berger, Joachim; Sztal, Tamar; Currie, Peter D.

2012-01-01

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

Effect of radiation dose-rate on hematopoietic cell engraftment in adult zebrafish.

Directory of Open Access Journals (Sweden)

Tiffany J Glass

Full Text Available Although exceptionally high radiation dose-rates are currently attaining clinical feasibility, there have been relatively few studies reporting the biological consequences of these dose-rates in hematopoietic cell transplant (HCT. In zebrafish models of HCT, preconditioning before transplant is typically achieved through radiation alone. We report the comparison of outcomes in adult zebrafish irradiated with 20 Gy at either 25 or 800 cGy/min in the context of experimental HCT. In non-transplanted irradiated fish we observed no substantial differences between dose-rate groups as assessed by fish mortality, cell death in the kidney, endogenous hematopoietic reconstitution, or gene expression levels of p53 and ddb2 (damage-specific DNA binding protein 2 in the kidney. However, following HCT, recipients conditioned with the higher dose rate showed significantly improved donor-derived engraftment at 9 days post transplant (p ≤ 0.0001, and improved engraftment persisted at 31 days post transplant. Analysis for sdf-1a expression, as well as transplant of hematopoietic cells from cxcr4b -/- zebrafish, (odysseus, cumulatively suggest that the sdf-1a/cxcr4b axis is not required of donor-derived cells for the observed dose-rate effect on engraftment. Overall, the adult zebrafish model of HCT indicates that exceptionally high radiation dose-rates can impact HCT outcome, and offers a new system for radiobiological and mechanistic interrogation of this phenomenon. Key words: Radiation dose rate, Total Marrow Irradiation (TMI, Total body irradiation (TBI, SDF-1, Zebrafish, hematopoietic cell transplant.

Regulation of zebrafish CYP3A65 transcription by AHR2

International Nuclear Information System (INIS)

Chang, Chin-Teng; Chung, Hsin-Yu; Su, Hsiao-Ting; Tseng, Hua-Pin; Tzou, Wen-Shyong; Hu, Chin-Hwa

2013-01-01

CYP3A proteins are the most abundant CYPs in the liver and intestines, and they play a pivotal role in drug metabolism. In mammals, CYP3A genes are induced by various xenobiotics through processes mediated by PXR. We previously identified zebrafish CYP3A65 as a CYP3A ortholog that is constitutively expressed in gastrointestinal tissues, and is upregulated by treatment with dexamethasone, rifampicin or tetrachlorodibenzo-p-dioxin (TCDD). However, the underlying mechanism of TCDD-mediated CYP3A65 transcription is unclear. Here we generated two transgenic zebrafish, Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP), which contain 2.1 and 5.4 kb 5′ flanking sequences, respectively, of the CYP3A65 gene upstream of EGFP. Both transgenic lines express EGFP in larval gastrointestinal tissues in a pattern similar to that of the endogenous CYP3A65 gene. Moreover, EGFP expression can be significantly induced by TCDD exposure during the larval stage. In addition, EGFP expression can be stimulated by kynurenine, a putative AHR ligand produced during tryptophan metabolism. AHRE elements in the upstream regulatory region of the CYP3A65 gene are indispensible for basal and TCDD-induced transcription. Furthermore, the AHR2 DNA and ligand-binding domains are required to mediate effective CYP3A65 transcription. AHRE sequences are present in the promoters of many teleost CYP3 genes, but not of mammalian CYP3 genes, suggesting that AHR/AHR2-mediated transcription is likely a common regulatory mechanism for teleost CYP3 genes. It may also reflect the different environments that terrestrial and aquatic organisms encounter. - Highlights: • Tg(CYP3A65:EGFP) and CYP3A65 exhibits identical expression pattern. • CYP3A65 can be significantly induced by TCDD or kynurenine. • The AHRE elements are required to mediate CYP3A65 transcription. • The AHR2 DNA and ligand-binding domains are required for CYP3A65 transcription. • AHRE elements are present in many teleost CYP3 genes, but not in

Regulation of zebrafish CYP3A65 transcription by AHR2

Energy Technology Data Exchange (ETDEWEB)

Chang, Chin-Teng; Chung, Hsin-Yu; Su, Hsiao-Ting; Tseng, Hua-Pin [Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan (China); Tzou, Wen-Shyong [Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan (China); Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan (China); Hu, Chin-Hwa, E-mail: chhu@mail.ntou.edu.tw [Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan (China); Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan (China)

2013-07-15

CYP3A proteins are the most abundant CYPs in the liver and intestines, and they play a pivotal role in drug metabolism. In mammals, CYP3A genes are induced by various xenobiotics through processes mediated by PXR. We previously identified zebrafish CYP3A65 as a CYP3A ortholog that is constitutively expressed in gastrointestinal tissues, and is upregulated by treatment with dexamethasone, rifampicin or tetrachlorodibenzo-p-dioxin (TCDD). However, the underlying mechanism of TCDD-mediated CYP3A65 transcription is unclear. Here we generated two transgenic zebrafish, Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP), which contain 2.1 and 5.4 kb 5′ flanking sequences, respectively, of the CYP3A65 gene upstream of EGFP. Both transgenic lines express EGFP in larval gastrointestinal tissues in a pattern similar to that of the endogenous CYP3A65 gene. Moreover, EGFP expression can be significantly induced by TCDD exposure during the larval stage. In addition, EGFP expression can be stimulated by kynurenine, a putative AHR ligand produced during tryptophan metabolism. AHRE elements in the upstream regulatory region of the CYP3A65 gene are indispensible for basal and TCDD-induced transcription. Furthermore, the AHR2 DNA and ligand-binding domains are required to mediate effective CYP3A65 transcription. AHRE sequences are present in the promoters of many teleost CYP3 genes, but not of mammalian CYP3 genes, suggesting that AHR/AHR2-mediated transcription is likely a common regulatory mechanism for teleost CYP3 genes. It may also reflect the different environments that terrestrial and aquatic organisms encounter. - Highlights: • Tg(CYP3A65:EGFP) and CYP3A65 exhibits identical expression pattern. • CYP3A65 can be significantly induced by TCDD or kynurenine. • The AHRE elements are required to mediate CYP3A65 transcription. • The AHR2 DNA and ligand-binding domains are required for CYP3A65 transcription. • AHRE elements are present in many teleost CYP3 genes, but not in

Temporally-controlled site-specific recombination in zebrafish.

Directory of Open Access Journals (Sweden)

Stefan Hans

Full Text Available Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreER(T2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM or its active metabolite, 4-hydroxy-tamoxifen (4-OHT. Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms.

Whole-body and multispectral photoacoustic imaging of adult zebrafish

Science.gov (United States)

Huang, Na; Xi, Lei

2016-10-01

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

Identification and expression analysis of zebrafish glypicans during embryonic development.

Directory of Open Access Journals (Sweden)

Mansi Gupta

Full Text Available Heparan sulfate Proteoglycans (HSPG are ubiquitous molecules with indispensable functions in various biological processes. Glypicans are a family of HSPG's, characterized by a Gpi-anchor which directs them to the cell surface and/or extracellular matrix where they regulate growth factor signaling during development and disease. We report the identification and expression pattern of glypican genes from zebrafish. The zebrafish genome contains 10 glypican homologs, as opposed to six in mammals, which are highly conserved and are phylogenetically related to the mammalian genes. Some of the fish glypicans like Gpc1a, Gpc3, Gpc4, Gpc6a and Gpc6b show conserved synteny with their mammalian cognate genes. Many glypicans are expressed during the gastrulation stage, but their expression becomes more tissue specific and defined during somitogenesis stages, particularly in the developing central nervous system. Existence of multiple glypican orthologs in fish with diverse expression pattern suggests highly specialized and/or redundant function of these genes during embryonic development.

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

Science.gov (United States)

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.

Neutrophil Reverse Migration Becomes Transparent with Zebrafish

Directory of Open Access Journals (Sweden)

Taylor W. Starnes

2012-01-01

Full Text Available The precise control of neutrophil-mediated inflammation is critical for both host defense and the prevention of immunopathology. In vivo imaging studies in zebrafish, and more recently in mice, have made the novel observation that neutrophils leave a site of inflammation through a process called neutrophil reverse migration. The application of advanced imaging techniques to the genetically tractable, optically transparent zebrafish larvae was critical for these advances. Still, the mechanisms underlying neutrophil reverse migration and its effects on the resolution or priming of immune responses remain unclear. Here, we review the current knowledge of neutrophil reverse migration, its potential roles in host immunity, and the live imaging tools that make zebrafish a valuable model for increasing our knowledge of neutrophil behavior in vivo.

The neurogenetic frontier--lessons from misbehaving zebrafish.

Science.gov (United States)

Burgess, Harold A; Granato, Michael

2008-11-01

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

Planar cell polarity proteins differentially regulate extracellular matrix organization and assembly during zebrafish gastrulation.

Science.gov (United States)

Dohn, Michael R; Mundell, Nathan A; Sawyer, Leah M; Dunlap, Julie A; Jessen, Jason R

2013-11-01

Zebrafish gastrulation cell movements occur in the context of dynamic changes in extracellular matrix (ECM) organization and require the concerted action of planar cell polarity (PCP) proteins that regulate cell elongation and mediolateral alignment. Data obtained using Xenopus laevis gastrulae have shown that integrin-fibronectin interactions underlie the formation of polarized cell protrusions necessary for PCP and have implicated PCP proteins themselves as regulators of ECM. By contrast, the relationship between establishment of PCP and ECM assembly/remodeling during zebrafish gastrulation is unclear. We previously showed that zebrafish embryos carrying a null mutation in the four-pass transmembrane PCP protein vang-like 2 (vangl2) exhibit increased matrix metalloproteinase activity and decreased immunolabeling of fibronectin. These data implicated for the first time a core PCP protein in the regulation of pericellular proteolysis of ECM substrates and raised the question of whether other zebrafish PCP proteins also impact ECM organization. In Drosophila melanogaster, the cytoplasmic PCP protein Prickle binds Van Gogh and regulates its function. Here we report that similar to vangl2, loss of zebrafish prickle1a decreases fibronectin protein levels in gastrula embryos. We further show that Prickle1a physically binds Vangl2 and regulates both the subcellular distribution and total protein level of Vangl2. These data suggest that the ability of Prickle1a to impact fibronectin organization is at least partly due to effects on Vangl2. In contrast to loss of either Vangl2 or Prickle1a function, we find that glypican4 (a Wnt co-receptor) and frizzled7 mutant gastrula embryos with disrupted non-canonical Wnt signaling exhibit the opposite phenotype, namely increased fibronectin assembly. Our data show that glypican4 mutants do not have decreased proteolysis of ECM substrates, but instead have increased cell surface cadherin protein expression and increased intercellular

Reversibility of endocrine disruption in zebrafish (Danio rerio) after discontinued exposure to the estrogen 17α-ethinylestradiol

Energy Technology Data Exchange (ETDEWEB)

Baumann, Lisa, E-mail: lisa.baumann@vetsuisse.unibe.ch [Centre for Fish and Wildlife Health, Vetsuisse Faculty, University of Bern, PO Box 8466, CH-3001 Bern (Switzerland); Aquatic Ecology and Toxicology Section, Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg (Germany); Knörr, Susanne, E-mail: susanne.knoerr@gmx.de [Aquatic Ecology and Toxicology Section, Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg (Germany); Keiter, Susanne, E-mail: susanne.keiter@cos.uni-heidelberg.de [Aquatic Ecology and Toxicology Section, Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg (Germany); Rehberger, Kristina, E-mail: k.rehberger@stud.uni-heidelberg.de [Aquatic Ecology and Toxicology Section, Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg (Germany); Volz, Sina, E-mail: s.volz@stud.uni-heidelberg.de [Aquatic Ecology and Toxicology Section, Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg (Germany); Schiller, Viktoria, E-mail: schiller@molbiotech.rwth-aachen.de [Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstr. 6, D-52074 Aachen (Germany); Fenske, Martina, E-mail: martina.fenske@ime.fraunhofer.de [Fraunhofer Institute for Molecular Biology and Applied Ecology, Forckenbeckstr. 6, D-52074 Aachen (Germany); Holbech, Henrik, E-mail: hol@biology.sdu.dk [Department of Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M (Denmark); Segner, Helmut, E-mail: helmut.segner@vetsuisse.unibe.ch [Centre for Fish and Wildlife Health, Vetsuisse Faculty, University of Bern, PO Box 8466, CH-3001 Bern (Switzerland); Braunbeck, Thomas, E-mail: braunbeck@uni-hd.de [Aquatic Ecology and Toxicology Section, Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg (Germany)

2014-08-01

The aim of the present study was to investigate the persistence of the feminizing effects of discontinued 17α-ethinylestradiol (EE2) exposure on zebrafish (Danio rerio). An exposure scenario covering the sensitive phase of sexual differentiation, as well as final gonad maturation was chosen to examine the estrogenic effects on sexual development of zebrafish. Two exposure scenarios were compared: continuous exposure to environmentally relevant concentrations (0.1–10 ng/L EE2) up to 100 days post-hatch (dph) and developmental exposure up to 60 dph, followed by 40 days of depuration in clean water. The persistence of effects was investigated at different biological organization levels from mRNA to population-relevant endpoints to cover a broad range of important parameters. EE2 had a strong feminizing and inhibiting effect on the sexual development of zebrafish. Brain aromatase (cyp19b) mRNA expression showed no clear response, but vitellogenin levels were significantly elevated, gonad maturation and body growth were inhibited in both genders, and sex ratios were skewed towards females and undifferentiated individuals. To a large extent, all of these effects were reversed after 40 days of recovery, leading to the conclusion that exposure to the estrogen EE2 results in very strong, but reversible underdevelopment and feminization of zebrafish. The present study is the first to show this reversibility at different levels of organization, which gives better insight into the mechanistic basis of estrogenic effects in zebrafish. - Highlights: • Zebrafish were exposed to 17α-ethinylestradiol during their sexual differentiation. • Reversibility of effects was investigated after depuration of 40 days. • Morphological and physiological parameters were compared. • Zebrafish were able to recover at all different levels from mRNA to population.

Reversibility of endocrine disruption in zebrafish (Danio rerio) after discontinued exposure to the estrogen 17α-ethinylestradiol

International Nuclear Information System (INIS)

Baumann, Lisa; Knörr, Susanne; Keiter, Susanne; Rehberger, Kristina; Volz, Sina; Schiller, Viktoria; Fenske, Martina; Holbech, Henrik; Segner, Helmut; Braunbeck, Thomas

2014-01-01

The aim of the present study was to investigate the persistence of the feminizing effects of discontinued 17α-ethinylestradiol (EE2) exposure on zebrafish (Danio rerio). An exposure scenario covering the sensitive phase of sexual differentiation, as well as final gonad maturation was chosen to examine the estrogenic effects on sexual development of zebrafish. Two exposure scenarios were compared: continuous exposure to environmentally relevant concentrations (0.1–10 ng/L EE2) up to 100 days post-hatch (dph) and developmental exposure up to 60 dph, followed by 40 days of depuration in clean water. The persistence of effects was investigated at different biological organization levels from mRNA to population-relevant endpoints to cover a broad range of important parameters. EE2 had a strong feminizing and inhibiting effect on the sexual development of zebrafish. Brain aromatase (cyp19b) mRNA expression showed no clear response, but vitellogenin levels were significantly elevated, gonad maturation and body growth were inhibited in both genders, and sex ratios were skewed towards females and undifferentiated individuals. To a large extent, all of these effects were reversed after 40 days of recovery, leading to the conclusion that exposure to the estrogen EE2 results in very strong, but reversible underdevelopment and feminization of zebrafish. The present study is the first to show this reversibility at different levels of organization, which gives better insight into the mechanistic basis of estrogenic effects in zebrafish. - Highlights: • Zebrafish were exposed to 17α-ethinylestradiol during their sexual differentiation. • Reversibility of effects was investigated after depuration of 40 days. • Morphological and physiological parameters were compared. • Zebrafish were able to recover at all different levels from mRNA to population

Efficacy of Cleaning and Disinfection Procedures in a Zebrafish (Danio rerio) Facility

OpenAIRE

Garcia, Rachel L; Sanders, George E

2011-01-01

Appropriate cleaning and disinfection procedures in zebrafish (Danio rerio) laboratories are crucial in preventing the spread of aquatic animal pathogens and minimizing the build-up of waste products and biologic matter. The procedures selected should accomplish these goals and incorporate the individual needs of various laboratories. In this study of a single zebrafish facility, we assessed the efficacy of 2 different cleaning and disinfection procedures for nets, tanks, and lids. ATP levels...

