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

A zebrafish model of lethal congenital contracture syndrome 1 reveals Gle1 function in spinal neural precursor survival and motor axon arborization.

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Jao, Li-En; Appel, Bruce; Wente, Susan R

2012-04-01

In humans, GLE1 is mutated in lethal congenital contracture syndrome 1 (LCCS1) leading to prenatal death of all affected fetuses. Although the molecular roles of Gle1 in nuclear mRNA export and translation have been documented, no animal models for this disease have been reported. To elucidate the function of Gle1 in vertebrate development, we used the zebrafish (Danio rerio) model system. gle1 mRNA is maternally deposited and widely expressed. Altering Gle1 using an insertional mutant or antisense morpholinos results in multiple defects, including immobility, small eyes, diminished pharyngeal arches, curved body axis, edema, underdeveloped intestine and cell death in the central nervous system. These phenotypes parallel those observed in LCCS1 human fetuses. Gle1 depletion also results in reduction of motoneurons and aberrant arborization of motor axons. Unexpectedly, the motoneuron deficiency results from apoptosis of neural precursors, not of differentiated motoneurons. Mosaic analyses further indicate that Gle1 activity is required extrinsically in the environment for normal motor axon arborization. Importantly, the zebrafish phenotypes caused by Gle1 deficiency are only rescued by expressing wild-type human GLE1 and not by the disease-linked Fin(Major) mutant form of GLE1. Together, our studies provide the first functional characterization of Gle1 in vertebrate development and reveal its essential role in actively dividing cells. We propose that defective GLE1 function in human LCCS1 results in both neurogenic and non-neurogenic defects linked to the apoptosis of proliferative organ precursors.

Ethanol Exposure Causes Muscle Degeneration in Zebrafish

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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.

Zebrafish eda and edar mutants reveal conserved and ancestral roles of ectodysplasin signaling in vertebrates.

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Matthew P Harris

2008-10-01

Full Text Available The genetic basis of the development and variation of adult form of vertebrates is not well understood. To address this problem, we performed a mutant screen to identify genes essential for the formation of adult skeletal structures of the zebrafish. Here, we describe the phenotypic and molecular characterization of a set of mutants showing loss of adult structures of the dermal skeleton, such as the rays of the fins and the scales, as well as the pharyngeal teeth. The mutations represent adult-viable, loss of function alleles in the ectodysplasin (eda and ectodysplasin receptor (edar genes. These genes are frequently mutated in the human hereditary disease hypohidrotic ectodermal dysplasia (HED; OMIM 224900, 305100 that affects the development of integumentary appendages such as hair and teeth. We find mutations in zebrafish edar that affect similar residues as mutated in human cases of HED and show similar phenotypic consequences. eda and edar are not required for early zebrafish development, but are rather specific for the development of adult skeletal and dental structures. We find that the defects of the fins and scales are due to the role of Eda signaling in organizing epidermal cells into discrete signaling centers of the scale epidermal placode and fin fold. Our genetic analysis demonstrates dose-sensitive and organ-specific response to alteration in levels of Eda signaling. In addition, we show substantial buffering of the effect of loss of edar function in different genetic backgrounds, suggesting canalization of this developmental system. We uncover a previously unknown role of Eda signaling in teleosts and show conservation of the developmental mechanisms involved in the formation and variation of both integumentary appendages and limbs. Lastly, our findings point to the utility of adult genetic screens in the zebrafish in identifying essential developmental processes involved in human disease and in morphological evolution.

Zebrafish eda and edar Mutants Reveal Conserved and Ancestral Roles of Ectodysplasin Signaling in Vertebrates

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Harris, Matthew P.; Rohner, Nicolas; Schwarz, Heinz; Perathoner, Simon; Konstantinidis, Peter; Nüsslein-Volhard, Christiane

2008-01-01

The genetic basis of the development and variation of adult form of vertebrates is not well understood. To address this problem, we performed a mutant screen to identify genes essential for the formation of adult skeletal structures of the zebrafish. Here, we describe the phenotypic and molecular characterization of a set of mutants showing loss of adult structures of the dermal skeleton, such as the rays of the fins and the scales, as well as the pharyngeal teeth. The mutations represent adult-viable, loss of function alleles in the ectodysplasin (eda) and ectodysplasin receptor (edar) genes. These genes are frequently mutated in the human hereditary disease hypohidrotic ectodermal dysplasia (HED; OMIM 224900, 305100) that affects the development of integumentary appendages such as hair and teeth. We find mutations in zebrafish edar that affect similar residues as mutated in human cases of HED and show similar phenotypic consequences. eda and edar are not required for early zebrafish development, but are rather specific for the development of adult skeletal and dental structures. We find that the defects of the fins and scales are due to the role of Eda signaling in organizing epidermal cells into discrete signaling centers of the scale epidermal placode and fin fold. Our genetic analysis demonstrates dose-sensitive and organ-specific response to alteration in levels of Eda signaling. In addition, we show substantial buffering of the effect of loss of edar function in different genetic backgrounds, suggesting canalization of this developmental system. We uncover a previously unknown role of Eda signaling in teleosts and show conservation of the developmental mechanisms involved in the formation and variation of both integumentary appendages and limbs. Lastly, our findings point to the utility of adult genetic screens in the zebrafish in identifying essential developmental processes involved in human disease and in morphological evolution. PMID:18833299

A novel zebrafish mutant with wavy-notochord: an effective biological index for monitoring the copper pollution of water from natural resources.

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Chen, Yau-Hung; Lin, Ji-Sheng

2011-02-01

We identified a novel zebrafish mutant that has wavy-notochord phenotypes, such as severely twisted notochord and posterior malformations, but has normal melanocytes. Histological evidences showed that proliferating vacuolar cells extended their growth to the muscle region, and consequently caused the wavy-notochord phenotypes. Interestingly, those malformations can be greatly reversed by exposure with copper, suggesting that copper plays an important role on wavy-notochord phenotypes. In addition, after long-term copper exposure, the surviving larvae derived from wavy-notochord mutants displayed bone malformations, such as twisted axial skeleton and osteophyte. These phenotypic changes and molecular evidences of wavy-notochord mutants are highly similar to those embryos whose lysyl oxidases activities have been inactivated. Taken together, we propose that (i) the putative mutated genes of this wavy-notochord mutant might be highly associated with the lysyl oxidase genes in zebrafish; and (ii) this fish model is an effective tool for monitoring copper pollution of water from natural resources. Copyright © 2009 Wiley Periodicals, Inc.

Zebrafish bandoneon mutants display behavioral defects due to a mutation in the glycine receptor β-subunit

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Hirata, Hiromi; Saint-Amant, Louis; Downes, Gerald B.; Cui, Wilson W.; Zhou, Weibin; Granato, Michael; Kuwada, John Y.

2005-01-01

Bilateral alternation of muscle contractions requires reciprocal inhibition between the two sides of the hindbrain and spinal cord, and disruption of this inhibition should lead to simultaneous activation of bilateral muscles. At 1 day after fertilization, wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. Similar simultaneous contractions are observed in wild-type embryos treated with strychnine, a blocker of the inhibitory glycine receptor (GlyR). This result suggests that glycinergic synaptic transmission is defective in beo mutants. Muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the CNS. Recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. Furthermore, immunostaining with an antibody against GlyR showed clusters in wild-type neurons but not in beo neurons. These data suggest that the failure of GlyRs to aggregate at synaptic sites causes impairment of glycinergic transmission and abnormal behavior in beo mutants. Indeed, mutations in the GlyR β-subunit, which are thought to be required for proper localization of GlyRs, were identified as the basis for the beo mutation. These data demonstrate that GlyRβ is essential for physiologically relevant clustering of GlyRs in vivo. Because GlyR mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, the zebrafish beo mutant should be a useful animal model for this condition. PMID:15928085

Forward Genetic Screening Using Behavioral Tests in Zebrafish: A Proof of Concept Analysis of Mutants.

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Gerlai, Robert; Poshusta, Tanya L; Rampersad, Mindy; Fernandes, Yohaan; Greenwood, Tammy M; Cousin, Margot A; Klee, Eric W; Clark, Karl J

2017-01-01

The zebrafish enjoys several advantages over other model organisms. It is small, easy to maintain, prolific, and numerous genetic tools are available for it. For example, forward genetic screens have allowed investigators to identify important genes potentially involved in a variety of functions from embryogenesis to cancer. However, despite its sophisticated behavioral repertoire, behavioral methods have rarely been utilized in forward genetic screens. Here, we employ a two-tiered strategy, a proof of concept study, to explore the feasibility of behavioral screens. We generated mutant lines using transposon-based insertional mutagenesis, allowing us to bias mutant selection with target genes expressed within the brain. Furthermore, we employed an efficient and fast behavioral pre-selection in which we investigated the locomotory response of 5-day post-fertilization old larval fish to hyperosmotic shock. Based on this assay, we selected five lines for our lower throughput secondary adult behavioral screen. The latter screen utilized tests in which computer animated image presentation and video-tracking-based automated quantification of behavior allowed us to compare heterozygous zebrafish with their wild-type siblings on their responses to a variety of stimuli. We found significant mutation induced adult behavioral alterations in 4 out of the 5 lines analyzed, including changes in response to social or fear inducing stimuli, to handling and novelty, or in habituation to novelty. We discuss the pros and cons of behavioral phenotyping and of the use of different forward genetic methods in biomedical research with zebrafish.

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

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

2016-07-01

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

Distinct and overlapping functions of ptpn11 genes in Zebrafish development.

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Monica Bonetti

Full Text Available The PTPN11 (protein-tyrosine phosphatase, non-receptor type 11 gene encodes SHP2, a cytoplasmic PTP that is essential for vertebrate development. Mutations in PTPN11 are associated with Noonan and LEOPARD syndrome. Human patients with these autosomal dominant disorders display various symptoms, including short stature, craniofacial defects and heart abnormalities. We have used the zebrafish as a model to investigate the role of Shp2 in embryonic development. The zebrafish genome encodes two ptpn11 genes, ptpn11a and ptpn11b. Here, we report that ptpn11a is expressed constitutively and ptpn11b expression is strongly upregulated during development. In addition, the products of both ptpn11 genes, Shp2a and Shp2b, are functional. Target-selected inactivation of ptpn11a and ptpn11b revealed that double homozygous mutants are embryonic lethal at 5-6 days post fertilization (dpf. Ptpn11a-/-ptpn11b-/- embryos showed pleiotropic defects from 4 dpf onwards, including reduced body axis extension and craniofacial defects, which was accompanied by low levels of phosphorylated Erk at 5 dpf. Interestingly, defects in homozygous ptpn11a-/- mutants overlapped with defects in the double mutants albeit they were milder, whereas ptpn11b-/- single mutants did not show detectable developmental defects and were viable and fertile. Ptpn11a-/-ptpn11b-/- mutants were rescued by expression of exogenous ptpn11a and ptpn11b alike, indicating functional redundance of Shp2a and Shp2b. The ptpn11 mutants provide a good basis for further unravelling of the function of Shp2 in vertebrate development.

Effect of NaN3 on oxygen-dependent lethality of UV-A in Escherichia coli mutants lacking active oxygen-defence and DNA-repair systems

International Nuclear Information System (INIS)

Yamada, Kazumasa; Ono, Tetsuyoshi; Nishioka, Hajime

1996-01-01

Escherichia coli mutants which lack defence systems against such active oxygen forms as OxyR (ΔoxyR), superoxide dismutase (SOD) (sodA and sodB) and catalase (katE and katG) are sensitive to UV-A lethality under aerobic conditions, whereas OxyR- and SOD-mutants have resistance under anaerobic conditions and in the presence of sodium azide (NaN 3 ) during irradiation. UV-A induces lipid peroxidation in the ΔoxyR mutant, which is suppressed by NaN 3 . These results suggest that UV-A generates 1 O 2 or the hydroxyl radical to produce lipid peroxides intracellularly in the ΔoxyR mutant and that O 2 - stress may be generated in the sodAB mutant after 8 hr of exposure to UV-A. The sensitivities of such DNA repair-deficient mutants as recA ind- and uvrA to UV-A also were examined and compared. These mutants are sensitive to UV-A lethality under aerobic conditions but show only slight resistance under anaerobic conditions or in the presence of NaN 3 during irradiation. We conclude that NaN 3 protects these mutant cells from oxygen-dependent UV-A lethality. (author)

Zebrafish Lacking Circadian Gene per2 Exhibit Visual Function Deficiency

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Deng-feng Huang

2018-03-01

Full Text Available The retina has an intrinsic circadian clock, but the importance of this clock for vision is unknown. Zebrafish offer many advantages for studying vertebrate vision and circadian rhythm. Here, we explored the role of zebrafish per2, a light-regulated gene, in visual behavior and the underlying mechanisms. We observed that per2 mutant zebrafish larvae showed decreased contrast sensitivity and visual acuity using optokinetic response (OKR assays. Using a visual motor response (VMR assay, we observed normal OFF responses but abnormal ON responses in mutant zebrafish larvae. Immunofluorescence showed that mutants had a normal morphology of cone photoreceptor cells and retinal organization. However, electron microscopy showed that per2 mutants displayed abnormal and decreased photoreceptor ribbon synapses with arciform density, which resulted in retinal ON pathway defect. We also examined the expression of three cone opsins by quantitative real-time PCR (qRT-PCR, and the expression of long-wave-sensitive opsin (opn1lw and short-wave-sensitive opsin (opn1sw was reduced in mutant zebrafish larvae. qRT-PCR analyses also showed a down-regulation of the clock genes cry1ba and bmal1b in the adult eye of per2 mutant zebrafish. This study identified a mechanism by which a clock gene affects visual function and defined important roles of per2 in retinal information processing.

Defects of the Glycinergic Synapse in Zebrafish

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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

Wolbachia Protein TomO Targets nanos mRNA and Restores Germ Stem Cells in Drosophila Sex-lethal Mutants.

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Ote, Manabu; Ueyama, Morio; Yamamoto, Daisuke

2016-09-12

Wolbachia, endosymbiotic bacteria prevalent in invertebrates, manipulate their hosts in a variety of ways: they induce cytoplasmic incompatibility, male lethality, male-to-female transformation, and parthenogenesis. However, little is known about the molecular basis for host manipulation by these bacteria. In Drosophila melanogaster, Wolbachia infection makes otherwise sterile Sex-lethal (Sxl) mutant females capable of producing mature eggs. Through a functional genomic screen for Wolbachia genes with growth-inhibitory effects when expressed in cultured Drosophila cells, we identified the gene WD1278 encoding a novel protein we call toxic manipulator of oogenesis (TomO), which phenocopies some of the Wolbachia effects in Sxl mutant D. melanogaster females. We demonstrate that TomO enhances the maintenance of germ stem cells (GSCs) by elevating Nanos (Nos) expression via its interaction with nos mRNA, ultimately leading to the restoration of germ cell production in Sxl mutant females that are otherwise without GSCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Zebrafish mutants in the von Hippel-Lindau tumor suppressor display a hypoxic response and recapitulate key aspects of Chuvash polycythemia

NARCIS (Netherlands)

van Rooijen, E.; Voest, E.E.; Logister, I.; Korving, J.; Schwerte, T.; Schulte-Merker, S.; Giles, R.H.; van Eeden, F.J.

2009-01-01

We have generated 2 zebrafish lines carrying inactivating germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene ortholog vhl. Mutant embryos display a general systemic hypoxic response, including the up-regulation of hypoxia-induced genes by 1 day after fertilization and a severe

A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development.

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Covassin, L D; Siekmann, A F; Kacergis, M C; Laver, E; Moore, J C; Villefranc, J A; Weinstein, B M; Lawson, N D

2009-05-15

In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development.

Mutant human FUS Is ubiquitously mislocalized and generates persistent stress granules in primary cultured transgenic zebrafish cells.

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Jamie Rae Acosta

Full Text Available FUS mutations can occur in familial amyotrophic lateral sclerosis (fALS, a neurodegenerative disease with cytoplasmic FUS inclusion bodies in motor neurons. To investigate FUS pathology, we generated transgenic zebrafish expressing GFP-tagged wild-type or fALS (R521C human FUS. Cell cultures were made from these zebrafish and the subcellular localization of human FUS and the generation of stress granule (SG inclusions examined in different cell types, including differentiated motor neurons. We demonstrate that mutant FUS is mislocalized from the nucleus to the cytosol to a similar extent in motor neurons and all other cell types. Both wild-type and R521C FUS localized to SGs in zebrafish cells, demonstrating an intrinsic ability of human FUS to accumulate in SGs irrespective of the presence of disease-associated mutations or specific cell type. However, elevation in relative cytosolic to nuclear FUS by the R521C mutation led to a significant increase in SG assembly and persistence within a sub population of vulnerable cells, although these cells were not selectively motor neurons.

Loss of Apela Peptide in Mice Causes Low Penetrance Embryonic Lethality and Defects in Early Mesodermal Derivatives

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Laina Freyer

2017-08-01

Full Text Available Apela (also known as Elabela, Ende, and Toddler is a small signaling peptide that activates the G-protein-coupled receptor Aplnr to stimulate cell migration during zebrafish gastrulation. Here, using CRISPR/Cas9 to generate a null, reporter-expressing allele, we study the role of Apela in the developing mouse embryo. We found that loss of Apela results in low-penetrance cardiovascular defects that manifest after the onset of circulation. Three-dimensional micro-computed tomography revealed a higher penetrance of vascular remodeling defects, from which some mutants recover, and identified extraembryonic anomalies as the earliest morphological distinction in Apela mutant embryos. Transcriptomics at late gastrulation identified aberrant upregulation of erythroid and myeloid markers in mutant embryos prior to the appearance of physical malformations. Double-mutant analyses showed that loss of Apela signaling impacts early Aplnr-expressing mesodermal populations independently of the alternative ligand Apelin, leading to lethal cardiac defects in some Apela null embryos.

The heartstrings mutation in zebrafish causes heart/fin Tbx5 deficiency syndrome.

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Garrity, Deborah M; Childs, Sarah; Fishman, Mark C

2002-10-01

Holt-Oram syndrome is one of the autosomal dominant human "heart-hand" disorders, with a combination of upper limb malformations and cardiac defects. Holt-Oram syndrome is caused by mutations in the TBX5 gene, a member of a large family of T-box transcription factors that play important roles in cell-type specification and morphogenesis. In a screen for mutations affecting zebrafish cardiac function, we isolated the recessive lethal mutant heartstrings, which lacks pectoral fins and exhibits severe cardiac dysfunction, beginning with a slow heart rate and progressing to a stretched, non-functional heart. We mapped and cloned the heartstrings mutation and find it to encode the zebrafish ortholog of the TBX5 gene. The heartstrings mutation causes premature termination at amino acid 316. Homozygous mutant embryos never develop pectoral fin buds and do not express several markers of early fin differentiation. The total absence of any fin bud differentiation distinguishes heartstrings from most other mutations that affect zebrafish fin development, suggesting that Tbx5 functions very early in the pectoral fin induction pathway. Moderate reduction of Tbx5 by morpholino causes fin malformations, revealing an additional early requirement for Tbx5 in coordinating the axes of fin outgrowth. The heart of heartstrings mutant embryos appears to form and function normally through the early heart tube stage, manifesting only a slight bradycardia compared with wild-type siblings. However, the heart fails to loop and then progressively deteriorates, a process affecting the ventricle as well as the atrium. Relative to mammals, fish require lower levels of Tbx5 to produce malformed appendages and display whole-heart rather than atrial-predominant cardiac defects. However, the syndromic deficiencies of tbx5 mutation are remarkably well retained between fish and mammals.

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.

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

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

2016-04-01

Nano-copper oxides are a versatile inorganic material. As a result of their versatility, the immense applications and usage end up in the environment causing a concern for the lifespan of various beings. The ambiguities surround globally on the toxic effects of copper oxide nanoparticles (CuO-NPs). Hence, the present study endeavored to study the sub-lethal acute exposure effects on the developing zebrafish embryos. The 48 hpf LC50 value was about 64 ppm. Therefore, we have chosen the sub-lethal dose of 40 and 60 ppm for the study. Accumulation of CuO-NPs was evidenced from the SEM-EDS and AAS analyzes. The alterations in the AChE and Na(+)/K(+)-ATPase activities disrupted the development process. An increment in the levels of oxidants with a concomitant decrease in the antioxidant enzymes confirmed the induction of oxidative stress. Oxidative stress triggered apoptosis in the exposed embryos. Developmental anomalies were observed with CuO-NPs exposure in addition to oxidative stress in the developing embryos. Decreased heart rate and hatching delay hindered the normal developmental processes. Our work has offered valuable data on the connection between oxidative stress and teratogenicity leading to lethality caused by CuO-NPs. A further molecular mechanism unraveling the uncharted connection between oxidative stress and teratogenicity will aid in the safe use of CuO-NPs. Copyright © 2015 John Wiley & Sons, Ltd.

Disruption of the folate pathway in zebrafish causes developmental defects

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Lee Marina S

2012-04-01

Full Text Available Abstract Background Folic acid supplementation reduces the risk of neural tube defects and congenital heart defects. The biological mechanisms through which folate prevents birth defects are not well understood. We explore the use of zebrafish as a model system to investigate the role of folate metabolism during development. Results We first identified zebrafish orthologs of 12 human folate metabolic genes. RT-PCR and in situ analysis indicated maternal transcripts supply the embryo with mRNA so that the embryo has an intact folate pathway. To perturb folate metabolism we exposed zebrafish embryos to methotrexate (MTX, a potent inhibitor of dihydrofolate reductase (Dhfr an essential enzyme in the folate metabolic pathway. Embryos exposed to high doses of MTX exhibited developmental arrest prior to early segmentation. Lower doses of MTX resulted in embryos with a shortened anterior-posterior axis and cardiac defects: linear heart tubes or incomplete cardiac looping. Inhibition of dhfr mRNA with antisense morpholino oligonucleotides resulted in embryonic lethality. One function of the folate pathway is to provide essential one-carbon units for dTMP synthesis, a rate-limiting step of DNA synthesis. After 24 hours of exposure to high levels of MTX, mutant embryos continue to incorporate the thymidine analog BrdU. However, additional experiments indicate that these embryos have fewer mitotic cells, as assayed with phospho-histone H3 antibodies, and that treated embryos have perturbed cell cycles. Conclusions Our studies demonstrate that human and zebrafish utilize similar one-carbon pathways. Our data indicate that folate metabolism is essential for early zebrafish development. Zebrafish studies of the folate pathway and its deficiencies could provide insight into the underlying etiology of human birth defects and the natural role of folate in development.

Lethal mutants and truncated selection together solve a paradox of the origin of life.

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David B Saakian

Full Text Available BACKGROUND: Many attempts have been made to describe the origin of life, one of which is Eigen's cycle of autocatalytic reactions [Eigen M (1971 Naturwissenschaften 58, 465-523], in which primordial life molecules are replicated with limited accuracy through autocatalytic reactions. For successful evolution, the information carrier (either RNA or DNA or their precursor must be transmitted to the next generation with a minimal number of misprints. In Eigen's theory, the maximum chain length that could be maintained is restricted to 100-1000 nucleotides, while for the most primitive genome the length is around 7000-20,000. This is the famous error catastrophe paradox. How to solve this puzzle is an interesting and important problem in the theory of the origin of life. METHODOLOGY/PRINCIPAL FINDINGS: We use methods of statistical physics to solve this paradox by carefully analyzing the implications of neutral and lethal mutants, and truncated selection (i.e., when fitness is zero after a certain Hamming distance from the master sequence for the critical chain length. While neutral mutants play an important role in evolution, they do not provide a solution to the paradox. We have found that lethal mutants and truncated selection together can solve the error catastrophe paradox. There is a principal difference between prebiotic molecule self-replication and proto-cell self-replication stages in the origin of life. CONCLUSIONS/SIGNIFICANCE: We have applied methods of statistical physics to make an important breakthrough in the molecular theory of the origin of life. Our results will inspire further studies on the molecular theory of the origin of life and biological evolution.

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.

Heart-specific expression of laminopathic mutations in transgenic zebrafish.

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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.

Abnormal Nuclear Pore Formation Triggers Apoptosis in the Intestinal Epithelium of elys-Deficient Zebrafish

NARCIS (Netherlands)

de Jong-Curtain, Tanya A.; Parslow, Adam C.; Trotter, Andrew J.; Hall, Nathan E.; Verkade, Heather; Tabone, Tania; Christie, Elizabeth L.; Crowhurst, Meredith O.; Layton, Judith E.; Shepherd, Iain T.; Nixon, Susan J.; Parton, Robert G.; Zon, Leonard I.; Stainier, Didier Y. R.; Lieschke, Graham J.; Heath, Joan K.

Background & Aims: Zebrafish mutants generated by ethylnitrosourea-mutagenesis provide a powerful toot for dissecting the genetic regulation of developmental processes, including organogenesis. One zebrafish mutant, "flotte lotte" (flo), displays striking defects in intestinal, liver, pancreas, and

Mismatch repair deficiency does not enhance ENU mutagenesis in the zebrafish germ line.

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Feitsma, Harma; de Bruijn, Ewart; van de Belt, Jose; Nijman, Isaac J; Cuppen, Edwin

2008-07-01

S(N)1-type alkylating agents such as N-ethyl-N-nitrosourea (ENU) are very potent mutagens. They act by transferring their alkyl group to DNA bases, which, upon mispairing during replication, can cause single base pair mutations in the next replication cycle. As DNA mismatch repair (MMR) proteins are involved in the recognition of alkylation damage, we hypothesized that ENU-induced mutation rates could be increased in a MMR-deficient background, which would be beneficial for mutagenesis approaches. We applied a standard ENU mutagenesis protocol to adult zebrafish deficient in the MMR gene msh6 and heterozygous controls to study the effect of MMR on ENU-induced DNA damage. Dose-dependent lethality was found to be similar for homozygous and heterozygous mutants, indicating that there is no difference in ENU resistance. Mutation discovery by high-throughput dideoxy resequencing of genomic targets in outcrossed progeny of the mutagenized fish did also not reveal any differences in germ line mutation frequency. These results may indicate that the maximum mutation load for zebrafish has been reached with the currently used, highly optimized ENU mutagenesis protocol. Alternatively, the MMR system in the zebrafish germ line may be saturated very rapidly, thereby having a limited effect on high-dose ENU mutagenesis.

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

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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.

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

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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.

mRNA processing in mutant zebrafish lines generated by chemical and CRISPR-mediated mutagenesis produces unexpected transcripts that escape nonsense-mediated decay.

Directory of Open Access Journals (Sweden)

Jennifer L Anderson

2017-11-01

Full Text Available As model organism-based research shifts from forward to reverse genetics approaches, largely due to the ease of genome editing technology, a low frequency of abnormal phenotypes is being observed in lines with mutations predicted to lead to deleterious effects on the encoded protein. In zebrafish, this low frequency is in part explained by compensation by genes of redundant or similar function, often resulting from the additional round of teleost-specific whole genome duplication within vertebrates. Here we offer additional explanations for the low frequency of mutant phenotypes. We analyzed mRNA processing in seven zebrafish lines with mutations expected to disrupt gene function, generated by CRISPR/Cas9 or ENU mutagenesis methods. Five of the seven lines showed evidence of altered mRNA processing: one through a skipped exon that did not lead to a frame shift, one through nonsense-associated splicing that did not lead to a frame shift, and three through the use of cryptic splice sites. These results highlight the need for a methodical analysis of the mRNA produced in mutant lines before making conclusions or embarking on studies that assume loss of function as a result of a given genomic change. Furthermore, recognition of the types of adaptations that can occur may inform the strategies of mutant generation.

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

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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.

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 ...

Characterization of Helicobacter pylori dapE and construction of a conditionally lethal dapE mutant.

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Karita, M; Etterbeek, M L; Forsyth, M H; Tummuru, M K; Blaser, M J

1997-10-01

Helicobacter pylori colonizes the human gastric mucosa and causes gastritis, ulceration, or gastric cancer. A previously uncharacterized region of the H. pylori genome was identified and sequenced. This region includes a putative operon containing three open reading frames termed gidA (1,866 bp), dapE (1,167 bp), and orf2 (753 bp); the gidA and dapE products are highly homologous to other bacterial proteins. In E. coli, dapE encodes N-succinyl-L-diaminopimelic acid desuccinylase, which catalyzes the hydrolysis of N-succinyl-L-diaminopimelic acid to L-diaminopimelic acid (L-DAP) and succinate. When wild-type H. pylori strains were transformed to select for dapE mutagenesis, mutants were present when plates were supplemented with DAP but not with lysine; orf2 mutants were selected without DAP supplementation. Consistent with the finding that GidA is essential in Escherichia coli, we were unable to obtain a gidA mutant in H. pylori despite evidence that insertional mutagenesis had occurred. The positions of gidA, dapE, and orf2 suggest that they form an operon, which was supported by slot blot RNA hybridization and reverse transcriptase PCR studies. The data imply that the H. pylori dapE mutant may be useful as a conditionally lethal vaccine.

Biophysical analysis of a lethal laminin alpha-1 mutation reveals altered self-interaction

KAUST Repository

Patel, Trushar R.; Nikodemus, Denise; Besong, Tabot M.D.; Reuten, Raphael; Meier, Markus; Harding, Stephen E.; Winzor, Donald J.; Koch, Manuel; Stetefeld, Jö rg

2015-01-01

Laminins are key basement membrane molecules that influence several biological activities and are linked to a number of diseases. They are secreted as heterotrimeric proteins consisting of one α, one β, and one γ chain, followed by their assembly into a polymer-like sheet at the basement membrane. Using sedimentation velocity, dynamic light scattering, and surface plasmon resonance experiments, we studied self-association of three laminin (LM) N-terminal fragments α-1 (hLM α-1 N), α-5 (hLM α-5 N) and β-3 (hLM β-3 N) originating from the short arms of the human laminin αβγ heterotrimer. Corresponding studies of the hLM α-1 N C49S mutant, equivalent to the larval lethal C56S mutant in zebrafish, have shown that this mutation causes enhanced self-association behavior, an observation that provides a plausible explanation for the inability of laminin bearing this mutation to fulfill functional roles in vivo, and hence for the deleterious pathological consequences of the mutation on lens function.

Biophysical analysis of a lethal laminin alpha-1 mutation reveals altered self-interaction

KAUST Repository

Patel, Trushar R.

2015-07-26

Laminins are key basement membrane molecules that influence several biological activities and are linked to a number of diseases. They are secreted as heterotrimeric proteins consisting of one α, one β, and one γ chain, followed by their assembly into a polymer-like sheet at the basement membrane. Using sedimentation velocity, dynamic light scattering, and surface plasmon resonance experiments, we studied self-association of three laminin (LM) N-terminal fragments α-1 (hLM α-1 N), α-5 (hLM α-5 N) and β-3 (hLM β-3 N) originating from the short arms of the human laminin αβγ heterotrimer. Corresponding studies of the hLM α-1 N C49S mutant, equivalent to the larval lethal C56S mutant in zebrafish, have shown that this mutation causes enhanced self-association behavior, an observation that provides a plausible explanation for the inability of laminin bearing this mutation to fulfill functional roles in vivo, and hence for the deleterious pathological consequences of the mutation on lens function.

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.

PFOS, PFNA, and PFOA sub-lethal exposure to embryonic zebrafish have different toxicity profiles in terms of morphometrics, behavior and gene expression.

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Jantzen, Carrie E; Annunziato, Kate A; Bugel, Sean M; Cooper, Keith R

2016-06-01

Polyfluorinated compounds (PFC) are a class of anthropogenic, persistent and toxic chemicals. PFCs are detected worldwide and consist of fluorinated carbon chains of varying length, terminal groups, and industrial uses. Previous zebrafish studies in the literature as well as our own studies have shown that exposure to these chemicals at a low range of concentrations (0.02-2.0μM; 20-2000ppb) resulted in chemical specific developmental defects and reduced post hatch survival. It was hypothesized that sub-lethal embryonic exposure to perfluorooctanesulfonic acid (PFOS), perfluorononanoic acid (PFNA), or perfluorooctanoic acid (PFOA) would result in different responses with regard to morphometric, behavior, and gene expression in both yolk sac fry and larval zebrafish. Zebrafish were exposed to PFOS, PFOA, and PFNA (0.02, 0.2, 2.0μM) for the first five days post fertilization (dpf) and analyzed for morphometrics (5 dpf, 14 dpf), targeted gene expression (5 dpf, 14 dpf), and locomotive behavior (14 dpf). All three PFCs commonly resulted in a decrease in total body length, increased tfc3a (muscle development) expression and decreased ap1s (protein transport) expression at 5dpf, and hyperactive locomotor activity 14 dpf. All other endpoints measured at both life-stage time points varied between each of the PFCs. PFOS, PFNA, and PFOA exposure resulted in significantly altered responses in terms of morphometric, locomotion, and gene expression endpoints, which could be manifested in field exposed teleosts. Copyright © 2016 Elsevier B.V. All rights reserved.

Zebrafish enpp1 mutants exhibit pathological mineralization, mimicking features of generalized arterial calcification of infancy (GACI and pseudoxanthoma elasticum (PXE

Directory of Open Access Journals (Sweden)

Alexander Apschner

2014-07-01

Full Text Available In recent years it has become clear that, mechanistically, biomineralization is a process that has to be actively inhibited as a default state. This inhibition must be released in a rigidly controlled manner in order for mineralization to occur in skeletal elements and teeth. A central aspect of this concept is the tightly controlled balance between phosphate, a constituent of the biomineral hydroxyapatite, and pyrophosphate, a physiochemical inhibitor of mineralization. Here, we provide a detailed analysis of a zebrafish mutant, dragonfish (dgf, which is mutant for ectonucleoside pyrophosphatase/phosphodiesterase 1 (Enpp1, a protein that is crucial for supplying extracellular pyrophosphate. Generalized arterial calcification of infancy (GACI is a fatal human disease, and the majority of cases are thought to be caused by mutations in ENPP1. Furthermore, some cases of pseudoxanthoma elasticum (PXE have recently been linked to ENPP1. Similar to humans, we show here that zebrafish enpp1 mutants can develop ectopic calcifications in a variety of soft tissues – most notably in the skin, cartilage elements, the heart, intracranial space and the notochord sheet. Using transgenic reporter lines, we demonstrate that ectopic mineralizations in these tissues occur independently of the expression of typical osteoblast or cartilage markers. Intriguingly, we detect cells expressing the osteoclast markers Trap and CathepsinK at sites of ectopic calcification at time points when osteoclasts are not yet present in wild-type siblings. Treatment with the bisphosphonate etidronate rescues aspects of the dgf phenotype, and we detected deregulated expression of genes that are involved in phosphate homeostasis and mineralization, such as fgf23, npt2a, entpd5 and spp1 (also known as osteopontin. Employing a UAS-GalFF approach, we show that forced expression of enpp1 in blood vessels or the floorplate of mutant embryos is sufficient to rescue the notochord

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

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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.

Biosecurity and Health Monitoring at the Zebrafish International Resource Center.

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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.

Innate Color Preference of Zebrafish and Its Use in Behavioral Analyses.

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Park, Jong-Su; Ryu, Jae-Ho; Choi, Tae-Ik; Bae, Young-Ki; Lee, Suman; Kang, Hae Jin; Kim, Cheol-Hee

2016-10-01

Although innate color preference of motile organisms may provide clues to behavioral biases, it has remained a longstanding question. In this study, we investigated innate color preference of zebrafish larvae. A cross maze with different color sleeves around each arm was used for the color preference test (R; red, G; green, B; blue, Y; yellow). The findings showed that 5 dpf zebrafish larvae preferred blue over other colors (B > R > G > Y). To study innate color recognition further, tyrosinase mutants were generated using CRISPR/Cas9 system. As a model for oculocutaneous albinism (OCA) and color vision impairment, tyrosinase mutants demonstrated diminished color sensation, indicated mainly by hypopigmentation of the retinal pigment epithelium (RPE). Due to its relative simplicity and ease, color preference screening using zebrafish larvae is suitable for high-throughput screening applications. This system may potentially be applied to the analysis of drug effects on larval behavior or the detection of sensory deficits in neurological disorder models, such as autism-related disorders, using mutant larvae generated by the CRISPR/Cas9 technique.