Carbonic anhydrase 5 regulates acid-base homeostasis in zebrafish.

Directory of Open Access Journals (Sweden)

Ruben Postel

Full Text Available The regulation of the acid-base balance in cells is essential for proper cellular homeostasis. Disturbed acid-base balance directly affects cellular physiology, which often results in various pathological conditions. In every living organism, the protein family of carbonic anhydrases regulate a broad variety of homeostatic processes. Here we describe the identification, mapping and cloning of a zebrafish carbonic anhydrase 5 (ca5 mutation, collapse of fins (cof, which causes initially a collapse of the medial fins followed by necrosis and rapid degeneration of the embryo. These phenotypical characteristics can be mimicked in wild-type embryos by acetazolamide treatment, suggesting that CA5 activity in zebrafish is essential for a proper development. In addition we show that CA5 regulates acid-base balance during embryonic development, since lowering the pH can compensate for the loss of CA5 activity. Identification of selective modulators of CA5 activity could have a major impact on the development of new therapeutics involved in the treatment of a variety of disorders.

Zebrafish in Toxicology and Environmental Health.

Science.gov (United States)

Bambino, Kathryn; Chu, Jaime

2017-01-01

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

Axonal regeneration in zebrafish spinal cord

Science.gov (United States)

Hui, Subhra Prakash

2018-01-01

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

Effects of alpha particles on zebrafish embryos

International Nuclear Information System (INIS)

Yum, E.H.W.; Choi, V.W.Y.; Yu, K.N.; Li, V.W.T.; Cheng, S.H.

2008-01-01

Full text: Ionizing radiation such as X-ray and alpha particles can damage cellular macromolecules, which can lead to DNA single- and double-strand breaks. In the present work, we studied the effects of alpha particles on dechorionated zebrafish embryos. Thin polyallyldiglycol carbonate (PADC) films with a thickness of 16 μm were prepared from commercially available PADC films (with thickness of 100 μm) by chemical etching and used as support substrates for holding zebrafish embryos for alpha-particle irradiation. These films recorded alpha-particle hit positions, quantified the number and energy of alpha particles actually incident on the embryo cells, and thus enabled the calculation of the dose absorbed by the embryo cells. Irradiation was made at 1.25 hours post fertilization (hpf) with various absorbed dose. TdT-mediated dUTP Nick-End Labeling (TUNEL) assay was performed on the embryos at different time stages after irradiation. Marked apoptosis was detected only in embryos at earlier time stages. The results showed that DNA double-strand break during zebrafish embryogenesis can be induced by alpha-particle irradiation, which suggests that zebrafish is a potential model for assessing the effects of alpha-particle radiation

Toxicity evaluation of biodegradable chitosan nanoparticles using a zebrafish embryo model

Directory of Open Access Journals (Sweden)

Hu YL

2011-12-01

Full Text Available Yu-Lan Hu1, Wang Qi1, Feng Han2, Jian-Zhong Shao3, Jian-Qing Gao11Institute of Pharmaceutics, College of Pharmaceutical Sciences, 2Institute of Pharmacology, Toxicology and Biochemical Pharmaceutics, 3College of Life Sciences, Key Laboratory for Cell and Gene Engineering of Zhejiang Province, Zhejiang University, Hangzhou, Zhejiang, People's Republic of ChinaBackground: Although there are a number of reports regarding the toxicity evaluation of inorganic nanoparticles, knowledge on biodegradable nanomaterials, which have always been considered safe, is still limited. For example, the toxicity of chitosan nanoparticles, one of the most widely used drug/gene delivery vehicles, is largely unknown. In the present study, the zebrafish model was used for a safety evaluation of this nanocarrier.Methods: Chitosan nanoparticles with two particle sizes were prepared by ionic cross-linking of chitosan with sodium tripolyphosphate. Chitosan nanoparticles of different concentrations were incubated with zebrafish embryos, and ZnO nanoparticles were used as the positive control.Results: Embryo exposure to chitosan nanoparticles and ZnO nanoparticles resulted in a decreased hatching rate and increased mortality, which was concentration-dependent. Chitosan nanoparticles at a size of 200 nm caused malformations, including a bent spine, pericardial edema, and an opaque yolk in zebrafish embryos. Furthermore, embryos exposed to chitosan nanoparticles showed an increased rate of cell death, high expression of reactive oxygen species, as well as overexpression of heat shock protein 70, indicating that chitosan nanoparticles can cause physiological stress in zebrafish. The results also suggest that the toxicity of biodegradable nanocarriers such as chitosan nanoparticles must be addressed, especially considering the in vivo distribution of these nanoscaled particles.Conclusion: Our results add new insights into the potential toxicity of nanoparticles produced by

Homeostatic response to sleep/rest deprivation by constant water flow in larval zebrafish in both dark and light conditions.

Science.gov (United States)

Aho, Vilma; Vainikka, Maija; Puttonen, Henri A J; Ikonen, Heidi M K; Salminen, Tiia; Panula, Pertti; Porkka-Heiskanen, Tarja; Wigren, Henna-Kaisa

2017-06-01

Sleep-or sleep-like states-have been reported in adult and larval zebrafish using behavioural criteria. These reversible quiescent periods, displaying circadian rhythmicity, have been used in pharmacological, genetic and neuroanatomical studies of sleep-wake regulation. However, one of the important criteria for sleep, namely sleep homeostasis, has not been demonstrated unequivocally. To study rest homeostasis in zebrafish larvae, we rest-deprived 1-week-old larvae with a novel, ecologically relevant method: flow of water. Stereotyped startle responses to sensory stimuli were recorded after the rest deprivation to study arousal threshold using a high-speed camera, providing an appropriate time resolution to detect species-specific behavioural responses occurring in a millisecond time-scale. Rest-deprived larvae exhibited fewer startle responses than control larvae during the remaining dark phase and the beginning of the light phase, which can be interpreted as a sign of rest homeostasis-often used as equivalent of sleep homeostasis. To address sleep homeostasis further, we probed the adenosinergic system, which in mammals regulates sleep homeostasis. The adenosine A1 receptor agonist, cyclohexyladenosine, administered during the light period, decreased startle responses and increased immobility bouts, while the adenosine antagonist, caffeine, administered during the dark period, decreased immobility bouts. These results suggest that the regulation of sleep homeostasis in zebrafish larvae consists of the same elements as that of other species. © 2017 European Sleep Research Society.

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

Science.gov (United States)

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

2013-09-17

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

A New Anaesthetic Protocol for Adult Zebrafish (Danio rerio: Propofol Combined with Lidocaine.

Directory of Open Access Journals (Sweden)

Ana M Valentim

Full Text Available The increasing use of zebrafish model has not been accompanied by the evolution of proper anaesthesia for this species in research. The most used anaesthetic in fishes, MS222, may induce aversion, reduction of heart rate, and consequently high mortality, especially during long exposures. Therefore, we aim to explore new anaesthetic protocols to be used in zebrafish by studying the quality of anaesthesia and recovery induced by different concentrations of propofol alone and in combination with different concentrations of lidocaine.In experiment A, eighty-three AB zebrafish were randomly assigned to 7 different groups: control, 2.5 (2.5P, 5 (5P or 7.5 μg/ml (7.5P of propofol; and 2.5 μg/ml of propofol combined with 50, (P/50L, 100 (P/100L or 150 μg/ml (P/150L of lidocaine. Zebrafish were placed in an anaesthetic water bath and time to lose the equilibrium, reflex to touch, reflex to a tail pinch, and respiratory rate were measured. Time to gain equilibrium was also assessed in a clean tank. Five and 24 hours after anaesthesia recovery, zebrafish were evaluated concerning activity and reactivity. Afterwards, in a second phase of experiments (experiment B, the best protocol of the experiment A was compared with a new group of 8 fishes treated with 100 mg/L of MS222 (100M.In experiment A, only different concentrations of propofol/lidocaine combination induced full anaesthesia in all animals. Thus only these groups were compared with a standard dose of MS222 in experiment B. Propofol/lidocaine induced a quicker loss of equilibrium, and loss of response to light and painful stimuli compared with MS222. However zebrafish treated with MS222 recovered quickly than the ones treated with propofol/lidocaine.In conclusion, propofol/lidocaine combination and MS222 have advantages in different situations. MS222 is ideal for minor procedures when a quick recovery is important, while propofol/lidocaine is best to induce a quick and complete anaesthesia.

Zebrafish Expression Ontology of Gene Sets (ZEOGS): A Tool to Analyze Enrichment of Zebrafish Anatomical Terms in Large Gene Sets

Science.gov (United States)

Marsico, Annalisa

2013-01-01

Abstract The zebrafish (Danio rerio) is an established model organism for developmental and biomedical research. It is frequently used for high-throughput functional genomics experiments, such as genome-wide gene expression measurements, to systematically analyze molecular mechanisms. However, the use of whole embryos or larvae in such experiments leads to a loss of the spatial information. To address this problem, we have developed a tool called Zebrafish Expression Ontology of Gene Sets (ZEOGS) to assess the enrichment of anatomical terms in large gene sets. ZEOGS uses gene expression pattern data from several sources: first, in situ hybridization experiments from the Zebrafish Model Organism Database (ZFIN); second, it uses the Zebrafish Anatomical Ontology, a controlled vocabulary that describes connected anatomical structures; and third, the available connections between expression patterns and anatomical terms contained in ZFIN. Upon input of a gene set, ZEOGS determines which anatomical structures are overrepresented in the input gene set. ZEOGS allows one for the first time to look at groups of genes and to describe them in terms of shared anatomical structures. To establish ZEOGS, we first tested it on random gene selections and on two public microarray datasets with known tissue-specific gene expression changes. These tests showed that ZEOGS could reliably identify the tissues affected, whereas only very few enriched terms to none were found in the random gene sets. Next we applied ZEOGS to microarray datasets of 24 and 72 h postfertilization zebrafish embryos treated with beclomethasone, a potent glucocorticoid. This analysis resulted in the identification of several anatomical terms related to glucocorticoid-responsive tissues, some of which were stage-specific. Our studies highlight the ability of ZEOGS to extract spatial information from datasets derived from whole embryos, indicating that ZEOGS could be a useful tool to automatically analyze gene

Zebrafish Expression Ontology of Gene Sets (ZEOGS): a tool to analyze enrichment of zebrafish anatomical terms in large gene sets.

Science.gov (United States)

Prykhozhij, Sergey V; Marsico, Annalisa; Meijsing, Sebastiaan H

2013-09-01

The zebrafish (Danio rerio) is an established model organism for developmental and biomedical research. It is frequently used for high-throughput functional genomics experiments, such as genome-wide gene expression measurements, to systematically analyze molecular mechanisms. However, the use of whole embryos or larvae in such experiments leads to a loss of the spatial information. To address this problem, we have developed a tool called Zebrafish Expression Ontology of Gene Sets (ZEOGS) to assess the enrichment of anatomical terms in large gene sets. ZEOGS uses gene expression pattern data from several sources: first, in situ hybridization experiments from the Zebrafish Model Organism Database (ZFIN); second, it uses the Zebrafish Anatomical Ontology, a controlled vocabulary that describes connected anatomical structures; and third, the available connections between expression patterns and anatomical terms contained in ZFIN. Upon input of a gene set, ZEOGS determines which anatomical structures are overrepresented in the input gene set. ZEOGS allows one for the first time to look at groups of genes and to describe them in terms of shared anatomical structures. To establish ZEOGS, we first tested it on random gene selections and on two public microarray datasets with known tissue-specific gene expression changes. These tests showed that ZEOGS could reliably identify the tissues affected, whereas only very few enriched terms to none were found in the random gene sets. Next we applied ZEOGS to microarray datasets of 24 and 72 h postfertilization zebrafish embryos treated with beclomethasone, a potent glucocorticoid. This analysis resulted in the identification of several anatomical terms related to glucocorticoid-responsive tissues, some of which were stage-specific. Our studies highlight the ability of ZEOGS to extract spatial information from datasets derived from whole embryos, indicating that ZEOGS could be a useful tool to automatically analyze gene expression

Dietary β-glucan enhances the contents of complement component 3 and factor B in eggs of zebrafish.

Science.gov (United States)

Jiang, Chengyan; Wang, Peng; Li, Mengyang; Liu, Shousheng; Zhang, Shicui

2016-12-01

β-glucan has been shown to increase non-specific immunity and resistance against infections or pathogenic bacteria in several fish species, but no information is available regarding its trans-generational effects to date. Here we clearly demonstrated that β-glucan enhanced the contents of immune-relevant molecules C3 and Bf in eggs of zebrafish, and the embryos derived from β-1,3 glucan-treated zebrafish were more resistant to bacterial challenge than control embryos. Moreover, the transferred C3 and Bf were directly associated with the antimicrobial defense of early embryos. In addition, feeding female zebrafish with β-glucan had little detrimental effects on the number of spawned eggs and their embryonic development. Collectively, these data show for the first time that β-glucan can be safely used to promote the non-specific immunity in offspring of fishes. Copyright © 2016 Elsevier Ltd. All rights reserved.

Defects of the Glycinergic Synapse in Zebrafish

Science.gov (United States)

Ogino, Kazutoyo; Hirata, Hiromi

2016-01-01

Glycine mediates fast inhibitory synaptic transmission. Physiological importance of the glycinergic synapse is well established in the brainstem and the spinal cord. In humans, the loss of glycinergic function in the spinal cord and brainstem leads to hyperekplexia, which is characterized by an excess startle reflex to sudden acoustic or tactile stimulation. In addition, glycinergic synapses in this region are also involved in the regulation of respiration and locomotion, and in the nociceptive processing. The importance of the glycinergic synapse is conserved across vertebrate species. A teleost fish, the zebrafish, offers several advantages as a vertebrate model for research of glycinergic synapse. Mutagenesis screens in zebrafish have isolated two motor defective mutants that have pathogenic mutations in glycinergic synaptic transmission: bandoneon (beo) and shocked (sho). Beo mutants have a loss-of-function mutation of glycine receptor (GlyR) β-subunit b, alternatively, sho mutant is a glycinergic transporter 1 (GlyT1) defective mutant. These mutants are useful animal models for understanding of glycinergic synaptic transmission and for identification of novel therapeutic agents for human diseases arising from defect in glycinergic transmission, such as hyperekplexia or glycine encephalopathy. Recent advances in techniques for genome editing and for imaging and manipulating of a molecule or a physiological process make zebrafish more attractive model. In this review, we describe the glycinergic defective zebrafish mutants and the technical advances in both forward and reverse genetic approaches as well as in vivo visualization and manipulation approaches for the study of the glycinergic synapse in zebrafish. PMID:27445686

Small GTPase R-Ras participates in neural tube formation in zebrafish embryonic spinal cord.

Science.gov (United States)

Ohata, Shinya; Uga, Hideko; Okamoto, Hitoshi; Katada, Toshiaki

2018-06-27

Ras related (R-Ras), a small GTPase, is involved in the maintenance of apico-basal polarity in neuroepithelial cells of the zebrafish hindbrain, axonal collapse in cultured murine hippocampal neurons, and maturation of blood vessels in adult mice. However, the role of R-Ras in neural tube formation remains unknown. Using antisense morpholino oligonucleotides (AMOs), we found that in the spinal cord of zebrafish embryos, the lumen was formed bilaterally in rras morphants, whereas it was formed at the midline in control embryos. As AMO can cause off-target effects, we generated rras mutant zebrafish lines using CRISPR/Cas9 technology. Although these rras mutant embryos did not have a bilateral lumen in the spinal cord, the following findings suggest that the phenotype is unlikely due to an off-target effect of rras AMO: 1) The rras morphant phenotype was rescued by an injection of AMO-resistant rras mRNA, and 2) a bilaterally segregated spinal cord was not observed in rras mutant embryos injected with rras AMO. The results suggest that the function of other ras family genes may be redundant in rras mutants. Previous research reported a bilaterally formed lumen in the spinal cord of zebrafish embryos with a mutation in a planar cell polarity (PCP) gene, van gogh-like 2 (vangl2). In the present study, in cultured cells, R-Ras was co-immunoprecipitated with Vangl2 but not with another PCP regulator, Pricke1. Interestingly, the interaction between R-Ras and Vangl2 was stronger in guanine-nucleotide free point mutants of R-Ras than in wild-type or constitutively active (GTP-bound) forms of R-Ras. R-Ras may regulate neural tube formation in cooperation with Vangl2 in the developing zebrafish spinal cord. Copyright © 2018 Elsevier Inc. All rights reserved.

The zebrafish progranulin gene family and antisense transcripts

Directory of Open Access Journals (Sweden)

Baranowski David

2005-11-01

Full Text Available Abstract Background Progranulin is an epithelial tissue growth factor (also known as proepithelin, acrogranin and PC-cell-derived growth factor that has been implicated in development, wound healing and in the progression of many cancers. The single mammalian progranulin gene encodes a glycoprotein precursor consisting of seven and one half tandemly repeated non-identical copies of the cystine-rich granulin motif. A genome-wide duplication event hypothesized to have occurred at the base of the teleost radiation predicts that mammalian progranulin may be represented by two co-orthologues in zebrafish. Results The cDNAs encoding two zebrafish granulin precursors, progranulins-A and -B, were characterized and found to contain 10 and 9 copies of the granulin motif respectively. The cDNAs and genes encoding the two forms of granulin, progranulins-1 and -2, were also cloned and sequenced. Both latter peptides were found to be encoded by precursors with a simplified architecture consisting of one and one half copies of the granulin motif. A cDNA encoding a chimeric progranulin which likely arises through the mechanism of trans-splicing between grn1 and grn2 was also characterized. A non-coding RNA gene with antisense complementarity to both grn1 and grn2 was identified which may have functional implications with respect to gene dosage, as well as in restricting the formation of the chimeric form of progranulin. Chromosomal localization of the four progranulin (grn genes reveals syntenic conservation for grna only, suggesting that it is the true orthologue of mammalian grn. RT-PCR and whole-mount in situ hybridization analysis of zebrafish grns during development reveals that combined expression of grna and grnb, but not grn1 and grn2, recapitulate many of the expression patterns observed for the murine counterpart. This includes maternal deposition, widespread central nervous system distribution and specific localization within the epithelial

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

Science.gov (United States)

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

2015-01-01

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

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

Science.gov (United States)

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

2017-09-01

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

Developmental toxicity and oxidative stress induced by gamma irradiation in zebrafish embryos.

Science.gov (United States)

Hu, Miao; Hu, Nan; Ding, Dexin; Zhao, Weichao; Feng, Yongfu; Zhang, Hui; Li, Guangyue; Wang, Yongdong

2016-11-01

This study aimed to evaluate the biological effects of gamma irradiation on zebrafish embryos. Different doses of gamma rays (0.01, 0.05, 0.1, 0.5 and 1 Gy) were used to irradiate zebrafish embryos at three developmental stages (stage 1, 6 h post-fertilization (hpf); stage 2, 12 hpf; stage three, 24 hpf), respectively. The survival, malformation and hatching rates of the zebrafish embryos were measured at the morphological endpoint of 96 hpf. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST) were assayed. Morphology analysis showed that gamma irradiation inhibited hatching and induced developmental toxicity in a dose-dependent manner. Interestingly, after irradiation the malformation rate changed not only in a dose-dependent manner but also in a developmental stage-dependent manner, indicating that the zebrafish embryos at stage 1 were more sensitive to gamma rays than those at other stages. Biochemical analysis showed that gamma irradiation modulated the activities of antioxidant enzymes in a dose-dependent manner. A linear relationship was found between GPx activity and irradiation dose in 0.1-1 Gy group, and GPx was a suitable biomarker for gamma irradiation in the dose range from 0.1 to 1 Gy. Furthermore, the activities of SOD, CAT, GR and GPx of the zebrafish embryos at stage 3 were found to be much higher than those at other stages, indicating that the zebrafish embryos at stage 3 had a greater ability to protect against gamma rays than those at other stages, and thus the activities of antioxidant enzymes changed in a developmental stage-dependent manner.

Identification of Chemical Inhibitors of β-Catenin-Driven Liver Tumorigenesis in Zebrafish.

Directory of Open Access Journals (Sweden)

Kimberley J Evason

2015-07-01

Full Text Available Hepatocellular carcinoma (HCC is one of the most lethal human cancers. The search for targeted treatments has been hampered by the lack of relevant animal models for the genetically diverse subsets of HCC, including the 20-40% of HCCs that are defined by activating mutations in the gene encoding β-catenin. To address this chemotherapeutic challenge, we created and characterized transgenic zebrafish expressing hepatocyte-specific activated β-catenin. By 2 months post fertilization (mpf, 33% of transgenic zebrafish developed HCC in their livers, and 78% and 80% of transgenic zebrafish showed HCC at 6 and 12 mpf, respectively. As expected for a malignant process, transgenic zebrafish showed significantly decreased mean adult survival compared to non-transgenic control siblings. Using this novel transgenic model, we screened for druggable pathways that mediate β-catenin-induced liver growth and identified two c-Jun N-terminal kinase (JNK inhibitors and two antidepressants (one tricyclic antidepressant, amitriptyline, and one selective serotonin reuptake inhibitor that suppressed this phenotype. We further found that activated β-catenin was associated with JNK pathway hyperactivation in zebrafish and in human HCC. In zebrafish larvae, JNK inhibition decreased liver size specifically in the presence of activated β-catenin. The β-catenin-specific growth-inhibitory effect of targeting JNK was conserved in human liver cancer cells. Our other class of hits, antidepressants, has been used in patient treatment for decades, raising the exciting possibility that these drugs could potentially be repurposed for cancer treatment. In support of this proposal, we found that amitriptyline decreased tumor burden in a mouse HCC model. Our studies implicate JNK inhibitors and antidepressants as potential therapeutics for β-catenin-induced liver tumors.

Developmental toxicity and oxidative stress induced by gamma irradiation in zebrafish embryos

International Nuclear Information System (INIS)

Hu, Miao; Hu, Nan; Ding, Dexin; Zhao, Weichao; Feng, Yongfu; Zhang, Hui; Li, Guangyue; Wang, Yongdong

2016-01-01

This study aimed to evaluate the biological effects of gamma irradiation on zebrafish embryos. Different doses of gamma rays (0.01, 0.05, 0.1, 0.5 and 1 Gy) were used to irradiate zebrafish embryos at three developmental stages (stage 1, 6 h post-fertilization (hpf); stage 2, 12 hpf; stage three, 24 hpf), respectively. The survival, malformation and hatching rates of the zebrafish embryos were measured at the morphological endpoint of 96 hpf. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST) were assayed. Morphology analysis showed that gamma irradiation inhibited hatching and induced developmental toxicity in a dose-dependent manner. Interestingly, after irradiation the malformation rate changed not only in a dose-dependent manner but also in a developmental stage-dependent manner, indicating that the zebrafish embryos at stage 1 were more sensitive to gamma rays than those at other stages. Biochemical analysis showed that gamma irradiation modulated the activities of antioxidant enzymes in a dose-dependent manner. A linear relationship was found between GPx activity and irradiation dose in 0.1-1 Gy group, and GPx was a suitable biomarker for gamma irradiation in the dose range from 0.1 to 1 Gy. Furthermore, the activities of SOD, CAT, GR and GPx of the zebrafish embryos at stage 3 were found to be much higher than those at other stages, indicating that the zebrafish embryos at stage 3 had a greater ability to protect against gamma rays than those at other stages, and thus the activities of antioxidant enzymes changed in a developmental stage-dependent manner. (orig.)

Developmental toxicity and oxidative stress induced by gamma irradiation in zebrafish embryos

Energy Technology Data Exchange (ETDEWEB)

Hu, Miao; Hu, Nan; Ding, Dexin; Zhao, Weichao; Feng, Yongfu; Zhang, Hui; Li, Guangyue; Wang, Yongdong [University of South China, Key Discipline Laboratory for National Defense for Biotechnology in Uranium Mining and Hydrometallurgy, Hengyang, Hunan Province (China)

2016-11-15

This study aimed to evaluate the biological effects of gamma irradiation on zebrafish embryos. Different doses of gamma rays (0.01, 0.05, 0.1, 0.5 and 1 Gy) were used to irradiate zebrafish embryos at three developmental stages (stage 1, 6 h post-fertilization (hpf); stage 2, 12 hpf; stage three, 24 hpf), respectively. The survival, malformation and hatching rates of the zebrafish embryos were measured at the morphological endpoint of 96 hpf. The activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione S-transferase (GST) were assayed. Morphology analysis showed that gamma irradiation inhibited hatching and induced developmental toxicity in a dose-dependent manner. Interestingly, after irradiation the malformation rate changed not only in a dose-dependent manner but also in a developmental stage-dependent manner, indicating that the zebrafish embryos at stage 1 were more sensitive to gamma rays than those at other stages. Biochemical analysis showed that gamma irradiation modulated the activities of antioxidant enzymes in a dose-dependent manner. A linear relationship was found between GPx activity and irradiation dose in 0.1-1 Gy group, and GPx was a suitable biomarker for gamma irradiation in the dose range from 0.1 to 1 Gy. Furthermore, the activities of SOD, CAT, GR and GPx of the zebrafish embryos at stage 3 were found to be much higher than those at other stages, indicating that the zebrafish embryos at stage 3 had a greater ability to protect against gamma rays than those at other stages, and thus the activities of antioxidant enzymes changed in a developmental stage-dependent manner. (orig.)

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

Directory of Open Access Journals (Sweden)

Emma D. Spikol

2016-03-01

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

ESX-5-deficient Mycobacterium marinum is hypervirulent in adult zebrafish

KAUST Repository

Weerdenburg, Eveline M.

2012-02-15

ESX-5 is a mycobacterial type VII protein secretion system responsible for transport of numerous PE and PPE proteins. It is involved in the induction of host cell death and modulation of the cytokine response in vitro. In this work, we studied the effects of ESX-5 in embryonic and adult zebrafish using Mycobacterium marinum. We found that ESX-5-deficient M.marinum was slightly attenuated in zebrafish embryos. Surprisingly, the same mutant showed highly increased virulence in adult zebrafish, characterized by increased bacterial loads and early onset of granuloma formation with rapid development of necrotic centres. This early onset of granuloma formation was accompanied by an increased expression of pro-inflammatory cytokines and tissue remodelling genes in zebrafish infected with the ESX-5 mutant. Experiments using RAG-1-deficient zebrafish showed that the increased virulence of the ESX-5 mutant was not dependent on the adaptive immune system. Mixed infection experiments with wild-type and ESX-5 mutant bacteria showed that the latter had a specific advantage in adult zebrafish and outcompeted wild-type bacteria. Together our experiments indicate that ESX-5-mediated protein secretion is used by M.marinum to establish a moderate and persistent infection. © 2012 Blackwell Publishing Ltd.

The neurogenetic frontier—lessons from misbehaving zebrafish

Science.gov (United States)

Granato, Michael

2008-01-01

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

Long-Term Social Recognition Memory in Zebrafish.

Science.gov (United States)

Madeira, Natália; Oliveira, Rui F

2017-08-01

In species in which individuals live in stable social groups, individual recognition is expected to evolve to allow individuals to remember past interactions with different individuals and adjust future behavior toward them accordingly. Thus, social memory is expected to be a ubiquitous component of social cognition of social species. However, few studies have investigated the occurrence of social memory in non-mammals. Here we evaluated the ability of zebrafish (Danio rerio) to recognize different conspecifics and to retain this information in long lasting (i.e. 24 h) memories. We used a social discrimination paradigm, adapted from mouse studies, in which the focal individual meets two pairs of conspecifics in two consecutive days: one conspecific is the same in both days and the other is different between days 1 and 2. If animals have the ability to discriminate between different conspecifics, it is predicted that they will spend more time exploring the novel than the familiar (i.e. already seen in day 1) conspecific. In this study, zebrafish with access to both olfactory and visual conspecific cues exhibited consistent recognition of a previously encountered (familiar) conspecific after a 24 h delay. This result supports the hypothesis that long-term social memory, previously described in mammals, is also present in zebrafish, hence extending the evidence for the presence of this type of memory to teleost fish.

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

Science.gov (United States)

Varshney, Gaurav Kumar

2014-01-01

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

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

Science.gov (United States)

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.

Functional Investigation of a Non-coding Variant Associated with Adolescent Idiopathic Scoliosis in Zebrafish: Elevated Expression of the Ladybird Homeobox Gene Causes Body Axis Deformation.

Directory of Open Access Journals (Sweden)

Long Guo

2016-01-01

Full Text Available Previously, we identified an adolescent idiopathic scoliosis susceptibility locus near human ladybird homeobox 1 (LBX1 and FLJ41350 by a genome-wide association study. Here, we characterized the associated non-coding variant and investigated the function of these genes. A chromosome conformation capture assay revealed that the genome region with the most significantly associated single nucleotide polymorphism (rs11190870 physically interacted with the promoter region of LBX1-FLJ41350. The promoter in the direction of LBX1, combined with a 590-bp region including rs11190870, had higher transcriptional activity with the risk allele than that with the non-risk allele in HEK 293T cells. The ubiquitous overexpression of human LBX1 or either of the zebrafish lbx genes (lbx1a, lbx1b, and lbx2, but not FLJ41350, in zebrafish embryos caused body curvature followed by death prior to vertebral column formation. Such body axis deformation was not observed in transcription activator-like effector nucleases mediated knockout zebrafish of lbx1b or lbx2. Mosaic expression of lbx1b driven by the GATA2 minimal promoter and the lbx1b enhancer in zebrafish significantly alleviated the embryonic lethal phenotype to allow observation of the later onset of the spinal curvature with or without vertebral malformation. Deformation of the embryonic body axis by lbx1b overexpression was associated with defects in convergent extension, which is a component of the main axis-elongation machinery in gastrulating embryos. In embryos overexpressing lbx1b, wnt5b, a ligand of the non-canonical Wnt/planar cell polarity (PCP pathway, was significantly downregulated. Injection of mRNA for wnt5b or RhoA, a key downstream effector of Wnt/PCP signaling, rescued the defective convergent extension phenotype and attenuated the lbx1b-induced curvature of the body axis. Thus, our study presents a novel pathological feature of LBX1 and its zebrafish homologs in body axis deformation at

Generation of FGF reporter transgenic zebrafish and their utility in chemical screens

Directory of Open Access Journals (Sweden)

Tsang Michael

2007-06-01

Full Text Available Abstract Background Fibroblast Growth Factors (FGFs represent a large family of secreted proteins that are required for proper development and physiological processes. Mutations in mouse and zebrafish FGFs result in abnormal embryogenesis and lethality. A key to understanding the precise role for these factors is to determine their spatial and temporal activity during embryogenesis. Results Expression of Dual Specificity Phosphatase 6 (dusp6, also known as Mkp3 is controlled by FGF signalling throughout development. The Dusp6 promoter was isolated from zebrafish and used to drive expression of destabilized green fluorescent protein (d2EGFP in transgenic embryos (Tg(Dusp6:d2EGFP. Expression of d2EGFP is initiated as early as 4 hours post-fertilization (hpf within the future dorsal region of the embryo, where fgf3 and fgf8 are initially expressed. At later stages, d2EGFP is detected within structures that correlate with the expression of Fgf ligands and their receptors. This includes the mid-hindbrain boundary (MHB, pharyngeal endoderm, otic vesicle, hindbrain, and Kupffer's vesicle. The expression of d2EGFP is under the control of FGF signalling as treatment with FGF Receptor (FGFR inhibitors results in the suppression of d2EGFP expression. In a pilot screen of commercially available small molecules we have evaluated the effectiveness of the transgenic lines to identify specific FGF inhibitors within the class of indolinones. These compounds were counter screened with the transgenic line Tg(Fli1:EGFPy1, that serves as an indirect read-out for Vascular Endothelial Growth Factor (VEGF signalling in order to determine the specificity between related receptor tyrosine kinases (RTKs. From these assays it is possible to determine the specificity of these indolinones towards specific RTK signalling pathways. This has enabled the identification of compounds that can block specifically the VEGFR or the FGFR signalling pathway. Conclusion The generation of

Evaluation of color preference in zebrafish for learning and memory.