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

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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.

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

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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.

Zebrafish scarb2a insertional mutant reveals a novel function for the Scarb2/Limp2 receptor in notochord development.

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Diaz-Tellez, Abigail; Zampedri, Cecilia; Ramos-Balderas, Jose L; García-Hernández, Fernando; Maldonado, Ernesto

2016-04-01

Scarb2 or Limp2 belong to a subfamily of Scavenger receptors described as lysosomal transmembrane glycosylated receptors, that are mutated in the human syndrome AMRF (action myoclonus-renal failure). The zebrafish insertional mutant scarb2a(hi1463Tg) has notochord defects, the notochord is a defining feature of chordates running along the center of the longitudinal axis and it is essential for forming the spinal column in all vertebrates. There are three paralogous scarb2 genes in zebrafish; scarb2a, scarb2b, and scarb2c. Both Scarb2a and Scarb2b proteins lack the classical di-leucine motif. We found that scarb2a(hi1463Tg) homozygous zebrafish embryos have a null mutation impairing vacuole formation in the notochord and simultaneously disrupting proper formation of the basement membrane resulting in its thickening at the ventral side of the notochord, which may be the cause for the anomalous upward bending observed in the trunk. Through whole-mount in situ hybridization, we detected scarb2a mRNA expression in the notochord and in the brain early in development. However, it is puzzling that scarb2a notochord mRNA expression is short-lived in the presumptive notochord and precedes the complete differentiation of the notochord. This work describes a novel function for the Scarb2 receptor as an essential glycoprotein for notochord development. © 2016 Wiley Periodicals, Inc.

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

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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.

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

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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 neurogenetic frontier--lessons from misbehaving zebrafish.

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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.

Essential role for fibrillin-2 in zebrafish notochord and vascular morphogenesis.

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Gansner, John M; Madsen, Erik C; Mecham, Robert P; Gitlin, Jonathan D

2008-10-01

Recent studies demonstrate that lysyl oxidase cuproenzymes are critical for zebrafish notochord formation, but the molecular mechanisms of copper-dependent notochord morphogenesis are incompletely understood. We, therefore, conducted a forward genetic screen for zebrafish mutants that exhibit notochord sensitivity to lysyl oxidase inhibition, yielding a mutant with defects in notochord and vascular morphogenesis, puff daddygw1 (pfdgw1). Meiotic mapping and cloning reveal that the pfdgw1 phenotype results from disruption of the gene encoding the extracellular matrix protein fibrillin-2, and the spatiotemporal expression of fibrillin-2 is consistent with the pfdgw1 phenotype. Furthermore, each aspect of the pfdgw1 phenotype is recapitulated by morpholino knockdown of fibrillin-2. Taken together, the data reveal a genetic interaction between fibrillin-2 and the lysyl oxidases in notochord formation and demonstrate the importance of fibrillin-2 in specific early developmental processes in zebrafish. Copyright (c) 2008 Wiley-Liss, Inc.

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

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

2014-05-01

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

ZNStress: a high-throughput drug screening protocol for identification of compounds modulating neuronal stress in the transgenic mutant sod1G93R zebrafish model of amyotrophic lateral sclerosis

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McGown, Alexander; Shaw, Dame Pamela J.; Ramesh, Tennore

2016-01-01

Background Amyotrophic lateral sclerosis (ALS) is a lethal neurodegenerative disease with death on average within 2?3 years of symptom onset. Mutations in superoxide dismutase 1 (SOD1) have been identified to cause ALS. Riluzole, the only neuroprotective drug for ALS provides life extension of only 3 months on average. Thishighlights the need for compound screening in disease models to identify new neuroprotective therapies for this disease. Zebrafish is an emerging model system that is well ...

A zebrafish screen for craniofacial mutants identifies wdr68 as a highly conserved gene required for endothelin-1 expression

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Amsterdam Adam

2006-06-01

Full Text Available Abstract Background Craniofacial birth defects result from defects in cranial neural crest (NC patterning and morphogenesis. The vertebrate craniofacial skeleton is derived from cranial NC cells and the patterning of these cells occurs within the pharyngeal arches. Substantial efforts have led to the identification of several genes required for craniofacial skeletal development such as the endothelin-1 (edn1 signaling pathway that is required for lower jaw formation. However, many essential genes required for craniofacial development remain to be identified. Results Through screening a collection of insertional zebrafish mutants containing approximately 25% of the genes essential for embryonic development, we present the identification of 15 essential genes that are required for craniofacial development. We identified 3 genes required for hyomandibular development. We also identified zebrafish models for Campomelic Dysplasia and Ehlers-Danlos syndrome. To further demonstrate the utility of this method, we include a characterization of the wdr68 gene. We show that wdr68 acts upstream of the edn1 pathway and is also required for formation of the upper jaw equivalent, the palatoquadrate. We also present evidence that the level of wdr68 activity required for edn1 pathway function differs between the 1st and 2nd arches. Wdr68 interacts with two minibrain-related kinases, Dyrk1a and Dyrk1b, required for embryonic growth and myotube differentiation, respectively. We show that a GFP-Wdr68 fusion protein localizes to the nucleus with Dyrk1a in contrast to an engineered loss of function mutation Wdr68-T284F that no longer accumulated in the cell nucleus and failed to rescue wdr68 mutant animals. Wdr68 homologs appear to exist in all eukaryotic genomes. Notably, we found that the Drosophila wdr68 homolog CG14614 could substitute for the vertebrate wdr68 gene even though insects lack the NC cell lineage. Conclusion This work represents a systematic

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

Small molecule screening identifies targetable zebrafish pigmentation pathways

DEFF Research Database (Denmark)

Colanesi, Sarah; Taylor, Kerrie L; Temperley, Nicholas D

2012-01-01

Small molecules complement genetic mutants and can be used to probe pigment cell biology by inhibiting specific proteins or pathways. Here, we present the results of a screen of active compounds for those that affect the processes of melanocyte and iridophore development in zebrafish and investig......Small molecules complement genetic mutants and can be used to probe pigment cell biology by inhibiting specific proteins or pathways. Here, we present the results of a screen of active compounds for those that affect the processes of melanocyte and iridophore development in zebrafish...... and investigate the effects of a few of these compounds in further detail. We identified and confirmed 57 compounds that altered pigment cell patterning, number, survival, or differentiation. Additional tissue targets and toxicity of small molecules are also discussed. Given that the majority of cell types...

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

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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.

Yoshiki Hotta and the dawn of zebrafish molecular neurogenetics in Japan.

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Higashijima, Shin-Ichi; Okamoto, Hitoshi

2012-03-01

Abstract: After coming back to Japan to work in the Department of Physics at the University of Tokyo, Yoshiki Hotta spent a year or so on searching for behavioral mutants of goldfish. Although this endeavor did not succeed, he remained an adamant supporter of the development of zebrafish research in Japan. Here we review how his support helped zebrafish neurogenetics in Japan gain a unique position in the world research community.

Mitochondrial uncoupler exerts a synthetic lethal effect against β-catenin mutant tumor cells.

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Shikata, Yuki; Kiga, Masaki; Futamura, Yushi; Aono, Harumi; Inoue, Hiroyuki; Kawada, Manabu; Osada, Hiroyuki; Imoto, Masaya

2017-04-01

The wingless/int-1 (Wnt) signal transduction pathway plays a central role in cell proliferation, survival, differentiation and apoptosis. When β-catenin: a component of the Wnt pathway, is mutated into an active form, cell growth signaling is hyperactive and drives oncogenesis. As β-catenin is mutated in a wide variety of tumors, including up to 10% of all sporadic colon carcinomas and 20% of hepatocellular carcinomas, it has been considered a promising target for therapeutic interventions. Therefore, we screened an in-house natural product library for compounds that exhibited synthetic lethality towards β-catenin mutations and isolated nonactin, an antibiotic mitochondrial uncoupler, as a hit compound. Nonactin, as well as other mitochondrial uncouplers, induced apoptosis selectively in β-catenin mutated tumor cells. Significant tumor regression was observed in the β-catenin mutant HCT 116 xenograft model, but not in the β-catenin wild type A375 xenograft model, in response to daily administration of nonactin in vivo. Furthermore, we found that expression of an active mutant form of β-catenin induced a decrease in the glycolysis rate. Taken together, our results demonstrate that tumor cells with mutated β-catenin depend on mitochondrial oxidative phosphorylation for survival. Therefore, they undergo apoptosis in response to mitochondrial dysfunction following the addition of mitochondrial uncouplers, such as nonactin. These results suggest that targeting mitochondria is a potential chemotherapeutic strategy for tumor cells that harbor β-catenin mutations. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

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

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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

Mechanisms of prickle1a function in zebrafish epilepsy and retinal neurogenesis

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Xue Mei

2013-05-01

Epilepsy is a complex neurological disorder characterized by unprovoked seizures. The etiology is heterogeneous with both genetic and environmental causes. Genes that regulate neurotransmitters and ion channels in the central nervous system have been associated with epilepsy. However, a recent screening in human epilepsy patients identified mutations in the PRICKLE1 (PK1 locus, highlighting a potentially novel mechanism underlying seizures. PK1 is a core component of the planar cell polarity network that regulates tissue polarity. Zebrafish studies have shown that Pk1 coordinates cell movement, neuronal migration and axonal outgrowth during embryonic development. Yet how dysfunction of Pk1 relates to epilepsy is unknown. To address the mechanism underlying epileptogenesis, we used zebrafish to characterize Pk1a function and epilepsy-related mutant forms. We show that knockdown of pk1a activity sensitizes zebrafish larva to a convulsant drug. To model defects in the central nervous system, we used the retina and found that pk1a knockdown induces neurite outgrowth defects; yet visual function is maintained. Furthermore, we characterized the functional and biochemical properties of the PK1 mutant forms identified in human patients. Functional analyses demonstrate that the wild-type Pk1a partially suppresses the gene knockdown retinal defects but not the mutant forms. Biochemical analysis reveals increased ubiquitylation of one mutant form and decreased translational efficiency of another mutant form compared with the wild-type Pk1a. Taken together, our results indicate that mutation of human PK1 could lead to defects in neurodevelopment and signal processing, providing insight into seizure predisposition in these patients.

Mutation of a vitelline membrane protein, BmEP80, is responsible for the silkworm "Ming" lethal egg mutant.

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Chen, Anli; Gao, Peng; Zhao, Qiaoling; Tang, Shunming; Shen, Xingjia; Zhang, Guozheng; Qiu, Zhiyong; Xia, Dingguo; Huang, Yongping; Xu, Yunmin; He, Ningjia

2013-02-25

The egg stage is an important stage in the silkworm (Bombyx mori) life cycle. Normal silkworm eggs are usually short, elliptical, and laterally flattened, with a sometimes hollowed surface on the lateral side. However, the eggs laid by homozygous recessive "Ming" lethal egg mutants (l-e(m)) lose water and become concaved around 1h, ultimately exhibiting a triangular shape on the egg surfaces. We performed positional cloning, and narrowed down the region containing the gene responsible for the l-e(m) mutant to 360 kb on chromosome 10 using 2287 F(2) individuals. Using expression analysis and RNA interference, the best l-e(m) candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that an ~1.9 kb region from the 3' untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a >100 kb DNA fragment in the l-e(m) mutant. Several eggs laid by the normal moths injected with BmEP80 small interfering RNAs were evidently depressed and exhibited a triangular shape on the surface. The phenotype exhibited was consistent with the eggs laid by the l-e(m) mutant. Moreover, two-dimensional gel electrophoresis showed that the BmEP80 protein was expressed in the ovary from the 9th day of the pupa stage to eclosion in the wild-type silkworm, but was absent in the l-e(m) mutant. These results indicate that BmEP80 is responsible for the l-e(m) mutation. Copyright © 2012 Elsevier B.V. All rights reserved.

Generation and characterization of Kctd15 mutations in zebrafish.

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Alison Heffer

Full Text Available Potassium channel tetramerization domain containing 15 (Kctd15 was previously found to have a role in early neural crest (NC patterning, specifically delimiting the region where NC markers are expressed via repression of transcription factor AP-2a and inhibition of Wnt signaling. We used transcription activator-like effector nucleases (TALENs to generate null mutations in zebrafish kctd15a and kctd15b paralogs to study the in vivo role of Kctd15. We found that while deletions producing frame-shift mutations in each paralog showed no apparent phenotype, kctd15a/b double mutant zebrafish are smaller in size and show several phenotypes including some affecting the NC, such as expansion of the early NC domain, increased pigmentation, and craniofacial defects. Both melanophore and xanthophore pigment cell numbers and early markers are up-regulated in the double mutants. While we find no embryonic craniofacial defects, adult mutants have a deformed maxillary segment and missing barbels. By confocal imaging of mutant larval brains we found that the torus lateralis (TLa, a region implicated in gustatory networks in other fish, is absent. Ablation of this brain tissue in wild type larvae mimics some aspects of the mutant growth phenotype. Thus kctd15 mutants show deficits in the development of both neural crest derivatives, and specific regions within the central nervous system, leading to a strong reduction in normal growth rates.

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

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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

Redundant roles of PRDM family members in zebrafish craniofacial development.

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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.

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

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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.

Norepinephrine is required to promote wakefulness and for hypocretin-induced arousal in zebrafish.

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Singh, Chanpreet; Oikonomou, Grigorios; Prober, David A

2015-09-16

Pharmacological studies in mammals suggest that norepinephrine (NE) plays an important role in promoting arousal. However, the role of endogenous NE is unclear, with contradicting reports concerning the sleep phenotypes of mice lacking NE due to mutation of dopamine β-hydroxylase (dbh). To investigate NE function in an alternative vertebrate model, we generated dbh mutant zebrafish. In contrast to mice, these animals exhibit dramatically increased sleep. Surprisingly, despite an increase in sleep, dbh mutant zebrafish have a reduced arousal threshold. These phenotypes are also observed in zebrafish treated with small molecules that inhibit NE signaling, suggesting that they are caused by the lack of NE. Using genetic overexpression of hypocretin (Hcrt) and optogenetic activation of hcrt-expressing neurons, we also find that NE is important for Hcrt-induced arousal. These results establish a role for endogenous NE in promoting arousal and indicate that NE is a critical downstream effector of Hcrt neurons.

Phleomycin-induced lethality and DNA degradation in Escherichia coli K12

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Nakayama, H

1975-01-01

The cell lethality and DNA fragmentation caused by phleomycin (PM) were studied in E. coli K12 strains with special reference to the effects of repair or recombination deficiencies and metabolic inhibitors. Unlike excision-defective derivatives of E. coli B, uvrA, uvrB, and uvrC mutants of strain K12 showed no peculiarities compared with wild type in regard to cell survival. Likewise, mutant alleles at uvrD and polA loci had no effect. In contrast, rec mutants were more sensitive to PM-killing than were rec/sup +/ strains. PM-induced strand breakage in DNA was observed in all strains tested including the above-mentioned mutants. There was no significant distinction between the uvr mutants and the wild type strain, indicating that the uvr-endonuclease was not responsible for the strand breaks. Involvement of endonuclease I was also ruled out. At least some of the PM-induced strand breaks were repairable. PM-induced lethality and strand breakage were totally dependent on energy supply. Inhibition of protein synthesis resulted in a partial and parallel suppression of the two effects. Our results suggest that the lethality is due to DNA strand breakage and the repair of such damage is postulated to be controlled by rec genes.

Functional and Genetic Analysis of Choroid Plexus Development in Zebrafish

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Hannah Elizabeth Henson

2014-11-01

Full Text Available The choroid plexus, an epithelial-based structure localized in the brain ventricle, is the major component of the blood-cerebrospinal fluid barrier. The choroid plexus produces the cerebrospinal fluid and regulates the components of the cerebrospinal fluid. Abnormal choroid plexus function is associated with neurodegenerative diseases, tumor formation in the choroid plexus epithelium, and hydrocephaly. In this study, we used zebrafish (Danio rerio as a model system to understand the genetic components of choroid plexus development. We generated an enhancer trap line, Et(cp:EGFPsj2, that expresses enhanced green fluorescent protein (EGFP in the choroid plexus epithelium. Using immunohistochemistry and fluorescent tracers, we demonstrated that the zebrafish choroid plexus possesses brain barrier properties such as tight junctions and transporter activity. Thus, we have established zebrafish as a functionally relevant model to study choroid plexus development. Using an unbiased approach, we performed a forward genetic dissection of the choroid plexus to identify genes essential for its formation and function. Using Et(cp:EGFPsj2, we isolated 10 recessive mutant lines with choroid plexus abnormalities, which were grouped into five classes based on GFP intensity, epithelial localization, and overall choroid plexus morphology. We also mapped the mutation for two mutant lines to chromosomes 4 and 21, respectively. The mutants generated in this study can be used to elucidate specific genes and signaling pathways essential for choroid plexus development, function, and/or maintenance and will provide important insights into how these genetic mutations contribute to disease.

The role of Sox6 in zebrafish muscle fiber type specification.

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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

Definition of the zebrafish genome using flow cytometry and cytogenetic mapping

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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.

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.

Expression of Thiaminase in Zebrafish (Danio rerio) is Lethal and Has Implications for Use as a Biocontainment Strategy in Aquaculture and Invasive Species.

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Noble, Sandra; Saxena, Vishal; Ekker, Marc; Devlin, Robert

2017-12-01

As the world increasingly relies on aquaculture operations to meet rising seafood demands, reliable biocontainment measures for farmed fish stocks are desired to minimize ecological impacts arising from interactions of cultured fish with wild populations. One possible biocontainment strategy is to induce a dietary dependence on a vitamin, such as thiamine (vitamin B1), required for survival. Fish expressing thiaminase (an enzyme that degrades thiamine) within a confined aquaculture facility could receive supplemental thiamine to allow survival and normal growth, whereas escapees lacking this dietary rescue would die from thiamine deficiency. To test the concept and efficacy of such a dietary dependency system (for potential future use in larger aquaculture species), we expressed thiaminase in zebrafish as a test model. We drove the expression of thiaminase under the strong ubiquitous and constitutive control of the CMV promoter which resulted in non-viable fish, indicating that the thiaminase sequence kills fish. However, the CMV promoter is too strong to allow conditional survival since the lethality could not be rescued by exogenous thiamine provided as a supplement to typical food. In addition, microinjection of 0.5 pg of thiaminase mRNA in zebrafish embryos at the one-cell stage resulted in 50% larval mortality at 5 days post-fertilization (dpf), which was partially rescued by thiamine supplementation. Evaluating the efficacy of biocontainment strategies helps assess which methods can reliably prevent ecological impacts arising from breaches in physical containment systems that release engineered organisms to nature, and consequently provides critical information for use in regulatory risk assessment processes.

Whole-body and multispectral photoacoustic imaging of adult zebrafish

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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.

Noonan syndrome gain-of-function mutations in NRAS cause zebrafish gastrulation defects

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Vincent Runtuwene

2011-05-01

Noonan syndrome is a relatively common developmental disorder that is characterized by reduced growth, wide-set eyes and congenital heart defects. Noonan syndrome is associated with dysregulation of the Ras–mitogen-activated-protein-kinase (MAPK signaling pathway. Recently, two mutations in NRAS were reported to be associated with Noonan syndrome, T50I and G60E. Here, we report a mutation in NRAS, resulting in an I24N amino acid substitution, that we identified in an individual bearing typical Noonan syndrome features. The I24N mutation activates N-Ras, resulting in enhanced downstream signaling. Expression of N-Ras-I24N, N-Ras-G60E or the strongly activating mutant N-Ras-G12V, which we included as a positive control, results in developmental defects in zebrafish embryos, demonstrating that these activating N-Ras mutants are sufficient to induce developmental disorders. The defects in zebrafish embryos are reminiscent of symptoms in individuals with Noonan syndrome and phenocopy the defects that other Noonan-syndrome-associated genes induce in zebrafish embryos. MEK inhibition completely rescued the activated N-Ras-induced phenotypes, demonstrating that these defects are mediated exclusively by Ras-MAPK signaling. In conclusion, mutations in NRAS from individuals with Noonan syndrome activated N-Ras signaling and induced developmental defects in zebrafish embryos, indicating that activating mutations in NRAS cause Noonan syndrome.

Noonan syndrome gain-of-function mutations in NRAS cause zebrafish gastrulation defects

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Runtuwene, Vincent; van Eekelen, Mark; Overvoorde, John; Rehmann, Holger; Yntema, Helger G.; Nillesen, Willy M.; van Haeringen, Arie; van der Burgt, Ineke; Burgering, Boudewijn; den Hertog, Jeroen

2011-01-01

SUMMARY Noonan syndrome is a relatively common developmental disorder that is characterized by reduced growth, wide-set eyes and congenital heart defects. Noonan syndrome is associated with dysregulation of the Ras–mitogen-activated-protein-kinase (MAPK) signaling pathway. Recently, two mutations in NRAS were reported to be associated with Noonan syndrome, T50I and G60E. Here, we report a mutation in NRAS, resulting in an I24N amino acid substitution, that we identified in an individual bearing typical Noonan syndrome features. The I24N mutation activates N-Ras, resulting in enhanced downstream signaling. Expression of N-Ras-I24N, N-Ras-G60E or the strongly activating mutant N-Ras-G12V, which we included as a positive control, results in developmental defects in zebrafish embryos, demonstrating that these activating N-Ras mutants are sufficient to induce developmental disorders. The defects in zebrafish embryos are reminiscent of symptoms in individuals with Noonan syndrome and phenocopy the defects that other Noonan-syndrome-associated genes induce in zebrafish embryos. MEK inhibition completely rescued the activated N-Ras-induced phenotypes, demonstrating that these defects are mediated exclusively by Ras-MAPK signaling. In conclusion, mutations in NRAS from individuals with Noonan syndrome activated N-Ras signaling and induced developmental defects in zebrafish embryos, indicating that activating mutations in NRAS cause Noonan syndrome. PMID:21263000

TRPM7 is required within zebrafish sensory neurons for the activation of touch-evoked escape behaviors

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Low, Sean E.; Amburgey, Kimberly; Horstick, Eric; Linsley, Jeremy; Sprague, Shawn M.; Cui, Wilson W.; Zhou, Weibin; Hirata, Hiromi; Saint-Amant, Louis; Hume, Richard I.; Kuwada, John Y.

2011-01-01

Mutations in the gene encoding TRPM7 (trpm7), a member of the TRP superfamily of cation channels that possesses an enzymatically active kinase at its carboxyl terminus, cause the touch-unresponsive zebrafish mutant touchdown. We identified and characterized a new allele of touchdown, as well as two previously reported alleles, and found that all three alleles harbor mutations which abolish channel activity. Through the selective restoration of TRPM7 expression in sensory neurons we found that TRPM7’s kinase activity, and selectivity for divalent cations over monovalent cations, were dispensable for touch-evoked activation of escape behaviors in zebrafish. Additional characterization revealed that sensory neurons were present and capable of responding to tactile stimuli in touchdown mutants, indicating that TRPM7 is not required for sensory neuron survival or mechanosensation. Finally, exposure to elevated concentrations of divalent cations was found to restore touch-evoked behaviors in touchdown mutants. Collectively these findings are consistent with a role for zebrafish TRPM7 within sensory neurons in the modulation of neurotransmitter release at central synapses, similar to that proposed for mammalian TRPM7 at peripheral synapses. PMID:21832193

Theories of Lethal Mutagenesis: From Error Catastrophe to Lethal Defection.

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Tejero, Héctor; Montero, Francisco; Nuño, Juan Carlos

2016-01-01

RNA viruses get extinct in a process called lethal mutagenesis when subjected to an increase in their mutation rate, for instance, by the action of mutagenic drugs. Several approaches have been proposed to understand this phenomenon. The extinction of RNA viruses by increased mutational pressure was inspired by the concept of the error threshold. The now classic quasispecies model predicts the existence of a limit to the mutation rate beyond which the genetic information of the wild type could not be efficiently transmitted to the next generation. This limit was called the error threshold, and for mutation rates larger than this threshold, the quasispecies was said to enter into error catastrophe. This transition has been assumed to foster the extinction of the whole population. Alternative explanations of lethal mutagenesis have been proposed recently. In the first place, a distinction is made between the error threshold and the extinction threshold, the mutation rate beyond which a population gets extinct. Extinction is explained from the effect the mutation rate has, throughout the mutational load, on the reproductive ability of the whole population. Secondly, lethal defection takes also into account the effect of interactions within mutant spectra, which have been shown to be determinant for the understanding the extinction of RNA virus due to an augmented mutational pressure. Nonetheless, some relevant issues concerning lethal mutagenesis are not completely understood yet, as so survival of the flattest, i.e. the development of resistance to lethal mutagenesis by evolving towards mutationally more robust regions of sequence space, or sublethal mutagenesis, i.e., the increase of the mutation rate below the extinction threshold which may boost the adaptability of RNA virus, increasing their ability to develop resistance to drugs (including mutagens). A better design of antiviral therapies will still require an improvement of our knowledge about lethal

The search for evolutionary developmental origins of aging in zebrafish: a novel intersection of developmental and senescence biology in the zebrafish model system.

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Kishi, Shuji

2011-09-01

Senescence may be considered the antithesis of early development, but yet there may be factors and mechanisms in common between these two phenomena during the process of aging. We investigated whether any relationship exists between the regulatory mechanisms that function in early development and in senescence using the zebrafish (Danio rerio), a small freshwater fish and a useful model animal for genetic studies. We conducted experiments to isolate zebrafish mutants expressing an apparent senescence phenotype during embryogenesis (embryonic senescence). Some of the genes we thereby identified had already been associated with cellular senescence and chronological aging in other organisms, but many had not yet been linked to these processes. Complete loss-of-function of developmentally essential genes induce embryonic (or larval) lethality, whereas it seems like their partial loss-of-function (i.e., decrease-of-function by heterozygote or hypomorphic mutations) still remains sufficient to go through the early developmental process because of its adaptive plasticity or rather heterozygote advantage. However, in some cases, such partial loss-of-function of genes compromise normal homeostasis due to haploinsufficiency later in adult life having many environmental stress challenges. By contrast, any heterozygote-advantageous genes might gain a certain benefit(s) (much more fitness) by such partial loss-of-function later in life. Physiological senescence may evolutionarily arise from both genetic and epigenetic drifts as well as from losing adaptive developmental plasticity in face of stress signals from the external environment that interacts with functions of multiple genes rather than effects of only a single gene mutation or defect. Previously uncharacterized developmental genes may thus mediate the aging process and play a pivotal role in senescence. Moreover, unexpected senescence-related genes might also be involved in the early developmental process and

In Vivo Toxicity of Silver Nanoparticles and Silver Ions in Zebrafish (Danio rerio

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Katrine Bilberg

2012-01-01

Full Text Available The influence of water chemistry on characterised polyvinyl pyrrolidone- (PVP- coated silver nanoparticles (81 nm was investigated. NaCl solution series of 100–800 mg L−1 lead to initial and temporal increase in nanoparticles size, but agglomeration was limited. pH variation (5–8 had only minor influence on the hydrodynamic particle size. Acute toxicity of nanosivler to zebrafish (Danio rerio was investigated in a 48-hour static renewal study and compared with the toxicity of silver ions (AgNO3. The nanosilver and silver ion 48-hour median lethal concentration (LC50 values were 84 μg L−1 and 25 μg L−1, respectively. To investigate exposure-related stress, the fish behaviour was observed visually after 0, 3, 6, 12, 24, 27, 30, and 48 hours of both nanosilver and ionic silver treatments. These observations revealed increased rate of operculum movement and surface respiration after nanosilver exposure, suggesting respiratory toxicity. The present study demonstrates that silver nanoparticles are lethal to zebrafish.

Isolation and characterization of radioresistant mutants in Bacillus subtilis and Bacillus thuringiensis

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Kalinin, V.L.; Petrov, V.N.; Petrova, T.M.

1981-01-01

Vegetative cells of Bac. thuringiensis var. galleriae (the wild-type strain 351) are much more sensitive to lethal effects of UV light and 60 Co-γ-rays than those of Bac. subtilis (the wild-type strain 168). This difference is less pronounced for spores of these strains. By means of repeated γ-irradiation-regrowth cycles radioresistant mutants Bac. thuringiensis Gamsup(r) 14 and Bac. subtilis Gamsup(r) 9 were selected. The vegetative cells of these mutants are correspondingly 19 times and 3.9 times more resistant to lethal effects of γ-radiation than the cells of the parental strains. The resistance of the Gamsup(r) mutant cells to lethal effects of UV light and H 2 O 2 is also increased. The spores of the Gamsup(r) 14 mutant are 1.5-1.7 times more resistant to γ-radiation and UV light than the wild-type spores. The radioresistant mutants and the parental strains do not vary in their capacity for host-cell reactivation of UV- or γ-irradiated phages Tg13 and 105

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

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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.

New Insights Into the Role of Estrogens in Male Fertility Based on Findings in Aromatase-Deficient Zebrafish.

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Tang, Haipei; Chen, Yu; Liu, Yun; Yin, Yike; Li, Gaofei; Guo, Yin; Liu, Xiaochun; Lin, Haoran

2017-09-01

It has been demonstrated that estrogens are indispensable for male fertility in mammals. Aromatase (encoded by CYP19) catalyzes the final step of estradiol biosynthesis. However, less is known about the role of aromatase in male fertility in nonmammalian species. Fish aromatase is encoded by two separate genes: the gonad-specific cyp19a1a and the brain-specific cyp19a1b. In a recent study, we used transcription activatorlike effector nucleases to systematically generate cyp19a1a and cyp19a1b mutant lines and a cyp19a1a;cyp19a1b double-mutant line in zebrafish and demonstrated that cyp19a1a was indispensable for sex differentiation. In this study, we focused on male fertility in these aromatase-deficient zebrafish. Our results showed that all aromatase-deficient male fish had normal fertility even at 1 year after fertilization. Interestingly, we observed more spermatozoa in the cyp19a1a and double-mutant males than in the wild-type and cyp19a1b mutant males. The whole-body androgen levels, follicle-stimulating hormone β and luteinizing hormone β protein levels in the pituitary, and transcript levels of genes known to be involved in spermatogenesis and steroidogenesis in the testes were significantly higher in the cyp19a1a mutant and aromatase double-mutant males than in the wild-type and cyp19a1b mutant males. These results might explain why more spermatozoa were observed in these fish. Collectively, our findings indicate that estrogens are not needed to achieve and maintain normal fertility in male zebrafish. This finding challenges the traditional view that estrogens are indispensable for male fertility. Copyright © 2017 Endocrine Society.

Genome-wide gene expression profiling of acute metal exposures in male zebrafish

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Christine E. Baer

2014-12-01

Full Text Available To capture global responses to metal poisoning and mechanistic insights into metal toxicity, gene expression changes were evaluated in whole adult male zebrafish following acute 24 h high dose exposure to three metals with known human health risks. Male adult zebrafish were exposed to nickel chloride, cobalt chloride or sodium dichromate at concentrations corresponding to their respective 96 h LC20, LC40 and LC60 (i.e. 96 h concentrations at which 20%, 40% and 60% lethality is expected, respectively. Histopathology was performed on a subset of metal-exposed zebrafish to phenotypically anchor transcriptional changes associated with each metal exposure. Here we describe in detail the contents and quality controls for the gene expression and other data associated with the study published by Hussainzada and colleagues in BMC Pharmacology and Toxicology (Hussainzada et al., 2014 with the data uploaded to Gene Expression Omnibus (accession number GSE50648.

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

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

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

2016-07-01

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

Molecular genetics of pituitary development in zebrafish.

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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.

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

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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

Multi-organ abnormalities and mTORC1 activation in zebrafish model of multiple acyl-CoA dehydrogenase deficiency.

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Seok-Hyung Kim

2013-06-01

Full Text Available Multiple Acyl-CoA Dehydrogenase Deficiency (MADD is a severe mitochondrial disorder featuring multi-organ dysfunction. Mutations in either the ETFA, ETFB, and ETFDH genes can cause MADD but very little is known about disease specific mechanisms due to a paucity of animal models. We report a novel zebrafish mutant dark xavier (dxa(vu463 that has an inactivating mutation in the etfa gene. dxa(vu463 recapitulates numerous pathological and biochemical features seen in patients with MADD including brain, liver, and kidney disease. Similar to children with MADD, homozygote mutant dxa(vu463 zebrafish have a spectrum of phenotypes ranging from moderate to severe. Interestingly, excessive maternal feeding significantly exacerbated the phenotype. Homozygous mutant dxa(vu463 zebrafish have swollen and hyperplastic neural progenitor cells, hepatocytes and kidney tubule cells as well as elevations in triacylglycerol, cerebroside sulfate and cholesterol levels. Their mitochondria were also greatly enlarged, lacked normal cristae, and were dysfunctional. We also found increased signaling of the mechanistic target of rapamycin complex 1 (mTORC1 with enlarged cell size and proliferation. Treatment with rapamycin partially reversed these abnormalities. Our results indicate that etfa gene function is remarkably conserved in zebrafish as compared to humans with highly similar pathological, biochemical abnormalities to those reported in children with MADD. Altered mTORC1 signaling and maternal nutritional status may play critical roles in MADD disease progression and suggest novel treatment approaches that may ameliorate disease severity.

Metastatic behaviour of primary human tumours in a zebrafish xenotransplantation model

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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

A novel role for MAPKAPK2 in morphogenesis during zebrafish development.

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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.

Effects of phytosterols on zebrafish reproduction in multigeneration test

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Nakari, Tarja; Erkomaa, Kirsti

2003-01-01

A multigeneration test is used to show disruption of the reproductive system by phytosterols. - Zebrafish from mixed sex populations were exposed continuously across three generations to two phytosterol preparations both containing β-sitosterol. The phytosterols were isolated from wood and soy beans. Blood vitellogenin levels and sex ratio changes were used as intermediate indicators of the reproduction failures. Both sterol preparations caused vitellogenin induction in the exposed fish. The wood sterol changed the sex ratio of the exposed fish. In generation F1, the predominant sex was male, and in generation F2 it was female. The soy sterol in the used test concentration was lethal to the exposed fish in generation F1. This multigeneration test evidenced that phytosterols containing β-sitosterol disrupt the reproduction system of zebrafish by changing the sex ratios and by inducing the vitellogenin production in the exposed fish

Effects of phytosterols on zebrafish reproduction in multigeneration test

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Nakari, Tarja; Erkomaa, Kirsti

2003-05-01

A multigeneration test is used to show disruption of the reproductive system by phytosterols. - Zebrafish from mixed sex populations were exposed continuously across three generations to two phytosterol preparations both containing {beta}-sitosterol. The phytosterols were isolated from wood and soy beans. Blood vitellogenin levels and sex ratio changes were used as intermediate indicators of the reproduction failures. Both sterol preparations caused vitellogenin induction in the exposed fish. The wood sterol changed the sex ratio of the exposed fish. In generation F1, the predominant sex was male, and in generation F2 it was female. The soy sterol in the used test concentration was lethal to the exposed fish in generation F1. This multigeneration test evidenced that phytosterols containing {beta}-sitosterol disrupt the reproduction system of zebrafish by changing the sex ratios and by inducing the vitellogenin production in the exposed fish.

Myotonia congenita-associated mutations in chloride channel-1 affect zebrafish body wave swimming kinematics.

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Cheng, Wei; Tian, Jing; Burgunder, Jean-Marc; Hunziker, Walter; Eng, How-Lung

2014-01-01

Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1). Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita.

Myotonia congenita-associated mutations in chloride channel-1 affect zebrafish body wave swimming kinematics.