Science.gov (United States)

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

2012-01-01

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

The rise of photoresponsive protein technologies applications in vivo: a spotlight on zebrafish developmental and cell biology [version 1; referees: 2 approved

Directory of Open Access Journals (Sweden)

Renee Wei-Yan Chow

2017-04-01

Full Text Available The zebrafish (Danio rerio is a powerful vertebrate model to study cellular and developmental processes in vivo. The optical clarity and their amenability to genetic manipulation make zebrafish a model of choice when it comes to applying optical techniques involving genetically encoded photoresponsive protein technologies. In recent years, a number of fluorescent protein and optogenetic technologies have emerged that allow new ways to visualize, quantify, and perturb developmental dynamics. Here, we explain the principles of these new tools and describe some of their representative applications in zebrafish.

Earth-strength magnetic field affects the rheotactic threshold of zebrafish swimming in shoals.

Science.gov (United States)

Cresci, Alessandro; De Rosa, Rosario; Putman, Nathan F; Agnisola, Claudio

2017-02-01

Rheotaxis, the unconditioned orienting response to water currents, is a main component of fish behavior. Rheotaxis is achieved using multiple sensory systems, including visual and tactile cues. Rheotactic orientation in open or low-visibility waters might also benefit from the stable frame of reference provided by the geomagnetic field, but this possibility has not been explored before. Zebrafish (Danio rerio) form shoals living in freshwater systems with low visibility, show a robust positive rheotaxis, and respond to geomagnetic fields. Here, we investigated whether a static magnetic field in the Earth-strength range influenced the rheotactic threshold of zebrafish in a swimming tunnel. The direction of the horizontal component of the magnetic field relative to water flow influenced the rheotactic threshold of fish as part of a shoal, but not of fish tested alone. Results obtained after disabling the lateral line of shoaling individuals with Co 2+ suggest that this organ system is involved in the observed magneto-rheotactic response. These findings constitute preliminary evidence that magnetic fields influence rheotaxis and suggest new avenues for further research. Copyright © 2016 Elsevier Inc. All rights reserved.

Knocking Down Snrnp200 Initiates Demorphogenesis of Rod Photoreceptors in Zebrafish

Directory of Open Access Journals (Sweden)

Yuan Liu

2015-01-01

Full Text Available Purpose. The small nuclear ribonucleoprotein 200 kDa (SNRNP200 gene is a fundamental component for precursor message RNA (pre-mRNA splicing and has been implicated in the etiology of autosomal dominant retinitis pigmentosa (adRP. This study aims to determine the consequences of knocking down Snrnp200 in zebrafish. Methods. Expression of the Snrnp200 transcript in zebrafish was determined via whole mount in situ hybridization. Morpholino oligonucleotide (MO aiming to knock down the expression of Snrnp200 was injected into zebrafish embryos, followed by analyses of aberrant splicing and expression of the U4/U6-U5 tri-small nuclear ribonucleoproteins (snRNPs components and retina-specific transcripts. Systemic changes and retinal phenotypes were further characterized by histological study and immunofluorescence staining. Results. Snrnp200 was ubiquitously expressed in zebrafish. Knocking down Snrnp200 in zebrafish triggered aberrant splicing of the cbln1 gene, upregulation of other U4/U6-U5 tri-snRNP components, and downregulation of a panel of retina-specific transcripts. Systemic defects were found correlated with knockdown of Snrnp200 in zebrafish. Only demorphogenesis of rod photoreceptors was detected in the initial stage, mimicking the disease characteristics of RP. Conclusions. We conclude that knocking down Snrnp200 in zebrafish could alter regular splicing and expression of a panel of genes, which may eventually trigger rod defects.

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

Directory of Open Access Journals (Sweden)

Mai Nguyen-Chi

2014-07-01

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

A novel transgenic zebrafish model for blood-brain and blood-retinal barrier development

Directory of Open Access Journals (Sweden)

Sugimoto Masahiko

2010-07-01

Full Text Available Abstract Background Development and maintenance of the blood-brain and blood-retinal barrier is critical for the homeostasis of brain and retinal tissue. Despite decades of research our knowledge of the formation and maintenance of the blood-brain (BBB and blood-retinal (BRB barrier is very limited. We have established an in vivo model to study the development and maintenance of these barriers by generating a transgenic zebrafish line that expresses a vitamin D-binding protein fused with enhanced green fluorescent protein (DBP-EGFP in blood plasma, as an endogenous tracer. Results The temporal establishment of the BBB and BRB was examined using this transgenic line and the results were compared with that obtained by injection of fluorescent dyes into the sinus venosus of embryos at various stages of development. We also examined the expression of claudin-5, a component of tight junctions during the first 4 days of development. We observed that the BBB of zebrafish starts to develop by 3 dpf, with expression of claudin-5 in the central arteries preceding it at 2 dpf. The hyaloid vasculature in the zebrafish retina develops a barrier function at 3 dpf, which endows the zebrafish with unique advantages for studying the BRB. Conclusion Zebrafish embryos develop BBB and BRB function simultaneously by 3 dpf, which is regulated by tight junction proteins. The Tg(l-fabp:DBP-EGFP zebrafish will have great advantages in studying development and maintenance of the blood-neural barrier, which is a new application for the widely used vertebrate model.

Characterization of Proliferating Neural Progenitors after Spinal Cord Injury in Adult Zebrafish.

Directory of Open Access Journals (Sweden)

Subhra Prakash Hui

Full Text Available Zebrafish can repair their injured brain and spinal cord after injury unlike adult mammalian central nervous system. Any injury to zebrafish spinal cord would lead to increased proliferation and neurogenesis. There are presences of proliferating progenitors from which both neuronal and glial loss can be reversed by appropriately generating new neurons and glia. We have demonstrated the presence of multiple progenitors, which are different types of proliferating populations like Sox2+ neural progenitor, A2B5+ astrocyte/ glial progenitor, NG2+ oligodendrocyte progenitor, radial glia and Schwann cell like progenitor. We analyzed the expression levels of two common markers of dedifferentiation like msx-b and vimentin during regeneration along with some of the pluripotency associated factors to explore the possible role of these two processes. Among the several key factors related to pluripotency, pou5f1 and sox2 are upregulated during regeneration and associated with activation of neural progenitor cells. Uncovering the molecular mechanism for endogenous regeneration of adult zebrafish spinal cord would give us more clues on important targets for future therapeutic approach in mammalian spinal cord repair and regeneration.

Genome-wide analysis of Tol2 transposon reintegration in zebrafish.

Science.gov (United States)

Kondrychyn, Igor; Garcia-Lecea, Marta; Emelyanov, Alexander; Parinov, Sergey; Korzh, Vladimir

2009-09-08

Tol2, a member of the hAT family of transposons, has become a useful tool for genetic manipulation of model animals, but information about its interactions with vertebrate genomes is still limited. Furthermore, published reports on Tol2 have mainly been based on random integration of the transposon system after co-injection of a plasmid DNA harboring the transposon and a transposase mRNA. It is important to understand how Tol2 would behave upon activation after integration into the genome. We performed a large-scale enhancer trap (ET) screen and generated 338 insertions of the Tol2 transposon-based ET cassette into the zebrafish genome. These insertions were generated by remobilizing the transposon from two different donor sites in two transgenic lines. We found that 39% of Tol2 insertions occurred in transcription units, mostly into introns. Analysis of the transposon target sites revealed no strict specificity at the DNA sequence level. However, Tol2 was prone to target AT-rich regions with weak palindromic consensus sequences centered at the insertion site. Our systematic analysis of sequential remobilizations of the Tol2 transposon from two independent sites within a vertebrate genome has revealed properties such as a tendency to integrate into transcription units and into AT-rich palindrome-like sequences. This information will influence the development of various applications involving DNA transposons and Tol2 in particular.

Developmental lead exposure causes startle response deficits in zebrafish.

Science.gov (United States)

Rice, Clinton; Ghorai, Jugal K; Zalewski, Kathryn; Weber, Daniel N

2011-10-01

Lead (Pb(2+)) exposure continues to be an important concern for fish populations. Research is required to assess the long-term behavioral effects of low-level concentrations of Pb(2+) and the physiological mechanisms that control those behaviors. Newly fertilized zebrafish embryos (max) and escape time. With increasing exposure concentrations, a larger number of larvae failed to respond to even the initial tap and, for those that did respond, ceased responding earlier than control larvae. These differences were more pronounced at a frequency of 4 taps/s. (2) Response to a visual stimulus: Fish, exposed as embryos (2-24 hpf) to Pb(2+) (0-10 μM) were tested as adults under low light conditions (≈ 60 μW/m(2)) for visual responses to a rotating black bar. Visual responses were significantly degraded at Pb(2+) concentrations of 30 nM. These data suggest that zebrafish are viable models for short- and long-term sensorimotor deficits induced by acute, low-level developmental Pb(2+) exposures. Copyright © 2011 Elsevier B.V. All rights reserved.

Triclabendazole sulfoxide causes stage-dependent embryolethality in zebrafish and mouse in vitro.

Directory of Open Access Journals (Sweden)

Nuria Boix

Full Text Available Fascioliasis and paragonimiasis are widespread foodborne trematode diseases, affecting millions of people in more than 75 countries. The treatment of choice for these parasitic diseases is based on triclabendazole, a benzimidazole derivative which has been suggested as a promising drug to treat pregnant women and children. However, at the moment, this drug is not approved for human use in most countries. Its potential adverse effects on embryonic development have been scarcely studied, and it has not been assigned a pregnancy category by the FDA. Thus, to help in the process of risk-benefit decision making upon triclabendazole treatment during pregnancy, a better characterization of its risks during gestation is needed.The zebrafish embryo test, a preimplantation and a postimplantation rodent whole embryo culture were used to investigate the potential embryotoxicity/teratogenicity of triclabendazole and its first metabolite triclabendazole sulfoxide. Albendazole and albendazole sulfoxide were included as positive controls.Triclabendazole was between 10 and 250 times less potent than albendazole in inducing dysmorphogenic effects in zebrafish or postimplantation rodent embryos, respectively. However, during the preimplantation period, both compounds, triclabendazole and triclabendazole sulfoxide, induced a dose-dependent embryolethal effect after only 24 h of exposure in rodent embryos and zebrafish (lowest observed adverse effect concentrations = 10 μM.In humans, after ingestion of the recommended doses of triclabendazole to treat fascioliasis and paragonimiasis (10 mg/kg, the main compound found in plasma is triclabendazole sulfoxide (maximum concentration 38.6 μM, while triclabendazole concentrations are approximately 30 times lower (1.16 μM. From our results it can be concluded that triclabendazole, at concentrations of the same order of magnitude as the clinically relevant ones, does not entail teratogenic potential in vitro during the

Evaluation of MWNT toxic effects on daphnia and zebrafish embryos

Energy Technology Data Exchange (ETDEWEB)

Olasagasti, Maider; Rainieri, Sandra [AZTI-TECNALIA, Parque Tecnologico de Bizkaia 609, 48160 Derio (Spain)], E-mail: srainieri@azti.es; Alvarez, Noelia; Vera, Carolina [INASMET-TECNALIA, Mikeletegi pasealekua, 2, Parque Tecnologico, 20009 San Sebastian (Spain)

2009-05-01

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

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

Science.gov (United States)

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

2015-12-01

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

Behavioral changes in response to sound exposure and no spatial avoidance of noisy conditions in captive zebrafish.

Science.gov (United States)

Neo, Yik Yaw; Parie, Lisa; Bakker, Frederique; Snelderwaard, Peter; Tudorache, Christian; Schaaf, Marcel; Slabbekoorn, Hans

2015-01-01

Auditory sensitivity in fish serves various important functions, but also makes fish susceptible to noise pollution. Human-generated sounds may affect behavioral patterns of fish, both in natural conditions and in captivity. Fish are often kept for consumption in aquaculture, on display in zoos and hobby aquaria, and for medical sciences in research facilities, but little is known about the impact of ambient sounds in fish tanks. In this study, we conducted two indoor exposure experiments with zebrafish (Danio rerio). The first experiment demonstrated that exposure to moderate sound levels (112 dB re 1 μPa) can affect the swimming behavior of fish by changing group cohesion, swimming speed and swimming height. Effects were brief for both continuous and intermittent noise treatments. In the second experiment, fish could influence exposure to higher sound levels by swimming freely between an artificially noisy fish tank (120-140 dB re 1 μPa) and another with ambient noise levels (89 dB re 1 μPa). Despite initial startle responses, and a brief period in which many individuals in the noisy tank dived down to the bottom, there was no spatial avoidance or noise-dependent tank preference at all. The frequent exchange rate of about 60 fish passages per hour between tanks was not affected by continuous or intermittent exposures. In conclusion, small groups of captive zebrafish were able to detect sounds already at relatively low sound levels and adjust their behavior to it. Relatively high sound levels were at least at the on-set disturbing, but did not lead to spatial avoidance. Further research is needed to show whether zebrafish are not able to avoid noisy areas or just not bothered. Quantitatively, these data are not directly applicable to other fish species or other fish tanks, but they do indicate that sound exposure may affect fish behavior in any captive condition.

L-histidine enhances learning in stressed zebrafish

Directory of Open Access Journals (Sweden)

L.P.V. Cofiel

2009-01-01

Full Text Available The aim of the present study was to determine the effect of the histaminergic precursor L-histidine and the H3 receptor antagonist thioperamide on the learning process of zebrafish submitted or not to confinement stress. On each of the 5 consecutive days of experiment (D1, D2, D3, D4, D5, animals had to associate an interruption of the aquarium air supply with food offering. Non-stressed zebrafish received an intraperitoneal injection of 100 mg/kg L-histidine, 10 mg/kg thioperamide or saline after training. Stressed animals received drug treatment and then were submitted to confinement stress for 1 h before the learning procedure. Time to approach the feeder was measured (in seconds and was considered to be indicative of learning. A decrease in time to approach the feeder was observed in the saline-treated group (D1 = 141.92 ± 13.57; D3 = 55 ± 13.54, indicating learning. A delay in learning of stressed animals treated with saline was observed (D1 = 217.5 ± 25.66. L-histidine facilitated learning in stressed (D1 = 118.68 ± 13.9; D2 = 45.88 ± 8.2 and non-stressed (D1 = 151.11 ± 19.20; D5 = 62 ± 14.68 animals. Thioperamide inhibited learning in non-stressed (D1 = 110.38 ± 9.49; D4 = 58.79 ± 16.83 and stressed animals (D1 = 167.3 ± 26.39; D5 = 172.15 ± 27.35. L-histidine prevented the increase in blood glucose after one session of confinement (L-histidine = 65.88 ± 4.50; control = 53 ± 3.50 mg/dL. These results suggest that the histaminergic system enhances learning and modulates stress responses in zebrafish.

Specific nanotoxicity of graphene oxide during zebrafish embryogenesis.

Science.gov (United States)

Chen, Yuming; Hu, Xiangang; Sun, Jing; Zhou, Qixing

2016-01-01

Graphene oxide (GO) has shown great potential for biological, medical, energy and electronic applications. As a consequence of these diverse applications, GO release into the ecosystem is inevitable; however, the corresponding risks are largely unknown, particularly with respect to the critical period of embryogenesis. This study revealed that GO adhered to and enveloped the chorion of zebrafish embryos mainly via hydroxyl group interactions, blocked the pore canals of the chorionic membrane, and caused marked hypoxia and hatching delay. Furthermore, GO spontaneously penetrated the chorion, entered the embryo via endocytosis, damaged the mitochondria and primarily translocated to the eye, heart and yolk sac regions, which are involved in the circulatory system of zebrafish. In these organs, GO induced excessive generation of reactive oxygen species and increased oxidative stress, DNA damage and apoptosis. Graphene oxide also induced developmental malformation of the eye, cardiac/yolk sac edema, tail flexure and heart rate reduction. In contrast to the common dose-effect relationships of nanoparticles, the adverse effects of GO on heart rate and tail/spinal cord flexure increased and then decreased as the GO concentration increased. These findings emphasize the specific adverse effects of GO on embryogenesis and highlight the potential ecological and health risks of GO.