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Wei Cheng

Full Text Available Myotonia congenita is a human muscle disorder caused by mutations in CLCN1, which encodes human chloride channel 1 (CLCN1. Zebrafish is becoming an increasingly useful model for human diseases, including muscle disorders. In this study, we generated transgenic zebrafish expressing, under the control of a muscle specific promoter, human CLCN1 carrying mutations that have been identified in human patients suffering from myotonia congenita. We developed video analytic tools that are able to provide precise quantitative measurements of movement abnormalities in order to analyse the effect of these CLCN1 mutations on adult transgenic zebrafish swimming. Two new parameters for body-wave kinematics of swimming reveal changes in body curvature and tail offset in transgenic zebrafish expressing the disease-associated CLCN1 mutants, presumably due to their effect on muscle function. The capability of the developed video analytic tool to distinguish wild-type from transgenic zebrafish could provide a useful asset to screen for compounds that reverse the disease phenotype, and may be applicable to other movement disorders besides myotonia congenita.

A zebrafish model of glucocorticoid resistance shows serotonergic modulation of the stress response

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Brian eGriffiths

2012-10-01

Full Text Available One function of glucocorticoids is to restore homeostasis after an acute stress response by providing negative feedback to stress circuits in the brain. Loss of this negative feedback leads to elevated physiological stress and may contribute to depression, anxiety and post-traumatic stress disorder. We investigated the early, developmental effects of glucocorticoid signaling deficits on stress physiology and related behaviors using a mutant zebrafish, grs357, with non-functional glucocorticoid receptors. These mutants are morphologically inconspicuous and adult-viable. A previous study of adult grs357 mutants showed loss of glucocorticoid-mediated negative feedback and elevated physiological and behavioral stress markers. Already at five days post-fertilization, mutant larvae had elevated whole body cortisol, increased expression of pro-opiomelanocortin (POMC, the precursor of adrenocorticotropic hormone (ACTH, and failed to show normal suppression of stress markers after dexamethasone treatment. Mutant larvae had larger auditory-evoked startle responses compared to wildtype sibling controls (grwt, despite having lower spontaneous activity levels. Fluoxetine (Prozac treatment in mutants decreased startle responding and increased spontaneous activity, making them behaviorally similar to wildtype. This result mirrors known effects of selective serotonin reuptake inhibitors (SSRIs in modifying glucocorticoid signaling and alleviating stress disorders in human patients. Our results suggest that larval grs357 zebrafish can be used to study behavioral, physiological and molecular aspects of stress disorders. Most importantly, interactions between glucocorticoid and serotonin signaling appear to be highly conserved among vertebrates, suggesting deep homologies at the neural circuit level and opening up new avenues for research into psychiatric conditions.

Spatial pattern of cell geometry and cell-division orientation in zebrafish lens epithelium

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Toshiaki Mochizuki

2014-09-01

Full Text Available Cell proliferation is a key regulator of tissue morphogenesis. We examined cell proliferation and cell division in zebrafish lens epithelium by visualizing cell-cycle phases and nuclear positions, using fluorescent-labeled geminin and histone proteins. Proliferation was low in the anterior region of lens epithelium and higher in the marginal zone anterior to the equator, suggesting that the proliferation zone, called the germinative zone, is formed in zebrafish lens. Interestingly, cell-division orientation was biased longitudinally in the anterior region, shifted from longitudinal to circumferential along the anterior–posterior axis of lens sphere, and was biased circumferentially in the peripheral region. These data suggest that cell-division orientation is spatially regulated in zebrafish lens epithelium. The Hertwig rule indicates that cells tend to divide along their long axes. Orientation of long axes and cell division were biased similarly in zebrafish lens epithelium, suggesting that cell geometry correlates with cell-division orientation. A cell adhesion molecule, E-cadherin, is expressed in lens epithelium. In a zebrafish e-cadherin mutant, the long axes and cell-division orientation were shifted more longitudinally. These data suggest that E-cadherin is required for the spatial pattern of cell geometry and cell-division orientation in zebrafish lens epithelium.

Zebrafish usp39 mutation leads to rb1 mRNA splicing defect and pituitary lineage expansion.

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Yesenia Ríos

2011-01-01

Full Text Available Loss of retinoblastoma (Rb tumor suppressor function is associated with human malignancies. Molecular and genetic mechanisms responsible for tumorigenic Rb downregulation are not fully defined. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish ubiquitin specific peptidase 39 (usp39 mutation, the yeast and human homolog of which encodes a component of RNA splicing machinery. Zebrafish usp39 mutants exhibit microcephaly and adenohypophyseal cell lineage expansion without apparent changes in major hypothalamic hormonal and regulatory signals. Gene expression profiling of usp39 mutants revealed decreased rb1 and increased e2f4, rbl2 (p130, and cdkn1a (p21 expression. Rb1 mRNA overexpression, or antisense morpholino knockdown of e2f4, partially reversed embryonic pituitary expansion in usp39 mutants. Analysis of pre-mRNA splicing status of critical cell cycle regulators showed misspliced Rb1 pre-mRNA resulting in a premature stop codon. These studies unravel a novel mechanism for rb1 regulation by a neuronal mRNA splicing factor, usp39. Zebrafish usp39 regulates embryonic pituitary homeostasis by targeting rb1 and e2f4 expression, respectively, contributing to increased adenohypophyseal sensitivity to these altered cell cycle regulators. These results provide a mechanism for dysregulated rb1 and e2f4 pathways that may result in pituitary tumorigenesis.

A zebrafish model for Waardenburg syndrome type IV reveals diverse roles for Sox10 in the otic vesicle.

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Dutton, Kirsten; Abbas, Leila; Spencer, Joanne; Brannon, Claire; Mowbray, Catriona; Nikaido, Masataka; Kelsh, Robert N; Whitfield, Tanya T

2009-01-01

In humans, mutations in the SOX10 gene are a cause of the auditory-pigmentary disorder Waardenburg syndrome type IV (WS4) and related variants. SOX10 encodes an Sry-related HMG box protein essential for the development of the neural crest; deafness in WS4 and other Waardenburg syndromes is usually attributed to loss of neural-crest-derived melanocytes in the stria vascularis of the cochlea. However, SOX10 is strongly expressed in the developing otic vesicle and so direct roles for SOX10 in the otic epithelium might also be important. Here, we examine the otic phenotype of zebrafish sox10 mutants, a model for WS4. As a cochlea is not present in the fish ear, the severe otic phenotype in these mutants cannot be attributed to effects on this tissue. In zebrafish sox10 mutants, we see abnormalities in all otic placodal derivatives. Gene expression studies indicate deregulated expression of several otic genes, including fgf8, in sox10 mutants. Using a combination of mutant and morphant data, we show that the three sox genes belonging to group E (sox9a, sox9b and sox10) provide a link between otic induction pathways and subsequent otic patterning: they act redundantly to maintain sox10 expression throughout otic tissue and to restrict fgf8 expression to anterior macula regions. Single-cell labelling experiments indicate a small and transient neural crest contribution to the zebrafish ear during normal development, but this is unlikely to account for the strong defects seen in the sox10 mutant. We discuss the implication that the deafness in WS4 patients with SOX10 mutations might reflect a haploinsufficiency for SOX10 in the otic epithelium, resulting in patterning and functional abnormalities in the inner ear.

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

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Ram Manohar Basnet

2017-03-01

Full Text Available Zebrafish embryo is emerging as an important tool for behavior analysis as well as toxicity testing. In this study, we compared the effect of nine different methylxanthine drugs using zebrafish embryo as a model. We performed behavioral analysis, biochemical assay and Fish Embryo Toxicity (FET test in zebrafish embryos after treatment with methylxanthines. Each drug appeared to behave in different ways and showed a distinct pattern of results. Embryos treated with seven out of nine methylxanthines exhibited epileptic-like pattern of movements, the severity of which varied with drugs and doses used. Cyclic AMP measurement showed that, despite of a significant increase in cAMP with some compounds, it was unrelated to the observed movement behavior changes. FET test showed a different pattern of toxicity with different methylxanthines. Each drug could be distinguished from the other based on its effect on mortality, morphological defects and teratogenic effects. In addition, there was a strong positive correlation between the toxic doses (TC50 calculated in zebrafish embryos and lethal doses (LD50 in rodents obtained from TOXNET database. Taken together, all these findings elucidate the potentiality of zebrafish embryos as an in vivo model for behavioral and toxicity testing of methylxanthines and other related compounds.

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

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

2017-04-01

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

Innovative Disease Model: Zebrafish as an In Vivo Platform for Intestinal Disorder and Tumors

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Jeng-Wei Lu

2017-09-01

Full Text Available Colorectal cancer (CRC is one of the world’s most common cancers and is the second leading cause of cancer deaths, causing more than 50,000 estimated deaths each year. Several risk factors are highly associated with CRC, including being overweight, eating a diet high in red meat and over-processed meat, having a history of inflammatory bowel disease, and smoking. Previous zebrafish studies have demonstrated that multiple oncogenes and tumor suppressor genes can be regulated through genetic or epigenetic alterations. Zebrafish research has also revealed that the activation of carcinogenesis-associated signal pathways plays an important role in CRC. The biology of cancer, intestinal disorders caused by carcinogens, and the morphological patterns of tumors have been found to be highly similar between zebrafish and humans. Therefore, the zebrafish has become an important animal model for translational medical research. Several zebrafish models have been developed to elucidate the characteristics of gastrointestinal diseases. This review article focuses on zebrafish models that have been used to study human intestinal disorders and tumors, including models involving mutant and transgenic fish. We also report on xenograft models and chemically-induced enterocolitis. This review demonstrates that excellent zebrafish models can provide novel insights into the pathogenesis of gastrointestinal diseases and help facilitate the evaluation of novel anti-tumor drugs.

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

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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.

Shp2 knockdown and Noonan/LEOPARD mutant Shp2-induced gastrulation defects.

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Chris Jopling

2007-12-01

Full Text Available Shp2 is a cytoplasmic protein-tyrosine phosphatase that is essential for normal development. Activating and inactivating mutations have been identified in humans to cause the related Noonan and LEOPARD syndromes, respectively. The cell biological cause of these syndromes remains to be determined. We have used the zebrafish to assess the role of Shp2 in early development. Here, we report that morpholino-mediated knockdown of Shp2 in zebrafish resulted in defects during gastrulation. Cell tracing experiments demonstrated that Shp2 knockdown induced defects in convergence and extension cell movements. In situ hybridization using a panel of markers indicated that cell fate was not affected by Shp2 knock down. The Shp2 knockdown-induced defects were rescued by active Fyn and Yes and by active RhoA. We generated mutants of Shp2 with mutations that were identified in human patients with Noonan or LEOPARD Syndrome and established that Noonan Shp2 was activated and LEOPARD Shp2 lacked catalytic protein-tyrosine phosphatase activity. Expression of Noonan or LEOPARD mutant Shp2 in zebrafish embryos induced convergence and extension cell movement defects without affecting cell fate. Moreover, these embryos displayed craniofacial and cardiac defects, reminiscent of human symptoms. Noonan and LEOPARD mutant Shp2s were not additive nor synergistic, consistent with the mutant Shp2s having activating and inactivating roles in the same signaling pathway. Our results demonstrate that Shp2 is required for normal convergence and extension cell movements during gastrulation and that Src family kinases and RhoA were downstream of Shp2. Expression of Noonan or LEOPARD Shp2 phenocopied the craniofacial and cardiac defects of human patients. The finding that defective Shp2 signaling induced cell movement defects as early as gastrulation may have implications for the monitoring and diagnosis of Noonan and LEOPARD syndrome.

Loss of Slc4a1b chloride/bicarbonate exchanger function protects mechanosensory hair cells from aminoglycoside damage in the zebrafish mutant persephone.

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Dale W Hailey

Full Text Available Mechanosensory hair cell death is a leading cause of hearing and balance disorders in the human population. Hair cells are remarkably sensitive to environmental insults such as excessive noise and exposure to some otherwise therapeutic drugs. However, individual responses to damaging agents can vary, in part due to genetic differences. We previously carried out a forward genetic screen using the zebrafish lateral line system to identify mutations that alter the response of larval hair cells to the antibiotic neomycin, one of a class of aminoglycoside compounds that cause hair cell death in humans. The persephone mutation confers resistance to aminoglycosides. 5 dpf homozygous persephone mutants are indistinguishable from wild-type siblings, but differ in their retention of lateral line hair cells upon exposure to neomycin. The mutation in persephone maps to the chloride/bicarbonate exchanger slc4a1b and introduces a single Ser-to-Phe substitution in zSlc4a1b. This mutation prevents delivery of the exchanger to the cell surface and abolishes the ability of the protein to import chloride across the plasma membrane. Loss of function of zSlc4a1b reduces hair cell death caused by exposure to the aminoglycosides neomycin, kanamycin, and gentamicin, and the chemotherapeutic drug cisplatin. Pharmacological block of anion transport with the disulfonic stilbene derivatives DIDS and SITS, or exposure to exogenous bicarbonate, also protects hair cells against damage. Both persephone mutant and DIDS-treated wild-type larvae show reduced uptake of labeled aminoglycosides. persephone mutants also show reduced FM1-43 uptake, indicating a potential impact on mechanotransduction-coupled activity in the mutant. We propose that tight regulation of the ionic environment of sensory hair cells, mediated by zSlc4a1b activity, is critical for their sensitivity to aminoglycoside antibiotics.

ATM loss leads to synthetic lethality in BRCA1 BRCT mutant mice associated with exacerbated defects in homology-directed repair.

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Chen, Chun-Chin; Kass, Elizabeth M; Yen, Wei-Feng; Ludwig, Thomas; Moynahan, Mary Ellen; Chaudhuri, Jayanta; Jasin, Maria

2017-07-18

BRCA1 is essential for homology-directed repair (HDR) of DNA double-strand breaks in part through antagonism of the nonhomologous end-joining factor 53BP1. The ATM kinase is involved in various aspects of DNA damage signaling and repair, but how ATM participates in HDR and genetically interacts with BRCA1 in this process is unclear. To investigate this question, we used the Brca1 S1598F mouse model carrying a mutation in the BRCA1 C-terminal domain of BRCA1. Whereas ATM loss leads to a mild HDR defect in adult somatic cells, we find that ATM inhibition leads to severely reduced HDR in Brca1 S1598F cells. Consistent with a critical role for ATM in HDR in this background, loss of ATM leads to synthetic lethality of Brca1 S1598F mice. Whereas both ATM and BRCA1 promote end resection, which can be regulated by 53BP1, 53bp1 deletion does not rescue the HDR defects of Atm mutant cells, in contrast to Brca1 mutant cells. These results demonstrate that ATM has a role in HDR independent of the BRCA1-53BP1 antagonism and that its HDR function can become critical in certain contexts.

Bushy Park Nursing Home, Nenagh Road, Borrisokane, Tipperary

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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.

Ard Na Rí Nursing Home, Holycross, Bruff, Limerick.

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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.

Reanalysis of parabiosis of obesity mutants in the age of leptin.

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Zeng, Wenwen; Lu, Yi-Hsueh; Lee, Jonah; Friedman, Jeffrey M

2015-07-21

In this study we set out to explain the differing effects of parabiosis with genetically diabetic (db) mice versus administration of recombinant leptin. Parabiosis of db mutant, which overexpress leptin, to wildtype (WT) or genetically obese (ob) mice has been reported to cause death by starvation, whereas leptin infusions do not produce lethality at any dose or mode of delivery tested. Leptin is not posttranslationally modified other than a single disulphide bond, raising the possibility that it might require additional factor(s) to exert the maximal appetite-suppressing effect. We reconfirmed the lethal effect of parabiosis of db mutant on WT mice and further showed that this lethality could not be rescued by administration of ghrelin or growth hormone. We then initiated a biochemical fractionation of a high-molecular-weight leptin complex from human plasma and identified clusterin as a major component of this leptin-containing complex. However, in contrast to previous reports, we failed to observe a leptin-potentiating effect of either exogenous or endogenous clusterin, and parabiosis of db clusterin(-/-) double-mutant to WT mice still caused lethality. Intriguingly, in parabiotic pairs of two WT mice, leptin infusion into one of the mice led to an enhanced starvation response during calorie restriction as evidenced by increased plasma ghrelin and growth-hormone levels. Moreover, leptin treatment resulted in death of the parabiotic pairs. These data suggest that the appetite suppression in WT mice after parabiosis to db mutants is the result of induced hyperleptinemia combined with the stress or other aspect(s) of the parabiosis procedure.

DNA polymerase I is crucial for the repair of potentially lethal damage caused by the indirect effects of X irradiation in Escherichia coli

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Billen, D.

1985-01-01

The radiosensitivity of an Escherichia coli mutant deficient in DNA polymerase I was measured in the presence of OH radical scavengers. The extreme X-ray sensitivity of the mutant could be abolished by OH radical scavengers if a sufficiently high level of radioprotector was present. There was a direct correlation between the OH radical scavenging activity of the chemicals tested (NO 2 - , n-butanol, glycerol, t-amyl alcohol, and t-butanol) and their protective ability. The author interprets the data as showing that the indirect actions of X rays (primarily OH radicals) result in major damage to the bacterial DNA which in large part consists of potentially lethal lesions. This potentially lethal damage is repaired through an enzymatic pathway requiring DNA polymerase I. I. In the mutant lacking DNA polymerase I, these potentially lethal lesions are expressed as cell lethality

Bacillus subtilis mutants deficient in the adaptive response to simple alkylating agents

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Morohoshi, F.; Munakata, N.

1985-03-01

Three mutant strains exhibiting hyper-sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine, but not to methyl methanesulfonate, were selected by a replica method from mutagenized spores of Bacillus subtilis. All three were totally deficient in the adaptive response to N-methyl-N'-nitro-N-nitrosoguanidine with regard to both lethality and mutagenesis. The activity to destroy O/sup 6/-methylguanine residues in the methylated DNA was not elevated in the mutant cells by the pretreatment with sublethal concentrations of N-methyl-N-nitro-N-nitrosoguanidine. This deficiency corresponded to the persistance of O/sup 6/-methylguanine residues in the DNA of both control and pretreated mutant cells challenged with the drug. The lethal and mutagenic sensitivity of the mutant strains were observed only for methyl- or ethyl-nitroso compounds that are thought to be active as inducers and are also active in O-alkylation. Except for the insensitivity to methyl methanesulfonate, the phenotypes of these mutants look very similar to those of ada mutants isolated previously in Escherichia coli.

Effect of lethality on the extinction and on the error threshold of quasispecies.

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Tejero, Hector; Marín, Arturo; Montero, Francisco

2010-02-21

In this paper the effect of lethality on error threshold and extinction has been studied in a population of error-prone self-replicating molecules. For given lethality and a simple fitness landscape, three dynamic regimes can be obtained: quasispecies, error catastrophe, and extinction. Using a simple model in which molecules are classified as master, lethal and non-lethal mutants, it is possible to obtain the mutation rates of the transitions between the three regimes analytically. The numerical resolution of the extended model, in which molecules are classified depending on their Hamming distance to the master sequence, confirms the results obtained in the simple model and shows how an error catastrophe regime changes when lethality is taken in account. (c) 2009 Elsevier Ltd. All rights reserved.

A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

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Sergey V. Prykhozhij

2017-06-01

Full Text Available Clustered regularly interspaced palindromic repeats (CRISPR/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in pycr1a, chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in chd7, suggesting activity of alternative translation sites in the other two mutants even though pycr1a exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible.

Circadian rhythms in the pineal organ persist in zebrafish larvae that lack ventral brain

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Goldstein-Kral Lauren

2011-01-01

Full Text Available Abstract Background The mammalian suprachiasmatic nucleus (SCN, located in the ventral hypothalamus, is a major regulator of circadian rhythms in mammals and birds. However, the role of the SCN in lower vertebrates remains poorly understood. Zebrafish cyclops (cyc mutants lack ventral brain, including the region that gives rise to the SCN. We have used cyc embryos to define the function of the zebrafish SCN in regulating circadian rhythms in the developing pineal organ. The pineal organ is the major source of the circadian hormone melatonin, which regulates rhythms such as daily rest/activity cycles. Mammalian pineal rhythms are controlled almost exclusively by the SCN. In zebrafish and many other lower vertebrates, the pineal has an endogenous clock that is responsible in part for cyclic melatonin biosynthesis and gene expression. Results We find that pineal rhythms are present in cyc mutants despite the absence of an SCN. The arginine vasopressin-like protein (Avpl, formerly called Vasotocin is a peptide hormone expressed in and around the SCN. We find avpl mRNA is absent in cyc mutants, supporting previous work suggesting the SCN is missing. In contrast, expression of the putative circadian clock genes, cryptochrome 1b (cry1b and cryptochrome 3 (cry3, in the brain of the developing fish is unaltered. Expression of two pineal rhythmic genes, exo-rhodopsin (exorh and serotonin-N-acetyltransferase (aanat2, involved in photoreception and melatonin synthesis, respectively, is also similar between cyc embryos and their wildtype (WT siblings. The timing of the peaks and troughs of expression are the same, although the amplitude of expression is slightly decreased in the mutants. Cyclic gene expression persists for two days in cyc embryos transferred to constant light or constant dark, suggesting a circadian clock is driving the rhythms. However, the amplitude of rhythms in cyc mutants kept in constant conditions decreased more quickly than in their

Highly variable penetrance of abnormal phenotypes in embryonic lethal knockout mice

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Wilson, Robert; Geyer, Stefan H.; Reissig, Lukas; Rose, Julia; Szumska, Dorota; Hardman, Emily; Prin, Fabrice; McGuire, Christina; Ramirez-Solis, Ramiro; White, Jacqui; Galli, Antonella; Tudor, Catherine; Tuck, Elizabeth; Mazzeo, Cecilia Icoresi; Smith, James C.; Robertson, Elizabeth; Adams, David J.; Mohun, Timothy; Weninger, Wolfgang J.

2017-01-01

Background: Identifying genes that are essential for mouse embryonic development and survival through term is a powerful and unbiased way to discover possible genetic determinants of human developmental disorders. Characterising the changes in mouse embryos that result from ablation of lethal genes is a necessary first step towards uncovering their role in normal embryonic development and establishing any correlates amongst human congenital abnormalities. Methods: Here we present results gathered to date in the Deciphering the Mechanisms of Developmental Disorders (DMDD) programme, cataloguing the morphological defects identified from comprehensive imaging of 220 homozygous mutant and 114 wild type embryos from 42 lethal and subviable lines, analysed at E14.5. Results: Virtually all mutant embryos show multiple abnormal phenotypes and amongst the 42 lines these affect most organ systems. Within each mutant line, the phenotypes of individual embryos form distinct but overlapping sets. Subcutaneous edema, malformations of the heart or great vessels, abnormalities in forebrain morphology and the musculature of the eyes are all prevalent phenotypes, as is loss or abnormal size of the hypoglossal nerve. Conclusions: Overall, the most striking finding is that no matter how profound the malformation, each phenotype shows highly variable penetrance within a mutant line. These findings have challenging implications for efforts to identify human disease correlates. PMID:27996060

Pigment pattern in jaguar/obelix zebrafish is caused by a Kir7.1 mutation: implications for the regulation of melanosome movement.

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Motoko Iwashita

2006-11-01

Full Text Available Many animals have a variety of pigment patterns, even within a species, and these patterns may be one of the driving forces of speciation. Recent molecular genetic studies on zebrafish have revealed that interaction among pigment cells plays a key role in pattern formation, but the mechanism of pattern formation is unclear. The zebrafish jaguar/obelix mutant has broader stripes than wild-type fish. In this mutant, the development of pigment cells is normal but their distribution is altered, making these fish ideal for studying the process of pigment pattern formation. Here, we utilized a positional cloning method to determine that the inwardly rectifying potassium channel 7.1 (Kir7.1 gene is responsible for pigment cell distribution among jaguar/obelix mutant fish. Furthermore, in jaguar/obelix mutant alleles, we identified amino acid changes in the conserved region of Kir7.1, each of which affected K(+ channel activity as demonstrated by patch-clamp experiments. Injection of a bacterial artificial chromosome containing the wild-type Kir7.1 genomic sequence rescued the jaguar/obelix phenotype. From these results, we conclude that mutations in Kir7.1 are responsible for jaguar/obelix. We also determined that the ion channel function defect of melanophores expressing mutant Kir7.1 altered the cellular response to external signals. We discovered that mutant melanophores cannot respond correctly to the melanosome dispersion signal derived from the sympathetic neuron and that melanosome aggregation is constitutively activated. In zebrafish and medaka, it is well known that melanosome aggregation and subsequent melanophore death increase when fish are kept under constant light conditions. These observations indicate that melanophores of jaguar/obelix mutant fish have a defect in the signaling pathway downstream of the alpha2-adrenoceptor. Taken together, our results suggest that the cellular defect of the Kir7.1 mutation is directly responsible for

Modeling tuberculous meningitis in zebrafish using Mycobacterium marinum

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Lisanne M. van Leeuwen

2014-09-01

Full Text Available Tuberculous meningitis (TBM is one of the most severe extrapulmonary manifestations of tuberculosis, with a high morbidity and mortality. Characteristic pathological features of TBM are Rich foci, i.e. brain- and spinal-cord-specific granulomas formed after hematogenous spread of pulmonary tuberculosis. Little is known about the early pathogenesis of TBM and the role of Rich foci. We have adapted the zebrafish model of Mycobacterium marinum infection (zebrafish–M. marinum model to study TBM. First, we analyzed whether TBM occurs in adult zebrafish and showed that intraperitoneal infection resulted in granuloma formation in the meninges in 20% of the cases, with occasional brain parenchyma involvement. In zebrafish embryos, bacterial infiltration and clustering of infected phagocytes was observed after infection at three different inoculation sites: parenchyma, hindbrain ventricle and caudal vein. Infection via the bloodstream resulted in the formation of early granulomas in brain tissue in 70% of the cases. In these zebrafish embryos, infiltrates were located in the proximity of blood vessels. Interestingly, no differences were observed when embryos were infected before or after early formation of the blood-brain barrier (BBB, indicating that bacteria are able to cross this barrier with relatively high efficiency. In agreement with this observation, infected zebrafish larvae also showed infiltration of the brain tissue. Upon infection of embryos with an M. marinum ESX-1 mutant, only small clusters and scattered isolated phagocytes with high bacterial loads were present in the brain tissue. In conclusion, our adapted zebrafish–M. marinum infection model for studying granuloma formation in the brain will allow for the detailed analysis of both bacterial and host factors involved in TBM. It will help solve longstanding questions on the role of Rich foci and potentially contribute to the development of better diagnostic tools and therapeutics.

Neurodegeneration in drop-dead mutant drosophila melanogaster is associated with the respiratory system but not with Hypoxia.

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Christine Lynn Sansone

Full Text Available Mutations in the gene drop-dead (drd cause diverse phenotypes in adult Drosophila melanogaster including early lethality, neurodegeneration, tracheal defects, gut dysfunction, reduced body mass, and female sterility. Despite the identification of the drd gene itself, the causes of early lethality and neurodegeneration in the mutant flies remain unknown. To determine the pattern of drd expression associated with the neurodegenerative phenotype, knockdown of drd with various Gal4 drivers was performed. Early adult lethality and neurodegeneration were observed upon knockdown of drd in the tracheal system with two independent insertions of the breathless-Gal4 driver and upon knockdown in the tracheal system and elsewhere with the DJ717-Gal4 driver. Surprisingly, rescue of drd expression exclusively in the tracheae in otherwise mutant flies rescued the neurodegenerative phenotype but not adult lethality. Gut dysfunction, as measured by defecation rate, was not rescued in these flies, and gut function appeared normal upon tracheal-specific knockdown of drd. Finally, the hypothesis that tracheal dysfunction in drd mutants results in hypoxia was tested. Hypoxia-sensitive reporter transgenes (LDH-Gal4 and LDH-LacZ were placed on a drd mutant background, but enhanced expression of these reporters was not observed. In addition, manipulation of drd expression in the tracheae did not affect expression of the hypoxia-induced genes LDH, tango, and similar. Overall, these results indicate that there are at least two causes of adult lethality in drd mutants, that gut dysfunction and neurodegeneration are independent phenotypes, and that neurodegeneration is associated with tracheal expression of drd but not with hypoxia.

X-rays sensitive mammalian cell mutant

International Nuclear Information System (INIS)

Utsumi, Hiroshi

1982-01-01

A phenomenon that in x-ray-sensitive mammalian-cell mutants, cellular death due to x-ray radiation was not increased by caffeine, but on the contrary, the dead cells were resuscitated by it was discussed. The survival rate of mutant cells increased by caffein in a low concentration. This suggested that caffeine may have induced some mechanism to produce x-ray resistant mutant cells. Postirradiation treatment with caffeine increased considerably the survival rate of the mutant cells, and this suggested the existence of latent caffeine-sensitive potentially lethal damage repair system. This system, after a few hours, is thought to be substituted by caffeine-resistant repair system which is induced by caffeine, and this may be further substituted by x-ray-resistant repair system. The repair system was also induced by adenine. (Ueda, J.)

Zebrafish integrin-linked kinase is required in skeletal muscles for strengthening the integrin-ECM adhesion complex.

NARCIS (Netherlands)

Postel, R.; Vakeel, P.; Topczewski, J.; Knoll, R.; Bakkers, J.

2008-01-01

Mechanical instability of skeletal muscle cells is the major cause of congenital muscular dystrophy. Here we show that the zebrafish lost-contact mutant, that lacks a functional integrin-linked kinase (ilk) gene, suffers from mechanical instability of skeletal muscle fibres. With genetic and

Efficient methods for targeted mutagenesis in zebrafish using zinc-finger nucleases: data from targeting of nine genes using CompoZr or CoDA ZFNs.

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Raman Sood

Full Text Available Recently, it has been shown that targeted mutagenesis using zinc-finger nucleases (ZFNs and transcription activator-like effector nucleases (TALENs can be used to generate knockout zebrafish lines for analysis of their function and/or developing disease models. A number of different methods have been developed for the design and assembly of gene-specific ZFNs and TALENs, making them easily available to most zebrafish researchers. Regardless of the choice of targeting nuclease, the process of generating mutant fish is similar. It is a time-consuming and multi-step process that can benefit significantly from development of efficient high throughput methods. In this study, we used ZFNs assembled through either the CompoZr (Sigma-Aldrich or the CoDA (context-dependent assembly platforms to generate mutant zebrafish for nine genes. We report our improved high throughput methods for 1 evaluation of ZFNs activity by somatic lesion analysis using colony PCR, eliminating the need for plasmid DNA extractions from a large number of clones, and 2 a sensitive founder screening strategy using fluorescent PCR with PIG-tailed primers that eliminates the stutter bands and accurately identifies even single nucleotide insertions and deletions. Using these protocols, we have generated multiple mutant alleles for seven genes, five of which were targeted with CompoZr ZFNs and two with CoDA ZFNs. Our data also revealed that at least five-fold higher mRNA dose was required to achieve mutagenesis with CoDA ZFNs than with CompoZr ZFNs, and their somatic lesion frequency was lower (<5% when compared to CopmoZr ZFNs (9-98%. This work provides high throughput protocols for efficient generation of zebrafish mutants using ZFNs and TALENs.

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.

Expression of ALS/FTD-linked mutant CCNF in zebrafish leads to increased cell death in the spinal cord and an aberrant motor phenotype.

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Hogan, Alison L; Don, Emily K; Rayner, Stephanie L; Lee, Albert; Laird, Angela S; Watchon, Maxinne; Winnick, Claire; Tarr, Ingrid S; Morsch, Marco; Fifita, Jennifer A; Gwee, Serene S L; Formella, Isabel; Hortle, Elinor; Yuan, Kristy C; Molloy, Mark P; Williams, Kelly L; Nicholson, Garth A; Chung, Roger S; Blair, Ian P; Cole, Nicholas J

2017-07-15

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive, fatal neurodegenerative disease characterised by the death of upper and lower motor neurons. Approximately 10% of cases have a known family history of ALS and disease-linked mutations in multiple genes have been identified. ALS-linked mutations in CCNF were recently reported, however the pathogenic mechanisms associated with these mutations are yet to be established. To investigate possible disease mechanisms, we developed in vitro and in vivo models based on an ALS-linked missense mutation in CCNF. Proteomic analysis of the in vitro models identified the disruption of several cellular pathways in the mutant model, including caspase-3 mediated cell death. Transient overexpression of human CCNF in zebrafish embryos supported this finding, with fish expressing the mutant protein found to have increased levels of cleaved (activated) caspase-3 and increased cell death in the spinal cord. The mutant CCNF fish also developed a motor neuron axonopathy consisting of shortened primary motor axons and increased frequency of aberrant axonal branching. Importantly, we demonstrated a significant correlation between the severity of the CCNF-induced axonopathy and a reduced motor response to a light stimulus (photomotor response). This is the first report of an ALS-linked CCNF mutation in vivo and taken together with the in vitro model identifies the disruption of cell death pathways as a significant consequence of this mutation. Additionally, this study presents a valuable new tool for use in ongoing studies investigating the pathobiology of ALS-linked CCNF mutations. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Lethal and mutagenic effects of ion beams and γ-rays in Aspergillus oryzae.

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Toyoshima, Yoshiyuki; Takahashi, Akemi; Tanaka, Hisaki; Watanabe, Jun; Mogi, Yoshinobu; Yamazaki, Tatsuo; Hamada, Ryoko; Iwashita, Kazuhiro; Satoh, Katsuya; Narumi, Issay

2012-12-01

Aspergillus oryzae is a fungus that is used widely in traditional Japanese fermentation industries. In this study, the lethal and mutagenic effects of different linear energy transfer (LET) radiation in freeze-dried conidia of A. oryzae were investigated. The lethal effect, which was evaluated by a 90% lethal dose, was dependent on the LET value of the ionizing radiation. The most lethal ionizing radiation among that tested was (12)C(5+) ion beams with an LET of 121keV/μm. The (12)C(5+) ion beams had a 3.6-times higher lethal effect than low-LET (0.2keV/μm) γ-rays. The mutagenic effect was evaluated by the frequency of selenate resistant mutants. (12)C(6+) ion beams with an LET of 86keV/μm were the most effective in inducing selenate resistance. The mutant frequency following exposure to (12)C(6+) ion beams increased with an increase in dose and reached 3.47×10(-3) at 700Gy. In the dose range from 0 to 700Gy, (12)C(5+) ion beams were the second most effective in inducing selenate resistance, the mutant frequency of which reached a maximum peak (1.67×10(-3)) at 400Gy. To elucidate the characteristics of mutation induced by ionizing radiation, mutations in the sulphate permease gene (sB) and ATP sulfurylase gene (sC) loci, the loss of function of which results in a selenate resistant phenotype, were compared between (12)C(5+) ion beams and γ-rays. We detected all types of transversions and transitions. For frameshifts, the frequency of a +1 frameshift was the highest in all cases. Although the incidence of deletions >2bp was generally low, deletions >20bp were characteristic for (12)C(5+) ion beams. γ-rays had a tendency to generate mutants carrying a multitude of mutations in the same locus. Both forms of radiation also induced genome-wide large-scale mutations including chromosome rearrangements and large deletions. These results provide new basic insights into the mutation breeding of A. oryzae using ionizing radiation. Crown Copyright © 2012. Published

The ventralizing activity of Radar, a maternally expressed bone morphogenetic protein, reveals complex bone morphogenetic protein interactions controlling dorso-ventral patterning in zebrafish.