Thyroid hormone regulates muscle function during cold acclimation in zebrafish (Danio rerio).

Science.gov (United States)

Little, Alexander G; Seebacher, Frank

2013-09-15

Thyroid hormone (TH) is a universal regulator of growth, development and metabolism during cold exposure in mammals. In zebrafish (Danio rerio), TH regulates locomotor performance and metabolism during cold acclimation. The influence of TH on locomotor performance may be via its effect on metabolism or, as has been shown in mammals, by modulating muscle phenotypes. Our aim was to determine whether TH influences muscle phenotypes in zebrafish, and whether this could explain changes in swimming capacity in response to thermal acclimation. We used propylthiouracil and iopanoic acid to induce hypothyroidism in zebrafish over a 3-week acclimation period to either 18 or 28°C. To verify that physiological changes following hypothyroid treatment were in fact due to the action of TH, we supplemented hypothyroid fish with 3,5-diiodothryronine (T2) or 3,5,3'-triiodothyronine (T3). Cold-acclimated fish had significantly greater sustained swimming performance (Ucrit) but not burst speed. Greater Ucrit was accompanied by increased tail beat frequency, but there was no change in tail beat amplitude. Hypothyroidism significantly decreased Ucrit and burst performance, as well as tail beat frequency and SERCA activity in cold-acclimated fish. However, myofibrillar ATPase activity increased in cold-acclimated hypothyroid fish. Hypothyroid treatment also decreased mRNA concentrations of myosin heavy chain fast isoforms and SERCA 1 isoform in cold-acclimated fish. SERCA 1 mRNA increased in warm-acclimated hypothyroid fish, and SERCA 3 mRNA decreased in both cold- and warm-acclimated hypothyroid fish. Supplementation with either T2 or T3 restored Ucrit, burst speed, tail beat frequency, SERCA activity and myosin heavy chain and SERCA 1 and 3 mRNA levels of hypothyroid fish back to control levels. We show that in addition to regulating development and metabolism in vertebrates, TH also regulates muscle physiology in ways that affect locomotor performance in fish. We suggest that the

Discovery of Quinoline-Derived Trifluoromethyl Alcohols, Determination of Their in vivo Toxicity and Anticancer Activity in a Zebrafish Embryo Model.

Science.gov (United States)

Sittaramane, Vinoth; Padgett, Jihan; Salter, Philip; Williams, Ashley; Luke, Shauntelle; McCall, Rebecca; Arambula, Jonathan F; Graves, Vincent B; Blocker, Mark; Van Leuven, David; Bowe, Keturah; Heimberger, Julia; Cade, Hannah C; Immaneni, Supriya; Shaikh, Abid

2015-11-01

In this study the rational design, synthesis, and anticancer activity of quinoline-derived trifluoromethyl alcohols were evaluated. Members of this novel class of trifluoromethyl alcohols were identified as potent growth inhibitors in a zebrafish embryo model. Synthesis of these compounds was carried out with an sp(3) -C-H functionalization strategy of methyl quinolines with trifluoromethyl ketones. A zebrafish embryo model was also used to explore the toxicity of ethyl 4,4,4-trifluoro-3-hydroxy-3-(quinolin-2-ylmethyl)butanoate (1), 2-benzyl-1,1,1-trifluoro-3-(quinolin-2-yl)propan-2-ol (2), and trifluoro-3-(isoquinolin-1-yl)-2-(thiophen-2-yl)propan-2-ol (3). Compounds 2 and 3 were found to be more toxic than compound 1; apoptotic staining assays indicated that compound 3 causes increased cell death. In vitro cell proliferation assays showed that compound 2, with an LC50 value of 14.14 μm, has more potent anticancer activity than cisplatin. This novel class of inhibitors provides a new direction in the discovery of effective anticancer agents. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Simple, economical heat-shock devices for zebrafish housing racks.

Science.gov (United States)

Duszynski, Robert J; Topczewski, Jacek; LeClair, Elizabeth E

2011-12-01

One reason for the popularity of the zebrafish (Danio rerio) as a model vertebrate is the ability to manipulate gene expression in this organism. A common method is to induce gene expression transiently under control of a heat-shock promoter (e.g., hsp70l). By making simple mechanical adjustments to small aquarium heaters (25-50W), we were able to produce consistent and reliable heat-shock conditions within a conventional zebrafish housing system. Up to two heat-shock intervals per day (>37°C) could be maintained under conditions of continuous flow (5-25 mL/min). Temperature logging every 30 s indicated rapid warm up times, consistent heat-shock lengths, and accurate and precise peak water temperatures (mean±SD=38°C±0.2°C). The biological effects of these heat-shock treatments were confirmed by observing inducible expression of enhanced green fluorescent protein (EGFP) and inhibition of caudal fin regeneration in a transgenic fish line expressing a dominant negative fibroblast growth factor receptor (Tg(hsp70l:dnfgfr1-EGFP)(pd1)). These devices are inexpensive, easily modified, and can be calibrated to accommodate a variety of experimental designs. After setup on a programmable timer, the heaters require no intervention to produce consistent daily heat shocks, and all other standard care protocols can be followed in the fish facility. The simplicity and stability of these devices make them suitable for long-term heat shocks at any stage of the zebrafish lifecycle (>7 days postfertilization), and useful for both laboratory and classroom experiments on transgenic zebrafish.

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

Science.gov (United States)

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.

Exercise quantity-dependent muscle hypertrophy in adult zebrafish (Danio rerio).

Science.gov (United States)

Hasumura, Takahiro; Meguro, Shinichi

2016-07-01

Exercise is very important for maintaining and increasing skeletal muscle mass, and is particularly important to prevent and care for sarcopenia and muscle disuse atrophy. However, the dose-response relationship between exercise quantity, duration/day, and overall duration and muscle mass is poorly understood. Therefore, we investigated the effect of exercise duration on skeletal muscle to reveal the relationship between exercise quantity and muscle hypertrophy in zebrafish forced to exercise. Adult male zebrafish were exercised 6 h/day for 4 weeks, 6 h/day for 2 weeks, or 3 h/day for 2 weeks. Flow velocity was adjusted to maximum velocity during continual swimming (initial 43 cm/s). High-speed consecutive photographs revealed that zebrafish mainly drove the caudal part. Additionally, X-ray micro computed tomography measurements indicated muscle hypertrophy of the mid-caudal half compared with the mid-cranial half part. The cross-sectional analysis of the mid-caudal half muscle revealed that skeletal muscle (red, white, or total) mass increased with increasing exercise quantity, whereas that of white muscle and total muscle increased only under the maximum exercise load condition of 6 h/day for 4 weeks. Additionally, the muscle fiver size distributions of exercised fish were larger than those from non-exercised fish. We revealed that exercise quantity, duration/day, and overall duration were correlated with skeletal muscle hypertrophy. The forced exercise model enabled us to investigate the relationship between exercise quantity and skeletal muscle mass. These results open up the possibility for further investigations on the effects of exercise on skeletal muscle in adult zebrafish.

Nitric oxide permits hypoxia-induced lymphatic perfusion by controlling arterial-lymphatic conduits in zebrafish and glass catfish

DEFF Research Database (Denmark)

Dahl Ejby Jensen, Lasse; Cao, Renhai; Hedlund, Eva-Maria

2009-01-01

and lymphatic perfusion. Here we show in the adult zebrafish and glass catfish (Kryptopterus bicirrhis) that blood-lymphatic conduits directly connect arterial vessels to the lymphatic system. Under hypoxic conditions, arterial-lymphatic conduits (ALCs) became highly dilated and linearized by NO...

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

International Nuclear Information System (INIS)

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

2014-01-01

Highlights: • TBEP exposure decreased the survival of zebrafish embryos/larvae. • TBEP exposure led to its bioconcentration in zebrafish lavare. • TBEP caused developmental toxicity by inhibiting the degradation and utilization of nutrients. • TBEP exposure caused developmental toxicity by inducing apoptosis. - Abstract: 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 (0.8–100 mg/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

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

Energy Technology Data Exchange (ETDEWEB)

Han, Zhihua [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing 210023 (China); Wang, Qiangwei [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China); Fu, Jie [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing 210023 (China); Chen, Hongshan [State Environmental Protection Key Laboratory of Wetland Ecology and Vegetation Restoration, School of the Environment, Northeast Normal University, Changchun 130024 (China); Department of Biological Sciences, National University of Singapore (Singapore); Zhao, Ye [Department of Biological Sciences, National University of Singapore (Singapore); Zhou, Bingsheng [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072 (China); Gong, Zhiyuan [Department of Biological Sciences, National University of Singapore (Singapore); Wei, Si; Li, Jun; Liu, Hongling; Zhang, Xiaowei [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing 210023 (China); Liu, Chunsheng, E-mail: liuchunshengidid@126.com [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing 210023 (China); Department of Biological Sciences, National University of Singapore (Singapore); College of Fisheries, Huazhong Agricultural University, Wuhan 430070 (China); Yu, Hongxia, E-mail: yuhx@nju.edu.cn [State Key Laboratory of Pollution Control and Resource Reuse and School of the Environment, Nanjing University, Nanjing 210023 (China)

2014-05-01

Highlights: • TBEP exposure decreased the survival of zebrafish embryos/larvae. • TBEP exposure led to its bioconcentration in zebrafish lavare. • TBEP caused developmental toxicity by inhibiting the degradation and utilization of nutrients. • TBEP exposure caused developmental toxicity by inducing apoptosis. - Abstract: 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 (0.8–100 mg/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.

Dissection of vertebrate hematopoiesis using zebrafish thrombopoietin

Czech Academy of Sciences Publication Activity Database

Svoboda, Ondřej; Stachura, D.L.; Machoňová, Olga; Pajer, Petr; Brynda, Jiří; Zon, L.I.; Traver, D.; Bartůněk, Petr

2014-01-01

Roč. 124, č. 2 (2014), s. 220-228 ISSN 0006-4971 R&D Projects: GA ČR GAP305/10/0953 Grant - others:NIH(US) K01-DK087814-01A1; NIH(US) R01-DK074482 Keywords : Zebrafish * hematopoiesis * progenitors * thrombopoietin * erythropoietin Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 10.452, year: 2014

Cryopreservation of zebrafish (Danio rerio) oocytes by vitrification.

Science.gov (United States)

Guan, M; Rawson, D M; Zhang, T

2010-01-01

Cryopreservation of fish oocytes is challenging because these oocytes have low membrane permeability to water and cryoprotectant and are highly chilling sensitive. Vitrification is considered to be a promising approach for their cryopreservation as it involves rapid freezing and thawing of the oocytes and therefore minimising the chilling injury. In the present study, vitrification properties and the toxicity of a range of vitrification solutions containing different concentrations of Me2SO, methanol, propylene glycol and ethylene glycol were investigated. Two different base media and vitrification methods were compared. The effect of different post-thaw dilution solutions together with incubation periods on oocyte viability were also investigated. Stage III zebrafish oocytes were equilibrated in increasing concentrations of cryoprotectants for 30 min in 3 steps. Oocytes were thawed rapidly in a water bath and cryoprotectants were removed in 4 steps. Oocyte viability was assessed using trypan blue staining. The results showed that vitrification solutions V3 and V4 in KCl buffer had low toxicity and vitrified well. The survivals of oocytes after stepwise dilution using solutions containing permeable cryoprotectants were significant higher than those diluted in 0.5M glucose, and the use of CVA65 vitrification system improved oocyte survival when compared with plastic straws after 30 min at 22 degrees C post-thawing. Cryopreservation of zebrafish oocytes by vitrification is reported here for the first time, although oocyte survivals after cryopreservation assessed by trypan blue staining were relatively high shortly after thawing, they became swollen and translucent after incubation in KCl buffer. Further studies are needed to optimise the post-thaw culturing conditions.

A bioenergetic model for zebrafish Danio rerio (Hamilton)

Science.gov (United States)

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

2008-01-01

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

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

Science.gov (United States)

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

2015-12-05

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

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

Science.gov (United States)

Mwaffo, Violet; Butail, Sachit; Porfiri, Maurizio

2017-01-01

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

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

Science.gov (United States)

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

2017-09-29

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

Zebrafish transgenic constructs label specific neurons in Xenopus laevis spinal cord and identify frog V0v spinal neurons.

Science.gov (United States)

Juárez-Morales, José L; Martinez-De Luna, Reyna I; Zuber, Michael E; Roberts, Alan; Lewis, Katharine E

2017-09-01

A correctly functioning spinal cord is crucial for locomotion and communication between body and brain but there are fundamental gaps in our knowledge of how spinal neuronal circuitry is established and functions. To understand the genetic program that regulates specification and functions of this circuitry, we need to connect neuronal molecular phenotypes with physiological analyses. Studies using Xenopus laevis tadpoles have increased our understanding of spinal cord neuronal physiology and function, particularly in locomotor circuitry. However, the X. laevis tetraploid genome and long generation time make it difficult to investigate how neurons are specified. The opacity of X. laevis embryos also makes it hard to connect functional classes of neurons and the genes that they express. We demonstrate here that Tol2 transgenic constructs using zebrafish enhancers that drive expression in specific zebrafish spinal neurons label equivalent neurons in X. laevis and that the incorporation of a Gal4:UAS amplification cassette enables cells to be observed in live X. laevis tadpoles. This technique should enable the molecular phenotypes, morphologies and physiologies of distinct X. laevis spinal neurons to be examined together in vivo. We have used an islet1 enhancer to label Rohon-Beard sensory neurons and evx enhancers to identify V0v neurons, for the first time, in X. laevis spinal cord. Our work demonstrates the homology of spinal cord circuitry in zebrafish and X. laevis, suggesting that future work could combine their relative strengths to elucidate a more complete picture of how vertebrate spinal cord neurons are specified, and function to generate behavior. © 2017 Wiley Periodicals, Inc. Develop Neurobiol 77: 1007-1020, 2017. © 2017 Wiley Periodicals, Inc.

The Hypocretin/Orexin Neuronal Networks in Zebrafish.

Science.gov (United States)

Elbaz, Idan; Levitas-Djerbi, Talia; Appelbaum, Lior

2017-01-01

The hypothalamic Hypocretin/Orexin (Hcrt) neurons secrete two Hcrt neuropeptides. These neurons and peptides play a major role in the regulation of feeding, sleep wake cycle, reward-seeking, addiction, and stress. Loss of Hcrt neurons causes the sleep disorder narcolepsy. The zebrafish has become an attractive model to study the Hcrt neuronal network because it is a transparent vertebrate that enables simple genetic manipulation, imaging of the structure and function of neuronal circuits in live animals, and high-throughput monitoring of behavioral performance during both day and night. The zebrafish Hcrt network comprises ~16-60 neurons, which similar to mammals, are located in the hypothalamus and widely innervate the brain and spinal cord, and regulate various fundamental behaviors such as feeding, sleep, and wakefulness. Here we review how the zebrafish contributes to the study of the Hcrt neuronal system molecularly, anatomically, physiologically, and pathologically.

Zebrafish as a Model for the Study of Human Myeloid Malignancies

Directory of Open Access Journals (Sweden)

Jeng-Wei Lu

2015-01-01

Full Text Available Myeloid malignancies are heterogeneous disorders characterized by uncontrolled proliferation or/and blockage of differentiation of myeloid progenitor cells. Although a substantial number of gene alterations have been identified, the mechanism by which these abnormalities interact has yet to be elucidated. Over the past decades, zebrafish have become an important model organism, especially in biomedical research. Several zebrafish models have been developed to recapitulate the characteristics of specific myeloid malignancies that provide novel insight into the pathogenesis of these diseases and allow the evaluation of novel small molecule drugs. This report will focus on illustrative examples of applications of zebrafish models, including transgenesis, zebrafish xenograft models, and cell transplantation approaches, to the study of human myeloid malignancies.

Cadmium(Cd)-induced oxidative stress down-regulates the gene expression of DNA mismatch recognition proteins MutS homolog 2 (MSH2) and MSH6 in zebrafish (Danio rerio) embryos

Energy Technology Data Exchange (ETDEWEB)

Hsu, Todd, E-mail: toddhsu@mail.ntou.edu.tw [Institute of Bioscience and Biotechnology and Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan (China); Huang, Kuan-Ming; Tsai, Huei-Ting; Sung, Shih-Tsung; Ho, Tsung-Nan [Institute of Bioscience and Biotechnology and Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung 20224, Taiwan (China)

2013-01-15

DNA mismatch repair (MMR) of simple base mismatches and small insertion-deletion loops in eukaryotes is initiated by the binding of the MutS homolog 2 (MSH2)-MSH6 heterodimer to mismatched DNA. Cadmium (Cd) is a genotoxic heavy metal that has been recognized as a human carcinogen. Oxidant stress and inhibition of DNA repair have been proposed as major factors underlying Cd genotoxicity. Our previous studies indicated the ability of Cd to disturb the gene expression of MSH6 in zebrafish (Danio rerio) embryos. This study was undertaken to explore if Cd-induced oxidative stress down-regulated MSH gene activities. Following the exposure of zebrafish embryos at 1 h post fertilization (hpf) to sublethal concentrations of Cd at 3-5 {mu}M for 4 or 9 h, a parallel down-regulation of MSH2, MSH6 and Cu/Zn superoxide dismutase (Cu/Zn-SOD) gene expression was detected by real-time RT-PCR and the expression levels were 40-50% of control after a 9-h exposure. Cd exposure also induced oxidative stress, yet no inhibition of catalase gene activity was observed. Whole mount in situ hybridization revealed a wide distribution of msh6 mRNA in the head regions of 10 hpf embryos and pretreatment of embryos with antioxidants butylhydroxytoluene (BHT), D-mannitol or N-acetylcysteine (NAC) at 1-10 {mu}M restored Cd-suppressed msh6 expression. QPCR confirmed the protective effects of antioxidants on Cd-suppressed msh2/msh6 mRNA production. Down-regulated MSH gene activities reaching about 50% of control were also induced in embryos exposed to paraquat, a reactive oxygen species (ROS)-generating herbicide, or hydrogen peroxide at 200 {mu}M. Hence, Cd at sublethal levels down-regulates msh2/msh6 expression primarily via ROS as signaling molecules. The transcriptional activation of human msh6 is known to be fully dependent on the specificity factor 1 (Sp1). Cd failed to inhibit the DNA binding activity of zebrafish Sp1 unless at lethal concentrations based on band shift assay, therefore

A novel anti-tumor inhibitor identified by virtual screen with PLK1 structure and zebrafish assay.

Directory of Open Access Journals (Sweden)

Jing Lu

Full Text Available Polo-like kinase 1 (PLK1, one of the key regulators of mitosis, is a target for cancer therapy due to its abnormally high activity in several tumors. Plk1 is highly conserved and shares a nearly identical 3-D structure between zebrafish and humans. The initial 10 mitoses of zebrafish embryonic cleavages occur every∼30 minutes, and therefore provide a rapid assay to evaluate mitosis inhibitors including those targeting Plk1. To increase efficiency and specificity, we first performed a computational virtual screen of∼60000 compounds against the human Plk1 3-D structure docked to both its kinase and Polo box domain. 370 candidates with the top free-energy scores were subjected to zebrafish assay and 3 were shown to inhibit cell division. Compared to general screen for compounds inhibiting zebrafish embryonic cleavage, computation increased the efficiency by 11 folds. One of the 3 compounds, named I2, was further demonstrated to effectively inhibit multiple tumor cell proliferation in vitro and PC3 prostate cancer growth in Xenograft mouse model in vivo. Furthermore, I2 inhibited Plk1 enzyme activity in a dose dependent manner. The IC50 values of I2 in these assays are compatible to those of ON-01910, a Plk1 inhibitor currently in Phase III clinic trials. Our studies demonstrate that zebrafish assays coupled with computational screening significantly improves the efficiency of identifying specific regulators of biological targets. The PLK1 inhibitor I2, and its analogs, may have potential in cancer therapeutics.

Uncoupling nicotine mediated motoneuron axonal pathfinding errors and muscle degeneration in zebrafish

International Nuclear Information System (INIS)

Welsh, Lillian; Tanguay, Robert L.; Svoboda, Kurt R.

2009-01-01

Zebrafish embryos offer a unique opportunity to investigate the mechanisms by which nicotine exposure impacts early vertebrate development. Embryos exposed to nicotine become functionally paralyzed by 42 hpf suggesting that the neuromuscular system is compromised in exposed embryos. We previously demonstrated that secondary spinal motoneurons in nicotine-exposed embryos were delayed in development and that their axons made pathfinding errors (Svoboda, K.R., Vijayaraghaven, S., Tanguay, R.L., 2002. Nicotinic receptors mediate changes in spinal motoneuron development and axonal pathfinding in embryonic zebrafish exposed to nicotine. J. Neurosci. 22, 10731-10741). In that study, we did not consider the potential role that altered skeletal muscle development caused by nicotine exposure could play in contributing to the errors in spinal motoneuron axon pathfinding. In this study, we show that an alteration in skeletal muscle development occurs in tandem with alterations in spinal motoneuron development upon exposure to nicotine. The alteration in the muscle involves the binding of nicotine to the muscle-specific AChRs. The nicotine-induced alteration in muscle development does not occur in the zebrafish mutant (sofa potato, [sop]), which lacks muscle-specific AChRs. Even though muscle development is unaffected by nicotine exposure in sop mutants, motoneuron axonal pathfinding errors still occur in these mutants, indicating a direct effect of nicotine exposure on nervous system development.

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

Science.gov (United States)

Scata, Kimberly A; El-Deiry, Wafik S

2004-06-01

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

Functional validation of GWAS gene candidates for abnormal liver function during zebrafish liver development

Directory of Open Access Journals (Sweden)

Leah Y. Liu

2013-09-01

Genome-wide association studies (GWAS have revealed numerous associations between many phenotypes and gene candidates. Frequently, however, further elucidation of gene function has not been achieved. A recent GWAS identified 69 candidate genes associated with elevated liver enzyme concentrations, which are clinical markers of liver disease. To investigate the role of these genes in liver homeostasis, we narrowed down this list to 12 genes based on zebrafish orthology, zebrafish liver expression and disease correlation. To assess the function of gene candidates during liver development, we assayed hepatic progenitors at 48 hours post fertilization (hpf and hepatocytes at 72 hpf using in situ hybridization following morpholino knockdown in zebrafish embryos. Knockdown of three genes (pnpla3, pklr and mapk10 decreased expression of hepatic progenitor cells, whereas knockdown of eight genes (pnpla3, cpn1, trib1, fads2, slc2a2, pklr, mapk10 and samm50 decreased cell-specific hepatocyte expression. We then induced liver injury in zebrafish embryos using acetaminophen exposure and observed changes in liver toxicity incidence in morphants. Prioritization of GWAS candidates and morpholino knockdown expedites the study of newly identified genes impacting liver development and represents a feasible method for initial assessment of candidate genes to instruct further mechanistic analyses. Our analysis can be extended to GWAS for additional disease-associated phenotypes.

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

Science.gov (United States)

Pan, Keyao; Deem, Michael

2011-03-01

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

Simultaneous quantification of T4, T3, rT3, 3,5-T2 and 3,3'-T2 in larval zebrafish (Danio rerio) as a model to study exposure to polychlorinated biphenyls.

Science.gov (United States)

Chen, Xiaopeng; Walter, Kyla M; Miller, Galen W; Lein, Pamela J; Puschner, Birgit

2018-06-01

Environmental toxicants that interfere with thyroid hormone (TH) signaling can impact growth and development in animals and humans. Zebrafish represent a model to study chemically induced TH disruption, prompting the need for sensitive detection of THs. Simultaneous quantification of 3,3',5-triiodo-l-thyronine (T3), thyroxine (T4), 3,3',5'-triiodo-l-thyronine (rT3), 3,5-diiodo-l-thyronine (3,5-T2) and 3,3'-diiodo-l-thyronine (3,3'-T2) in zebrafish larvae was achieved by ultra-performance liquid chromatography-tandem mass spectrometry in positive ion mode. Solid-phase extraction with SampliQ cartridges and derivatization with 3 m hydrochloric acid in n-butanol reduced matrix effects. Derivatized compounds were separated on an Acquity UPLC BEH C 18 column with mobile phases consisting of 0.1% acetic acid in deionized water and 0.1% acetic acid in methanol. The limits of detection ranged from 0.5 to 0.6 pg injected on column. The method was validated by evaluating recovery (77.1-117.2%), accuracy (87.3-123.9%) and precision (0.5-12.4%) using diluted homogenized zebrafish embryos spiked with all target compounds. This method was then applied to zebrafish larvae collected after 114 h of exposure to polychlorinated biphenyls (PCBs), including PCB 28, PCB 66 and PCB 95, or the technical mixture Aroclor 1254. Exposure to PCB 28 and PCB 95 increased the T4:T3 ratio and decreased the T3:rT3 ratio, demonstrating that this method can effectively detect PCB-induced alterations in THs. Copyright © 2018 John Wiley & Sons, Ltd.

Activation of the Nkx2.5–Calr–p53 signaling pathway by hyperglycemia induces cardiac remodeling and dysfunction in adult zebrafish

Directory of Open Access Journals (Sweden)

Yanyi Sun

2017-10-01

Full Text Available Hyperglycemia is an independent risk factor for diabetic cardiomyopathy in humans; however, the underlying mechanisms have not been thoroughly elucidated. Zebrafish (Danio rerio was used in this study as a novel vertebrate model to explore the signaling pathways of human adult cardiomyopathy. Hyperglycemia was induced by alternately immersing adult zebrafish in a glucose solution or water. The hyperglycemic fish gradually exhibited some hallmarks of cardiomyopathy such as myocardial hypertrophy and apoptosis, myofibril loss, fetal gene reactivation, and severe arrhythmia. Echocardiography of the glucose-treated fish demonstrated diastolic dysfunction at an early stage and systolic dysfunction at a later stage, consistent with what is observed in diabetic patients. Enlarged hearts with decreased myocardial density, accompanied by decompensated cardiac function, indicated that apoptosis was critical in the pathological process. Significant upregulation of the expression of Nkx2.5 and its downstream targets calreticulin (Calr and p53 was noted in the glucose-treated fish. High-glucose stimulation in vitro evoked marked apoptosis of primary cardiomyocytes, which was rescued by the p53 inhibitor pifithrin-μ. In vitro experiments were performed using compound treatment and genetically via cell infection. Genetically, knockout of Nkx2.5 induced decreased expression of Nkx2.5, Calr and p53. Upregulation of Calr resulted in increased p53 expression, whereas the level of Nkx2.5 remained unchanged. An adult zebrafish model of hyperglycemia-induced cardiomyopathy was successfully established. Hyperglycemia-induced myocardial apoptosis was mediated, at least in part, by activation of the Nkx2.5–Calr–p53 pathway in vivo, resulting in cardiac dysfunction and hyperglycemia-induced cardiomyopathy.

ZebrafishMiner: an open source software for interactive evaluation of domain-specific fluorescence in zebrafish

Directory of Open Access Journals (Sweden)

Reischl Markus

2017-09-01

Full Text Available High-throughput microscopy makes it possible to observe the morphology of zebrafish on large scale to quantify genetic, toxic or drug effects. The image acquisition is done by automated microscopy, images are evaluated automatically by image processing pipelines, tailored specifically to the requirements of the scientific question. The transfer of such algorithms to other projects, however, is complex due to missing guidelines and lack of mathematical or programming knowledge. In this work, we implement an image processing pipeline for automatic fluorescence quantification in user-defined domains of zebrafish embryos and larvae of different age. The pipeline is capable of detecting embryos and larvae in image stacks and quantifying domain activity. To make this protocol available to the community, we developed an open source software package called „ZebrafishMiner“ which guides the user through all steps of the processing pipeline and makes the algorithms available and easy to handle. We implemented all routines in an MATLAB-based graphical user interface (GUI that gives the user control over all image processing parameters. The software is shipped with a manual of 30 pages and three tutorial datasets, which guide the user through the manual step by step. It can be downloaded at https://sourceforge.net/projects/scixminer/.

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

Science.gov (United States)

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

2014-07-01

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

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

Science.gov (United States)

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

2017-01-01

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

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

Science.gov (United States)

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.

HISTOPATHOLOGICAL EFFECTS OF (4S)-2-(4-HYDROXY-3-METHOXYPHENYL) THIAZOLIDINE-4-CARBOXYLIC ACID ON ZEBRAFISH (Danio rerio) HEART TISSUE

OpenAIRE

YÖN, Nazan Deniz; ÖZTÜRK, Burcu; ZENGİN, Mustafa; AKBULUT, Cansu

2018-01-01

Examination thehistopathological effects of (4s)-2-(4-hydroxy-3-methoxyphenyl) thiazolidine-4-carboxylicacid on heart tissue of zebrafish were aimed.Introduction:(4S)-2-(4-hydroxy-3-methoxyphenyl) thiazolidine-4-carboxylic acid is newsynthesized substance which obtained from cysteine and valine. Because ofthiazolidine derivates have important biological responses scientist workintensively on these compounds recent years. It is obvious that thiazolidinecontained compounds will be used in futur...

Genome-wide analysis of Tol2 transposon reintegration in zebrafish

Directory of Open Access Journals (Sweden)

Parinov Sergey

2009-09-01

Full Text Available Abstract Background Tol2, a member of the hAT family of transposons, has become a useful tool for genetic manipulation of model animals, but information about its interactions with vertebrate genomes is still limited. Furthermore, published reports on Tol2 have mainly been based on random integration of the transposon system after co-injection of a plasmid DNA harboring the transposon and a transposase mRNA. It is important to understand how Tol2 would behave upon activation after integration into the genome. Results We performed a large-scale enhancer trap (ET screen and generated 338 insertions of the Tol2 transposon-based ET cassette into the zebrafish genome. These insertions were generated by remobilizing the transposon from two different donor sites in two transgenic lines. We found that 39% of Tol2 insertions occurred in transcription units, mostly into introns. Analysis of the transposon target sites revealed no strict specificity at the DNA sequence level. However, Tol2 was prone to target AT-rich regions with weak palindromic consensus sequences centered at the insertion site. Conclusion Our systematic analysis of sequential remobilizations of the Tol2 transposon from two independent sites within a vertebrate genome has revealed properties such as a tendency to integrate into transcription units and into AT-rich palindrome-like sequences. This information will influence the development of various applications involving DNA transposons and Tol2 in particular.

Effects of prolonged exposure to perchlorate on thyroid and reproductive function in zebrafish

Science.gov (United States)

Mukhi, S.; Patino, R.

2007-01-01

The objectives of this study were to determine the effects of prolonged exposure to perchlorate on (1) thyroid status and reproductive performance of adult zebrafish (Danio rerio) and (2) F1 embryo survival and early larval development. Using a static-renewal procedure, mixed sex populations of adult zebrafish were exposed to 0, 10, and 100 mg/l nominal concentrations of waterborne perchlorate for 10 weeks. Thyroid histology was qualitatively assessed, and females and males were separated and further exposed to their respective treatments for six additional weeks. Eight females in each tank replicate (n = 3) were paired weekly with four males from the same respective treatment, and packed-egg (spawn) volume (PEV) was measured each of the last five weeks. At least once during weeks 14-16 of exposure, other end points measured included fertilization rate, fertilized egg diameter, hatching rate, standard length, and craniofacial development of 4-day-postfertilization larvae and thyroid hormone content of 3.5-h embryos and of exposed mothers. At 10 weeks of exposure, perchlorate at both concentrations caused thyroidal hypertrophy and colloid depletion. A marked reduction in PEV was observed toward the end of the 6-week spawning period, but fertilization and embryo hatching rates were unaffected. Fertilized egg diameter and larval length were increased by parental exposure to perchlorate. Larval head depth was unaffected but the forward protrusion of the lower jaw-associated cartilage complexes, Meckel's and ceratohyal, was decreased. Exposure to both concentrations of perchlorate inhibited whole-body thyroxine content in mothers and embryos, but triiodothyronine content was unchanged. In conclusion, prolonged exposure of adult zebrafish to perchlorate not only disrupts their thyroid endocrine system but also impairs reproduction and influences early F1 development. ?? 2007 Oxford University Press.