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Goutel, C; Kishimoto, Y; Schulte-Merker, S; Rosa, F

2000-12-01

In Xenopus and zebrafish, BMP2, 4 and 7 have been implicated, after the onset of zygotic expression, in inducing and maintaining ventro-lateral cell fate during early development. We provide evidence here that a maternally expressed bone morphogenetic protein (BMP), Radar, may control early ventral specification in zebrafish. We show that Radar ventralizes zebrafish embryos and induces the early expression of bmp2b and bmp4. The analysis of Radar overexpression in both swirl/bmp2b mutants and embryos expressing truncated BMP receptors shows that Radar-induced ventralization is dependent on functional BMP2/4 pathways, and may initially rely on an Alk6-related signaling pathway. Finally, we show that while radar-injected swirl embryos still exhibit a strongly dorsalized phenotype, the overexpression of Radar into swirl/bmp2b mutant embryos restores ventral marker expression, including bmp4 expression. Our results suggest that a complex regulation of different BMP pathways controls dorso-ventral (DV) patterning from early cleavage stages until somitogenesis.

ngs (Notochord Granular Surface) Gene Encodes a Novel Type of Intermediate Filament Family Protein Essential for Notochord Maintenance in Zebrafish*

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Tong, Xiangjun; Xia, Zhidan; Zu, Yao; Telfer, Helena; Hu, Jing; Yu, Jingyi; Liu, Huan; Zhang, Quan; Sodmergen; Lin, Shuo; Zhang, Bo

2013-01-01

The notochord is an important organ involved in embryonic patterning and locomotion. In zebrafish, the mature notochord consists of a single stack of fully differentiated, large vacuolated cells called chordocytes, surrounded by a single layer of less differentiated notochordal epithelial cells called chordoblasts. Through genetic analysis of zebrafish lines carrying pseudo-typed retroviral insertions, a mutant exhibiting a defective notochord with a granular appearance was isolated, and the corresponding gene was identified as ngs (notochord granular surface), which was specifically expressed in the notochord. In the mutants, the notochord started to degenerate from 32 hours post-fertilization, and the chordocytes were then gradually replaced by smaller cells derived from chordoblasts. The granular notochord phenotype was alleviated by anesthetizing the mutant embryos with tricaine to prevent muscle contraction and locomotion. Phylogenetic analysis showed that ngs encodes a new type of intermediate filament (IF) family protein, which we named chordostatin based on its function. Under the transmission electron microcopy, bundles of 10-nm-thick IF-like filaments were enriched in the chordocytes of wild-type zebrafish embryos, whereas the chordocytes in ngs mutants lacked IF-like structures. Furthermore, chordostatin-enhanced GFP (EGFP) fusion protein assembled into a filamentous network specifically in chordocytes. Taken together, our work demonstrates that ngs encodes a novel type of IF protein and functions to maintain notochord integrity for larval development and locomotion. Our work sheds light on the mechanisms of notochord structural maintenance, as well as the evolution and biological function of IF family proteins. PMID:23132861

ngs (notochord granular surface) gene encodes a novel type of intermediate filament family protein essential for notochord maintenance in zebrafish.

Science.gov (United States)

Tong, Xiangjun; Xia, Zhidan; Zu, Yao; Telfer, Helena; Hu, Jing; Yu, Jingyi; Liu, Huan; Zhang, Quan; Sodmergen; Lin, Shuo; Zhang, Bo

2013-01-25

The notochord is an important organ involved in embryonic patterning and locomotion. In zebrafish, the mature notochord consists of a single stack of fully differentiated, large vacuolated cells called chordocytes, surrounded by a single layer of less differentiated notochordal epithelial cells called chordoblasts. Through genetic analysis of zebrafish lines carrying pseudo-typed retroviral insertions, a mutant exhibiting a defective notochord with a granular appearance was isolated, and the corresponding gene was identified as ngs (notochord granular surface), which was specifically expressed in the notochord. In the mutants, the notochord started to degenerate from 32 hours post-fertilization, and the chordocytes were then gradually replaced by smaller cells derived from chordoblasts. The granular notochord phenotype was alleviated by anesthetizing the mutant embryos with tricaine to prevent muscle contraction and locomotion. Phylogenetic analysis showed that ngs encodes a new type of intermediate filament (IF) family protein, which we named chordostatin based on its function. Under the transmission electron microcopy, bundles of 10-nm-thick IF-like filaments were enriched in the chordocytes of wild-type zebrafish embryos, whereas the chordocytes in ngs mutants lacked IF-like structures. Furthermore, chordostatin-enhanced GFP (EGFP) fusion protein assembled into a filamentous network specifically in chordocytes. Taken together, our work demonstrates that ngs encodes a novel type of IF protein and functions to maintain notochord integrity for larval development and locomotion. Our work sheds light on the mechanisms of notochord structural maintenance, as well as the evolution and biological function of IF family proteins.

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

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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

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

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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.

Pregnancy-associated plasma protein-A (PAPP-A) modulates early developmental rate in zebrafish independent of its proteolytic activity

DEFF Research Database (Denmark)

Kjær-Sørensen, Kasper; Engholm, Ditte Høyer; Kamei, Hiroyasu

2013-01-01

the developmental rate beginning during gastrulation without affecting the normal patterning of the embryo. This phenotype is different from those resulting from deficiency of Igf receptor or ligand in zebrafish, suggesting a function of Papp-a outside the Igf system. Biochemical analysis of recombinant zebrafish...... Papp-a demonstrates conservation of proteolytic activity, specificity, and intrinsic regulatory mechanism. However, in vitro transcribed mRNA, which encodes a proteolytically inactive Papp-a mutant, recues the papp-a knockdown phenotype as efficient as wild-type Papp-a. Thus, the developmental...

RAF Suppression Synergizes with MEK Inhibition in KRAS Mutant Cancer Cells

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Simona Lamba

2014-09-01

Full Text Available KRAS is the most frequently mutated oncogene in human cancer, yet no therapies are available to treat KRAS mutant cancers. We used two independent reverse genetic approaches to identify components of the RAS-signaling pathways required for growth of KRAS mutant tumors. Small interfering RNA (siRNA screening of 37 KRAS mutant colorectal cancer cell lines showed that RAF1 suppression was synthetic lethal with MEK inhibition. An unbiased kinome short hairpin RNA (shRNA-based screen confirmed this synthetic lethal interaction in colorectal as well as in lung cancer cells bearing KRAS mutations. Compounds targeting RAF kinases can reverse resistance to the MEK inhibitor selumetinib. MEK inhibition induces RAS activation and BRAF-RAF1 dimerization and sustains MEK-ERK signaling, which is responsible for intrinsic resistance to selumetinib. Prolonged dual blockade of RAF and MEK leads to persistent ERK suppression and efficiently induces apoptosis. Our data underlie the relevance of developing combinatorial regimens of drugs targeting the RAF-MEK pathway in KRAS mutant tumors.

Toxicological effects of graphene oxide on adult zebrafish (Danio rerio)

Energy Technology Data Exchange (ETDEWEB)

Souza, Jaqueline P., E-mail: souza.jaqueline@gmail.com; Baretta, Jéssica F.; Santos, Fabrício; Paino, Ieda M.M.; Zucolotto, Valtencir

2017-05-15

Highlights: • Graphene oxide exposure caused apoptotic and necrotic stages in zebrafish gill cells. • Graphene oxide induced reactive oxygen generation in zebrafish gill cells. • Gill and liver tissues suffered injuries after graphene oxide chronic exposure. • Zebrafish blood cells did not present DNA damages after graphene oxide exposure. - Abstract: Graphene exhibits unique physical and chemical properties that facilitate its application in many fields, including electronics and biomedical areas. However, the use of graphene and its derivatives could result in accumulation in aquatic environments, and the risks posed by these compounds for organisms are not completely understood. In this study, we investigated the effects of graphene oxide (GO) on adult zebrafish (Danio rerio). Experimental fish were exposed to 2, 10 or 20 mg L{sup −1} GO, and the cytotoxicity, genotoxicity and oxidative stress were assessed. The morphology of the gills and liver tissues was also analyzed. Graphene oxide exposure led to an increase in the number of gill cells that were in early apoptotic and necrotic stages, but genotoxicity was not observed in blood cells. We also observed the generation of Reactive Oxygen Species (ROS) in gill cells. Structural analysis revealed injuries to gill tissues, including a dilated marginal channel, lamellar fusion, clubbed tips, swollen mucocytes, epithelial lifting, aneurysms, and necrosis. Liver tissues also presented lesions such as peripherally located nuclei. Furthermore, hepatocytes exhibited a non-uniform shape, picnotic nuclei, vacuole formation, cell rupture, and necrosis. Our results showed that sub-lethal doses of graphene oxide could be harmful to fish species and thus represent risks for the aquatic food chain.

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

Directory of Open Access Journals (Sweden)

Francisco X Mora-Zamorano

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

Molecular Mechanisms Regulating Ocular Apoptosis in Zebrafish gdf6a Mutants

DEFF Research Database (Denmark)

Pant, Sameer D.; March, Lindsey D.; Famulski, Jakub K.

2013-01-01

intrinsic or extrinsic apoptotic mechanisms were involved, morpholino antisense oligonucleotides targeting baxa, baxb, and p53 were employed. Caspase-3 immunohistochemistry (IHC) was performed to assay apoptosis. Pharmacologic inhibition (using SB203580) and IHC were used to investigate the role of p38...... occurs 28 hours post fertilization (hpf) in gdf6a(-/-) mutants that is mediated independently of p53 by intrinsic mechanisms involving Bax proteins. Also, gdf6a(-/-) mutants exhibit markedly increased p38 MAP kinase activation that can be inhibited to significantly reduce retinal apoptosis. A reduction...... in retinal smad1 expression was also noted in gdf6a(-/-) mutants. CONCLUSIONS. gdf6a(-/-)-induced apoptosis is characterized by the involvement of intrinsic apoptotic pathways, p38 MAP kinases, and dysregulated smad expression. Modulation of key mediators can inhibit retinal apoptosis offering potential...

Molecular cloning of a novel glucuronokinase/putative pyrophosphorylase from zebrafish acting in an UDP-glucuronic acid salvage pathway.

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Roman Gangl

Full Text Available In animals, the main precursor for glycosaminoglycan and furthermore proteoglycan biosynthesis, like hyaluronic acid, is UDP-glucuronic acid, which is synthesized via the nucleotide sugar oxidation pathway. Mutations in this pathway cause severe developmental defects (deficiency in the initiation of heart valve formation. In plants, UDP-glucuronic acid is synthesized via two independent pathways. Beside the nucleotide sugar oxidation pathway, a second minor route to UDP-glucuronic acid exist termed the myo-inositol oxygenation pathway. Within this myo-inositol is ring cleaved into glucuronic acid, which is subsequently converted to UDP-glucuronic acid by glucuronokinase and UDP-sugar pyrophosphorylase. Here we report on a similar, but bifunctional enzyme from zebrafish (Danio rerio which has glucuronokinase/putative pyrophosphorylase activity. The enzyme can convert glucuronic acid into UDP-glucuronic acid, required for completion of the alternative pathway to UDP-glucuronic acid via myo-inositol and thus establishes a so far unknown second route to UDP-glucuronic acid in animals. Glucuronokinase from zebrafish is a member of the GHMP-kinase superfamily having unique substrate specificity for glucuronic acid with a Km of 31 ± 8 µM and accepting ATP as the only phosphate donor (Km: 59 ± 9 µM. UDP-glucuronic acid pyrophosphorylase from zebrafish has homology to bacterial nucleotidyltransferases and requires UTP as nucleosid diphosphate donor. Genes for bifunctional glucuronokinase and putative UDP-glucuronic acid pyrophosphorylase are conserved among some groups of lower animals, including fishes, frogs, tunicates, and polychaeta, but are absent from mammals. The existence of a second pathway for UDP-glucuronic acid biosynthesis in zebrafish likely explains some previous contradictory finding in jekyll/ugdh zebrafish developmental mutants, which showed residual glycosaminoglycans and proteoglycans in knockout mutants of UDP

Gamma-ray induced mutants in castor (Ricinus communis L.)

International Nuclear Information System (INIS)

Janila, P.; Ashok Kumar, A.; Rajashekar Reddy, N.; Hemalatha, V.

2007-01-01

We report isolation of three recessive mutants in castor using dry seed irradiation with gamma rays. The crinkled leaf mutant (crf) was identified in K-55-112 M2 family and leafy mutant (lea) in H-55-577 M2 family; both are recessive lethal and thus maintained as heterozygotes. The cri mutant has highly wrinkled leaves resembling finger millet head and failed to enter reproductive phase, consequently did not produce seeds. The number of leaf lobes is reduced in lea mutant and though it produced spikes, the male and female flowers are converted to leafy appendages. The third mutant, fused (Ius) stem identified in H-55-617 M2 family is a recessive mutant. The branches of which are fused at the base and though each branch terminates in to monoceous spike like normal plant, the spike is highly condensed. The three mutants under report are valuable genetic stocks for development of linkage maps in castor, which is at infancy. (author)

Decreased Dissolution of ZnO by Iron Doping Yields Nanoparticles with Reduced Toxicity in the Rodent Lung and Zebrafish Embryos

Science.gov (United States)

Xia, Tian; Zhao, Yan; Sager, Tina; George, Saji; Pokhrel, Suman; Li, Ning; Schoenfeld, David; Meng, Huan; Lin, Sijie; Wang, Xiang; Wang, Meiying; Ji, Zhaoxia; Zink, Jeffrey I.; Mädler, Lutz; Castranova, Vincent; Lin, Shuo; Nel, Andre E.

2014-01-01

We have recently shown that the dissolution of ZnO nanoparticles and Zn2+ shedding leads to a series of sub-lethal and lethal toxicological responses at cellular level that can be alleviated by iron-doping. Iron-doping changes the particle matrix and slows the rate of particle dissolution. To determine whether iron doping of ZnO also leads to lesser toxic effects in vivo, toxicity studies were performed in rodent and zebrafish models. First, we synthesized a fresh batch of ZnO nanoparticles doped with 1–10 wt % of Fe. These particles were extensively characterized to confirm their doping status, reduced rate of dissolution in an exposure medium and reduced toxicity in a cellular screen. Subsequent studies compared the effects of undoped to doped particles in the rat lung, mouse lung and the zebrafish embryo. The zebrafish studies looked at embryo hatching and mortality rates as well as the generation of morphological defects, while the endpoints in the rodent lung included an assessment of inflammatory cell infiltrates, LDH release and cytokine levels in the bronchoalveolar lavage fluid. Iron doping, similar to the effect of the metal chelator, DTPA, interfered in the inhibitory effects of Zn2+ on zebrafish hatching. In the oropharyngeal aspiration model in the mouse, iron doping was associated with decreased polymorphonuclear cell counts and IL-6 mRNA production. Doped particles also elicited decreased heme oxygenase 1 expression in the murine lung. In the intratracheal instillation studies in the rat, Fe-doping was associated with decreased polymorphonuclear cell counts, LDH and albumin levels. All considered, the above data show that Fe-doping is a possible safe design strategy for preventing ZnO toxicity in animals and the environment. PMID:21250651

Completion of meiosis in male zebrafish (Danio rerio) despite lack of DNA mismatch repair gene mlh1

NARCIS (Netherlands)

Leal, M.C.; Feitsma, H.; Cuppen, E.; França, L.R.; Schulz, R.W.

2008-01-01

Mlh1 is a member of DNA mismatch repair (MMR) machinery and is also essential for the stabilization of crossovers during the first meiotic division. Recently, we have shown that zebrafish mlh1 mutant males are completely infertile because of a block in metaphase I, whereas females are fertile

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.

Completion of meiosis in male zebrafish (Danio rerio) despite lack of DNA mismatch repair gene mlh1.

NARCIS (Netherlands)

Leal, M.C.; Feitsma, H.; Cuppen, E.; Franca, L.R.; Schulz, R.W.

2008-01-01

Mlh1 is a member of DNA mismatch repair (MMR) machinery and is also essential for the stabilization of crossovers during the first meiotic division. Recently, we have shown that zebrafish mlh1 mutant males are completely infertile because of a block in metaphase I, whereas females are fertile but

Quantification of vestibular-induced eye movements in zebrafish larvae

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Mo Weike

2010-09-01

Full Text Available Abstract Background Vestibular reflexes coordinate movements or sensory input with changes in body or head position. Vestibular-evoked responses that involve the extraocular muscles include the vestibulo-ocular reflex (VOR, a compensatory eye movement to stabilize retinal images. Although an angular VOR attributable to semicircular canal stimulation was reported to be absent in free-swimming zebrafish larvae, recent studies reveal that vestibular-induced eye movements can be evoked in zebrafish larvae by both static tilts and dynamic rotations that tilt the head with respect to gravity. Results We have determined herein the basis of sensitivity of the larval eye movements with respect to vestibular stimulus, developmental stage, and sensory receptors of the inner ear. For our experiments, video recordings of larvae rotated sinusoidally at 0.25 Hz were analyzed to quantitate eye movements under infrared illumination. We observed a robust response that appeared as early as 72 hours post fertilization (hpf, which increased in amplitude over time. Unlike rotation about an earth horizontal axis, rotation about an earth vertical axis at 0.25 Hz did not evoke eye movements. Moreover, vestibular-induced responses were absent in mutant cdh23 larvae and larvae lacking anterior otoliths. Conclusions Our results provide evidence for a functional vestibulo-oculomotor circuit in 72 hpf zebrafish larvae that relies upon sensory input from anterior/utricular otolith organs.

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

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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.

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

Science.gov (United States)

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

2015-01-01

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

Evaluation of the toxic effect on zebrafish (Danio rerio) exposed to uranium mill tailings leaching solution

International Nuclear Information System (INIS)

Fang Geng; Nan Hu; Ji-Fang Zheng; Cheng-Lei Wang; Xin Chen; Jia Yu; De-Xin Ding

2012-01-01

The objective of this study was to evaluate the potential ecological danger and toxic effect of uranium mill tailings leaching solution (UMTLS) on aquatic animals. UMTLS was identified to contain two radioactive elements, nine heavy metal elements, and five non-metallic materials. The acute toxicity test indicated that the 1, 12, 24, 48, 72, 96 h LC 50 values of UMTLS to the zebrafish were 12.1, 7.1, 4.4, 3.8, 3.4, and 2.9%, respectively. In sub-lethal toxicity tests, superoxide dismutase, catalase, Na + -K + -ATPase activities, and malondialdehyde content were respectively determined and analyzed in the zebrafish gill, gonad, muscle, and liver after exposed to four different concentration levels of UMTLS for 7 and 14 days, respectively. The result showed that the most sensitivity of the antioxidant system in zebrafish tissues in UMTLS was gill, and then decreased in gonad, muscle and liver respectively. Na + -K + -ATPase activity in the liver and gonad may be considered as a reference biomarker of UMTLS stress. The data in this study may be valuable that the toxicity of such as the leaching solution of potentially hazardous material was compared with that of each constituent. (author)

Progranulin is neurotrophic in vivo and protects against a mutant TDP-43 induced axonopathy.

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Angela S Laird

Full Text Available Mislocalization, aberrant processing and aggregation of TAR DNA-binding protein 43 (TDP-43 is found in the neurons affected by two related diseases, amyotrophic lateral sclerosis (ALS and frontotemporal lobe dementia (FTLD. These TDP-43 abnormalities are seen when TDP-43 is mutated, such as in familial ALS, but also in FTLD, caused by null mutations in the progranulin gene. They are also found in many patients with sporadic ALS and FTLD, conditions in which only wild type TDP-43 is present. The common pathological hallmarks and symptomatic cross over between the two diseases suggest that TDP-43 and progranulin may be mechanistically linked. In this study we aimed to address this link by establishing whether overexpression of mutant TDP-43 or knock-down of progranulin in zebrafish embryos results in motor neuron phenotypes and whether human progranulin is neuroprotective against such phenotypes. Mutant TDP-43 (A315T mutation induced a motor axonopathy characterized by short axonal outgrowth and aberrant branching, similar, but more severe, than that induced by mutant SOD1. Knockdown of the two zebrafish progranulin genes, grna and grnb, produced a substantial decrease in axonal length, with knockdown of grna alone producing a greater decrease in axonal length than grnb. Progranulin overexpression rescued the axonopathy induced by progranulin knockdown. Interestingly, progranulin also rescued the mutant TDP-43 induced axonopathy, whilst it failed to affect the mutant SOD1-induced phenotype. TDP-43 was found to be nuclear in all conditions described. The findings described here demonstrate that progranulin is neuroprotective in vivo and may have therapeutic potential for at least some forms of motor neuron degeneration.

Disruption of the hypothalamic-pituitary-thyroid axis in zebrafish embryo–larvae following waterborne exposure to BDE-47, TBBPA and BPA

International Nuclear Information System (INIS)

Chan, Winson K.; Chan, King Ming

2012-01-01

We performed waterborne exposures of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47), tetrabromobisphenol A (TBBPA) or bisphenol A (BPA) on zebrafish (Danio rerio) embryo–larvae and quantitatively measured the expression of genes belonging to the hypothalamic-pituitary-thyroid (HPT) axis to assess for adverse thyroid function. For analysis on the effects of BDE-47, TBBPA and BPA on the hypothalamic-pituitary-thyroid genes, zebrafish embryo–larvae were acutely exposed to lethal concentrations of the chemical agents in order to determine the 96 h-LC50 (96 h lethal median concentration) and 96 h-EC50 (96 h effective median concentration) values. Further exposures at sub-lethal concentrations were then carried out and total RNA samples were extracted to quantify the mRNA expression levels of the genes of interest. In larvae, BDE-47 was found to have significantly induced many genes of interest, namely thyroglobulin, thyroid peroxidase, thyroid receptors α and β, thyroid stimulating hormone, and transthyretin. TBBPA only significantly induced three genes of interest (thyroid receptor α, thyroid stimulating hormone, and transthyretin) while BPA only induced thyroid stimulating hormone. In embryos, BDE-47 significantly induced the sodium iodide symporter and thyroid stimulating hormone. TBBPA significantly induced thyroid receptor α and thyroid stimulating hormone, while BPA did not significantly induce any of the genes. Most genes were only induced at the 75% 96 h-LC50 or 96 h-EC50 value; however, thyroid peroxidase and thyroid stimulating hormone demonstrated upregulation in a level as little as the 10% 96 h-LC50 value. The present study provides a new set of data on zebrafish mRNA induction of hypothalamic-pituitary-thyroid genes from exposure to BDE-47, TBBPA, or BPA. This information would serve useful for elucidating the toxicological mechanism of brominated flame retardants, assessing appropriate safety levels in the environment for these compounds, as well

Disruption of the hypothalamic-pituitary-thyroid axis in zebrafish embryo-larvae following waterborne exposure to BDE-47, TBBPA and BPA

Energy Technology Data Exchange (ETDEWEB)

Chan, Winson K. [Biochemistry Program, School of Life Sciences, Chinese University of Hong Kong, Sha Tin, Hong Kong (Hong Kong); Chan, King Ming, E-mail: kingchan@cuhk.edu.hk [Biochemistry Program, School of Life Sciences, Chinese University of Hong Kong, Sha Tin, Hong Kong (Hong Kong); Environmental Science Program, School of Life Sciences, Chinese University of Hong Kong, Sha Tin, Hong Kong (Hong Kong)

2012-02-15

We performed waterborne exposures of 2,2 Prime ,4,4 Prime -tetrabromodiphenyl ether (BDE-47), tetrabromobisphenol A (TBBPA) or bisphenol A (BPA) on zebrafish (Danio rerio) embryo-larvae and quantitatively measured the expression of genes belonging to the hypothalamic-pituitary-thyroid (HPT) axis to assess for adverse thyroid function. For analysis on the effects of BDE-47, TBBPA and BPA on the hypothalamic-pituitary-thyroid genes, zebrafish embryo-larvae were acutely exposed to lethal concentrations of the chemical agents in order to determine the 96 h-LC50 (96 h lethal median concentration) and 96 h-EC50 (96 h effective median concentration) values. Further exposures at sub-lethal concentrations were then carried out and total RNA samples were extracted to quantify the mRNA expression levels of the genes of interest. In larvae, BDE-47 was found to have significantly induced many genes of interest, namely thyroglobulin, thyroid peroxidase, thyroid receptors {alpha} and {beta}, thyroid stimulating hormone, and transthyretin. TBBPA only significantly induced three genes of interest (thyroid receptor {alpha}, thyroid stimulating hormone, and transthyretin) while BPA only induced thyroid stimulating hormone. In embryos, BDE-47 significantly induced the sodium iodide symporter and thyroid stimulating hormone. TBBPA significantly induced thyroid receptor {alpha} and thyroid stimulating hormone, while BPA did not significantly induce any of the genes. Most genes were only induced at the 75% 96 h-LC50 or 96 h-EC50 value; however, thyroid peroxidase and thyroid stimulating hormone demonstrated upregulation in a level as little as the 10% 96 h-LC50 value. The present study provides a new set of data on zebrafish mRNA induction of hypothalamic-pituitary-thyroid genes from exposure to BDE-47, TBBPA, or BPA. This information would serve useful for elucidating the toxicological mechanism of brominated flame retardants, assessing appropriate safety levels in the environment for

Lethal and mutagenic effects of ion beams and γ-rays in Aspergillus oryzae

International Nuclear Information System (INIS)

Toyoshima, Yoshiyuki; Takahashi, Akemi; Tanaka, Hisaki; Watanabe, Jun; Mogi, Yoshinobu; Yamazaki, Tatsuo; Hamada, Ryoko; Iwashita, Kazuhiro; Satoh, Katsuya; Narumi, Issay

2012-01-01

Highlights: ► We investigated the effects of different LET radiation in A. oryzae. ► Both γ-rays and ion beams induced base substitutions, frameshifts, deletions. ► Both γ-rays and ion beams induced genome-wide large-scale mutations in A. oryzae. ► Some differences in the types and frequencies of mutations were found. ► Our results provide new basic insights into the mutation breeding of A. oryzae. - Abstract: Aspergillus oryzae is a fungus that is used widely in traditional Japanese fermentation industries. In this study, the lethal and mutagenic effects of different linear energy transfer (LET) radiation in freeze-dried conidia of A. oryzae were investigated. The lethal effect, which was evaluated by a 90% lethal dose, was dependent on the LET value of the ionizing radiation. The most lethal ionizing radiation among that tested was 12 C 5+ ion beams with an LET of 121 keV/μm. The 12 C 5+ ion beams had a 3.6-times higher lethal effect than low-LET (0.2 keV/μm) γ-rays. The mutagenic effect was evaluated by the frequency of selenate resistant mutants. 12 C 6+ ion beams with an LET of 86 keV/μm were the most effective in inducing selenate resistance. The mutant frequency following exposure to 12 C 6+ ion beams increased with an increase in dose and reached 3.47 × 10 −3 at 700 Gy. In the dose range from 0 to 700 Gy, 12 C 5+ ion beams were the second most effective in inducing selenate resistance, the mutant frequency of which reached a maximum peak (1.67 × 10 −3 ) at 400 Gy. To elucidate the characteristics of mutation induced by ionizing radiation, mutations in the sulphate permease gene (sB) and ATP sulfurylase gene (sC) loci, the loss of function of which results in a selenate resistant phenotype, were compared between 12 C 5+ ion beams and γ-rays. We detected all types of transversions and transitions. For frameshifts, the frequency of a +1 frameshift was the highest in all cases. Although the incidence of deletions >2 bp was generally low

Improved somatic mutagenesis in zebrafish using transcription activator-like effector nucleases (TALENs.

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Finola E Moore

Full Text Available Zinc Finger Nucleases (ZFNs made by Context-Dependent Assembly (CoDA and Transcription Activator-Like Effector Nucleases (TALENs provide robust and user-friendly technologies for efficiently inactivating genes in zebrafish. These designer nucleases bind to and cleave DNA at particular target sites, inducing error-prone repair that can result in insertion or deletion mutations. Here, we assess the relative efficiencies of these technologies for inducing somatic DNA mutations in mosaic zebrafish. We find that TALENs exhibited a higher success rate for obtaining active nucleases capable of inducing mutations than compared with CoDA ZFNs. For example, all six TALENs tested induced DNA mutations at genomic target sites while only a subset of CoDA ZFNs exhibited detectable rates of mutagenesis. TALENs also exhibited higher mutation rates than CoDA ZFNs that had not been pre-screened using a bacterial two-hybrid assay, with DNA mutation rates ranging from 20%-76.8% compared to 1.1%-3.3%. Furthermore, the broader targeting range of TALENs enabled us to induce mutations at the methionine translation start site, sequences that were not targetable using the CoDA ZFN platform. TALENs exhibited similar toxicity to CoDA ZFNs, with >50% of injected animals surviving to 3 days of life. Taken together, our results suggest that TALEN technology provides a robust alternative to CoDA ZFNs for inducing targeted gene-inactivation in zebrafish, making it a preferred technology for creating targeted knockout mutants in zebrafish.

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

Zebrafish con/disp1 reveals multiple spatiotemporal requirements for Hedgehog-signaling in craniofacial development

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Schwend Tyler

2009-11-01

Full Text Available Abstract Background The vertebrate head skeleton is derived largely from cranial neural crest cells (CNCC. Genetic studies in zebrafish and mice have established that the Hedgehog (Hh-signaling pathway plays a critical role in craniofacial development, partly due to the pathway's role in CNCC development. Disruption of the Hh-signaling pathway in humans can lead to the spectral disorder of Holoprosencephaly (HPE, which is often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia 12. Previous work has uncovered a role for Hh-signaling in zebrafish dorsal neurocranium patterning and chondrogenesis, however Hh-signaling mutants have not been described with respect to the ventral pharyngeal arch (PA skeleton. Lipid-modified Hh-ligands require the transmembrane-spanning receptor Dispatched 1 (Disp1 for proper secretion from Hh-synthesizing cells to the extracellular field where they act on target cells. Here we study chameleon mutants, lacking a functional disp1(con/disp1. Results con/disp1 mutants display reduced and dysmorphic mandibular and hyoid arch cartilages and lack all ceratobranchial cartilage elements. CNCC specification and migration into the PA primorida occurs normally in con/disp1 mutants, however disp1 is necessary for post-migratory CNCC patterning and differentiation. We show that disp1 is required for post-migratory CNCC to become properly patterned within the first arch, while the gene is dispensable for CNCC condensation and patterning in more posterior arches. Upon residing in well-formed pharyngeal epithelium, neural crest condensations in the posterior PA fail to maintain expression of two transcription factors essential for chondrogenesis, sox9a and dlx2a, yet continue to robustly express other neural crest markers. Histology reveals that posterior arch residing-CNCC differentiate into fibrous-connective tissue, rather than becoming chondrocytes. Treatments with Cyclopamine, to

Dusp6 attenuates Ras/MAPK signaling to limit zebrafish heart regeneration.

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Missinato, Maria A; Saydmohammed, Manush; Zuppo, Daniel A; Rao, Krithika S; Opie, Graham W; Kühn, Bernhard; Tsang, Michael

2018-03-06

Zebrafish regenerate cardiac tissue through proliferation of pre-existing cardiomyocytes and neovascularization. Secreted growth factors such as FGFs, IGF, PDGFs and Neuregulin play essential roles in stimulating cardiomyocyte proliferation. These factors activate the Ras/MAPK pathway, which is tightly controlled by the feedback attenuator Dual specificity phosphatase 6 (Dusp6), an ERK phosphatase. Here, we show that suppressing Dusp6 function enhances cardiac regeneration. Inactivation of Dusp6 by small molecules or by gene inactivation increased cardiomyocyte proliferation, coronary angiogenesis, and reduced fibrosis after ventricular resection. Inhibition of Erbb or PDGF receptor signaling suppressed cardiac regeneration in wild-type zebrafish, but had a milder effect on regeneration in dusp6 mutants. Moreover, in rat primary cardiomyocytes, NRG1-stimulated proliferation can be enhanced upon chemical inhibition of Dusp6 with BCI. Our results suggest that Dusp6 attenuates Ras/MAPK signaling during regeneration and that suppressing Dusp6 can enhance cardiac repair. © 2018. Published by The Company of Biologists Ltd.

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

Science.gov (United States)

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

2012-01-01

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

The genetics of hair-cell function in zebrafish.

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Nicolson, Teresa

2017-09-01

Our ears are remarkable sensory organs, providing the important senses of balance and hearing. The complex structure of the inner ear, or 'labyrinth', along with the assorted neuroepithelia, have evolved to detect head movements and sounds with impressive sensitivity. The rub is that the inner ear is highly vulnerable to genetic lesions and environmental insults. According to National Institute of Health estimates, hearing loss is one of the most commonly inherited or acquired sensorineural diseases. To understand the causes of deafness and balance disorders, it is imperative to understand the underlying biology of the inner ear, especially the inner workings of the sensory receptors. These receptors, which are termed hair cells, are particularly susceptible to genetic mutations - more than two dozen genes are associated with defects in this cell type in humans. Over the past decade, a substantial amount of progress has been made in working out the molecular basis of hair-cell function using vertebrate animal models. Given the transparency of the inner ear and the genetic tools that are available, zebrafish have become an increasingly popular animal model for the study of deafness and vestibular dysfunction. Mutagenesis screens for larval defects in hearing and balance have been fruitful in finding key components, many of which have been implicated in human deafness. This review will focus on the genes that are required for hair-cell function in zebrafish, with a particular emphasis on mechanotransduction. In addition, the generation of new tools available for the characterization of zebrafish hair-cell mutants will be discussed.

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

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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.

Usefulness of zebrafish larvae to evaluate drug-induced functional and morphological renal tubular alterations.

Science.gov (United States)

Gorgulho, Rita; Jacinto, Raquel; Lopes, Susana S; Pereira, Sofia A; Tranfield, Erin M; Martins, Gabriel G; Gualda, Emilio J; Derks, Rico J E; Correia, Ana C; Steenvoorden, Evelyne; Pintado, Petra; Mayboroda, Oleg A; Monteiro, Emilia C; Morello, Judit

2018-01-01

Prediction and management of drug-induced renal injury (DIRI) rely on the knowledge of the mechanisms of drug insult and on the availability of appropriate animal models to explore it. Zebrafish (Danio rerio) offers unique advantages for assessing DIRI because the larval pronephric kidney has a high homology with its human counterpart and it is fully mature at 3.5 days post-fertilization. Herein, we aimed to evaluate the usefulness of zebrafish larvae as a model of renal tubular toxicity through a comprehensive analysis of the renal alterations induced by the lethal concentrations for 10% of the larvae for gentamicin, paracetamol and tenofovir. We evaluated drug metabolic profile by mass spectrometry, renal function with the inulin clearance assay, the 3D morphology of the proximal convoluted tubule by two-photon microscopy and the ultrastructure of proximal convoluted tubule mitochondria by transmission electron microscopy. Paracetamol was metabolized by conjugation and oxidation with further detoxification with glutathione. Renal clearance was reduced with gentamicin and paracetamol. Proximal tubules were enlarged with paracetamol and tenofovir. All drugs induced mitochondrial alterations including dysmorphic shapes ("donuts", "pancakes" and "rods"), mitochondrial swelling, cristae disruption and/or loss of matrix granules. These results are in agreement with the tubular effects of gentamicin, paracetamol and tenofovir in man and demonstrate that zebrafish larvae might be a good model to assess functional and structural damage associated with DIRI.