Tributyltin and Zebrafish: Swimming in Dangerous Water

Directory of Open Access Journals (Sweden)

Clemilson Berto-Júnior

2018-04-01

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

Tributyltin and Zebrafish: Swimming in Dangerous Water

Science.gov (United States)

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

2018-01-01

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

Disease modeling in genetic kidney diseases: zebrafish.

Science.gov (United States)

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

2017-07-01

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

Use of zebrafish to study Shigella infection

Science.gov (United States)

Duggan, Gina M.

2018-01-01

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

Use of zebrafish to study Shigella infection

Directory of Open Access Journals (Sweden)

Gina M. Duggan

2018-02-01

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

Glyphosate and Roundup® alter morphology and behavior in zebrafish.

Science.gov (United States)

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

2017-12-01

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

Overexpression of miR-19b Impairs Cardiac Development in Zebrafish by Targeting ctnnb1

Directory of Open Access Journals (Sweden)

Mengmeng Li

2014-07-01

Full Text Available Background: MicroRNAs are broadly accepted as crucial regulators of cardiovascular development, and dysregulation of their expression has been linked to cardiac disease. MicroRNA cluster miR-17-92 has been implicated in cardiac development and function, yet its defined mechanisms of action in this context are uncertain. Here, we focused on miR-19b, a key component of the miR-17-92 cluster proven to induce cardiomyocyte proliferation in vitro. We aimed to identify the biological significance of miR-19b in cardiac development and its underlying molecular mechanism of action in vivo. Methods: We micro-injected zebrafish embryos with different concentrations (0, 2, 4 and 8 μm of miR-19b mimics or a negative control, and assessed the embryo malformation rate, mortality rate, hatching rate and heart abnormalities at 72 hours post-fertilization (72 hpf. Results: We found that overexpression of miR-19b impacted left-right symmetry and cardiac development of zebrafish embryos, characterized by pericardial edema, slower heart rate and cardiac looping defects in a dose-dependent manner. Moreover, several important signaling molecules in the Wnt signaling pathway were abnormally expressed, suggesting that overexpression of miR-19b induces the inhibition of the Wnt signaling pathway by directly targeting ctnnb1. Interestingly, the deformed cardiac phenotype was partially rescued by treatment with the GSK3β inhibitor lithium chloride. Conclusion: Our findings suggest that miR-19b regulates laterality development and heart looping in zebrafish embryos by targeting ctnnb1.

Effects of short-term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on microRNA expression in zebrafish embryos

International Nuclear Information System (INIS)

Jenny, Matthew J.; Aluru, Neelakanteswar; Hahn, Mark E.

2012-01-01

Although many drugs and environmental chemicals are teratogenic, the mechanisms by which most toxicants disrupt embryonic development are not well understood. MicroRNAs, single-stranded RNA molecules of ∼ 22 nt that regulate protein expression by inhibiting mRNA translation and promoting mRNA sequestration or degradation, are important regulators of a variety of cellular processes including embryonic development and cellular differentiation. Recent studies have demonstrated that exposure to xenobiotics can alter microRNA expression and contribute to the mechanisms by which environmental chemicals disrupt embryonic development. In this study we tested the hypothesis that developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a well-known teratogen, alters microRNA expression during zebrafish development. We exposed zebrafish embryos to DMSO (0.1%) or TCDD (5 nM) for 1 h at 30 hours post fertilization (hpf) and measured microRNA expression using several methods at 36 and 60 hpf. TCDD caused strong induction of CYP1A at 36 hpf (62-fold) and 60 hpf (135-fold) as determined by real-time RT-PCR, verifying the effectiveness of the exposure. MicroRNA expression profiles were determined using microarrays (Agilent and Exiqon), next-generation sequencing (SOLiD), and real-time RT-PCR. The two microarray platforms yielded results that were similar but not identical; both showed significant changes in expression of miR-451, 23a, 23b, 24 and 27e at 60 hpf. Multiple analyses were performed on the SOLiD sequences yielding a total of 16 microRNAs as differentially expressed by TCDD in zebrafish embryos. However, miR-27e was the only microRNA to be identified as differentially expressed by all three methods (both microarrays, SOLiD sequencing, and real-time RT-PCR). These results suggest that TCDD exposure causes modest changes in expression of microRNAs, including some (miR-451, 23a, 23b, 24 and 27e) that are critical for hematopoiesis and cardiovascular

Bioluminescence Monitoring of Neuronal Activity in Freely Moving Zebrafish Larvae

Science.gov (United States)

Knafo, Steven; Prendergast, Andrew; Thouvenin, Olivier; Figueiredo, Sophie Nunes; Wyart, Claire

2017-01-01

The proof of concept for bioluminescence monitoring of neural activity in zebrafish with the genetically encoded calcium indicator GFP-aequorin has been previously described (Naumann et al., 2010) but challenges remain. First, bioluminescence signals originating from a single muscle fiber can constitute a major pitfall. Second, bioluminescence signals emanating from neurons only are very small. To improve signals while verifying specificity, we provide an optimized 4 steps protocol achieving: 1) selective expression of a zebrafish codon-optimized GFP-aequorin, 2) efficient soaking of larvae in GFP-aequorin substrate coelenterazine, 3) bioluminescence monitoring of neural activity from motor neurons in free-tailed moving animals performing acoustic escapes and 4) verification of the absence of muscle expression using immunohistochemistry. PMID:29130058

Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

OpenAIRE

Hirata, Hiromi; Carta, Eloisa; Yamanaka, Iori; Harvey, Robert J.; Kuwada, John Y.

2010-01-01

Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem. Recently, in vivo analysis of glycinergic synaptic transmission has been pursued in zebrafish using molecular genetics. An ENU mutagenesis screen identified two behavioral mutants that are defective in glycinergic synaptic transmission. Zebrafish bandoneon (beo) mutants have a defect in glrbb, one of the duplicated glycine receptor (GlyR) β subunit genes. These mutants exhibit a loss of glycinergic synaptic ...

Understanding the transformation, speciation, and hazard potential of copper particles in a model septic tank system using zebrafish to monitor the effluent.

Science.gov (United States)

Lin, Sijie; Taylor, Alicia A; Ji, Zhaoxia; Chang, Chong Hyun; Kinsinger, Nichola M; Ueng, William; Walker, Sharon L; Nel, André E

2015-02-24

Although copper-containing nanoparticles are used in commercial products such as fungicides and bactericides, we presently do not understand the environmental impact on other organisms that may be inadvertently exposed. In this study, we used the zebrafish embryo as a screening tool to study the potential impact of two nano Cu-based materials, CuPRO and Kocide, in comparison to nanosized and micron-sized Cu and CuO particles in their pristine form (0-10 ppm) as well as following their transformation in an experimental wastewater treatment system. This was accomplished by construction of a modeled domestic septic tank system from which effluents could be retrieved at different stages following particle introduction (10 ppm). The Cu speciation in the effluent was identified as nondissolvable inorganic Cu(H2PO2)2 and nondiffusible organic Cu by X-ray diffraction, inductively coupled plasma mass spectrometry (ICP-MS), diffusive gradients in thin-films (DGT), and Visual MINTEQ software. While the nanoscale materials, including the commercial particles, were clearly more potent (showing 50% hatching interference above 0.5 ppm) than the micron-scale particulates with no effect on hatching up to 10 ppm, the Cu released from the particles in the septic tank underwent transformation into nonbioavailable species that failed to interfere with the function of the zebrafish embryo hatching enzyme. Moreover, we demonstrate that the addition of humic acid, as an organic carbon component, could lead to a dose-dependent decrease in Cu toxicity in our high content zebrafish embryo screening assay. Thus, the use of zebrafish embryo screening, in combination with the effluents obtained from a modeled exposure environment, enables a bioassay approach to follow the change in the speciation and hazard potential of Cu particles instead of difficult-to-perform direct particle tracking.

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

Energy Technology Data Exchange (ETDEWEB)

Kwon, Hye-Joo, E-mail: hjkwon@pnu.edu.sa [Biology Department, Texas A& M University, College Station, TX77843-3258 (United States); Biology Department, Princess Nourah University, Riyadh 11671 (Saudi Arabia)

2016-02-12

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

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

International Nuclear Information System (INIS)

Kwon, Hye-Joo

2016-01-01

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

Effects of larval-juvenile treatment with perchlorate and co-treatment with thyroxine on zebrafish sex ratios

Science.gov (United States)

Mukhi, S.; Torres, L.; Patino, R.

2007-01-01

The objective of this study was to determine the effect of larval-juvenile exposure to perchlorate, a thyroid hormone synthesis inhibitor, on the establishment of gonadal sex ratios in zebrafish. Zebrafish were exposed to untreated water or water containing perchlorate at 100 or 250 ppm for a period of 30 days starting at 3 days postfertilization (dpf). Recovery treatments consisted of a combination of perchlorate and exogenous thyroxine (T4; 10 nM). Thyroid histology was assessed at the end of the treatment period (33 dpf), and gonadal histology and sex ratios were determined in fish that were allowed an additional 10-day period of growth in untreated water. As expected, exposure to perchlorate caused changes in thyroid histology consistent with hypothyroidism and these effects were reversed by co-treatment with exogenous T4. Perchlorate did not affect fish survival but co-treatment with T4 induced higher mortality. However, relative to the corresponding perchlorate concentration, co-treatment with T4 caused increased mortality only at a perchlorate concentration of 100 ppm. Perchlorate alone or in the presence of T4 suppressed body length at 43 dpf relative to control values. Perchlorate exposure skewed the sex ratio toward female in a concentration-dependent manner, and co-treatment with T4 not only blocked the feminizing effect of perchlorate but also overcompensated by skewing the sex ratio towards male. Moreover, co-treatment with T4 advanced the onset of spermatogenesis in males. There was no clear association between sex ratios and larval survival or growth. We conclude that endogenous thyroid hormone plays a role in the establishment of gonadal sex phenotype during early development in zebrafish. ?? 2006 Elsevier Inc. All rights reserved.

Life-stage dependent response in zebrafish (Danio rerio) to phototoxicity of TiO2 nanoparticles

Science.gov (United States)

The Zebrafish, and especially its embryo stage, has been increasingly used as a model to evaluate toxicity of manufactured nanomaterials. However, many studies have indicated that the chorion may protect developing embroys from the toxic effects of nanomaterials, suggesting that ...

Metastatic behaviour of primary human tumours in a zebrafish xenotransplantation model

International Nuclear Information System (INIS)

Marques, Ines J; Bagowski, Christoph P; Weiss, Frank Ulrich; Vlecken, Danielle H; Nitsche, Claudia; Bakkers, Jeroen; Lagendijk, Anne K; Partecke, Lars Ivo; Heidecke, Claus-Dieter; Lerch, Markus M

2009-01-01

Aberrant regulation of cell migration drives progression of many diseases, including cancer cell invasion and metastasis formation. Analysis of tumour invasion and metastasis in living organisms to date is cumbersome and involves difficult and time consuming investigative techniques. For primary human tumours we establish here a simple, fast, sensitive and cost-effective in vivo model to analyse tumour invasion and metastatic behaviour. We fluorescently labelled small explants from gastrointestinal human tumours and investigated their metastatic behaviour after transplantation into zebrafish embryos and larvae. The transparency of the zebrafish embryos allows to follow invasion, migration and micrometastasis formation in real-time. High resolution imaging was achieved through laser scanning confocal microscopy of live zebrafish. In the transparent zebrafish embryos invasion, circulation of tumour cells in blood vessels, migration and micrometastasis formation can be followed in real-time. Xenografts of primary human tumours showed invasiveness and micrometastasis formation within 24 hours after transplantation, which was absent when non-tumour tissue was implanted. Furthermore, primary human tumour cells, when organotopically implanted in the zebrafish liver, demonstrated invasiveness and metastatic behaviour, whereas primary control cells remained in the liver. Pancreatic tumour cells showed no metastatic behaviour when injected into cloche mutant embryos, which lack a functional vasculature. Our results show that the zebrafish is a useful in vivo animal model for rapid analysis of invasion and metastatic behaviour of primary human tumour specimen

Scopolamine methylbromide mitigates radiation induced damage and lethality in zebrafish

International Nuclear Information System (INIS)

Shrivastava, Nitisha; Joshi, Jayadev; Ghosh, Subhajit; Dimri, Manali; Prem Kumar, Indracanti; Sehgal, Neeta

2014-01-01

In view of the strategic importance radiation countermeasures hold, the present study was undertaken to screen a collection of small molecule clinical compounds for possible radioprotective action using zebrafish as a model system. Preliminary screening in developing zebrafish embryos (24 hour post fertilization, (hpf)) using damage manifestations and survival as end point identified scopolamine methylbromide (SMB), a muscarinic receptor antagonist, as a potential radiomitigator. It was found to be optimal (60% survival advantage after 6 th post irradiation day) at a dose of 80 μM when added 3 h post 20 Gy exposure. Mechanistic studies suggested that SMB though exhibited no significant antioxidant potential, but was found to limit radiation induced apoptosis (pre G1 population) quantified through flow cytometry (6 and 5% reduction after 8 or 24 h after treatments) and annexin V staining (8% reduction). Further, quantitative analysis, using caspase 3 assay, revealed a 2.46 fold increase in apoptosis in irradiated group and treatment of irradiated zebrafish embryos with SMB led to a significant reduction in global apoptosis (1.7 fold; p<0.05) when compared to irradiated group. In silico studies based on structural and functional similarity with known radioprotectors suggested similarities with atropine, a known anti-inflammatory agent with muscarinic antagonism and radioprotective potential. In view of this SMB was tested, in silico, for possible anti-inflammatory action. Molecular docking studies revealed that SMB interacts (B.E-8.0 Kcal/mole) with cycloxygenase-2 (COX-2). In lieu of this, anti-inflammation activity was assessed through ChIN (chemically induced inflammation) method in 3 dpf (days post fertilization) embryos and SMB was found to significantly inhibit inflammation at all doses studied from 20-200 μM at 3 and 6 hpi (hours post inflammation). Overall the result suggests that scopolamine methylbromide mitigates radiation induced injury and lethality in

Kita driven expression of oncogenic HRAS leads to early onset and highly penetrant melanoma in zebrafish.

Directory of Open Access Journals (Sweden)

Cristina Santoriello

2010-12-01

Full Text Available Melanoma is the most aggressive and lethal form of skin cancer. Because of the increasing incidence and high lethality of melanoma, animal models for continuously observing melanoma formation and progression as well as for testing pharmacological agents are needed.Using the combinatorial Gal4-UAS system, we have developed a zebrafish transgenic line that expresses oncogenic HRAS under the kita promoter. Already at 3 days transgenic kita-GFP-RAS larvae show a hyper-pigmentation phenotype as earliest evidence of abnormal melanocyte growth. By 2-4 weeks, masses of transformed melanocytes form in the tail stalk of the majority of kita-GFP-RAS transgenic fish. The adult tumors evident between 1-3 months of age faithfully reproduce the immunological, histological and molecular phenotypes of human melanoma, but on a condensed time-line. Furthermore, they show transplantability, dependence on mitfa expression and do not require additional mutations in tumor suppressors. In contrast to kita expressing melanocyte progenitors that efficiently develop melanoma, mitfa expressing progenitors in a second Gal4-driver line were 4 times less efficient in developing melanoma during the three months observation period.This indicates that zebrafish kita promoter is a powerful tool for driving oncogene expression in the right cells and at the right level to induce early onset melanoma in the presence of tumor suppressors. Thus our zebrafish model provides a link between kita expressing melanocyte progenitors and melanoma and offers the advantage of a larval phenotype suitable for large scale drug and genetic modifier screens.

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

Science.gov (United States)

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

2017-01-01

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

Imaging Subcellular Structures in the Living Zebrafish Embryo.

Science.gov (United States)

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.

Zebrafish genetics gets the Scube on Hedgehog secretion.

Science.gov (United States)

Ingham, Philip W

2012-11-15

Inspired by a zebrafish mutation, two recent studies by Creanga and colleagues (pp. 1312-1325) and Tukachinsky and colleagues have shed new light on the way in which lipidated Hedgehog proteins are secreted and released from expressing cells, suggesting a model for the sequential action of the Disp and Scube2 proteins in this process.