A zebrafish transgenic model of Ewing’s sarcoma reveals conserved mediators of EWS-FLI1 tumorigenesis

Directory of Open Access Journals (Sweden)

Stefanie W. Leacock

2012-01-01

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

Lethal and mutagenic effects of ion beams and γ-rays in Aspergillus oryzae

Energy Technology Data Exchange (ETDEWEB)

Toyoshima, Yoshiyuki, E-mail: toyoshima@yamasa.com [Soy Sauce Laboratory, Yamasa Corporation, 2-10-1 Araoicho, Choshi, Chiba 288-0056 (Japan); Takahashi, Akemi; Tanaka, Hisaki; Watanabe, Jun; Mogi, Yoshinobu; Yamazaki, Tatsuo [Soy Sauce Laboratory, Yamasa Corporation, 2-10-1 Araoicho, Choshi, Chiba 288-0056 (Japan); Hamada, Ryoko; Iwashita, Kazuhiro [Fundamental Research Division, National Research Institute of Brewing, 3-7-1 Kagamiyama, Higashihiroshima, Hiroshima 739-0046 (Japan); Satoh, Katsuya; Narumi, Issay [Ion Beam Mutagenesis Research Group, Quantum Beam Science Directorate, Japan Atomic Energy Agency, 1233 Watanuki, Takasaki, Gunma 370-1292 (Japan)

2012-12-15

Highlights: ► We investigated the effects of different LET radiation in A. oryzae. ► Both γ-rays and ion beams induced base substitutions, frameshifts, deletions. ► Both γ-rays and ion beams induced genome-wide large-scale mutations in A. oryzae. ► Some differences in the types and frequencies of mutations were found. ► Our results provide new basic insights into the mutation breeding of A. oryzae. - Abstract: Aspergillus oryzae is a fungus that is used widely in traditional Japanese fermentation industries. In this study, the lethal and mutagenic effects of different linear energy transfer (LET) radiation in freeze-dried conidia of A. oryzae were investigated. The lethal effect, which was evaluated by a 90% lethal dose, was dependent on the LET value of the ionizing radiation. The most lethal ionizing radiation among that tested was {sup 12}C{sup 5+} ion beams with an LET of 121 keV/μm. The {sup 12}C{sup 5+} ion beams had a 3.6-times higher lethal effect than low-LET (0.2 keV/μm) γ-rays. The mutagenic effect was evaluated by the frequency of selenate resistant mutants. {sup 12}C{sup 6+} ion beams with an LET of 86 keV/μm were the most effective in inducing selenate resistance. The mutant frequency following exposure to {sup 12}C{sup 6+} ion beams increased with an increase in dose and reached 3.47 × 10{sup −3} at 700 Gy. In the dose range from 0 to 700 Gy, {sup 12}C{sup 5+} ion beams were the second most effective in inducing selenate resistance, the mutant frequency of which reached a maximum peak (1.67 × 10{sup −3}) at 400 Gy. To elucidate the characteristics of mutation induced by ionizing radiation, mutations in the sulphate permease gene (sB) and ATP sulfurylase gene (sC) loci, the loss of function of which results in a selenate resistant phenotype, were compared between {sup 12}C{sup 5+} ion beams and γ-rays. We detected all types of transversions and transitions. For frameshifts, the frequency of a +1 frameshift was the highest in all

Uvm mutants of Escherichia coli K 12 deficient in UV mutagenesis. Pt. 1

International Nuclear Information System (INIS)

Steinborn, G.

1978-01-01

Selection for defective reversion induction, after UV treatment of E. coli K 12, yielded uvm mutants. These mutants exhibited highly reduced or no UV mutability for all loci tested although they were moderately and normally mutable by X-rays and EMS, respectively. Uvm mutations confer only a slight sensitivity to killing by UV and X-rays and no clear sensitivity to the lethal effect of HN2, EMS or MMS. Growth and viability of untreated uvm cells were normal. The properties of uvm mutants are discussed in relation to those of other relevant mutant types and to some actual problems of induced mutagenesis. (orig.) 891 AJ [de

The pathogenicity of thymidine kinase-deficient mutants of herpes simplex virus in mice.

Science.gov (United States)

Field, H J; Wildy, P

1978-10-01

The pathogenicity for mice of two mutants of herpes simplex virus (type 1 and type 2), which fail to induce thymidine kinase, were compared with their respective parent strains. The mutants were much less virulent than the parents following either intracerebral or peripheral inoculation. The replication of the virus at the site of inoculation and its progression into the nervous system were studied. Following a very large inoculum in the ear, the type 1 mutant was found to establish a latent infection in the cervical dorsal root ganglia. Mice inoculated intracerebrally with small doses of the mutant viruses were solidly immune to challenge with lethal doses of the parent strain.

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

NARCIS (Netherlands)

Knöbel, M.; Busser, F.J.M.; Rico-Rico, A.; Kramer, N.I.|info:eu-repo/dai/nl/304836125; Hermens, J.L.M.|info:eu-repo/dai/nl/069681384; Hafner, C.; Tanneberger, K.; Schirmer, K.; Scholz, S.

2012-01-01

The zebrafish embryo toxicity test has been proposed as an alternative for the acute fish toxicity test, which is required by various regulations for environmental risk assessment of chemicals. We investigated the reliability of the embryo test by probing organic industrial chemicals with a wide

Differential expression of the lethal gene Luteus-Pa in cacao of the Parinari series.

Science.gov (United States)

Rehem, B C; Almeida, A-A F; Figueiredo, G S F; Gesteira, A S; Santos, S C; Corrêa, R X; Yamada, M M; Valle, R R

2016-02-22

The recessive lethal character Luteus-Pa is found in cacao (Theobroma cacao) genotypes of the Parinari series (Pa) and is characterized by expression of leaf chlorosis and seedling death. Several genotypes of the Pa series are bearers of the gene responsible for the expression of the Luteus-Pa character, which can be used as a tool for determining relationships between genotypes of this group. To evaluate this phenomenon, we analyzed the differential expression of genes between mutant seedlings and wild-type hybrid Pa 30 x 169 seedlings, with the aim of elucidating the possible lethal mechanisms of the homozygous recessive character Luteus-Pa. Plant material was harvested from leaves of wild and mutant seedlings at different periods to construct a subtractive library and perform quantitative analysis using real-time PCR. The 649 sequences obtained from the subtractive library had an average length of 500 bp, forming 409 contigs. The probable proteins encoded were grouped into 10 functional categories. Data from ESTs identified genes associated with Rubisco, peroxidases, and other proteins and enzymes related to carbon assimilation, respiration, and photosystem 2. Mutant seedlings were characterized by synthesizing defective PsbO and PsbA proteins, which were overexpressed from 15 to 20 days after seedling emergence.

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

Science.gov (United States)

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

2015-10-01

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

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

Science.gov (United States)

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

2013-11-01

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

Impacts of triclosan exposure on zebrafish early-life stage: Toxicity and acclimation mechanisms.

Science.gov (United States)

Falisse, Elodie; Voisin, Anne-Sophie; Silvestre, Frédéric

2017-08-01

Triclosan (TCS) is a broad spectrum antibacterial agent widely used in personal care products and present in most aquatic ecosystems. This study investigated the occurrence of triclosan acclimation and the biological mechanisms underlying the stress response triggered in early-life stage of zebrafish. Zebrafish eggs were first exposed to four different sublethal concentrations of TCS (2, 20, 50 and 100μg/L) for 7days following fertilization and subsequently exposed to a lethal concentration of TCS (1000μg/L). During the time-to-death exposure (TTD), mortality was continuously recorded to evaluate if increased resistance occurred. Overall, larvae exposed to 50μg/L of TCS demonstrated higher sensitivity, with delayed hatching and increased mortality during the sub-lethal exposure and significant lower mean time-to-death (TTD) value compared to the other groups. Interestingly, fish exposed to the highest concentration of TCS (100μg/L) presented a similar mean TTD value as controls and a significantly better survival in comparison with embryos exposed to 50μg/L, suggesting that acclimation process has been triggered at this concentration. Proteomic and enzymatic analyses were conducted on 7days post fertilization (dpf) larvae exposed to 50μg/L and 100μg/L of TCS giving insights into the functional changes triggered at those specific concentrations. TCS seemed to affect proteins involved in cytoskeleton, stress response, eyes and neuronal development. This was endorsed by the enzymatic results, which suggest impairment in glutathione metabolism and acute neurotoxicity. A significant 2.5-fold and 3-fold increase of AChE activity was observed following TCS exposure. Moreover, GPx activity was significantly increased whereas a significant inhibition of GR activity was observed, suggesting that de novo synthesis of reduced GSH might occur in order to maintain the ratio between reduced and oxidized GSH. Proteomic results revealed possible candidate protein involved in

Bipyridine (2,2′-dipyridyl) potentiates Escherichia coli lethality induced by nitrogen mustard mechlorethamine

International Nuclear Information System (INIS)

De Alencar, T.A.M.; Wilmart-Gonçalves, T.C.; Vidal, L.S.; Fortunato, R.S.; Leitão, A.C.; Lage, C.

2014-01-01

Highlights: • Reduction of Fe 2+ ensues a respiratory burst to reduce the oxidized iron pool. • Through Harber–Weiss recycling, superoxide electrons can reduce oxidized iron. • Redox imbalance sensitized repair proficient Escherichia coli to mustard lethal crosslinks. • A stronger synergism impacted survival of a superoxide dismutase-deficient strain. • Anti-cancer cocktails added of an iron chelator may impact hypoxia and genotoxicity. - Abstract: Alkylating agents are used in anti-tumor chemotherapy because they bind covalently to DNA and generate adducts that may lead to cell death. Bifunctional (HN2) and monofunctional (HN1) nitrogen are two such agents, and HN2 was the first drug successfully employed in anti-leukemia chemotherapy. Currently, HN2 is used either alone or combined with other drugs to treat Hodgkin's disease. It is well known that several crosslinking agents require metabolic activation via reactive oxygen species (ROS) to exert their lethal effects. The objective of this work was therefore to determine whether the abovementioned mustards would also require metabolic activation to exert lethal action against Escherichia coli. For this purpose, we measured survival following exposure to HN2 in E. coli strains that were deficient in nucleotide excision repair (uvrA NER mutant), base excision repair (xthA nfo nth fpg BER mutant) or superoxide dismutase (sodAB mutant) activity. We also performed the same experiments in cells pretreated with an iron chelator (2,2′-dipyridyl, DIP). The NER and BER mutants were only sensitive to HN2 treatment (survival rates similar to those of the wild-type were achieved with 5-fold lower HN2 doses). However, wild-type and sodAB strains were not sensitive to treatment with HN2. In all tested strains, survival dropped by 2.5-fold following pretreatment with DIP compared to treatment with HN2 alone. Furthermore, DIP treatment increased ROS generation in both wild type and sodAB-deficient strains. Based

Bipyridine (2,2′-dipyridyl) potentiates Escherichia coli lethality induced by nitrogen mustard mechlorethamine

Energy Technology Data Exchange (ETDEWEB)

De Alencar, T.A.M.; Wilmart-Gonçalves, T.C.; Vidal, L.S.; Fortunato, R.S.; Leitão, A.C. [Laboratório de Radiobiologia Molecular (Brazil); Lage, C., E-mail: claudia_lage_dna@yahoo.com.br [Laboratório de Radiações em Biologia (Brazil)

2014-07-15

Highlights: • Reduction of Fe{sup 2+} ensues a respiratory burst to reduce the oxidized iron pool. • Through Harber–Weiss recycling, superoxide electrons can reduce oxidized iron. • Redox imbalance sensitized repair proficient Escherichia coli to mustard lethal crosslinks. • A stronger synergism impacted survival of a superoxide dismutase-deficient strain. • Anti-cancer cocktails added of an iron chelator may impact hypoxia and genotoxicity. - Abstract: Alkylating agents are used in anti-tumor chemotherapy because they bind covalently to DNA and generate adducts that may lead to cell death. Bifunctional (HN2) and monofunctional (HN1) nitrogen are two such agents, and HN2 was the first drug successfully employed in anti-leukemia chemotherapy. Currently, HN2 is used either alone or combined with other drugs to treat Hodgkin's disease. It is well known that several crosslinking agents require metabolic activation via reactive oxygen species (ROS) to exert their lethal effects. The objective of this work was therefore to determine whether the abovementioned mustards would also require metabolic activation to exert lethal action against Escherichia coli. For this purpose, we measured survival following exposure to HN2 in E. coli strains that were deficient in nucleotide excision repair (uvrA NER mutant), base excision repair (xthA nfo nth fpg BER mutant) or superoxide dismutase (sodAB mutant) activity. We also performed the same experiments in cells pretreated with an iron chelator (2,2′-dipyridyl, DIP). The NER and BER mutants were only sensitive to HN2 treatment (survival rates similar to those of the wild-type were achieved with 5-fold lower HN2 doses). However, wild-type and sodAB strains were not sensitive to treatment with HN2. In all tested strains, survival dropped by 2.5-fold following pretreatment with DIP compared to treatment with HN2 alone. Furthermore, DIP treatment increased ROS generation in both wild type and sodAB-deficient strains

A pharmacological screen for compounds that rescue the developmental lethality of a Drosophila ATM mutant.

Science.gov (United States)

Rimkus, Stacey A; Wassarman, David A

2018-01-01

Ataxia-telangiectasia (A-T) is a neurodegenerative disease caused by mutation of the A-T mutated (ATM) gene. ATM encodes a protein kinase that is activated by DNA damage and phosphorylates many proteins, including those involved in DNA repair, cell cycle control, and apoptosis. Characteristic biological and molecular functions of ATM observed in mammals are conserved in Drosophila melanogaster. As an example, conditional loss-of-function ATM alleles in flies cause progressive neurodegeneration through activation of the innate immune response. However, unlike in mammals, null alleles of ATM in flies cause lethality during development. With the goals of understanding biological and molecular roles of ATM in a whole animal and identifying candidate therapeutics for A-T, we performed a screen of 2400 compounds, including FDA-approved drugs, natural products, and bioactive compounds, for modifiers of the developmental lethality caused by a temperature-sensitive ATM allele (ATM8) that has reduced kinase activity at non-permissive temperatures. Ten compounds reproducibly suppressed the developmental lethality of ATM8 flies, including Ronnel, which is an organophosphate. Ronnel and other suppressor compounds are known to cause mitochondrial dysfunction or to inhibit the enzyme acetylcholinesterase, which controls the levels of the neurotransmitter acetylcholine, suggesting that detrimental consequences of reduced ATM kinase activity can be rescued by inhibiting the function of mitochondria or increasing acetylcholine levels. We carried out further studies of Ronnel because, unlike the other compounds that suppressed the developmental lethality of homozygous ATM8 flies, Ronnel was toxic to the development of heterozygous ATM8 flies. Ronnel did not affect the innate immune response of ATM8 flies, and it further increased the already high levels of DNA damage in brains of ATM8 flies, but its effects were not harmful to the lifespan of rescued ATM8 flies. These results provide

Enantioselective developmental toxicity and immunotoxicity of pyraclofos toward zebrafish (Danio rerio)

Energy Technology Data Exchange (ETDEWEB)

Zhuang, Shulin, E-mail: shulin@zju.edu.cn [Institute of Environmental Science, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058 (China); Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control, Hangzhou 310058 (China); Zhang, Zhisheng [Institute of Environmental Science, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058 (China); Zhang, Wenjing; Bao, Lingling [Institute of Environmental Science, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou 310058 (China); Zhejiang Provincial Key Laboratory of Organic Pollution Process and Control, Hangzhou 310058 (China); Xu, Chao, E-mail: chaoxu@zjut.edu.cn [Research Center of Environmental Science, College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032 (China); Zhang, Hu [Institute of Quality and Standard for Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou 210021 (China)

2015-02-15

Highlights: • Pyraclofos has significant enantioselective aquatic toxicities to zebrafish. • Pyraclofos induces time- and concentration-dependent developmental toxicity and immunotoxicity. • The mRNA level of IL-1β gene was significantly up-regulated by pyraclofos. • Pyraclofos binds potently to IL-1β, potentially affecting IL-1β-dependent proinflammatory signal transduction. • Our in vitro and in silico studies help to understand the molecular basis for aquatic toxicity of pyraclofos. - Abstract: Pyraclofos, a relatively new organophosphorus pesticide, has shown potential ecotoxicities, however, its aquatic toxicity, especially enantioselective aquatic toxicity, remains largely unknown. Using zebrafish (Danio rerio) as a preeminent vertebrate aquatic model, the enantioselective differences in the developmental toxicity and immunotoxicity of pyraclofos were evaluated. Following 96-h exposure, pyraclofos enantiomers exhibited acute toxicity and showed lethal concentration 50 of 2.23 and 3.99 mg/L for (R)-Pyraclofos and (S)-Pyraclofos, respectively. Exposure to pyraclofos caused time- and concentration-dependent malformations such as pericardial edema, yolk sac edema, crooked bodies and hatching during the embryonic development, with markedly higher percentages of malformation at higher concentrations. The concentration-dependent immunotoxicity to zebrafish embryo exposed to low level pyraclofos was induced with significant up-regulation of mRNA levels of immune-related interleukin-1β (IL-1β) gene. (R)-Pyraclofos was consistently more toxic than (S)-Pyraclofos for the acute toxicity, developmental toxicity and immunotoxicity to zebrafish. Molecular dynamics simulations revealed that at the atomic level, (R)-Pyraclofos binds more potently to IL-1β protein than (S)-Pyraclofos. This enantioselective binding is mainly contributed by the distinct binding mode of pyraclofos enantiomers and their electrostatic interactions with IL-1β, which potentially

Enantioselective developmental toxicity and immunotoxicity of pyraclofos toward zebrafish (Danio rerio)

International Nuclear Information System (INIS)

Zhuang, Shulin; Zhang, Zhisheng; Zhang, Wenjing; Bao, Lingling; Xu, Chao; Zhang, Hu

2015-01-01

Highlights: • Pyraclofos has significant enantioselective aquatic toxicities to zebrafish. • Pyraclofos induces time- and concentration-dependent developmental toxicity and immunotoxicity. • The mRNA level of IL-1β gene was significantly up-regulated by pyraclofos. • Pyraclofos binds potently to IL-1β, potentially affecting IL-1β-dependent proinflammatory signal transduction. • Our in vitro and in silico studies help to understand the molecular basis for aquatic toxicity of pyraclofos. - Abstract: Pyraclofos, a relatively new organophosphorus pesticide, has shown potential ecotoxicities, however, its aquatic toxicity, especially enantioselective aquatic toxicity, remains largely unknown. Using zebrafish (Danio rerio) as a preeminent vertebrate aquatic model, the enantioselective differences in the developmental toxicity and immunotoxicity of pyraclofos were evaluated. Following 96-h exposure, pyraclofos enantiomers exhibited acute toxicity and showed lethal concentration 50 of 2.23 and 3.99 mg/L for (R)-Pyraclofos and (S)-Pyraclofos, respectively. Exposure to pyraclofos caused time- and concentration-dependent malformations such as pericardial edema, yolk sac edema, crooked bodies and hatching during the embryonic development, with markedly higher percentages of malformation at higher concentrations. The concentration-dependent immunotoxicity to zebrafish embryo exposed to low level pyraclofos was induced with significant up-regulation of mRNA levels of immune-related interleukin-1β (IL-1β) gene. (R)-Pyraclofos was consistently more toxic than (S)-Pyraclofos for the acute toxicity, developmental toxicity and immunotoxicity to zebrafish. Molecular dynamics simulations revealed that at the atomic level, (R)-Pyraclofos binds more potently to IL-1β protein than (S)-Pyraclofos. This enantioselective binding is mainly contributed by the distinct binding mode of pyraclofos enantiomers and their electrostatic interactions with IL-1β, which potentially

Mutation of the Na+/K+-ATPase Atp1a1a.1 causes QT interval prolongation and bradycardia in zebrafish.

Science.gov (United States)

Pott, Alexander; Bock, Sarah; Berger, Ina M; Frese, Karen; Dahme, Tillman; Keßler, Mirjam; Rinné, Susanne; Decher, Niels; Just, Steffen; Rottbauer, Wolfgang

2018-05-08

The genetic underpinnings that orchestrate the vertebrate heart rate are not fully understood yet, but of high clinical importance, since diseases of cardiac impulse formation and propagation are common and severe human arrhythmias. To identify novel regulators of the vertebrate heart rate, we deciphered the pathogenesis of the bradycardia in the homozygous zebrafish mutant hiphop (hip) and identified a missense-mutation (N851K) in Na + /K + -ATPase α1-subunit (atp1a1a.1). N851K affects zebrafish Na + /K + -ATPase ion transport capacity, as revealed by in vitro pump current measurements. Inhibition of the Na + /K + -ATPase in vivo indicates that hip rather acts as a hypomorph than being a null allele. Consequently, reduced Na + /K + -ATPase function leads to prolonged QT interval and refractoriness in the hip mutant heart, as shown by electrocardiogram and in vivo electrical stimulation experiments. We here demonstrate for the first time that Na + /K + -ATPase plays an essential role in heart rate regulation by prolonging myocardial repolarization. Copyright © 2018. Published by Elsevier Ltd.

Contribution of the C-terminal region within the catalytic core domain of HIV-1 integrase to yeast lethality, chromatin binding and viral replication

Directory of Open Access Journals (Sweden)

Belhumeur Pierre

2008-11-01

Full Text Available Abstract Background HIV-1 integrase (IN is a key viral enzymatic molecule required for the integration of the viral cDNA into the genome. Additionally, HIV-1 IN has been shown to play important roles in several other steps during the viral life cycle, including reverse transcription, nuclear import and chromatin targeting. Interestingly, previous studies have demonstrated that the expression of HIV-1 IN induces the lethal phenotype in some strains of Saccharomyces cerevisiae. In this study, we performed mutagenic analyses of the C-terminal region of the catalytic core domain of HIV-1 IN in order to delineate the critical amino acid(s and/or motif(s required for the induction of the lethal phenotype in the yeast strain HP16, and to further elucidate the molecular mechanism which causes this phenotype. Results Our study identified three HIV-1 IN mutants, V165A, A179P and KR186,7AA, located in the C-terminal region of the catalytic core domain of IN that do not induce the lethal phenotype in yeast. Chromatin binding assays in yeast and mammalian cells demonstrated that these IN mutants were impaired for the ability to bind chromatin. Additionally, we determined that while these IN mutants failed to interact with LEDGF/p75, they retained the ability to bind Integrase interactor 1. Furthermore, we observed that VSV-G-pseudotyped HIV-1 containing these IN mutants was unable to replicate in the C8166 T cell line and this defect was partially rescued by complementation with the catalytically inactive D64E IN mutant. Conclusion Overall, this study demonstrates that three mutations located in the C-terminal region of the catalytic core domain of HIV-1 IN inhibit the IN-induced lethal phenotype in yeast by inhibiting the binding of IN to the host chromatin. These results demonstrate that the C-terminal region of the catalytic core domain of HIV-1 IN is important for binding to host chromatin and is crucial for both viral replication and the promotion of

Antigen Uptake during Different Life Stages of Zebrafish (Danio rerio Using a GFP-Tagged Yersinia ruckeri.

Directory of Open Access Journals (Sweden)

Rozalia Korbut

Full Text Available Immersion-vaccines (bacterins are routinely used for aquacultured rainbow trout to protect against Yersinia ruckeri (Yr. During immersion vaccination, rainbow trout take up and process the antigens, which induce protection. The zebrafish was used as a model organism to study uptake mechanisms and subsequent antigen transport in fish. A genetically modified Yr was developed to constitutively express green fluorescent protein (GFP and was used for bacterin production. Larval, juvenile and adult transparent zebrafish (tra:nac mutant received a bath in the bacterin for up to 30 minutes. Samples were taken after 1 min, 15 min, 30 min, 2 h, 12 h and 24 h. At each sampling point fish were used for live imaging of the uptake using a fluorescence stereomicroscope and for immunohistochemistry (IHC. In adult fish, the bacterin could be traced within 30 min in scale pockets, skin, oesophagus, intestine and fins. Within two hours post bath (pb Yr-antigens were visible in the spleen and at 24 h in liver and kidney. Bacteria were associated with the gills, but uptake at this location was limited. Antigens were rarely detected in the blood and never in the nares. In juvenile fish uptake of the bacterin was seen in the intestine 30 min pb and in the nares 2 hpb but never in scale pockets. Antigens were detected in the spleen 12 hpb. Zebrafish larvae exhibited major Yr uptake only in the mid-intestine enterocytes 24 hpb. The different life stages of zebrafish varied with regard to uptake locations, however the gut was consistently a major uptake site. Zebrafish and rainbow trout tend to have similar uptake mechanisms following immersion or bath vaccination, which points towards zebrafish as a suitable model organism for this aquacultured species.

Immersion infection of germ-free zebrafish with Listeria monocytogenes induces transient expression of innate immune response genes

Directory of Open Access Journals (Sweden)

Ying eShan

2015-04-01

Full Text Available Zebrafish, Denio rerio, could be an alternative to other classic animal models for human infectious diseases to examine the processes of microbial infections and host-pathogen interactions in vivo because of their small body dimension but large clutch size. We established germ-free zebrafish infection models of Listeria monocytogenes through different routes of infection: oral immersion and injection via yolk sac, brain ventricle and blood island. Immersion of zebrafish larva even with 1010CFU/mL L. monocytogenes EGDe strain in egg water was unable to cause mortality, but GFP-expressing bacteria in the gut lumen could be observed in frozen sections. Several selected maker genes of the innate immune system, including cyp1a, irg1l, il1b and mmp9, were significantly induced by oral immersion not only with strain EGDe, but also with strain M7 and L. innocua, though to a lesser degree (P < 0.01. Such induction appears to be transient with peak at 48 h post-infection, but returned to basal level at 72 h post-infection. Of the three injection routes, mortality after infection by yolk sac was 80% in early stage of infection. Few eggs could survive and hatch. Injection into zebrafish embryos via brain ventricle or blood island led to progressive lethal infection. L. mocytogenes EGDe showed steady replication in the fish embryos and was far more pathogenic than strain M7, which is consistent with findings in the murine model. We conclude that zebrafish could serve as susceptible and microscopically visible infection models for L. monocytogenes via different routes and could be applied to further studies on the interactions between bacterial virulence factors and host immune responses.

Cep70 and Cep131 contribute to ciliogenesis in zebrafish embryos

Directory of Open Access Journals (Sweden)

Carl Matthias

2009-03-01

Full Text Available Abstract Background The centrosome is the cell's microtubule organising centre, an organelle with important roles in cell division, migration and polarity. However, cells can divide and flies can, for a large part of development, develop without them. Many centrosome proteins have been identified but the roles of most are still poorly understood. The centrioles of the centrosome are similar to the basal bodies of cilia, hair-like extensions of many cells that have important roles in cell signalling and development. In a number of human diseases, such Bardet-Biedl syndrome, centrosome/cilium proteins are mutated, leading to polycystic kidney disease, situs inversus, and neurological problems, amongst other symptoms. Results We describe zebrafish (Danio rerio embryos depleted for two uncharacterised, centrosome proteins, Cep70 and Cep131. The phenotype of these embryos resembles that of zebrafish mutants for intraflagellar transport proteins (IFTs, with kidney and ear development affected and left-right asymmetry randomised. These organs and processes are those affected in Bardet-Biedl syndrome and other similar diseases. Like these diseases, the root cause of the phenotype lies, in fact, in dysfunctional cilia, which are shortened but not eliminated in several tissues in the morphants. Centrosomes and basal bodies, on the other hand, are present. Both Cep70 and Cep131 possess a putative HDAC (histone deacetylase interacting domain. However, we could not detect in yeast two-hybrid assays any interaction with the deacetylase that controls cilium length, HDAC6, or any of the IFTs that we tested. Conclusion Cep70 and Cep131 contribute to ciliogenesis in many tissues in the zebrafish embryo: cilia are made in cep70 and cep131 morphant zebrafish embryos but are shortened. We propose that the role of these centrosomal/basal body proteins is in making the cilium and that they are involved in determination of the length of the axoneme.

Quantitative measures of mutagenicity and multability based on mutant yield data

International Nuclear Information System (INIS)

Eckhardt, F.; Haynes, R.H.

1980-01-01

We describe, how mutant yield data (mutants per cell treated) can be used both to compare the mutagenenicity of different mutagens, and to characterize the mutability of different cell types. Yield curves reveal the net effect of the lethal and genetic actions of mutagens on cells. Normally, yields are the quantities measured in assays for mutagenesis, and rectilinear plots of such data baldly reveal the amount of experimental error and the extent of actual mutant induction above the background level. Plots of yield versus lethal hits can be used to quantify the relative mutagenenic efficiency (RME) of agents whose physical exposure doses otherwise would be incommensurable, as well as the relative mutability (Rmt) of different strains to the same mutagen. Plots of yield versus log dose provide an unambiguous way of assessing the relative mutational sensitivities (Rms) and mutational resolutions (Rmr) of different strains against a given mutagen. Such analysis is important for evaluation of the relative merits of excision-proficient and excision-deficient strains of the same organism as mutagen-testing systems. The mathematical approach outlined here is applied, by way of example, to measurements of UV and 4-NQO induced mutagenesis in both repair-deficient and repair-proficient haploid strains of the yeast Sacccharomyces cerevsiae. (orig.)

Disruption of the regulatory beta subunit of protein kinase CK2 in mice leads to a cell-autonomous defect and early embryonic lethality

DEFF Research Database (Denmark)

Buchou, Thierry; Vernet, Muriel; Blond, Olivier

2003-01-01

in mice leads to postimplantation lethality. Mutant embryos were reduced in size at embryonic day 6.5 (E6.5). They did not exhibit signs of apoptosis but did show reduced cell proliferation. Mutant embryos were resorbed at E7.5. In vitro, CK2beta(-/-) morula development stopped after the blastocyst stage...

Essential role for the alpha 1 chain of type VIII collagen in zebrafish notochord formation.

Science.gov (United States)

Gansner, John M; Gitlin, Jonathan D

2008-12-01

Several zebrafish mutants identified in large-scale forward genetic screens exhibit notochord distortion. We now report the cloning and further characterization of one such mutant, gulliver(m208) (gul(m208)). The notochord defect in gul(m208) mutants is exacerbated under conditions of copper depletion or lysyl oxidase cuproenzyme inhibition that are without a notochord effect on wild-type embryos. The gul(m208) phenotype results from a missense mutation in the gene encoding Col8a1, a lysyl oxidase substrate, and morpholino knockdown of col8a1 recapitulates the notochord distortion observed in gul(m208) mutants. Of interest, the amino acid mutated in gul(m208) Col8a1 is highly conserved, and the equivalent substitution in a closely related human protein, COL10A1, causes Schmid metaphyseal chondrodysplasia. Taken together, the data identify a new protein essential for notochord morphogenesis, extend our understanding of gene-nutrient interactions in early development, and suggest that human mutations in COL8A1 may cause structural birth defects. (c) 2008 Wiley-Liss, Inc.

Radiation induced mutants in cassava (Manihot esculenta Crantz)

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Nayar, G.G.; Rajendran, P.G.

1987-01-01

Full text: Stem cuttings and true seeds of three promising cultivars of cassava were exposed respectively to 1 to 5 kR and 10 to 50 kR acute gamma rays from a 60 Co source. Treatments of stem cuttings beyond 5 kR and seeds beyond 50 kR were lethal. One mutant each in the cultivars M4, H-165 and H-2304 was obtained from the stem irradiated populations. Another mutant was found in the seed irradiated progeny of H-2304. The mutant of M4 is characterised by light green (chlorina) leaves. The mutant of H-165 shows significantly shorter petiole (22,5 against 35.2 cm) and narrow leaf lobes, while the H-2304 mutant shows speckled leaves, branching and early flowering. The mutant found in the seed irradiated progeny of H-2304 is having yellow tuber flesh indicating the presence of carotene. The mutants may be useful in studies related to basic information as well as in practical breeding. The chlorina mutant in M4 showed slow growth and high HCN content in leaves. Late branching may be a useful trait in the traditionally non-branching clones of cassava to maintain the desirable leaf area index during high leaf fall period. Early flowering could be useful in a recombinant breeding programme. The tuber yield of the short petiole mutant in H-165 increased by 20% - 25% through closer planting. The narrow leaf lobes of this mutant permit better light penetration to lower leaves. (author)

Pilot study of large-scale production of mutant pigs by ENU mutagenesis.

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Hai, Tang; Cao, Chunwei; Shang, Haitao; Guo, Weiwei; Mu, Yanshuang; Yang, Shulin; Zhang, Ying; Zheng, Qiantao; Zhang, Tao; Wang, Xianlong; Liu, Yu; Kong, Qingran; Li, Kui; Wang, Dayu; Qi, Meng; Hong, Qianlong; Zhang, Rui; Wang, Xiupeng; Jia, Qitao; Wang, Xiao; Qin, Guosong; Li, Yongshun; Luo, Ailing; Jin, Weiwu; Yao, Jing; Huang, Jiaojiao; Zhang, Hongyong; Li, Menghua; Xie, Xiangmo; Zheng, Xuejuan; Guo, Kenan; Wang, Qinghua; Zhang, Shibin; Li, Liang; Xie, Fei; Zhang, Yu; Weng, Xiaogang; Yin, Zhi; Hu, Kui; Cong, Yimei; Zheng, Peng; Zou, Hailong; Xin, Leilei; Xia, Jihan; Ruan, Jinxue; Li, Hegang; Zhao, Weiming; Yuan, Jing; Liu, Zizhan; Gu, Weiwang; Li, Ming; Wang, Yong; Wang, Hongmei; Yang, Shiming; Liu, Zhonghua; Wei, Hong; Zhao, Jianguo; Zhou, Qi; Meng, Anming

2017-06-22

N-ethyl-N-nitrosourea (ENU) mutagenesis is a powerful tool to generate mutants on a large scale efficiently, and to discover genes with novel functions at the whole-genome level in Caenorhabditis elegans, flies, zebrafish and mice, but it has never been tried in large model animals. We describe a successful systematic three-generation ENU mutagenesis screening in pigs with the establishment of the Chinese Swine Mutagenesis Consortium. A total of 6,770 G1 and 6,800 G3 pigs were screened, 36 dominant and 91 recessive novel pig families with various phenotypes were established. The causative mutations in 10 mutant families were further mapped. As examples, the mutation of SOX10 (R109W) in pig causes inner ear malfunctions and mimics human Mondini dysplasia, and upregulated expression of FBXO32 is associated with congenital splay legs. This study demonstrates the feasibility of artificial random mutagenesis in pigs and opens an avenue for generating a reservoir of mutants for agricultural production and biomedical research.

Studying disorders of vertebrate iron and heme metabolism using zebrafish.

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van der Vorm, Lisa N; Paw, Barry H

2017-01-01

Iron is a crucial component of heme- and iron-sulfur clusters, involved in vital cellular functions such as oxygen transport, DNA synthesis, and respiration. Both excess and insufficient levels of iron and heme-precursors cause human disease, such as iron-deficiency anemia, hemochromatosis, and porphyrias. Hence, their levels must be tightly regulated, requiring a complex network of transporters and feedback mechanisms. The use of zebrafish to study these pathways and the underlying genetics offers many advantages, among others their optical transparency, ex-vivo development and high genetic and physiological conservations. This chapter first reviews well-established methods, such as large-scale mutagenesis screens that have led to the initial identification of a series of iron and heme transporters and the generation of a variety of mutant lines. Other widely used techniques are based on injection of RNA, including complementary morpholino knockdown and gene overexpression. In addition, we highlight several recently developed approaches, most notably endonuclease-based gene knockouts such as TALENs or the CRISPR/Cas9 system that have been used to study how loss of function can induce human disease phenocopies in zebrafish. Rescue by chemical complementation with iron-based compounds or small molecules can subsequently be used to confirm causality of the genetic defect for the observed phenotype. All together, zebrafish have proven to be - and will continue to serve as an ideal model to advance our understanding of the pathogenesis of human iron and heme-related diseases and to develop novel therapies to treat these conditions. Copyright © 2017 Elsevier Inc. All rights reserved.

Modification of radiation-induced sex-linked recessive lethal mutation frequency by tocopherol

International Nuclear Information System (INIS)

Beckman, C.; Roy, R.M.; Sproule, A.

1982-01-01

The present study evaluates the effect of supplementing culture medium with α-tocopherol acetate on the yield of sex-linked recessive lethal mutants induced by X-irradiation in mature sperm of Drosophila. Although tocopherol treatment of males had no impact on the yield of mutations, a drastic reduction in mutation frequency was observed when irradiated males were mated to females raised and subsequently maintained on tocopherol-enriched diet. (orig./MG)

Molybdenum cofactor deficiency causes translucent integument, male-biased lethality, and flaccid paralysis in the silkworm Bombyx mori.

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Fujii, Tsuguru; Yamamoto, Kimiko; Banno, Yutaka

2016-06-01

Uric acid accumulates in the epidermis of Bombyx mori larvae and renders the larval integument opaque and white. Yamamoto translucent (oya) is a novel spontaneous mutant with a translucent larval integument and unique phenotypic characteristics, such as male-biased lethality and flaccid larval paralysis. Xanthine dehydrogenase (XDH) that requires a molybdenum cofactor (MoCo) for its activity is a key enzyme for uric acid synthesis. It has been observed that injection of a bovine xanthine oxidase, which corresponds functionally to XDH and contains its own MoCo activity, changes the integuments of oya mutants from translucent to opaque and white. This finding suggests that XDH/MoCo activity might be defective in oya mutants. Our linkage analysis identified an association between the oya locus and chromosome 23. Because XDH is not linked to chromosome 23 in B. mori, MoCo appears to be defective in oya mutants. In eukaryotes, MoCo is synthesized by a conserved biosynthesis pathway governed by four loci (MOCS1, MOCS2, MOCS3, and GEPH). Through a candidate gene approach followed by sequence analysis, a 6-bp deletion was detected in an exon of the B. mori molybdenum cofactor synthesis-step 1 gene (BmMOCS1) in the oya strain. Moreover, recombination was not observed between the oya and BmMOCS1 loci. These results indicate that the BmMOCS1 locus is responsible for the oya locus. Finally, we discuss the potential cause of male-biased lethality and flaccid paralysis observed in the oya mutants. Copyright © 2016 Elsevier Ltd. All rights reserved.

Disruption of the Sec24d gene results in early embryonic lethality in the mouse.

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Andrea C Baines

Full Text Available Transport of newly synthesized proteins from the endoplasmic reticulum (ER to the Golgi is mediated by the coat protein complex COPII. The inner coat of COPII is assembled from heterodimers of SEC23 and SEC24. Though mice with mutations in one of the four Sec24 paralogs, Sec24b, exhibit a neural tube closure defect, deficiency in humans or mice has not yet been described for any of the other Sec24 paralogs. We now report characterization of mice with targeted disruption of Sec24d. Early embryonic lethality is observed in mice completely deficient in SEC24D, while a hypomorphic Sec24d allele permits survival to mid-embryogenesis. Mice haploinsufficient for Sec24d exhibit no phenotypic abnormality. A BAC transgene containing Sec24d rescues the embryonic lethality observed in Sec24d-null mice. These results demonstrate an absolute requirement for SEC24D expression in early mammalian development that is not compensated by the other three Sec24 paralogs. The early embryonic lethality resulting from loss of SEC24D in mice contrasts with the previously reported mild skeletal phenotype of SEC24D deficiency in zebrafish and restricted neural tube phenotype of SEC24B deficiency in mice. Taken together, these observations suggest that the multiple Sec24 paralogs have developed distinct functions over the course of vertebrate evolution.

Otolith tethering in the zebrafish otic vesicle requires Otogelin and α-Tectorin.

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Stooke-Vaughan, Georgina A; Obholzer, Nikolaus D; Baxendale, Sarah; Megason, Sean G; Whitfield, Tanya T

2015-03-15

Otoliths are biomineralised structures important for balance and hearing in fish. Their counterparts in the mammalian inner ear, otoconia, have a primarily vestibular function. Otoliths and otoconia form over sensory maculae and are attached to the otolithic membrane, a gelatinous extracellular matrix that provides a physical coupling between the otolith and the underlying sensory epithelium. In this study, we have identified two proteins required for otolith tethering in the zebrafish ear, and propose that there are at least two stages to this process: seeding and maintenance. The initial seeding step, in which otolith precursor particles tether directly to the tips of hair cell kinocilia, fails to occur in the einstein (eis) mutant. The gene disrupted in eis is otogelin (otog); mutations in the human OTOG gene have recently been identified as causative for deafness and vestibular dysfunction (DFNB18B). At later larval stages, maintenance of otolith tethering to the saccular macula is dependent on tectorin alpha (tecta) function, which is disrupted in the rolling stones (rst) mutant. α-Tectorin (Tecta) is a major constituent of the tectorial membrane in the mammalian cochlea. Mutations in the human TECTA gene can cause either dominant (DFNA8/12) or recessive (DFNB21) forms of deafness. Our findings indicate that the composition of extracellular otic membranes is highly conserved between mammals and fish, reinforcing the view that the zebrafish is an excellent model system for the study of deafness and vestibular disease. © 2015. Published by The Company of Biologists Ltd.

nr0b1 (DAX1) mutation in zebrafish causes female-to-male sex reversal through abnormal gonadal proliferation and differentiation.

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Chen, Sijie; Zhang, Hefei; Wang, Fenghua; Zhang, Wei; Peng, Gang

2016-09-15

Sex determinations are diverse in vertebrates. Although many sex-determining genes and pathways are conserved, the mechanistic roles of these genes and pathways in the genetic sex determination are not well understood. DAX1 (encoded by the NR0B1 gene) is a vertebrate specific orphan nuclear receptor that regulates gonadal development and sexual determination. In human, duplication of the NR0B1 gene leads to male-to-female sex reversal. In mice, Nr0b1 shows both pro-testis and anti-testis functions. We generated inheritable nr0b1 mutation in the zebrafish and found the nr0b1 mutation caused homozygous mutants to develop as fertile males due to female-to-male sex reversal. The nr0b1 mutation did not increase Caspase-3 labeling nor tp53 expression in the developing gonads. Introduction of a tp53 mutation into the nr0b1 mutant did not rescue the sex-reversal phenotype. Further examination revealed reduction in cell proliferation and abnormal somatic cell differentiation in the nr0b1 mutant gonads at the undifferentiated and bi-potential ovary stages. Together, our results suggest nr0b1 regulates somatic cell differentiation and cell proliferation to ensure normal sex development in the zebrafish. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Cobalt-induced genotoxicity in male zebrafish (Danio rerio), with implications for reproduction and expression of DNA repair genes

Energy Technology Data Exchange (ETDEWEB)

Reinardy, Helena C.; Syrett, James R. [School of Biomedical and Biological Sciences, University of Plymouth (United Kingdom); Jeffree, Ross A. [Faculty of Science, University of Technology, Sydney, PO Box 123, Broadway, NSW 2007 (Australia); Henry, Theodore B., E-mail: ted.henry@plymouth.ac.uk [School of Biomedical and Biological Sciences, University of Plymouth (United Kingdom); Center for Environmental Biotechnology, University of Tennessee, Knoxville, TN 37996 (United States); Department of Forestry, Wildlife and Fisheries, University of Tennessee, Knoxville, TN 37996. USA (United States); Jha, Awadhesh N. [School of Biomedical and Biological Sciences, The University of Plymouth (United Kingdom)

2013-01-15

Although cobalt (Co) is an environmental contaminant of surface waters in both radioactive (e.g. {sup 60}Co) and non-radioactive forms, there is relatively little information about Co toxicity in fishes. The objective of this study was to investigate acute and chronic toxicity of Co in zebrafish, with emphasis on male genotoxicity and implications for reproductive success. The lethal concentration for 50% mortality (LC{sub 50}) in larval zebrafish exposed (96 h) to 0-50 mg l{sup -1} Co was 35.3 {+-} 1.1 (95% C.I.) mg l{sup -1} Co. Adult zebrafish were exposed (13 d) to sub-lethal (0-25 mg l{sup -1}) Co and allowed to spawn every 4 d and embryos were collected. After 12-d exposure, fertilisation rate was reduced (6% total eggs fertilised, 25 mg l{sup -1}) and embryo survival to hatching decreased (60% fertilised eggs survived, 25 mg l{sup -1}). A concentration-dependent increase in DNA strand breaks was detected in sperm from males exposed (13 d) to Co, and DNA damage in sperm returned to control levels after males recovered for 6 d in clean water. Induction of DNA repair genes (rad51, xrcc5, and xrcc6) in testes was complex and not directly related to Co concentration, although there was significant induction in fish exposed to 15 and 25 mg l{sup -1} Co relative to controls. Induction of 4.0 {+-} 0.9, 2.5 {+-} 0.7, and 3.1 {+-} 0.7-fold change (mean {+-} S.E.M. for rad51, xrcc5, and xrcc6, respectively) was observed in testes at the highest Co concentration (25 mg l{sup -1}). Expression of these genes was not altered in offspring (larvae) spawned after 12-d exposure. Chronic exposure to Co resulted in DNA damage in sperm, induction of DNA repair genes in testes, and indications of reduced reproductive success.

Standardized orthotopic xenografts in zebrafish reveal glioma cell-line-specific characteristics and tumor cell heterogeneity

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Alessandra M. Welker

2016-02-01

Full Text Available Glioblastoma (GBM is a deadly brain cancer, for which few effective drug treatments are available. Several studies have used zebrafish models to study GBM, but a standardized approach to modeling GBM in zebrafish was lacking to date, preventing comparison of data across studies. Here, we describe a new, standardized orthotopic xenotransplant model of GBM in zebrafish. Dose-response survival assays were used to define the optimal number of cells for tumor formation. Techniques to measure tumor burden and cell spread within the brain over real time were optimized using mouse neural stem cells as control transplants. Applying this standardized approach, we transplanted two patient-derived GBM cell lines, serum-grown adherent cells and neurospheres, into the midbrain region of embryonic zebrafish and analyzed transplanted larvae over time. Progressive brain tumor growth and premature larval death were observed using both cell lines; however, fewer transplanted neurosphere cells were needed for tumor growth and lethality. Tumors were heterogeneous, containing both cells expressing stem cell markers and cells expressing markers of differentiation. A small proportion of transplanted neurosphere cells expressed glial fibrillary acidic protein (GFAP or vimentin, markers of more differentiated cells, but this number increased significantly during tumor growth, indicating that these cells undergo differentiation in vivo. By contrast, most serum-grown adherent cells expressed GFAP and vimentin at the earliest times examined post-transplant. Both cell types produced brain tumors that contained Sox2+ cells, indicative of tumor stem cells. Transplanted larvae were treated with currently used GBM therapeutics, temozolomide or bortezomib, and this resulted in a reduction in tumor volume in vivo and an increase in survival. The standardized model reported here facilitates robust and reproducible analysis of glioblastoma tumor cells in real time and provides a

A metabolomic study of fipronil for the anxiety-like behavior in zebrafish larvae at environmentally relevant levels

International Nuclear Information System (INIS)

Wang, Cui; Qian, Yi; Zhang, Xiaofeng; Chen, Fang; Zhang, Quan; Li, Zhuoyu; Zhao, Meirong

2016-01-01

Field residue of fipronil can interfere with the physiological characters of the domesticated fish; thus, lethal dose test and the general biomarker cannot delineate the low-level situation. Manipulating by video track, we observed an anxiety-like behavior including high speed and abnormal photoperiod accommodation after exposure to fipronil at environmental typical dose in zebrafish larvae. Examining the unbiased metabolomic profiles, we found perturbation in several metabolic pathways, including the increased contents of fatty acids and glycerol and the decreased levels of the glycine, serine, and branched amino acid. We presumed that observed enhanced fatty acid utility was in response to increase energy demands caused by anxiety like behavior. Additionally, the body burden of neurotransmitter such as glycine and L-glutamate may concurrently stimulate the swimming behavior. The insight of this study showed that integral perturbation such as metabolism helps us to further understand the risk to aquatic fish at the environmentally relevant levels. - Highlights: • Fipronil increased the swimming speed at 10 μg/L to zebrafish larvae. • Accommodation to light–dark photoperiod switch was disturbed by fipronil. • Metabolomics indicated an increase energy availability for anxiety-like behavior. • Anxiety-like behavior induced by fipronil may attribute to neurotransmitter changes. - Zebrafish larvae exposed to environmentally relevant concentrations of fipronil display anxiety like behavior that may attribute to observed changes in energy utilization and neurotransmitter disturbances.

Comparative metal oxide nanoparticle toxicity using embryonic zebrafish

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Leah C. Wehmas

2015-01-01

Full Text Available Engineered metal oxide nanoparticles (MO NPs are finding increasing utility in the medical field as anticancer agents. Before validation of in vivo anticancer efficacy can occur, a better understanding of whole-animal toxicity is required. We compared the toxicity of seven widely used semiconductor MO NPs made from zinc oxide (ZnO, titanium dioxide, cerium dioxide and tin dioxide prepared in pure water and in synthetic seawater using a five-day embryonic zebrafish assay. We hypothesized that the toxicity of these engineered MO NPs would depend on physicochemical properties. Significant agglomeration of MO NPs in aqueous solutions is common making it challenging to associate NP characteristics such as size and charge with toxicity. However, data from our agglomerated MO NPs suggests that the elemental composition and dissolution potential are major drivers of toxicity. Only ZnO caused significant adverse effects of all MO particles tested, and only when prepared in pure water (point estimate median lethal concentration = 3.5–9.1 mg/L. This toxicity was life stage dependent. The 24 h toxicity increased greatly (∼22.7 fold when zebrafish exposures started at the larval life stage compared to the 24 h toxicity following embryonic exposure. Investigation into whether dissolution could account for ZnO toxicity revealed high levels of zinc ion (40–89% of total sample were generated. Exposure to zinc ion equivalents revealed dissolved Zn2+ may be a major contributor to ZnO toxicity.

Bypass of lethality with mosaic mice generated by Cre-loxP-mediated recombination.

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Betz, U A; Vosshenrich, C A; Rajewsky, K; Müller, W

1996-10-01

The analysis of gene function based on the generation of mutant mice by homologous recombination in embryonic stem cells is limited if gene disruption results in embryonic lethality. Mosaic mice, which contain a certain proportion of mutant cells in all organs, allow lethality to be circumvented and the potential of mutant cells to contribute to different cell lineages to be analyzed. To generate mosaic animals, we used the bacteriophage P1-derived Cre-loxP recombination system, which allows gene alteration by Cre-mediated deletion of loxP-flanked gene segments. We generated nestin-cre transgenic mouse lines, which expressed the Cre recombinase under the control of the rat nestin promoter and its second intron enhancer. In crosses to animals carrying a loxP-flanked target gene, partial deletion of the loxP-flanked allele occurred before day 10.5 post coitum and was detectable in all adult organs examined, including germ-line cells. Using this approach, we generated mosaic mice containing cells deficient in the gamma-chain of the interleukin-2 receptor (IL-2R gamma); in these animals, the IL-2R gamma-deficient cells were underrepresented in the thymus and spleen. Because mice deficient in DNA polymerase beta die perinatally, we studied the effects of DNA polymerase beta deficiency in mosaic animals. We found that some of the mosaic polymerase beta-deficient animals were viable, but were often reduced in size and weight. The fraction of DNA polymerase beta-deficient cells in mosaic embryos decreased during embryonic development, presumably because wild-type cells had a competitive advantage. The nestin-cre transgenic mice can be used to generate mosaic animals in which target genes are mutated by Cre-mediated recombination of loxP-flanked target genes. By using mosaic animals, embryonic lethality can be bypassed and cell lineages for whose development a given target gene is critical can be identified. In the case of DNA polymerase beta, deficient cells are already

Highly variable penetrance of abnormal phenotypes in embryonic lethal knockout mice [version 2; referees: 1 approved, 2 approved with reservations

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Robert Wilson

2017-02-01

Full Text Available Background: Identifying genes that are essential for mouse embryonic development and survival through term is a powerful and unbiased way to discover possible genetic determinants of human developmental disorders. Characterising the changes in mouse embryos that result from ablation of lethal genes is a necessary first step towards uncovering their role in normal embryonic development and establishing any correlates amongst human congenital abnormalities. Methods: Here we present results gathered to date in the Deciphering the Mechanisms of Developmental Disorders (DMDD programme, cataloguing the morphological defects identified from comprehensive imaging of 220 homozygous mutant and 114 wild type embryos from 42 lethal and subviable lines, analysed at E14.5. Results: Virtually all mutant embryos show multiple abnormal phenotypes and amongst the 42 lines these affect most organ systems. Within each mutant line, the phenotypes of individual embryos form distinct but overlapping sets. Subcutaneous edema, malformations of the heart or great vessels, abnormalities in forebrain morphology and the musculature of the eyes are all prevalent phenotypes, as is loss or abnormal size of the hypoglossal nerve. Conclusions: Overall, the most striking finding is that no matter how profound the malformation, each phenotype shows highly variable penetrance within a mutant line. These findings have challenging implications for efforts to identify human disease correlates.

Improvement of heavy metal stress and toxicity assays by coupling a transgenic reporter in a mutant nematode strain

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Chu, K.-W. [Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China); Chan, Shirley K.W. [Atmospheric, Marine and Coastal Environment Program, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China); Chow, King L. [Department of Biology, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China) and Atmospheric, Marine and Coastal Environment Program, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong (China)]. E-mail: bokchow@ust.hk

2005-09-30

Previous studies have demonstrated that wild type Caenorhabditis elegans displays high sensitivity to heavy metals in a lethality test at a level comparable to that of other bioindicator organisms. Taking advantage of the genetics of this model organism, we have tested a number of mutant strains for enhanced sensitivity in heavy metal induced lethality and stress response. These mutants are defective in genes controlling dauer formation, longevity or response to reactive oxygen species (ROS). Among the tested mutants, a double mutant daf-16 unc-75 strain was identified to have superior sensitivity. It has a 6-, 3- and 2-fold increase in sensitivity to cadmium, copper and zinc, respectively, as compared with that of wild type animals. When a fluorescent reporter transgene was coupled with this double mutant for stress detection, a 10-fold enhancement of sensitivity to cadmium over the wild type strain was observed. These transgenic animals, superior to most of the model organisms currently used in bioassays for environmental pollutants, offer a fast and economic approach to reveal the bioavailability of toxic substance in field samples. This study also demonstrates that combination of genetic mutations and transgenesis is a viable approach to identify sensitive indicator animals for environmental monitoring.

Improvement of heavy metal stress and toxicity assays by coupling a transgenic reporter in a mutant nematode strain

International Nuclear Information System (INIS)

Chu, K.-W.; Chan, Shirley K.W.; Chow, King L.

2005-01-01

Previous studies have demonstrated that wild type Caenorhabditis elegans displays high sensitivity to heavy metals in a lethality test at a level comparable to that of other bioindicator organisms. Taking advantage of the genetics of this model organism, we have tested a number of mutant strains for enhanced sensitivity in heavy metal induced lethality and stress response. These mutants are defective in genes controlling dauer formation, longevity or response to reactive oxygen species (ROS). Among the tested mutants, a double mutant daf-16 unc-75 strain was identified to have superior sensitivity. It has a 6-, 3- and 2-fold increase in sensitivity to cadmium, copper and zinc, respectively, as compared with that of wild type animals. When a fluorescent reporter transgene was coupled with this double mutant for stress detection, a 10-fold enhancement of sensitivity to cadmium over the wild type strain was observed. These transgenic animals, superior to most of the model organisms currently used in bioassays for environmental pollutants, offer a fast and economic approach to reveal the bioavailability of toxic substance in field samples. This study also demonstrates that combination of genetic mutations and transgenesis is a viable approach to identify sensitive indicator animals for environmental monitoring

Distinct and Cooperative Roles of amh and dmrt1 in Self-Renewal and Differentiation of Male Germ Cells in Zebrafish.

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Lin, Qiaohong; Mei, Jie; Li, Zhi; Zhang, Xuemei; Zhou, Li; Gui, Jian-Fang

2017-11-01

Spermatogenesis is a fundamental process in male reproductive biology and depends on precise balance between self-renewal and differentiation of male germ cells. However, the regulative factors for controlling the balance are poorly understood. In this study, we examined the roles of amh and dmrt1 in male germ cell development by generating their mutants with Crispr/Cas9 technology in zebrafish. Amh mutant zebrafish displayed a female-biased sex ratio, and both male and female amh mutants developed hypertrophic gonads due to uncontrolled proliferation and impaired differentiation of germ cells. A large number of proliferating spermatogonium-like cells were observed within testicular lobules of the amh -mutated testes, and they were demonstrated to be both Vasa- and PH3-positive. Moreover, the average number of Sycp3- and Vasa-positive cells in the amh mutants was significantly lower than in wild-type testes, suggesting a severely impaired differentiation of male germ cells. Conversely, all the dmrt1 -mutated testes displayed severe testicular developmental defects and gradual loss of all Vasa-positive germ cells by inhibiting their self-renewal and inducing apoptosis. In addition, several germ cell and Sertoli cell marker genes were significantly downregulated, whereas a prominent increase of Insl3-positive Leydig cells was revealed by immunohistochemical analysis in the disorganized dmrt1 -mutated testes. Our data suggest that amh might act as a guardian to control the balance between proliferation and differentiation of male germ cells, whereas dmrt1 might be required for the maintenance, self-renewal, and differentiation of male germ cells. Significantly, this study unravels novel functions of amh gene in fish. Copyright © 2017 by the Genetics Society of America.

Loss of the homologous recombination gene rad51 leads to Fanconi anemia-like symptoms in zebrafish.

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Botthof, Jan Gregor; Bielczyk-Maczyńska, Ewa; Ferreira, Lauren; Cvejic, Ana

2017-05-30

RAD51 is an indispensable homologous recombination protein, necessary for strand invasion and crossing over. It has recently been designated as a Fanconi anemia (FA) gene, following the discovery of two patients carrying dominant-negative mutations. FA is a hereditary DNA-repair disorder characterized by various congenital abnormalities, progressive bone marrow failure, and cancer predisposition. In this report, we describe a viable vertebrate model of RAD51 loss. Zebrafish rad51 loss-of-function mutants developed key features of FA, including hypocellular kidney marrow, sensitivity to cross-linking agents, and decreased size. We show that some of these symptoms stem from both decreased proliferation and increased apoptosis of embryonic hematopoietic stem and progenitor cells. Comutation of p53 was able to rescue the hematopoietic defects seen in the single mutants, but led to tumor development. We further demonstrate that prolonged inflammatory stress can exacerbate the hematological impairment, leading to an additional decrease in kidney marrow cell numbers. These findings strengthen the assignment of RAD51 as a Fanconi gene and provide more evidence for the notion that aberrant p53 signaling during embryogenesis leads to the hematological defects seen later in life in FA. Further research on this zebrafish FA model will lead to a deeper understanding of the molecular basis of bone marrow failure in FA and the cellular role of RAD51.

Free radical scavenging and the expression of potentially lethal damage in X-irradiated repair-deficient Escherichia coli

International Nuclear Information System (INIS)

Billen, D.

1987-01-01

When cells are exposed to ionizing radiation, they suffer lethal damage (LD), potentially lethal damage (PLD), and sublethal damage (SLD). All three forms of damage may be caused by direct or indirect radiation action or by the interaction of indirect radiation products with direct DNA damage. In this report I examine the expression of LD and PLD caused by the indirect action of X rays in isogenic, repair-deficient Escherichia coli. The radiosensitivity of a recA mutant, deficient both in pre- and post replication recombination repair and SOS induction (inducible error-prone repair), was compared to that of a recB mutant which is recombination deficient but SOS proficient and to a previously studied DNA polymerase 1-deficient mutant (polA) which lacks the excision repair pathway. Indirect damage by water radicals (primarily OH radicals) was circumvented by the presence of 2 M glycerol during irradiation. Indirect X-ray damage by water radicals accounts for at least 85% of the PLD found in exposed repair-deficient cells. The DNA polymerase 1-deficient mutant is most sensitive to indirect damage with the order of sensitivity polA1 greater than recB greater than or equal to recA greater than wild type. For the direct effects of X rays the order of sensitivity is recA greater than recB greater than polA1 greater than wild type. The significance of the various repair pathways in mitigating PLD by direct and indirect damage is discussed

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

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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.

Characterization of conditionally expressed mutants affecting age-specific Drosophila melanogaster : Lethal conditions and temperature-sensitive periods

NARCIS (Netherlands)

Vermeulen, CJ; Bijlsma, R

The specific genetic basis of inbreeding depression is poorly understood. To address this question, two conditionally expressed lethal effects that were found to cause line-specific life span reductions in two separate inbred lines of Drosophila melanogaster. were characterized phenotypically and

Inhibition of vascular endothelial growth factor signaling facilitates liver repair from acute ethanol-induced injury in zebrafish

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Changwen Zhang

2016-11-01

Full Text Available Alcoholic liver disease (ALD results from alcohol overconsumption and is among the leading causes of liver-related morbidity and mortality worldwide. Elevated expression of vascular endothelial growth factor (VEGF and its receptors has been observed in ALD, but how it contributes to ALD pathophysiology is unclear. Here, we investigated the impact of VEGF signaling inhibition on an established zebrafish model of acute alcoholic liver injury. Kdrl activity was blocked by chemical inhibitor treatment or by genetic mutation. Exposing 4-day-old zebrafish larvae to 2% ethanol for 24 h induced hepatic steatosis, angiogenesis and fibrogenesis. The liver started self-repair once ethanol was removed. Although inhibiting Kdrl did not block the initial activation of hepatic stellate cells during ethanol treatment, it suppressed their proliferation, extracellular matrix protein deposition and fibrogenic gene expression after ethanol exposure, thus enhancing the liver repair. It also ameliorated hepatic steatosis and attenuated hepatic angiogenesis that accelerated after the ethanol treatment. qPCR showed that hepatic stellate cells are the first liver cell type to increase the expression of VEGF ligand and receptor genes in response to ethanol exposure. Both hepatic stellate cells and endothelial cells, but not hepatic parenchymal cells, expressed kdrl upon ethanol exposure and were likely the direct targets of Kdrl inhibition. Ethanol-induced steatosis and fibrogenesis still occurred in cloche mutants that have hepatic stellate cells but lack hepatic endothelial cells, and Kdrl inhibition suppressed both phenotypes in the mutants. These results suggest that VEGF signaling mediates interactions between activated hepatic stellate cells and hepatocytes that lead to steatosis. Our study demonstrates the involvement of VEGF signaling in regulating sustained liver injuries after acute alcohol exposure. It also provides a proof of principle of using the

Time-response characteristic and potential biomarker identification of heavy metal induced toxicity in zebrafish.

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Yin, Jian; Wang, Ai-Ping; Li, Wan-Fang; Shi, Rui; Jin, Hong-Tao; Wei, Jin-Feng

2018-01-01

The present work aims to explore the time-response (from 24 h to 96 h) characteristic and identify early potential sensitive biomarkers of copper (Cu) (as copper chloride dihydrate), cadmium (Cd) (as cadmium acetate), lead (Pb) (as lead nitrate) and chromium (Cr) (as potassium dichromate) exposure in adult zebrafish, focusing on reactive oxygen species (ROS), SOD activity, lipid peroxidation and gene expression related to oxidative stress and inflammatory response. Furthermore, the survival rate decreased apparently by a concentration-dependent manner after Cu, Cr, Cd and Pb exposure, and we selected non-lethal concentrations 0.05 mg/L for Cu, 15 mg/L for Cr, 3 mg/L for Cd and 93.75μg/L for Pb to test the effect on the following biological indicators. Under non-lethal concentration, the four heavy metals have no apparent histological change in adult zebrafish gills. Similar trends in ROS production, MDA level and SOD activity were up-regulated by the four heavy metals, while MDA level responded more sensitive to Pb by time-dependent manner than the other three heavy metals. In addition, mRNA levels related to antioxidant system (SOD1, SOD2 and Nrf2) were up-regulated by non-lethal concentration Cu, Cr, Cd and Pb exposure. MDA level and SOD1 gene have a more delayed response to heavy metals. Genes related to immunotoxicity were increased significantly after heavy metals exposure at non-lethal concentrations. TNF-α and IL-1β gene have similar sensibility to the four heavy metals, while IL-8 gene was more responsive to Cr, Cd and Pb exposure at 48 h groups and IFN-γ gene showed more sensitivity to Cu at 48 h groups than the other heavy metals. In conclusion, the present works have suggested that the IFN-γ gene may applied as early sensitive biomarker to identify Cu-induced toxicity, while MDA content and IL-8 gene may use as early sensitive biomarkers for evaluating the risk of Pb exposure. Moreover, IL-8 and IFN-γ gene were more responsive to heavy

Zebrafish and clean water technology: assessing soil bioretention as a protective treatment for toxic urban runoff.

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McIntyre, J K; Davis, J W; Incardona, J P; Stark, J D; Anulacion, B F; Scholz, N L

2014-12-01

Urban stormwater contains a complex mixture of contaminants that can be acutely toxic to aquatic biota. Green stormwater infrastructure (GSI) is a set of evolving technologies intended to reduce impacts on natural systems by slowing and filtering runoff. The extent to which GSI methods work as intended is usually assessed in terms of water quantity (hydrology) and quality (chemistry). Biological indicators of GSI effectiveness have received less attention, despite an overarching goal of protecting the health of aquatic species. Here we use the zebrafish (Danio rerio) experimental model to evaluate bioinfiltration as a relatively inexpensive technology for treating runoff from an urban highway with dense motor vehicle traffic. Zebrafish embryos exposed to untreated runoff (48-96h; six storm events) displayed an array of developmental abnormalities, including delayed hatching, reduced growth, pericardial edema, microphthalmia (small eyes), and reduced swim bladder inflation. Three of the six storms were acutely lethal, and sublethal toxicity was evident across all storms, even when stormwater was diluted by as much as 95% in clean water. As anticipated from exposure to cardiotoxic polycyclic aromatic hydrocarbons (PAHs), untreated runoff also caused heart failure, as indicated by circulatory stasis, pericardial edema, and looping defects. Bioretention treatment dramatically improved stormwater quality and reversed nearly all forms of developmental toxicity. The zebrafish model therefore provides a versatile experimental platform for rapidly assessing GSI effectiveness. Copyright © 2014 Elsevier B.V. All rights reserved.

Identification and characterization of zebrafish thrombocytes.

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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 lethal and sub-lethal concentrations of tobacco (Nicotiana ...

African Journals Online (AJOL)

Lethal and sub-lethal bioassays on Clarias gariepinus were conducted to evaluate the toxicity of tobacco (Nicotiana tobaccum) leaf dust on weight gain and haematological indices of Clarias gariepinus (mean weight 10.5±0.70g) in glass aquaria with aeration system. The concentrations used during the lethal exposure are: ...

Anthrax Toxin Receptor 2–Dependent Lethal Toxin Killing In Vivo

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Scobie, Heather M; Wigelsworth, Darran J; Marlett, John M; Thomas, Diane; Rainey, G. Jonah A; Lacy, D. Borden; Manchester, Marianne; Collier, R. John; Young, John A. T

2006-01-01

Anthrax toxin receptors 1 and 2 (ANTXR1 and ANTXR2) have a related integrin-like inserted (I) domain which interacts with a metal cation that is coordinated by residue D683 of the protective antigen (PA) subunit of anthrax toxin. The receptor-bound metal ion and PA residue D683 are critical for ANTXR1-PA binding. Since PA can bind to ANTXR2 with reduced affinity in the absence of metal ions, we reasoned that D683 mutant forms of PA might specifically interact with ANTXR2. We show here that this is the case. The differential ability of ANTXR1 and ANTXR2 to bind D683 mutant PA proteins was mapped to nonconserved receptor residues at the binding interface with PA domain 2. Moreover, a D683K mutant form of PA that bound specifically to human and rat ANTXR2 mediated killing of rats by anthrax lethal toxin, providing strong evidence for the physiological importance of ANTXR2 in anthrax disease pathogenesis. PMID:17054395

Candidate gene identification of ovulation-inducing genes by RNA sequencing with an in vivo assay in zebrafish.

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Wanlada Klangnurak

Full Text Available We previously reported the microarray-based selection of three ovulation-related genes in zebrafish. We used a different selection method in this study, RNA sequencing analysis. An additional eight up-regulated candidates were found as specifically up-regulated genes in ovulation-induced samples. Changes in gene expression were confirmed by qPCR analysis. Furthermore, up-regulation prior to ovulation during natural spawning was verified in samples from natural pairing. Gene knock-out zebrafish strains of one of the candidates, the starmaker gene (stm, were established by CRISPR genome editing techniques. Unexpectedly, homozygous mutants were fertile and could spawn eggs. However, a high percentage of unfertilized eggs and abnormal embryos were produced from these homozygous females. The results suggest that the stm gene is necessary for fertilization. In this study, we selected additional ovulation-inducing candidate genes, and a novel function of the stm gene was investigated.

Comparative ecotoxicity of potential biofuels to water flea (Daphnia magna), zebrafish (Danio rerio) and Chinese hamster (Cricetulus griseus) V79 cells.

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Heger, Sebastian; Du, Miaomiao; Bauer, Kevin; Schäffer, Andreas; Hollert, Henner

2018-08-01

The ecotoxicity of two biofuel candidates (1‑octanol and 2‑butanone) was investigated by an integrative test strategy using three bioassays: the acute immobilisation test with water flea (D. magna), the fish embryo acute toxicity test with zebrafish (Danio rerio) and the in vitro micronucleus assay with Chinese hamster (Cricetulus griseus) V79 cells. The median effective concentration (EC 50 ) values were 14.9±0.66mgL -1 for 1‑octanol, and 2152.1±44.6mgL -1 for 2‑butanone in the D. magna test. Both 1‑octanol and 2‑butanone caused teratogenic and lethal effects on zebrafish embryos, while exposure to 1‑octanol significantly induced these effects at concentrations ≥2.0mgL -1 . These results indicate that 1‑octanol exert much higher ecotoxicity than 2‑butanone to D. magna and zebrafish embryos. Moreover, both 1‑octanol and 2‑butanone did not cause significant genotoxic effects, while their metabolites significantly induced micronuclei in V79 cells. The present study proposed an integrative test approach to evaluate the potential ecotoxicity of biofuels using simple, quick and inexpensive bioassays. Copyright © 2018 Elsevier B.V. All rights reserved.

Swimming Effects on Developing Zebrafish

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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...

New tides: using zebrafish to study renal regeneration.

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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.

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

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

2014-11-01

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

Correlating gene expression with deformities caused by aryl hydrocarbon receptor agonists in zebrafish (Danio rerio)

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Bugiak, B.; Weber, L. [Saskatchewan Univ., Saskatoon, SK (Canada)

2009-07-01

Exposure to aryl hydrocarbon receptor (AhR) agonists in fish causes lethal disturbances in fish development, but the effects of acute AhR agonist exposure on the cardiovascular system and deformities remain unclear. This study addressed this issue by performing a series of experiments on zebrafish (Danio rerio). The authors hypothesized that genes needed for cardiovascular regulation (PTGS) would exhibit a stronger link to deformities than detoxification enzymes (CYPs). Zebrafish eggs were exposed aqueously until 4 days post-fertilization (dpf) to the AhR agonists benzo(a)pyrene (BaP) or 2,3,7,8-tetrachlorodibenzop-dioxin (TCDD) alone and in combination with the putative AhR antagonists resveratrol or alpha-naphthoflavone (ANF). Gene expression was measured using real-time, reverse transcriptase PCR in zebrafish at 5 and 10 dpf. Although the mortalities did not differ considerably among groups at 10 dpf, the deformities increased significantly after BaP-ANF at 5 dpf and after BaP at 10 dpf, but not after TCDD treatment. CYP and PTGS isozymes exhibited small, but statistically significant changes at 5 dpf. By 10 dpf, the expression returned to control values. In general, CYP1A and PTGS-1 expression at 5 dpf were positively correlated with deformities, while all other genes were negatively correlated with deformities. It was concluded that changes in CYP1A, CYP1C2, and PTGS-1 gene expression at 5 dpf are associated with developmental deformities, but additional work is needed to determine which has the most important mechanistic link.

Linkage analysis for the gametic lethal gene of a rice variety 'Koshihikari' and the semi-dwarfing gene induced in 'Koshihikari'

International Nuclear Information System (INIS)

Tomita, M.; Tanisaka, T.; Okumoto, Y.; Yamagata, H.