GROWTH AND BEHAVIOR OF LARVAL ZEBRAFISH Danio ...

Science.gov (United States)

Because Zebrafish (Danio rerio) have become a popular and important model for scientific research, the capability to rear larval zebrafish to adulthood is of great importance. Recently research examining the effects of diet (live versus processed) have been published. In the current study we examined whether the larvae can be reared on a processed diet alone, live food alone, or the combination while maintaining normal locomotor behavior, and acceptable survival, length and weight at 14 dpf in a static system. A 14 day feeding trial was conducted in glass crystallizing dishes containing 500 ml of 4 ppt Instant Ocean. On day 0 pdf 450 embryos were selected as potential study subjects and placed in a 26○C incubator on a 14:10 (light:dark) light cycle. At 4 dpf 120 normally developing embryos were selected per treatment and divided into 3 bowls of 40 embryos (for an n=3 per treatment; 9 bowls total). Treatment groups were: G (Gemma Micro 75 only), R (L-type marine rotifers (Brachionus plicatilis) only) or B (Gemma and rotifers). Growth (length), survival, water quality and rotifer density were monitored on days 5-14. On day 14, weight of larva in each bowl was measured and 8 larva per bowl were selected for use in locomotor testing. This behavior paradigm tests individual larval zebrafish under both light and dark conditions in a 24-well plate.After 14 dpf, survival among the groups was not different (92-98%). By days 7 -14 R and B larvae were ~2X longer

75 FR 36588 - Veterinary Feed Directive; Extension of Comment Period

Science.gov (United States)

2010-06-28

... 558 [Docket No. FDA-2010-N-0155] Veterinary Feed Directive; Extension of Comment Period AGENCY: Food... veterinary feed directive (VFD) regulation. The agency is taking this action in response to requests for an... CONTACT: Neal Bataller, Center for Veterinary Medicine (HFV-230), Food and Drug Administration, 7500...

Pbx and Prdm1a transcription factors differentially regulate subsets of the fast skeletal muscle program in zebrafish

Directory of Open Access Journals (Sweden)

Zizhen Yao

2013-04-01

The basic helix–loop–helix factor Myod initiates skeletal muscle differentiation by directly and sequentially activating sets of muscle differentiation genes, including those encoding muscle contractile proteins. We hypothesize that Pbx homeodomain proteins direct Myod to a subset of its transcriptional targets, in particular fast-twitch muscle differentiation genes, thereby regulating the competence of muscle precursor cells to differentiate. We have previously shown that Pbx proteins bind with Myod on the promoter of the zebrafish fast muscle gene mylpfa and that Pbx proteins are required for Myod to activate mylpfa expression and the fast-twitch muscle-specific differentiation program in zebrafish embryos. Here we have investigated the interactions of Pbx with another muscle fiber-type regulator, Prdm1a, a SET-domain DNA-binding factor that directly represses mylpfa expression and fast muscle differentiation. The prdm1a mutant phenotype, early and increased fast muscle differentiation, is the opposite of the Pbx-null phenotype, delayed and reduced fast muscle differentiation. To determine whether Pbx and Prdm1a have opposing activities on a common set of genes, we used RNA-seq analysis to globally assess gene expression in zebrafish embryos with single- and double-losses-of-function for Pbx and Prdm1a. We find that the levels of expression of certain fast muscle genes are increased or approximately wild type in pbx2/4-MO;prdm1a−/− embryos, suggesting that Pbx activity normally counters the repressive action of Prdm1a for a subset of the fast muscle program. However, other fast muscle genes require Pbx but are not regulated by Prdm1a. Thus, our findings reveal that subsets of the fast muscle program are differentially regulated by Pbx and Prdm1a. Our findings provide an example of how Pbx homeodomain proteins act in a balance with other transcription factors to regulate subsets of a cellular differentiation program.

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

Science.gov (United States)

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

2017-02-01

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

Phototoxicity of TiO2 nanoparticles to zebrafish (Danio rerio) is dependent on life stage

Science.gov (United States)

The zebrafish (Danio rerio) embryo has been increasingly used as a model to evaluate toxicity of manufactured nanomaterials. Many studies indicate that the embryo chorion may protect animals from toxic effects of nanomaterials, suggesting that post-hatch life stages may be more s...

Cross-Modal Learning between Visual and Vibration Signals in Zebrafish Danio Rerio

Directory of Open Access Journals (Sweden)

Mu-Yun Wang

2011-10-01

Full Text Available Animals are always integrating environmental information from multiple sensory modalities, but the mechanisms underneath are highly underexploited. Crossmodal interactions in animal perception have been found in several species including human, mice and flies. Here we subjected zebrafish as model because its genetic effects on brain and sense organ development are well studied, but the attentional processes are mainly unexplored. Zebrafish show impressive behaviour capabilities with relatively small or “simple” brains which make their nervous system relatively more accessible to experimentation than large-brained mammals. When conditioned with both vision and vibration signals, zebrafish were able to make higher correct choices than only one sensation. After multimodal training, zebrafish were also able to transfer the memory to unimodal conditioning when only given vision or vibration signals. This study provided basic findings for how animals process multimodal sensory from the environment, and showed crossmodal interactions in zebrafish for the first time.

A splice site mutation in laminin-α2 results in a severe muscular dystrophy and growth abnormalities in zebrafish.

Directory of Open Access Journals (Sweden)

Vandana A Gupta

Full Text Available Congenital muscular dystrophy (CMD is a clinically and genetically heterogeneous group of inherited muscle disorders. In patients, muscle weakness is usually present at or shortly after birth and is progressive in nature. Merosin deficient congenital muscular dystrophy (MDC1A is a form of CMD caused by a defect in the laminin-α2 gene (LAMA2. Laminin-α2 is an extracellular matrix protein that interacts with the dystrophin-dystroglycan (DGC complex in membranes providing stability to muscle fibers. In an N-ethyl-N-nitrosourea mutagenesis screen to develop zebrafish models of neuromuscular diseases, we identified a mutant fish that exhibits severe muscular dystrophy early in development. Genetic mapping identified a splice site mutation in the lama2 gene. This splice site is highly conserved in humans and this mutation results in mis-splicing of RNA and a loss of protein function. Homozygous lama2 mutant zebrafish, designated lama2(cl501/cl501, exhibited reduced motor function and progressive degeneration of skeletal muscles and died at 8-15 days post fertilization. The skeletal muscles exhibited damaged myosepta and detachment of myofibers in the affected fish. Laminin-α2 deficiency also resulted in growth defects in the brain and eye of the mutant fish. This laminin-α2 deficient mutant fish represents a novel disease model to develop therapies for modulating splicing defects in congenital muscular dystrophies and to restore the muscle function in human patients with CMD.

Effects of probiotic administration on zebrafish development and reproduction.

Science.gov (United States)

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

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

OpenAIRE

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

2010-01-01

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...

Characterization of heme oxygenase and biliverdin reductase gene expression in zebrafish (Danio rerio): Basal expression and response to pro-oxidant exposures

International Nuclear Information System (INIS)

Holowiecki, Andrew; O'Shields, Britton; Jenny, Matthew J.

2016-01-01

While heme is an important cofactor for numerous proteins, it is highly toxic in its unbound form and can perpetuate the formation of reactive oxygen species. Heme oxygenase enzymes (HMOX1 and HMOX2) degrade heme into biliverdin and carbon monoxide, with biliverdin subsequently being converted to bilirubin by biliverdin reductase (BVRa or BVRb). As a result of the teleost-specific genome duplication event, zebrafish have paralogs of hmox1 (hmox1a and hmox1b) and hmox2 (hmox2a and hmox2b). Expression of all four hmox paralogs and two bvr isoforms were measured in adult tissues (gill, brain and liver) and sexually dimorphic differences were observed, most notably in the basal expression of hmox1a, hmox2a, hmox2b and bvrb in liver samples. hmox1a, hmox2a and hmox2b were significantly induced in male liver tissues in response to 96 h cadmium exposure (20 μM). hmox2a and hmox2b were significantly induced in male brain samples, but only hmox2a was significantly reduced in male gill samples in response to the 96 h cadmium exposure. hmox paralogs displayed significantly different levels of basal expression in most adult tissues, as well as during zebrafish development (24 to 120 hpf). Furthermore, hmox1a, hmox1b and bvrb were significantly induced in zebrafish eleutheroembryos in response to multiple pro-oxidants (cadmium, hemin and tert-butylhydroquinone). Knockdown of Nrf2a, a transcriptional regulator of hmox1a, was demonstrated to inhibit the Cd-mediated induction of hmox1b and bvrb. These results demonstrate distinct mechanisms of hmox and bvr transcriptional regulation in zebrafish, providing initial evidence of the partitioning of function of the hmox paralogs. - Highlights: • hmox1a, hmox2a, hmox2b and bvrb are sexually dimorphic in expression. • hmox paralogs were induced in adult tissues by cadmium exposure. • hmox1a, hmox1b and bvrb were induced by multiple pro-oxidants zebrafish embryos. • Differential expression of zebrafish hmox paralogs suggest

Characterization of heme oxygenase and biliverdin reductase gene expression in zebrafish (Danio rerio): Basal expression and response to pro-oxidant exposures

Energy Technology Data Exchange (ETDEWEB)

Holowiecki, Andrew [Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487 (United States); Molecular Cardiovascular Biology Division and Heart Institute, Cincinnati Children' s Research Foundation, Cincinnati, OH (United States); O' Shields, Britton [Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487 (United States); Jenny, Matthew J., E-mail: mjjenny@ua.edu [Department of Biological Sciences, University of Alabama, Tuscaloosa, AL 35487 (United States)

2016-11-15

While heme is an important cofactor for numerous proteins, it is highly toxic in its unbound form and can perpetuate the formation of reactive oxygen species. Heme oxygenase enzymes (HMOX1 and HMOX2) degrade heme into biliverdin and carbon monoxide, with biliverdin subsequently being converted to bilirubin by biliverdin reductase (BVRa or BVRb). As a result of the teleost-specific genome duplication event, zebrafish have paralogs of hmox1 (hmox1a and hmox1b) and hmox2 (hmox2a and hmox2b). Expression of all four hmox paralogs and two bvr isoforms were measured in adult tissues (gill, brain and liver) and sexually dimorphic differences were observed, most notably in the basal expression of hmox1a, hmox2a, hmox2b and bvrb in liver samples. hmox1a, hmox2a and hmox2b were significantly induced in male liver tissues in response to 96 h cadmium exposure (20 μM). hmox2a and hmox2b were significantly induced in male brain samples, but only hmox2a was significantly reduced in male gill samples in response to the 96 h cadmium exposure. hmox paralogs displayed significantly different levels of basal expression in most adult tissues, as well as during zebrafish development (24 to 120 hpf). Furthermore, hmox1a, hmox1b and bvrb were significantly induced in zebrafish eleutheroembryos in response to multiple pro-oxidants (cadmium, hemin and tert-butylhydroquinone). Knockdown of Nrf2a, a transcriptional regulator of hmox1a, was demonstrated to inhibit the Cd-mediated induction of hmox1b and bvrb. These results demonstrate distinct mechanisms of hmox and bvr transcriptional regulation in zebrafish, providing initial evidence of the partitioning of function of the hmox paralogs. - Highlights: • hmox1a, hmox2a, hmox2b and bvrb are sexually dimorphic in expression. • hmox paralogs were induced in adult tissues by cadmium exposure. • hmox1a, hmox1b and bvrb were induced by multiple pro-oxidants zebrafish embryos. • Differential expression of zebrafish hmox paralogs suggest

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

Science.gov (United States)

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

2018-06-01

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

The α₂Na⁺/K⁺-ATPase is critical for skeletal and heart muscle function in zebrafish

DEFF Research Database (Denmark)

Doganli, Canan; Kjaer-Sørensen, Kasper; Knoeckel, Christopher

2012-01-01

The Na+/K+-ATPase generates ion gradients across the plasma membrane, essential for multiple cellular functions. In mammals, four different Na+/K+-ATPase α-subunit isoforms are associated with characteristic cell-type expression profiles and kinetics. We found the zebrafish α2Na+/K+-ATPase associ...

Developmental Deltamethrin Exposure Causes Persistent Changes in Dopaminergic Gene Expression, Neurochemistry, and Locomotor Activity in Zebrafish.

Science.gov (United States)

Kung, Tiffany S; Richardson, Jason R; Cooper, Keith R; White, Lori A

2015-08-01

Pyrethroids are commonly used insecticides that are considered to pose little risk to human health. However, there is an increasing concern that children are more susceptible to the adverse effects of pesticides. We used the zebrafish model to test the hypothesis that developmental exposure to low doses of the pyrethroid deltamethrin results in persistent alterations in dopaminergic gene expression, neurochemistry, and locomotor activity. Zebrafish embryos were treated with deltamethrin (0.25-0.50 μg/l), at concentrations below the LOAEL, during the embryonic period [3-72 h postfertilization (hpf)], after which transferred to fresh water until the larval stage (2-weeks postfertilization). Deltamethrin exposure resulted in decreased transcript levels of the D1 dopamine (DA) receptor (drd1) and increased levels of tyrosine hydroxylase at 72 hpf. The reduction in drd1 transcripts persisted to the larval stage and was associated with decreased D2 dopamine receptor transcripts. Larval fish, exposed developmentally to deltamethrin, had increased levels of homovanillic acid, a DA metabolite. Since the DA system is involved in locomotor activity, we measured the swim activity of larval fish following a transition to darkness. Developmental exposure to deltamethrin significantly increased larval swim activity which was attenuated by concomitant knockdown of the DA transporter. Acute exposure to methylphenidate, a DA transporter inhibitor, increased swim activity in control larva, while reducing swim activity in larva developmentally exposed to deltamethrin. Developmental exposure to deltamethrin causes locomotor deficits in larval zebrafish, which is likely mediated by dopaminergic dysfunction. This highlights the need to understand the persistent effects of low-dose neurotoxicant exposure during development. © The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Protective Effect of Phillyrin on Lethal LPS-Induced Neutrophil Inflammation in Zebrafish

Directory of Open Access Journals (Sweden)

Liling Yang

2017-10-01

Full Text Available Background/Aims: Forsythia suspensa Vahl. (Oleaceae fruits are widely used in traditional Chinese medicine to treat pneumonia, typhoid, dysentery, ulcers and oedema. Antibacterial and anti-inflammatory activities have been reported for phillyrin (PHN, the main ingredient in Forsythia suspensa Vahl fruits, in vitro. However, the underlying mechanisms in vivo remain poorly defined. In this study, we discovered that PHN exerted potent anti-inflammatory effects in lethal LPS-induced neutrophil inflammation by suppressing the MyD88-dependent signalling pathway in zebrafish. Methods: LPS-yolk microinjection was used to induce a lethal LPS-infected zebrafish model. The effect of PHN on the survival of zebrafish challenged with lethal LPS was evaluated using survival analysis. The effect of PHN on neutrophil inflammation grading in vivo was assessed by tracking neutrophils with a transgenic line. The effects of PHN on neutrophil production and migration were analysed by SB+ cell counts during consecutive hours after modelling. Additionally, key cytokines and members of the MyD88 signalling pathway that are involved in inflammatory response were detected using quantitative RT-PCR. To assess gene expression changes during consecutive hours after modelling, the IL-1β, IL-6, TNF-α, MyD88, TRIF, ERK1/2, JNK, IκBa and NF-κB expression levels were measured. Results: PHN could protect zebrafish against a lethal LPS challenge in a dose-dependent manner, as indicated by decreased neutrophil infltration, reduced tissue necrosis and increased survival rates. Up-regulated IL-1β, IL-6 and TNF-α expression also showed the same tendencies of depression by PHN. Critically, PHN significantly inhibited the LPS-induced activation of MyD88, IκBa, and NF-κB but did not affect the expression of ERK1/2 MAPKs or JNK MAPKs in LPS-stimulated zebrafish. Additionally, PHN regulated the MyD88/IκBα/NF-κB signalling pathway by controlling IκBα, IL-1β, IL-6, and TNF

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

Science.gov (United States)

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

2013-04-25

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

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

Science.gov (United States)

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

2009-06-01

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

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

Science.gov (United States)

Butail, Sachit; Mwaffo, Violet; Porfiri, Maurizio

2016-04-01

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