1990-01-01

Full text: 'Koshihikari', a Japanese tall variety, is now most widely cultivated in Japan because of its good quality and taste, but is extremely poor in lodging resistance. In order to create a semi-dwarf 'Koshihikari', large scale mutation breeding was carried out at Hokuriku Agricultural Experiment Station, resulting in the production of an excellent semi-dwarf mutant strain 'Hokuriku 100'. It has extensively been used as cross parent. Genetic analyses revealed that the semi-dwarfness of 'Hokuriku 100' is controlled by two mutant genes, a recessive semi-dwarfness gene sd(t) and a non-gametic lethal gene lt m mutated from the genetic lethal gene of 'Koshihikari' lt, which would cause abortion of both male and female gametes when it occurs together with sd(t). Further analyses led to conclude that It is located on chromosome 9, sd(t) on chromosome 10. (author)

tortuga refines Notch pathway gene expression in the zebrafish presomitic mesoderm at the post-transcriptional level.

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Dill, Kariena K; Amacher, Sharon L

2005-11-15

We have identified the zebrafish tortuga (tor) gene by an ENU-induced mutation that disrupts the presomitic mesoderm (PSM) expression of Notch pathway genes. In tor mutants, Notch pathway gene expression persists in regions of the PSM where expression is normally off in wild type embryos. The expression of hairy/Enhancer of split-related 1 (her1) is affected first, followed by the delta genes deltaC and deltaD, and finally, by another hairy/Enhancer of split-related gene, her7. In situ hybridization with intron-specific probes for her1 and deltaC indicates that transcriptional bursts of expression are normal in tor mutants, suggesting that tor normally functions to refine her1 and deltaC message levels downstream of transcription. Despite the striking defects in Notch pathway gene expression, somite boundaries form normally in tor mutant embryos, although somitic mesoderm defects are apparent later, when cells mature to form muscle fibers. Thus, while the function of Notch pathway genes is required for proper somite formation, the tor mutant phenotype suggests that precise oscillations of Notch pathway transcripts are not essential for establishing segmental pattern in the presomitic mesoderm.

Object recognition memory in zebrafish.

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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.

The importance of Zebrafish in biomedical research.

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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.

Zebrafish neurobehavioral phenomics for aquatic neuropharmacology and toxicology research.

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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.

The aspartate-semialdehyde dehydrogenase of Edwardsiella ictaluri and its use as balanced-lethal system in fish vaccinology.

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Javier Santander

Full Text Available asdA mutants of gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP, which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd(+ plasmid into a ΔasdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd(+ plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ΔasdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd(+ expression vectors with different origins of replication were transformed into E. ictaluri ΔasdA01. Asd(+ vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd(+ plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry.

The aspartate-semialdehyde dehydrogenase of Edwardsiella ictaluri and its use as balanced-lethal system in fish vaccinology.

Science.gov (United States)

Santander, Javier; Xin, Wei; Yang, Zhao; Curtiss, Roy

2010-12-29

asdA mutants of gram-negative bacteria have an obligate requirement for diaminopimelic acid (DAP), which is an essential constituent of the peptidoglycan layer of the cell wall of these organisms. In environments deprived of DAP, i.e., animal tissues, they will undergo lysis. Deletion of the asdA gene has previously been exploited to develop antibiotic-sensitive strains of live attenuated recombinant bacterial vaccines. Introduction of an Asd(+) plasmid into a ΔasdA mutant makes the bacterial strain plasmid-dependent. This dependence on the Asd(+) plasmid vector creates a balanced-lethal complementation between the bacterial strain and the recombinant plasmid. E. ictaluri is an enteric gram-negative fish pathogen that causes enteric septicemia in catfish. Because E. ictaluri is a nasal/oral invasive intracellular pathogen, this bacterium is a candidate to develop a bath/oral live recombinant attenuated Edwardsiella vaccine (RAEV) for the catfish aquaculture industry. As a first step to develop an antibiotic-sensitive RAEV strain, we characterized and deleted the E. ictaluri asdA gene. E. ictaluri ΔasdA01 mutants exhibit an absolute requirement for DAP to grow. The asdA gene of E. ictaluri was complemented by the asdA gene from Salmonella. Several Asd(+) expression vectors with different origins of replication were transformed into E. ictaluri ΔasdA01. Asd(+) vectors were compatible with the pEI1 and pEI2 E. ictaluri native plasmids. The balanced-lethal system was satisfactorily evaluated in vivo. Recombinant GFP, PspA, and LcrV proteins were synthesized by E. ictaluri ΔasdA01 harboring Asd(+) plasmids. Here we constructed a balanced-lethal system, which is the first step to develop an antibiotic-sensitive RAEV for the aquaculture industry.

Seedling lethality in Nicotiana plumbaginifolia conferred by Ds transposable element insertion into a plant-specific gene.

Science.gov (United States)

Majira, Amel; Domin, Monique; Grandjean, Olivier; Gofron, Krystyna; Houba-Hérin, Nicole

2002-10-01

A seedling lethal mutant of Nicotiana plumbaginifolia (sdl-1) was isolated by transposon tagging using a maize Dissociation (Ds) element. The insertion mutation was produced by direct co-transformation of protoplasts with two plasmids: one containing Ds and a second with an Ac transposase gene. sdl-1 seedlings exhibit several phenotypes: swollen organs, short hypocotyls in light and dark conditions, and enlarged and multinucleated cells, that altogether suggest cell growth defects. Mutant cells are able to proliferate under in vitro culture conditions. Genomic DNA sequences bordering the transposon were used to recover cDNA from the normal allele. Complementation of the mutant phenotype with the cDNA confirmed that the transposon had caused the mutation. The Ds element was inserted into the first exon of the open reading frame and the homozygous mutant lacked detectable transcript. Phenocopies of the mutant were obtained by an antisense approach. SDL-1 encodes a novel protein found in several plant genomes but apparently missingfrom animal and fungal genomes; the protein is highly conserved and has a potential plastid targeting motif.

Manifestation of x-radiation induced sex-linked recessive lethal mutation impairing the development of imaginal disks and gonads in Drosophila Melanogaster

International Nuclear Information System (INIS)

Abeleva, Eh.A.; Ivanov, A.I.

1982-01-01

A study was made of Drosophila melanogaster mutations impairing the development of imaginal disks. The state of gonads in these mutants was not studied. Using X-radiation a lethal mutation in X chromosome was obtained that induced degeneration of imaginal disks at the 3d stage of larva development. The gonads of the mutants at this stage of development vary in size. The transplantation tests showed that the mutation manifests itself in both the imaginal disks and the gonads

Episodic-like memory in zebrafish.

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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.

Sensitivity of Escherichia coli acrA Mutants to Psoralen plus Near-Ultraviolet Radiation

DEFF Research Database (Denmark)

Hansen, M. Trier

1982-01-01

The sensitivity to psoralen plus near-ultraviolet radiation (PUVA) was compared in a pair of E. coli strains differing at the acrA locus. Survival was determined for both bacteria and phage λ. AcrA mutant cells were 40 times more sensitive than wild type to the lethal effect of PUVA. Free λ phage...

Zebrafish swimming in the flow: a particle image velocimetry study

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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.

Lethality Index 2008-2014: Less shootings, same lethality, more opacity

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Carlos Silva Forné

2017-11-01

Full Text Available This article evaluates the use of lethal force by Mexican federal security forces during shootings with presumed members of organized crime from 2008-2014. The authors use official data and press reports on deaths and wounded in shootings to construct indicators such as the number of dead civilians over the number of dead officials from the federal security forces and the number of dead civilians over the number of wounded civilians. In a context where certain factors that contribute to an excessive use of force become more common, the results of the study show a growing use of lethal force. This raises questions over the possible excessive use of lethal force as a normal or systematic practice. The study also shows a growing context of opacity in the information available to evaluate the use of lethal force and the general lack of a legal framework to regulate the use of lethal force in Mexico.

Toxicity of hexahydro-1,3,5-trinitro-1,3,5-triazine to larval zebrafish (Danio rerio)

Science.gov (United States)

Mukhi, S.; Pan, X.; Cobb, G.P.; Patino, R.

2005-01-01

Hexahydro-1,3,5-trinitro-1,3,5-triazine, a cyclonitramine commonly known as RDX, is used in the production of military munitions. Contamination of soil, sediment, and ground and surface waters with RDX has been reported in different places around the world. Acute and subacute toxicities of RDX have been relatively well documented in terrestrial vertebrates, but among aquatic vertebrates the information available is limited. The objective of this study was to characterize the acute toxicity of RDX to larval zebrafish. Mortality (LC50) and incidence of vertebral column deformities (EC50) were two of the end points measured in this study. The 96-h LC50 was estimated at 22.98 and 25.64 mg l-1 in two different tests. The estimated no-observed-effective- concentration (NOEC) values of RDX on lethality were 13.27 ?? 0.05 and 15.32 ?? 0.30 mg l-1; and the lowest-observed-effective- concentration (LOEC) values were 16.52 ?? 0.05 and 19.09 ?? 0.23 mg l-1 in these two tests, respectively. The 96-h EC50 for vertebral deformities on survivors from one of the acute lethality tests was estimated at 20.84 mg l-1, with NOEC and LOEC of 9.75 ?? 0.34 and 12.84 ?? 0.34 mg l-1, respectively. Behavioral aberrations were also noted in this acute toxicity study, including the occurrence of whirling movement and lethargic behavior. The acute effects of RDX on survival, incidence of deformities, and behavior of larval zebrafish occurred at the high end of the most frequently reported concentrations of RDX in aquatic environments. The chronic effects of RDX in aquatic vertebrates need to be determined for an adequate assessment of the ecological risk of environmental RDX. ?? 2005 Elsevier Ltd. All rights reserved.

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...

On the roles and regulation of chondroitin sulfate and heparan sulfate in zebrafish pharyngeal cartilage morphogenesis

DEFF Research Database (Denmark)

Holmborn, Katarina; Habicher, Judith; Kasza, Zsolt

2012-01-01

The present study addresses the roles of heparan sulfate (HS) proteoglycans and chondroitin sulfate (CS) proteoglycans in the development of zebrafish pharyngeal cartilage structures. uxs1 and b3gat3 mutants, predicted to have impaired biosynthesis of both HS and CS because of defective formation...... levels of CS than control larvae, whereas morpholino-mediated suppression of csgalnact1/csgalnact2 resulted in increased HS biosynthesis. Thus, the balance of the Extl3 and Csgalnact1/Csgalnact2 proteins influences the HS/CS ratio. A characterization of the pharyngeal cartilage element morphologies...

A strong loss-of-function mutation in RAN1 results in constitutive activation of the ethylene response pathway as well as a rosette-lethal phenotype

Science.gov (United States)

Woeste, K. E.; Kieber, J. J.; Evans, M. L. (Principal Investigator)

2000-01-01

A recessive mutation was identified that constitutively activated the ethylene response pathway in Arabidopsis and resulted in a rosette-lethal phenotype. Positional cloning of the gene corresponding to this mutation revealed that it was allelic to responsive to antagonist1 (ran1), a mutation that causes seedlings to respond in a positive manner to what is normally a competitive inhibitor of ethylene binding. In contrast to the previously identified ran1-1 and ran1-2 alleles that are morphologically indistinguishable from wild-type plants, this ran1-3 allele results in a rosette-lethal phenotype. The predicted protein encoded by the RAN1 gene is similar to the Wilson and Menkes disease proteins and yeast Ccc2 protein, which are integral membrane cation-transporting P-type ATPases involved in copper trafficking. Genetic epistasis analysis indicated that RAN1 acts upstream of mutations in the ethylene receptor gene family. However, the rosette-lethal phenotype of ran1-3 was not suppressed by ethylene-insensitive mutants, suggesting that this mutation also affects a non-ethylene-dependent pathway regulating cell expansion. The phenotype of ran1-3 mutants is similar to loss-of-function ethylene receptor mutants, suggesting that RAN1 may be required to form functional ethylene receptors. Furthermore, these results suggest that copper is required not only for ethylene binding but also for the signaling function of the ethylene receptors.

A Simple and Rapid Gene Disruption Strategy in Mycobacterium abscessus: On the Design and Application of Glycopeptidolipid Mutants.

Science.gov (United States)

Viljoen, Albertus; Gutiérrez, Ana Victoria; Dupont, Christian; Ghigo, Eric; Kremer, Laurent

2018-01-01

Little is known about the disease-causing genetic determinants that are used by Mycobacterium abscessus , increasingly acknowledged as an important emerging pathogen, notably in cystic fibrosis. The presence or absence of surface exposed glycopeptidolipids (GPL) conditions the smooth (S) or rough (R) M. abscessus subsp. abscessus ( M. abscessus ) variants, respectively, which are characterized by distinct infective programs. However, only a handful of successful gene knock-out and conditional mutants have been reported in M. abscessus , testifying that genetic manipulation of this mycobacterium is difficult. To facilitate gene disruption and generation of conditional mutants in M. abscessus , we have designed a one-step single cross-over system that allows the rapid and simple generation of such mutants. Cloning of as small as 300 bp of the target gene allows for efficient homologous recombination to occur without additional exogenous recombination-promoting factors. The presence of tdTomato on the plasmids allows easily sifting out the large background of mutants spontaneously resistant to antibiotics. Using this strategy in the S genetic background and the target gene mmpL4a , necessary for GPL synthesis and transport, nearly 100% of red fluorescent clones exhibited a rough morphotype and lost GPL on the surface, suggesting that most red fluorescent colonies obtained after transformation incorporated the plasmid through homologous recombination into the chromosome. This system was further exploited to generate another strain with reduced GPL levels to explore how the presence of these cell wall-associated glycolipids influences M. abscessus hydrophobicity as well as virulence in the zebrafish model of infection. This mutant exhibited a more pronounced killing phenotype in zebrafish embryos compared to its S progenitor and this effect correlated with the production of abscesses in the central nervous system. Overall, these results suggest that the near

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.

Fibroblast growth factor signaling is required for early somatic gonad development in zebrafish.

Science.gov (United States)

Leerberg, Dena M; Sano, Kaori; Draper, Bruce W

2017-09-01

The vertebrate ovary and testis develop from a sexually indifferent gonad. During early development of the organism, primordial germ cells (the gamete lineage) and somatic gonad cells coalesce and begin to undergo growth and morphogenesis to form this bipotential gonad. Although this aspect of development is requisite for a fertile adult, little is known about the genetic regulation of early gonadogenesis in any vertebrate. Here, we provide evidence that fibroblast growth factor (Fgf) signaling is required for the early growth phase of a vertebrate bipotential gonad. Based on mutational analysis in zebrafish, we show that the Fgf ligand 24 (Fgf24) is required for proliferation, differentiation, and morphogenesis of the early somatic gonad, and as a result, most fgf24 mutants are sterile as adults. Additionally, we describe the ultrastructural elements of the early zebrafish gonad and show that distinct somatic cell populations can be identified soon after the gonad forms. Specifically, we show that fgf24 is expressed in an epithelial population of early somatic gonad cells that surrounds an inner population of mesenchymal somatic gonad cells that are in direct contact with the germ cells, and that fgf24 is required for stratification of the somatic tissue. Furthermore, based on gene expression analysis, we find that differentiation of the inner mesenchymal somatic gonad cells into functional cell types in the larval and early juvenile-stage gonad is dependent on Fgf24 signaling. Finally, we argue that the role of Fgf24 in zebrafish is functionally analogous to the role of tetrapod FGF9 in early gonad development.

A survey of new temperature-sensitive, embryonic-lethal mutations in C. elegans: 24 alleles of thirteen genes.

Directory of Open Access Journals (Sweden)

Sean M O'Rourke

2011-03-01

Full Text Available To study essential maternal gene requirements in the early C. elegans embryo, we have screened for temperature-sensitive, embryonic lethal mutations in an effort to bypass essential zygotic requirements for such genes during larval and adult germline development. With conditional alleles, multiple essential requirements can be examined by shifting at different times from the permissive temperature of 15°C to the restrictive temperature of 26°C. Here we describe 24 conditional mutations that affect 13 different loci and report the identity of the gene mutations responsible for the conditional lethality in 22 of the mutants. All but four are mis-sense mutations, with two mutations affecting splice sites, another creating an in-frame deletion, and one creating a premature stop codon. Almost all of the mis-sense mutations affect residues conserved in orthologs, and thus may be useful for engineering conditional mutations in other organisms. We find that 62% of the mutants display additional phenotypes when shifted to the restrictive temperature as L1 larvae, in addition to causing embryonic lethality after L4 upshifts. Remarkably, we also found that 13 out of the 24 mutations appear to be fast-acting, making them particularly useful for careful dissection of multiple essential requirements. Our findings highlight the value of C. elegans for identifying useful temperature-sensitive mutations in essential genes, and provide new insights into the requirements for some of the affected loci.

Genomic Organization of Zebrafish microRNAs

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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.

Influence Of Quinolone Lethality on Irradiated Anaerobic Growth of Escherichia Coli

International Nuclear Information System (INIS)

Ibrahim, I.M.; El-Kabbany, H.M.; El-Esseily, E.SH.

2012-01-01

Bacteriostatic and bactericidal activities were measured with wild type cells and isomerase mutants of Escherichia coli for ciprofloxacin, formation of quinolone-gyrase-DNA complexes, observed as a sodium dodecyl sulfate (SDS) dependent drop in cell lysate viscosity, occurred during aerobic and anaerobic growth and in the presence and in the absence of chloramphenicol. Quinolone activity against Escherichia coli was examined during aerobic growth, aerobic treatment with chloramphenicol, and anaerobic growth. Nalidixic acid, norfloxacin and ciprofloxacin were lethal for cultures growing aerobically, and the bacteriostatic activity of each quinolone was unaffected by anaerobic growth. However, lethal activity was distinct for each quinolone with cells treated aerobically with chloramphenicol or grown anaerobically. Nalidixic acid failed to kill cells under both conditions, norfloxacin killed cells when they were grown anaerobically but not when they were treated with chloramphenicol, ciprofloxacin killed cells under both conditions but required higher concentrations than those required with cells grown aerobically, C-methoxy fluoro quinolone was equally lethal under all conditions. However, lethal chromosome fragmentation, detected as a drop in viscosity in the absence of SDS, was occurred with nalidixic acid treatment only under aerobic conditions in the absence of chloramphenicol, thus, all quinolones tested appeared to form reversible bacteriostatic complexes containing broken DNA during aerobic growth, during anaerobic growth, and when protein synthesis is blocked. The ability to fragment chromosomes rapidly kill cells under these conditions depends on quinolone structure. The radiation of sublethal dose was 3 Gy at rate of 0.6 Gy/min was shown as non-significant result

Macrophage–Microbe Interactions: Lessons from the Zebrafish Model

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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.

Severity of mutant phenotype in a series of chlorophyll-deficient wheat mutants depends on light intensity and the severity of the block in chlorophyll synthesis.

Science.gov (United States)

Falbel, T G; Meehl, J B; Staehelin, L A

1996-10-01

Analyses of a series of allelic chlorina mutants of wheat (Triticum aestivum L.), which have partial blocks in chlorophyll (Chl) synthesis and, therefore, a limited Chl supply, reinforce the principle that Chl is required for the stable accumulation of Chl-binding proteins and that only reaction centers accumulate when the supply of Chl is severely limited. Depending on the rate of Chl accumulation (determined by the severity of the mutation) and on the rate of turnover of Chl and its precursors (determined by the environment in which the plant is grown), the mutants each reach an equilibrium of Chl synthesis and degradation. Together these mutants generate a spectrum of phenotypes. Under the harshest conditions (high illumination), plants with moderate blocks in Chl synthesis have membranes with very little Chl and Chl-proteins and membrane stacks resembling the thylakoids of the lethal xantha mutants of barely grown at low to medium light intensities (which have more severe blocks). In contrast, when grown under low-light conditions the same plants with moderate blocks have thylakoids resembling those of the wild type. The wide range of phenotypes of Chl b-deficient mutants has historically produced more confusion than enlightenment, but incomparable growth conditions can now explain the discrepancies reported in the literature.

Transcriptome Analysis of Chemically-Induced Sensory Neuron Ablation in Zebrafish.

Directory of Open Access Journals (Sweden)

Jane A Cox

Full Text Available Peripheral glia are known to have a critical role in the initial response to axon damage and degeneration. However, little is known about the cellular responses of non-myelinating glia to nerve injury. In this study, we analyzed the transcriptomes of wild-type and mutant (lacking peripheral glia zebrafish larvae that were treated with metronidazole. This treatment allowed us to conditionally and selectively ablate cranial sensory neurons whose axons are ensheathed only by non-myelinating glia. While transcripts representing over 27,000 genes were detected by RNAseq, only a small fraction (~1% of genes were found to be differentially expressed in response to neuronal degeneration in either line at either 2 hrs or 5 hrs of metronidazole treatment. Analysis revealed that most expression changes (332 out of the total of 458 differentially expressed genes occurred over a continuous period (from 2 to 5 hrs of metronidazole exposure, with a small number of genes showing changes limited to only the 2 hr (55 genes or 5 hr (71 genes time points. For genes with continuous alterations in expression, some of the most meaningful sets of enriched categories in the wild-type line were those involving the inflammatory TNF-alpha and IL6 signaling pathways, oxidoreductase activities and response to stress. Intriguingly, these changes were not observed in the mutant line. Indeed, cluster analysis indicated that the effects of metronidazole treatment on gene expression was heavily influenced by the presence or absence of glia, indicating that the peripheral non-myelinating glia play a significant role in the transcriptional response to sensory neuron degeneration. This is the first transcriptome study of metronidazole-induced neuronal death in zebrafish and the response of non-myelinating glia to sensory neuron degeneration. We believe this study provides important insight into the mechanisms by which non-myelinating glia react to neuronal death and degeneration in

FishNet: an online database of zebrafish anatomy

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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.

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

Toxicity of effluents from gasoline stations oil-water separators to early life stages of zebrafish Danio rerio.

Science.gov (United States)

Alves, Romulo Nepomuceno; Mariz, Célio Freire; Paulo, Driele Ventura de; Carvalho, Paulo S M

2017-07-01

Used petroleum hydrocarbons and gasoline stations runoff are significant sources of polycyclic aromatic hydrocarbons (PAHs) to aquatic ecosystems. Samples of the final effluent of oil-water-separators were collected at gasoline stations in the metropolitan region of Recife, Brazil, before release to sewage or rainwater systems. Effluent soluble fractions (ESF) were prepared and bioassays were performed according to the Fish Embryo Toxicity Test. The test involved exposing zebrafish Danio rerio embryos to dilutions of the ESFs for 96 h, with daily examination of lethality and sublethal morphological effects integrated through the General Morphology Score (GMS), based on the achievement of developmental hallmarks. Frequencies of abnormalities were recorded after exposures. ESF LC50-96h (lethal concentration to 50% of exposed embryos) in the most toxic effluent achieved 8.9% (v/v), equivalent to 11 μg phenanthrene equivalents L -1 . GMS scores indicated significantly delayed embryo-larval development at ESF dilutions of 10% and 20% from effluents of all gas stations. Major abnormalities detected after the 96 h exposure included the presence of a yolk sac not fully absorbed coupled with the lack of an inflated swim bladder, lack of both pectoral fins, and the failure to develop a protruding mouth. Effective equivalent PAH concentrations that induce a 50% frequency of larvae without an inflated swim bladder (EC50) were 4.9 μg phenanthrene L -1 , 21.8 μg naphthalene L -1 , and 34.1 μg chrysene L -1 . This study shows that PAHs in ESFs from gas stations oil water separators are toxic to zebrafish, contributing to the toxicity of urban storm waters. Copyright © 2017 Elsevier Ltd. All rights reserved.

Zebrafish: A Versatile Animal Model for Fertility Research

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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.

In vivo DNA mismatch repair measurement in zebrafish embryos and its use in screening of environmental carcinogens

International Nuclear Information System (INIS)

Chen, Yuanhong; Huang, Changjiang; Bai, Chenglian; Du, Changchun; Liao, Junhua; Dong, Qiaoxiang

2016-01-01

Highlights: • We developed an in vivo DNA mismatch repair (MMR) measurement assay in zebrafish embryos. • This assay involves microinjection of homo- and heteroduplex EGFP plasmids into zebrafish embryos. • This novel assay was validated with embryos from the MMR-deficient mlh1 mutant fish. • We successfully applied this assay for detecting environmental chemicals with carcinogenic effect. • This novel assay can be used for screening of environmental carcinogens. - Abstract: Impairment of DNA mismatch repair (MMR) function leads to the development and progression of certain cancers. Many environmental contaminants can target DNA MMR system. Currently, measurement of MMR activity is limited to in vitro or in vivo methods at the cell line level, and reports on measurement of MMR activity at the live organism level are lacking. Here, we report an efficient method to measure DNA MMR activity in zebrafish embryos. A G-T mismatch was introduced into enhanced green fluorescent protein (EGFP) gene. Repair of the G-T mismatch to G-C in the heteroduplex plasmid generates a functional EGFP expression. The heteroduplex plasmid and a similarly constructed homoduplex plasmid were injected in parallel into the same batch of embryos at 1-cell stage and EGFP expression in EGFP positive embryos was quantified at 24 h after injection. MMR efficiency was calculated as the total fluorescence intensity of embryos injected with the heteroduplex construct divided by that of embryos injected with the homoduplex construct. Our results showed 73% reduction of MMR activity in embryos derived from MMR-deficient mlh1 mutant fish (positive control) when compared with embryos from MMR-competent wild type AB line fish, indicating feasibility of in vivo MMR activity measurement in zebrafish embryos. We further applied this novel assay for measurement of MMR efficiency in embryos exposed to environmental chemicals such as cadmium chloride (CdCl_2), benzo[a]pyrene (BaP), and

In vivo DNA mismatch repair measurement in zebrafish embryos and its use in screening of environmental carcinogens

Energy Technology Data Exchange (ETDEWEB)

Chen, Yuanhong [Institute of Environmental Safety and Human Health, Wenzhou Medical University, Wenzhou 325035 (China); Huang, Changjiang, E-mail: cjhuang5711@163.com [Institute of Environmental Safety and Human Health, Wenzhou Medical University, Wenzhou 325035 (China); Bai, Chenglian; Du, Changchun; Liao, Junhua [Institute of Environmental Safety and Human Health, Wenzhou Medical University, Wenzhou 325035 (China); Dong, Qiaoxiang, E-mail: dqxdong@163.com [Institute of Environmental Safety and Human Health, Wenzhou Medical University, Wenzhou 325035 (China); School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou 325035 (China)

2016-01-25

Highlights: • We developed an in vivo DNA mismatch repair (MMR) measurement assay in zebrafish embryos. • This assay involves microinjection of homo- and heteroduplex EGFP plasmids into zebrafish embryos. • This novel assay was validated with embryos from the MMR-deficient mlh1 mutant fish. • We successfully applied this assay for detecting environmental chemicals with carcinogenic effect. • This novel assay can be used for screening of environmental carcinogens. - Abstract: Impairment of DNA mismatch repair (MMR) function leads to the development and progression of certain cancers. Many environmental contaminants can target DNA MMR system. Currently, measurement of MMR activity is limited to in vitro or in vivo methods at the cell line level, and reports on measurement of MMR activity at the live organism level are lacking. Here, we report an efficient method to measure DNA MMR activity in zebrafish embryos. A G-T mismatch was introduced into enhanced green fluorescent protein (EGFP) gene. Repair of the G-T mismatch to G-C in the heteroduplex plasmid generates a functional EGFP expression. The heteroduplex plasmid and a similarly constructed homoduplex plasmid were injected in parallel into the same batch of embryos at 1-cell stage and EGFP expression in EGFP positive embryos was quantified at 24 h after injection. MMR efficiency was calculated as the total fluorescence intensity of embryos injected with the heteroduplex construct divided by that of embryos injected with the homoduplex construct. Our results showed 73% reduction of MMR activity in embryos derived from MMR-deficient mlh1 mutant fish (positive control) when compared with embryos from MMR-competent wild type AB line fish, indicating feasibility of in vivo MMR activity measurement in zebrafish embryos. We further applied this novel assay for measurement of MMR efficiency in embryos exposed to environmental chemicals such as cadmium chloride (CdCl{sub 2}), benzo[a]pyrene (BaP), and

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

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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.

Arsenic transport by zebrafish aquaglyceroporins

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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

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

Running the Stop Sign: Readthrough of a Premature UAG Termination Signal in the Translation of a Zebrafish (Danio rerio) Taurine Biosynthetic Enzyme.

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Larkin, Mary E M; Place, Allen R

2017-06-03

The UAG termination codon is generally recognized as the least efficient and least frequently used of the three universal stop codons. This is substantiated by numerous studies in an array of organisms. We present here evidence of a translational readthrough of a mutant nonsense UAG codon in the transcript from the cysteine sulfinic acid decarboxylase ( csad ) gene (ENSDARG00000026348) in zebrafish. The csad gene encodes the terminal enzyme in the taurine biosynthetic pathway. Taurine is a critical amino acid for all animals, playing several essential roles throughout the body, including modulation of the immune system. The sa9430 zebrafish strain (ZDB-ALT-130411-5055) has a point mutation leading to a premature stop codon (UAG) 20 amino acids 5' of the normal stop codon, UGA. Data from immunoblotting, enzyme activity assays, and mass spectrometry provide evidence that the mutant is making a CSAD protein identical to that of the wild-type (XP_009295318.1) in terms of size, activity, and amino acid sequence. UAG readthrough has been described in several species, but this is the first presentation of a case in fish. Also presented are the first data substantiating the ability of a fish CSAD to utilize cysteic acid, an alternative to the standard substrate cysteine sulfinic acid, to produce taurine.

Normal anatomy and histology of the adult zebrafish.

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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.

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

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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.

Immunomodulatory properties of Concholepas concholepas hemocyanin against francisellosis in a zebrafish model.

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Lagos, Leidy; Tandberg, Julia I; Becker, María Inés; Winther-Larsen, Hanne C

2017-08-01

The development of vaccines for aquaculture has been an important milestone in providing a continuous and sustainable production. Most of the vaccines currently on the market for aquaculture include oil as adjuvant. Nevertheless, several studies reported an occurrence of side effects after their use in farmed fish. As a result, there is a need for new and improved adjuvants that can stimulate the immune system while causing as few side-effects as possible. Hemocyanins are versatile macromolecules with strong immunogenic and immunomodulatory properties. Due to these characteristics, hemocyanin from Concholepas concholepas (CCH) has been biochemically characterized and evaluated as vaccine adjuvant in mice and humans. Francisellosis is a chronic granulomatous disease, which can result in high mortality depending on the host. The disease is caused by the facultative intracellular Gram-negative bacteria Francisella noatunensis, which remains an unsolved problem for the aquaculture, as no efficient vaccines are available. The aim of the present work was to investigate the immunoregulatory properties of CCH against francisellosis in an experimental zebrafish model. When immunized with CCH, zebrafish were protected from subsequent challenge with a lethal dose of Francisella noatunensis subsp. orientalis. Subsequently the mRNA expression levels of several immune-related genes were studied, including mhcii, il12a, tnfα and ifng1-1. Taken together, the data report the immunoregulatory properties of CCH and its potential use as a vaccine adjuvant for aquaculture. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Morphological and Physiological Interactions Between GnRH3 and Hypocretin/Orexin Neuronal Systems in Zebrafish (Danio rerio).

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Zhao, Yali; Singh, Chanpreet; Prober, David A; Wayne, Nancy L

2016-10-01

GnRH neurons integrate internal and external cues to control sexual maturation and fertility. Homeostasis of energy balance and food intake correlates strongly with the status of reproduction. Neuropeptides secreted by the hypothalamus involved in modulating energy balance and feeding may play additional roles in the regulation of reproduction. Hypocretin (Hcrt) (also known as orexin) is one such peptide, primarily controlling sleep/wakefulness, food intake, and reward processing. There is a growing body of evidence indicating that Hcrt/orexin (Hcrt) modulates reproduction through interacting with the hypothalamo-pituitary-gonadal axis in mammals. To explore potential morphological and functional interactions between the GnRH and Hcrt neuronal systems, we employed a variety of experimental approaches including confocal imaging, immunohistochemistry, and electrophysiology in transgenic zebrafish, in which fluorescent proteins are genetically expressed in GnRH3 and Hcrt neurons. Our imaging data revealed close apposition and direct connection between GnRH3 and Hcrt neuronal systems in the hypothalamus during larval development through adulthood. Furthermore, the Hcrt receptor (HcrtR) is expressed in GnRH3 neurons. Electrophysiological data revealed a reversible inhibitory effect of Hcrt on GnRH3 neuron electrical activity, which was blocked by the HcrtR antagonist almorexant. In addition, Hcrt had no effect on the electrical activity of GnRH3 neurons in the HcrtR null mutant zebrafish (HcrtR -/- ). Our findings demonstrate a close anatomical and functional relationship between Hcrt and GnRH neuronal systems in zebrafish. It is the first demonstration of a link between neuronal circuits controlling sleeping/arousal/feeding and reproduction in zebrafish, an important animal model for investigating the molecular genetics of development.

Evaluation of visible implant elastomer tags in zebrafish (Danio rerio

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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.

Learning and memory in zebrafish larvae

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

2013-01-01

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

Exploring the regulatory role of isocitrate dehydrogenase mutant protein on glioma stem cell proliferation.

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Lu, H-C; Ma, J; Zhuang, Z; Qiu, F; Cheng, H-L; Shi, J-X

2016-08-01

Glioma is the most lethal form of cancer that originates mostly from the brain and less frequently from the spine. Glioma is characterized by abnormal regulation of glial cell differentiation. The severity of the glioma was found to be relaxed in isocitrate dehydrogenase 1 (IDH1) mutant. The present study focused on histological discrimination and regulation of cancer stem cell between IDH1 mutant and in non-IDH1 mutant glioma tissue. Histology, immunohistochemistry and Western blotting techniques are used to analyze the glioma nature and variation in glioma stem cells that differ between IDH1 mutant and in non-IDH1 mutant glioma tissue. The aggressive form of non-IDH1 mutant glioma shows abnormal cellular histological variation with prominent larger nucleus along with abnormal clustering of cells. The longer survival form of IDH1 mutant glioma has a control over glioma stem cell proliferation. Immunohistochemistry with stem cell markers, CD133 and EGFRvIII are used to demonstrate that the IDH1 mutant glioma shows limited dependence on cancer stem cells and it shows marked apoptotic signals in TUNEL assay to regulate abnormal cells. The non-IDH1 mutant glioma failed to regulate misbehaving cells and it promotes cancer stem cell proliferation. Our finding supports that the IDH1 mutant glioma has a regulatory role in glioma stem cells and their survival.

Histopathological effects of lethal and sub-lethal concentrations of ...

African Journals Online (AJOL)

The histopathological effects of lethal and sub-lethal concentrations of glyphosate on African catfish Clarias gariepinus were investigated. C. gariepinus juveniles were assessed in a static renewal bioassay for 96 hours (acute toxicity) and 28 days (chronic toxicity) using varying concentrations (0.0 mg/l 20.0 mg/l, 30.0 mg/l, ...

A dominant negative zebrafish Ahr2 partially protects developing zebrafish from dioxin toxicity.

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Kevin A Lanham

Full Text Available The toxicity by 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD is thought to be caused by activation of the aryl hydrocarbon receptor (AHR. However, our understanding of how AHR activation by TCDD leads to toxic effects is poor. Ideally we would like to manipulate AHR activity in specific tissues and at specific times. One route to this is expressing dominant negative AHRs (dnAHRs. This work describes the construction and characterization of dominant negative forms of the zebrafish Ahr2 in which the C-terminal transactivation domain was either removed, or replaced with the inhibitory domain from the Drosophila engrailed repressor protein. One of these dnAhr2s was selected for expression from the ubiquitously active e2fα promoter in transgenic zebrafish. We found that these transgenic zebrafish expressing dnAhr2 had reduced TCDD induction of the Ahr2 target gene cyp1a, as measured by 7-ethoxyresorufin-O-deethylase activity. Furthermore, the cardiotoxicity produced by TCDD, pericardial edema, heart malformation, and reduced blood flow, were all mitigated in the zebrafish expressing the dnAhr2. These results provide in vivo proof-of-principle results demonstrating the effectiveness of dnAHRs in manipulating AHR activity in vivo, and demonstrating that this approach can be a means for blocking TCDD toxicity.

Neurotransmitter-Regulated Regeneration in the Zebrafish Retina

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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

Zebrafish Models for the Mechanosensory Hair Cell Dysfunction in Usher Syndrome 3 Reveal That Clarin-1 Is an Essential Hair Bundle Protein.

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Gopal, Suhasini R; Chen, Daniel H-C; Chou, Shih-Wei; Zang, Jingjing; Neuhauss, Stephan C F; Stepanyan, Ruben; McDermott, Brian M; Alagramam, Kumar N

2015-07-15

Usher syndrome type III (USH3) is characterized by progressive loss of hearing and vision, and varying degrees of vestibular dysfunction. It is caused by mutations that affect the human clarin-1 protein (hCLRN1), a member of the tetraspanin protein family. The missense mutation CLRN1(N48K), which affects a conserved N-glycosylation site in hCLRN1, is a common causative USH3 mutation among Ashkenazi Jews. The affected individuals hear at birth but lose that function over time. Here, we developed an animal model system using zebrafish transgenesis and gene targeting to provide an explanation for this phenotype. Immunolabeling demonstrated that Clrn1 localized to the hair cell bundles (hair bundles). The clrn1 mutants generated by zinc finger nucleases displayed aberrant hair bundle morphology with diminished function. Two transgenic zebrafish that express either hCLRN1 or hCLRN1(N48K) in hair cells were produced to examine the subcellular localization patterns of wild-type and mutant human proteins. hCLRN1 localized to the hair bundles similarly to zebrafish Clrn1; in contrast, hCLRN1(N48K) largely mislocalized to the cell body with a small amount reaching the hair bundle. We propose that this small amount of hCLRN1(N48K) in the hair bundle provides clarin-1-mediated function during the early stages of life; however, the presence of hCLRN1(N48K) in the hair bundle diminishes over time because of intracellular degradation of the mutant protein, leading to progressive loss of hair bundle integrity and hair cell function. These findings and genetic tools provide an understanding and path forward to identify therapies to mitigate hearing loss linked to the CLRN1 mutation. Mutations in the clarin-1 gene affect eye and ear function in humans. Individuals with the CLRN1(N48K) mutation are born able to hear but lose that function over time. Here, we develop an animal model system using zebrafish transgenesis and gene targeting to provide an explanation for this phenotype

Lack of chemically induced mutation in repair-deficient mutants of yeast

International Nuclear Information System (INIS)

Prakash, L.

1974-01-01

Two genes, rad6 and rad9, that confer radiation sensitivity in the yeast Saccharomyces cerevisiae also greatly reduce the frequency of chemically-induced reversions of a tester mutant cyc1-131, which is a chain initiation mutant in the structural gene determining iso-1-cytochrome c. Mutations induced by ethyl methanesulfonate (EMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), dimethyl sulfate (DMS), nitroquinoline oxide (NQO), nitrosoguanidine (NTG), nitrogen mustard (HN2), β-propiolactone, and tritiated uridine, as well as mutations induced by ultraviolet light (UV) and ionizing radiation were greatly diminished in strains homozygous for either the rad6 or rad9 gene. Nitrous acid and nitrosoimidazolidone (NIL), on the other hand, were highly mutagenic in these repair-deficient mutants, and at low doses, these mutagens acted with about the same efficiency as in the normal RAD strain. At high doses of either nitrous acid or NIL, however, reversion frequencies were significantly reduced in the two rad mutants compared to normal strains. Although both rad mutants are immutable to about the same extent, the rad9 strains tend to be less sensitive to the lethal effect of chemical mutagens than rad6 strains. It is concluded that yeast requires a functional repair system for mutation induction by chemical agents. (auth)

Lack of chemically induced mutation in repair-deficient mutants of yeast.

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Prakash, L

1974-12-01

Two genes, rad6 and rad9, that confer radiation sensitivity in the yeast Saccharomyces cerevisiae also greatly reduce the frequency of chemically-induced reversions of a tester mutant cyc1-131, which is a chain initiation mutant in the structural gene determining iso-1-cytochrome c. Mutations induced by ethyl methanesulfonate (EMS), diethyl sulfate (DES), methyl methanesulfonate (MMS), dimethyl sulfate (DMS), nitroquinoline oxide (NQO), nitrosoguanidine (NTG), nitrogen mustard (HN2), beta-propiolactone, and tritiated uridine, as well as mutations induced by ultraviolet light (UV) and ionizing radiation were greatly diminished in strains homozygous for either the rad6 or rad9 gene. Nitrous acid and nitrosoimidazolidone (NIL), on the other hand, were highly mutagenic in these repair-deficient mutants, and at low doses, these mutagens acted with about the same efficiency as in the normal RAD strain. At high doses of either nitrous acid or NIL, however, reversion frequencies were significantly reduced in the two rad mutants compared to normal strains. Although both rad mutants are immutable to about the same extent, the rad9 strains tend to be less sensitive to the lethal effect of chemical mutagens than rad6 strains. It is concluded that yeast requires a functional repair system for mutation induction by chemical agents.

Triclosan Lacks (Anti-Estrogenic Effects in Zebrafish Cells but Modulates Estrogen Response in Zebrafish Embryos

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Hélène Serra

2018-04-01

Full Text Available Triclosan (TCS, an antimicrobial agent widely found in the aquatic environment, is suspected to act as an endocrine disrupting compound, however mechanistic information is lacking in regards to aquatic species. This study assessed the ability of TCS to interfere with estrogen receptor (ER transcriptional activity, in zebrafish-specific in vitro and in vivo reporter gene assays. We report that TCS exhibits a lack of either agonistic or antagonistic effects on a panel of ER-expressing zebrafish (ZELH-zfERα and -zfERβ and human (MELN cell lines. At the organism level, TCS at concentrations of up to 0.3 µM had no effect on ER-regulated brain aromatase gene expression in transgenic cyp19a1b-GFP zebrafish embryos. At a concentration of 1 µM, TCS interfered with the E2 response in an ambivalent manner by potentializing a low E2 response (0.625 nM, but decreasing a high E2 response (10 nM. Altogether, our study suggests that while modulation of ER-regulated genes by TCS may occur in zebrafish, it does so irrespective of a direct binding and activation of zfERs.

A Zebrafish Heart Failure Model for Assessing Therapeutic Agents.

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

2018-03-20

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

Transgenic fluorescent zebrafish Tg(fli1:EGFP)y¹ for the identification of vasotoxicity within the zFET.

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Delov, Vera; Muth-Köhne, Elke; Schäfers, Christoph; Fenske, Martina

2014-05-01

The fish embryo toxicity test (FET) is currently one of the most advocated animal alternative tests in ecotoxicology. To date, the application of the FET with zebrafish (zFET) has focused on acute toxicity assessment, where only lethal morphological effects are accounted for. An application of the zFET beyond acute toxicity, however, necessitates the establishment of more refined and quantifiable toxicological endpoints. A valuable tool in this context is the use of gene expression-dependent fluorescent markers that can even be measured in vivo. We investigated the application of embryos of Tg(fli1:EGFP)(y1) for the identification of vasotoxic substances within the zFET. Tg(fli1:EGFP)(y1) fish express enhanced GFP in the entire vasculature under the control of the fli1 promoter, and thus enable the visualization of vascular defects in live zebrafish embryos. We assessed the fli1 driven EGFP-expression in the intersegmental blood vessels (ISVs) qualitatively and quantitatively, and found an exposure concentration related increase in vascular damage for chemicals like triclosan, cartap and genistein. The fluorescence endpoint ISV-length allowed an earlier and more sensitive detection of vasotoxins than the bright field assessment method. In combination with the standard bright field morphological effect assessment, an increase in significance and value of the zFET for a mechanism-specific toxicity evaluation was achieved. This study highlights the benefits of using transgenic zebrafish as convenient tools for identifying toxicity in vivo and to increase sensitivity and specificity of the zFET. Copyright © 2014 Elsevier B.V. All rights reserved.

Loss of col8a1a Function during Zebrafish Embryogenesis Results in Congenital Vertebral Malformations

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Gray, Ryan S.; Wilm, Thomas; Smith, Jeff; Bagnat, Michel; Dale, Rodney M.; Topczewski, Jacek; Johnson, Stephen L.; Solnica-Krezel, Lilianna

2014-01-01

Congenital vertebral malformations (CVM) occur in 1 in 1,000 live births and in many cases can cause spinal deformities, such as scoliosis, and result in disability and distress of affected individuals. Many severe forms of the disease, such as spondylocostal dystostosis, are recessive monogenic traits affecting somitogenesis, however the etiologies of the majority of CVM cases remain undetermined. Here we demonstrate that morphological defects of the notochord in zebrafish can generate congenital-type spine defects. We characterize three recessive zebrafish leviathan/col8a1a mutant alleles (m531, vu41, vu105) that disrupt collagen type VIII alpha1a (col8a1a), and cause folding of the embryonic notochord and consequently adult vertebral column malformations. Furthermore, we provide evidence that a transient loss of col8a1a function or inhibition of Lysyl oxidases with drugs during embryogenesis was sufficient to generate vertebral fusions and scoliosis in the adult spine. Using periodic imaging of individual zebrafish, we correlate focal notochord defects of the embryo with vertebral malformations (VM) in the adult. Finally, we show that bends and kinks in the notochord can lead to aberrant apposition of osteoblasts normally confined to well-segmented areas of the developing vertebral bodies. Our results afford a novel mechanism for the formation of VM, independent of defects of somitogenesis, resulting from aberrant bone deposition at regions of misshapen notochord tissue. PMID:24333517

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.

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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

Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond.

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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.

Lethal and mutagenic effects of fast neutrons of different energy on Streptomyces griseus spores

International Nuclear Information System (INIS)

Podgorskaya, M.E.; Tulina, G.G.; Serdechnaya, A.I.; Matselyukh, B.P.

1986-01-01

A study was made of lethal and mutagenic effects of fast neutrons of different energy on spores of prototrophic and auxotrophic strains of Streptomyces griseus. Relative biological effectiveness of fast neutrons is higher than that of γ-rays and depends on beam energy. Neutrons of 22-50 MeV induce Streptomyces griseus mutations more frequently (by one order of magnitude) than neutrons of 1.4-1.6 MeV do. The obtained mutants can be used in studying Streptomyces griseus genetics

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

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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.

Muscle dysfunction in a zebrafish model of Duchenne muscular dystrophy.

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

2016-11-01

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

Examination of a Palatogenic Gene Program in Zebrafish

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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

Energetics of cellular repair processes in a respiratory-deficient mutant of yeast

International Nuclear Information System (INIS)

Jain, V.K.; Gupta, I.; Lata, K.

1982-01-01

Repair of potentially lethal damage induced by cytoxic agents like UV irradiation (254 nm), psorelen-plus-UVA (365 mn), and methyl methanesulfonate has been studied in the presence of a glucose analog, 2-deoxy-D-glucose, in yeast cells. Simultaneously, effects of 2-deoxy-D-glucose were also investigated on parameters of energy metabolism like glucose utilization, rate of ATP production, and ATP content of cells. The following results were obtained. (i) 2-Deoxy-D-glucose is able to inhibit repair of potentially lethal damage induced by all the cytotoxic agents tested. The 2-deoxy-D-glucose-induced inhibition of repair depends upon the type of lesion and the pattern of cellular energy metabolism, the inhibition being greater in respiratory-deficient mutants than in the wild type. (ii) A continuous energy flow is necessary for repair of potentially lethal damage in yeast cells. Energy may be supplied by the glycolytic and/or the respiratory pathway; respiratory metabolism is not essential for this purpose. (iii) The magnitude of repair correlates with the rate of ATP production in a sigmoid manner

Developmental Toxicity Assay for Food Additive Tartrazine Using Zebrafish ( Danio rerio) Embryo Cultures.

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Joshi, Vani; Katti, Pancharatna

Tartrazine (TTZ) is an azo dye used as a colorant in food products, drugs, and cosmetics. The present study evaluates the impacts of TTZ on embryonic development of zebrafish ( Danio rerio). Laboratory-raised D. rerio embryos (n = 20/concentration) were exposed to graded dilutions of TTZ (0, 0.1, 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 75, and 100 mM) from gastrulation stage (5.25 hours postfertilization [hpf]) until hatching and developmental trajectory was traced up to day 7. The no observed effect concentration (NOEC), median lethal concentration (LC 50 ), median effective concentration (EC 50 ), and teratogenic index (TI) were calculated. Exposure of embryos to effects; 20 to 30 mM TTZ caused tail bending, cardiac and yolk sac edema in 50% of larvae; in 30 to 50 mM TTZ-exposed embryos the heart rates declined along with the above mentioned deformities, causing mortality within 96 to 144 hpf; development ceased completely at 75 to 100 mM concentration. The NOEC and LC 50 were recorded at 5 and 29.4 mM dose, respectively. The EC 50 values for heart rate, cardiac edema, tail bending, and hatching success were at 59.60, 53.81, 98.28, and 58.97 mM with TI quotient 0.49, 0.54, 0.29, and 0.49, respectively. We conclude that TTZ is not embryo toxic/teratogenic for zebrafish embryos up to a dose level of 10 mM concentration.

Zebrafish models in neuropsychopharmacology and CNS drug discovery.

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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.

Analysis of fast neutron-generated mutants at the Arabidopsis thaliana HY4 locus

International Nuclear Information System (INIS)

Bruggemann, E.; Handwerger, K.; Essex, C.; Storz, G.

1996-01-01

Ionizing radiation is expected to produce mutants with deletions or other chromosomal rearrangements. These mutants are useful for a variety of purposes, such as creating null alleles and cloning genes whose existence is known only from their mutant phenotype; however, only a few mutations generated by ionizing radiation have been characterized at the molecular level in Arabidopsis thaliana. Twenty fast neutron-generated alleles of the Arabidopsis HY4 locus, which encodes a blue light receptor, CRY1, were isolated and characterized. Nine of the mutant alleles displayed normal genetic behavior. The other 11 mutant alleles were poorly transmitted through the male gametophyte and were lethal in homozygous plants. Southern blot analysis demonstrated that alleles of the first group generally contain small or moderate-sized deletions at HY4, while alleles of the second group contain large deletions at this locus. These results demonstrate that fast neutrons can produce a range of deletions at a single locus in Arabidopsis. Many of these deletions would be suitable for cloning by genomic subtraction or representational difference analysis. The results also suggest the presence of an essential locus adjacent to HY4. (author)

Identification of Spt5 target genes in zebrafish development reveals its dual activity in vivo.

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Keerthi Krishnan

Full Text Available Spt5 is a conserved essential protein that represses or stimulates transcription elongation in vitro. Immunolocalization studies on Drosophila polytene chromosomes suggest that Spt5 is associated with many loci throughout the genome. However, little is known about the prevalence and identity of Spt5 target genes in vivo during development. Here, we identify direct target genes of Spt5 using fog(sk8 zebrafish mutant, which disrupts the foggy/spt5 gene. We identified that fog(sk8 and their wildtype siblings differentially express less than 5% of genes examined. These genes participate in diverse biological processes from stress response to cell fate specification. Up-regulated genes exhibit shorter overall gene length compared to all genes examined. Through chromatin immunoprecipitation in zebrafish embryos, we identified a subset of developmentally critical genes that are bound by both Spt5 and RNA polymerase II. The protein occupancy patterns on these genes are characteristic of both repressive and stimulatory elongation regulation. Together our findings establish Spt5 as a dual regulator of transcription elongation in vivo and identify a small but diverse set of target genes critically dependent on Spt5 during development.

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

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

2017-01-01

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

Embryonic Lethality of Mitochondrial Pyruvate Carrier 1 Deficient Mouse Can Be Rescued by a Ketogenic Diet

OpenAIRE

Vanderperre, Beno?t; Herzig, S?bastien; Krznar, Petra; H?rl, Manuel; Ammar, Zeinab; Montessuit, Sylvie; Pierredon, Sandra; Zamboni, Nicola; Martinou, Jean-Claude

2016-01-01

Mitochondrial import of pyruvate by the mitochondrial pyruvate carrier (MPC) is a central step which links cytosolic and mitochondrial intermediary metabolism. To investigate the role of the MPC in mammalian physiology and development, we generated a mouse strain with complete loss of MPC1 expression. This resulted in embryonic lethality at around E13.5. Mouse embryonic fibroblasts (MEFs) derived from mutant mice displayed defective pyruvate-driven respiration as well as perturbed metabolic p...

Evaluating the Zebrafish Embryo Toxicity Test for Pesticide ...

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

Detection of vitellogenin incorporation into zebrafish oocytes by FITC fluorescence

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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.

Mutagenesis-mediated virus extinction: virus-dependent effect of viral load on sensitivity to lethal defection.

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Héctor Moreno

Full Text Available BACKGROUND: Lethal mutagenesis is a transition towards virus extinction mediated by enhanced mutation rates during viral genome replication, and it is currently under investigation as a potential new antiviral strategy. Viral load and virus fitness are known to influence virus extinction. Here we examine the effect or the multiplicity of infection (MOI on progeny production of several RNA viruses under enhanced mutagenesis. RESULTS: The effect of the mutagenic base analogue 5-fluorouracil (FU on the replication of the arenavirus lymphocytic choriomeningitis virus (LCMV can result either in inhibition of progeny production and virus extinction in infections carried out at low multiplicity of infection (MOI, or in a moderate titer decrease without extinction at high MOI. The effect of the MOI is similar for LCMV and vesicular stomatitis virus (VSV, but minimal or absent for the picornaviruses foot-and-mouth disease virus (FMDV and encephalomyocarditis virus (EMCV. The increase in mutation frequency and Shannon entropy (mutant spectrum complexity as a result of virus passage in the presence of FU was more accentuated at low MOI for LCMV and VSV, and at high MOI for FMDV and EMCV. We present an extension of the lethal defection model that agrees with the experimental results. CONCLUSIONS: (i Low infecting load favoured the extinction of negative strand viruses, LCMV or VSV, with an increase of mutant spectrum complexity. (ii This behaviour is not observed in RNA positive strand viruses, FMDV or EMCV. (iii The accumulation of defector genomes may underlie the MOI-dependent behaviour. (iv LCMV coinfections are allowed but superinfection is strongly restricted in BHK-21 cells. (v The dissimilar effects of the MOI on the efficiency of mutagenic-based extinction of different RNA viruses can have implications for the design of antiviral protocols based on lethal mutagenesis, presently under development.

Zebrafish models for the functional genomics of neurogenetic disorders.

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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.

Centromere replication timing determines different forms of genomic instability in Saccharomyces cerevisiae checkpoint mutants during replication stress.

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Feng, Wenyi; Bachant, Jeff; Collingwood, David; Raghuraman, M K; Brewer, Bonita J

2009-12-01

Yeast replication checkpoint mutants lose viability following transient exposure to hydroxyurea, a replication-impeding drug. In an effort to understand the basis for this lethality, we discovered that different events are responsible for inviability in checkpoint-deficient cells harboring mutations in the mec1 and rad53 genes. By monitoring genomewide replication dynamics of cells exposed to hydroxyurea, we show that cells with a checkpoint deficient allele of RAD53, rad53K227A, fail to duplicate centromeres. Following removal of the drug, however, rad53K227A cells recover substantial DNA replication, including replication through centromeres. Despite this recovery, the rad53K227A mutant fails to achieve biorientation of sister centromeres during recovery from hydroxyurea, leading to secondary activation of the spindle assembly checkpoint (SAC), aneuploidy, and lethal chromosome segregation errors. We demonstrate that cell lethality from this segregation defect could be partially remedied by reinforcing bipolar attachment. In contrast, cells with the mec1-1 sml1-1 mutations suffer from severely impaired replication resumption upon removal of hydroxyurea. mec1-1 sml1-1 cells can, however, duplicate at least some of their centromeres and achieve bipolar attachment, leading to abortive segregation and fragmentation of incompletely replicated chromosomes. Our results highlight the importance of replicating yeast centromeres early and reveal different mechanisms of cell death due to differences in replication fork progression.

Continual low-level MEK inhibition ameliorates cardio-facio-cutaneous phenotypes in zebrafish

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Corina Anastasaki

2012-07-01

Cardio-facio-cutaneous (CFC syndrome is caused by germline mutations in KRAS, BRAF and MEK1/2. The highly selective and potent MEK inhibitors that have been developed as anti-cancer agents hold potential as therapeutics for CFC syndrome. We have previously shown that the effects of CFC mutations on zebrafish gastrulation can be prevented by a 1-hour treatment with MEK inhibitors within a specific developmental time-window. However, MEK activity is essential for normal development and PD0325901 treatment outside this treatment window leads to additional developmental defects in MEK-dependent tissues. We now test ten different doses of PD0325901 at six developmental time points and assess the effects on body axis length, heart development and craniofacial structures in zebrafish embryos. Notably, we find that a continuous low-level dose of PD0325901 that has only minor inhibition of MEK activity can prevent the action of both the common CFC BRAFQ257R kinase-active allele and the BRAFG596V kinase-impaired mutant allele through the first 5 days of development. These results provide a detailed study of the effects of PD0325901 in development and show that, unlike in cancer, which requires robust inhibition of MAPK signalling, a partial reduction in phospho-ERK1/2 activity is sufficient to moderate the developmental effects of BRAFCFC mutations.

Characterization of behavioral and endocrine effects of LSD on zebrafish.

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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.

Gap junctions composed of connexins 41.8 and 39.4 are essential for colour pattern formation in zebrafish

Science.gov (United States)

Irion, Uwe; Frohnhöfer, Hans Georg; Krauss, Jana; Çolak Champollion, Tuǧba; Maischein, Hans-Martin; Geiger-Rudolph, Silke; Weiler, Christian; Nüsslein-Volhard, Christiane

2014-01-01

Interactions between all three pigment cell types are required to form the stripe pattern of adult zebrafish (Danio rerio), but their molecular nature is poorly understood. Mutations in leopard (leo), encoding Connexin 41.8 (Cx41.8), a gap junction subunit, cause a phenotypic series of spotted patterns. A new dominant allele, leotK3, leads to a complete loss of the pattern, suggesting a dominant negative impact on another component of gap junctions. In a genetic screen, we identified this component as Cx39.4 (luchs). Loss-of-function alleles demonstrate that luchs is required for stripe formation in zebrafish; however, the fins are almost not affected. Double mutants and chimeras, which show that leo and luchs are only required in xanthophores and melanophores, but not in iridophores, suggest that both connexins form heteromeric gap junctions. The phenotypes indicate that these promote homotypic interactions between melanophores and xanthophores, respectively, and those cells instruct the patterning of the iridophores. DOI: http://dx.doi.org/10.7554/eLife.05125.001 PMID:25535837

Harmonin (Ush1c is required in zebrafish Müller glial cells for photoreceptor synaptic development and function

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Jennifer B. Phillips

2011-11-01

Usher syndrome is the most prevalent cause of hereditary deaf-blindness, characterized by congenital sensorineural hearing impairment and progressive photoreceptor degeneration beginning in childhood or adolescence. Diagnosis and management of this disease are complex, and the molecular changes underlying sensory cell impairment remain poorly understood. Here we characterize two zebrafish models for a severe form of Usher syndrome, Usher syndrome type 1C (USH1C: one model is a mutant with a newly identified ush1c nonsense mutation, and the other is a morpholino knockdown of ush1c. Both have defects in hearing, balance and visual function from the first week of life. Histological analyses reveal specific defects in sensory cell structure that are consistent with these behavioral phenotypes and could implicate Müller glia in the retinal pathology of Usher syndrome. This study shows that visual defects associated with loss of ush1c function in zebrafish can be detected from the onset of vision, and thus could be applicable to early diagnosis for USH1C patients.

Characterization of the Zebrafish Homolog of Zipper Interacting Protein Kinase

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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.

HCV IRES-mediated core expression in zebrafish.

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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.

A zebrafish model of inflammatory lymphangiogenesis

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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.

Characterization of a mutant of Escherichia coli B/R defective in mutation frequency decline

International Nuclear Information System (INIS)

George, D.L.

1974-01-01

A mutant of Escherichia coli B/r designated mfd is very deficient in the ability to exhibit mutation frequency decline (MFD), the characteristic loss of potential suppressor mutations which occurs when protein synthesis is briefly inhibited after irradiation with ultraviolet light (uv). This mutant is known to excise pyrimidine dimers very slowly, although it is as uv-resistant as its mfd + B/r parent strain. We have found that the mfd mutant performs the initial incision step of excision repair normally, but repairs the resulting single-strand breaks much more slowly than the mfd + strain. In spite of the slow dimer excision in the mfd mutant, single-strand DNA breaks do not accumulate during postirradiation incubation, implying that incision and excision are well corrdinated. the prolonged postirradiation lag in cell division and DNA synthesis which accompany slow excision in the mfd strain indicates that resumption of these processes of optimal rates is linked to the timing of excision repair. The normal uv-resistance of the mfd mutant also suggests such coordination and shows that the rate of excision repair is independent of its ultimate efficiency in the removal of potentially lethal uv-induced damage. (U.S.)

On the relevance of genotoxicity for fish populations I: effects of a model genotoxicant on zebrafish (Danio rerio) in a complete life-cycle test.

Science.gov (United States)

Diekmann, Markus; Hultsch, Veit; Nagel, Roland

2004-05-28

Genotoxicity may be detected in surface waters by means of various genotoxicity assays. In order to enable an ecotoxicological assessment of the consequences of such genotoxic potential for fish populations, a complete life-cycle test with zebrafish (Danio rerio) and the model genotoxicant 4-nitroquinoline-1-oxide (NQO) was conducted. Zebrafish (f1) were continuously exposed to NQO (i.e. 0.1, 0.3, 1.1, 2.9, and 14.6 microg/l, respectively) from fertilised eggs until sexual maturity. In addition to reproduction studies in the f1-generation, f2-fish were exposed to NQO during the first 6 weeks of development. Up to 2.9 microg/l NQO, fish did not display differences in survival and growth (P < 0.05). A NQO concentration of 14.6 microg/l, however, was lethal. Female fish exposed to all NQO concentrations up to 2.9 microg/l displayed a significant reduction in egg production (P < 0.05). A mathematical simulation revealed that exposure to weak concentrations of NQO is leading to an elevated extinction risk. Copyright 2004 Elsevier B.V.

Silver nanoparticles induce endoplasmatic reticulum stress response in zebrafish

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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

Culturable gut microbiota diversity in zebrafish.

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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

Genetic determinants of hyaloid and retinal vasculature in zebrafish

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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.

Characterization of an MMS sensitive mutant of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Martin, P.S.

1979-01-01

We have characterized a methyl methanesulfonate sensitive mutant of the yeast Saccharomyces cerevisiae in order to learn more about DNA repair and mutagenesis in this organism. The mutation, designated mms3-1, also confers sensitivity to ultraviolet light and to ethyl methanesulfonate in both haploids and homozygous diploids. Its effect on γ-ray sensitivity, however, is a function of the ploidy of the cell and its effect on induced mutation is a function of both the ploidy of the cell and the nature of the inducing agent. Our major findings are discussed. Our data indicate that: (1) Saccharomyces cerevisiae has an error prone pathway for the repair of uv damage controlled by the MMS3 gene product operating in and only in, and possibly induced by conditions present only in, a/α diploids; (2) in diploids, at least, there exists at least one step in the error prone repair of uv induced damage which is different from a step in the error prone repair of EMS induced damage; (3) a/α mms3-1/mms3-1 diploids may be defective in a step common to the repair of mutagenic lesions following uv irradiation and lethal lesions following γ irradiation; and (4) there are steps in the repair of MMS induced lethal damage that are different from steps in the repair of EMS induced lethal damage

Toxicity assessment of zebrafish following exposure to CdTe QDs

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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.

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)

Impaired Eye-Blink Conditioning in waggler, a Mutant Mouse With Cerebellar BDNF Deficiency

OpenAIRE

Bao, Shaowen; Chen, Lu; Qiao, Xiaoxi; Knusel, Beat; Thompson, Richard F.

1998-01-01

In addition to their trophic functions, neurotrophins are also implicated in synaptic modulation and learning and memory. Although gene knockout techniques have been used widely in studying the roles of neurotrophins at molecular and cellular levels, behavioral studies using neurotrophin knockouts are limited by the early-onset lethality and various sensory deficits associated with the gene knockout mice. In the present study, we found that in a spontaneous mutant mouse, waggler, the expressi...

Suicide Lethality: A Concept Analysis.

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DeBastiani, Summer; De Santis, Joseph P

2018-02-01

Suicide is a significant health problem internationally. Those who complete suicide may have different behaviors and risk factors than those who attempt a non-fatal suicide. The purpose of this article is to analyze the concept of suicide lethality and propose a clear definition of the concept through the identification of antecedents, attributes, and consequences. A literature search for articles published in the English language between 1970 and 2016 was conducted using MEDLINE, the Cochrane Library, Pubmed, Psychlit, Ovid, PsycINFO, and Proquest. The bibliographies of all included studies were also reviewed to identify additional relevant citations. A concept analysis was conducted on the literature findings using six stages of Walker and Avant's method. The concept analysis differentiated between suicide, lethality, suicidal behavior, and suicide lethality. Presence of a suicide plan or a written suicide note was not found to be associated with the majority of completed suicides included in the definition of suicide lethality. There are a few scales that measure the lethality of a suicide attempt, but none that attempt to measure the concept of suicide lethality as described in this analysis. Clarifying the concept of suicide lethality encourages awareness of the possibility of different suicidal behaviors associated with different suicide outcomes and will inform the development of future nursing interventions. A clearer definition of the concept of suicide lethality will guide clinical practice, research, and policy development aimed at suicide prevention.

Myomaker mediates fusion of fast myocytes in zebrafish embryos

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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.

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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.

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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.

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.

Phenotypic and epistatic grouping of hypo- and hyper-rec mus mutants in Aspergillus.

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Kafer, E; Chae, S K

1994-03-01

The mutants musK to musS of Aspergillus nidulans are sensitive to methyl-methanesulfonate (MMS) and several of them are meiotic-defective and alter mitotic recombination frequencies. All were found to be cross-sensitive to 4-nitro-quinoline-N-oxide (4-NQO) but unexpectedly none of them was hypersensitive to gamma-rays and few to UV light. Double mus; uvs mutants were constructed to test for interactions with uvs mutations of the four epistatic groups of Aspergillus, "UvsF", "UvsC", "UvsI", and "UvsB". All meiotic-defective mus mutations caused some lethal interactions, usually with uvsF. None of them showed epistasis with UvsF or UvsB group mutants and one, musO, may represent a new group. Three mus mutations that affect recombination were assigned to the UvsC group, namely musN and K, and also musL which is recombination-defective and closely resembles uvsC. While uvsC mutants are mutators and lack UV-mutagenesis, most mus mutants had no effects on mutation. Only musR, which appeared epistatic with uvsI, showed reduced UV-reversion frequencies similar to uvsI. The recombination-proficient mus mutants appeared to be epistatic with more than one group, but in several cases sensitivities were slight and overlaps insufficient to obtain corroborating results with MMS and 4-NQO.

Zebrafish Axenic Larvae Colonization with Human Intestinal Microbiota.

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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.

Saccharomyces cerevisiae mutants with enhanced induced mutation and altered mitotic gene conversion.

Science.gov (United States)

Ivanov, E L; Kovaltzova, S V; Korolev, V G

1989-08-01

We have developed a method to isolate yeast (Saccharomyces cerevisiae) mutants with enhanced induced mutagenesis based on nitrous acid-induced reversion of the ade2-42 allele. Six mutants have been isolated and designated him (high induced mutagenesis), and 4 of them were studied in more detail. The him mutants displayed enhanced reversion of the ade2-42 allele, either spontaneous or induced by nitrous acid, UV light, and the base analog 6-N-hydroxylaminopurine, but not by gamma-irradiation. It is worth noting that the him mutants turned out not to be sensitive to the lethal effects of the mutagens used. The enhancement in mutation induced by nitrous acid, UV light, and 6-N-hydroxylaminopurine has been confirmed in a forward-mutation assay (induction of mutations in the ADE1, ADE2 genes). The latter agent revealed the most apparent differences between the him mutants and the wild-type strain and was, therefore, chosen for the genetic analysis of mutants, him mutations analyzed behaved as a single Mendelian trait; complementation tests indicated 3 complementation groups (HIM1, HIM2, and HIM3), each containing 1 mutant allele. Uracil-DNA glycosylase activity was determined in crude cell extracts, and no significant differences between the wild-type and him strains were detected. Spontaneous mitotic gene conversion at the ADE2 locus is altered in him1 strains, either increased or decreased, depending on the particular heteroallelic combination. Genetic evidence strongly suggests him mutations to be involved in a process of mismatch correction of molecular heteroduplexes.

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

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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.

Patterning and gastrulation defects caused by the tw18 lethal are due to loss of Ppp2r1a

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Lisette Lange

2017-06-01

Full Text Available The mouse t haplotype, a variant 20 cM genomic region on Chromosome 17, harbors 16 embryonic control genes identified by recessive lethal mutations isolated from wild mouse populations. Due to technical constraints so far only one of these, the tw5 lethal, has been cloned and molecularly characterized. Here we report the molecular isolation of the tw18 lethal. Embryos carrying the tw18 lethal die from major gastrulation defects commencing with primitive streak formation at E6.5. We have used transcriptome and marker gene analyses to describe the molecular etiology of the tw18 phenotype. We show that both WNT and Nodal signal transduction are impaired in the mutant epiblast, causing embryonic patterning defects and failure of primitive streak and mesoderm formation. By using a candidate gene approach, gene knockout by homologous recombination and genetic rescue, we have identified the gene causing the tw18 phenotype as Ppp2r1a, encoding the PP2A scaffolding subunit PR65alpha. Our work highlights the importance of phosphatase 2A in embryonic patterning, primitive streak formation, gastrulation, and mesoderm formation downstream of WNT and Nodal signaling.

The zebrafish maternal-effect gene cellular atoll encodes the centriolar component sas-6 and defects in its paternal function promote whole genome duplication.

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Yabe, Taijiro; Ge, Xiaoyan; Pelegri, Francisco

2007-12-01

A female-sterile zebrafish maternal-effect mutation in cellular atoll (cea) results in defects in the initiation of cell division starting at the second cell division cycle. This phenomenon is caused by defects in centrosome duplication, which in turn affect the formation of a bipolar spindle. We show that cea encodes the centriolar coiled-coil protein Sas-6, and that zebrafish Cea/Sas-6 protein localizes to centrosomes. cea also has a genetic paternal contribution, which when mutated results in an arrested first cell division followed by normal cleavage. Our data supports the idea that, in zebrafish, paternally inherited centrosomes are required for the first cell division while maternally derived factors are required for centrosomal duplication and cell divisions in subsequent cell cycles. DNA synthesis ensues in the absence of centrosome duplication, and the one-cycle delay in the first cell division caused by cea mutant sperm leads to whole genome duplication. We discuss the potential implications of these findings with regards to the origin of polyploidization in animal species. In addition, the uncoupling of developmental time and cell division count caused by the cea mutation suggests the presence of a time window, normally corresponding to the first two cell cycles, which is permissive for germ plasm recruitment.

Immobilization of Dystrophin and Laminin α2-Chain Deficient Zebrafish Larvae In Vivo Prevents the Development of Muscular Dystrophy.

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Mei Li

Full Text Available Muscular dystrophies are often caused by genetic alterations in the dystrophin-dystroglycan complex or its extracellular ligands. These structures are associated with the cell membrane and provide mechanical links between the cytoskeleton and the matrix. Mechanical stress is considered a pathological mechanism and muscle immobilization has been shown to be beneficial in some mouse models of muscular dystrophy. The zebrafish enables novel and less complex models to examine the effects of extended immobilization or muscle relaxation in vivo in different dystrophy models. We have examined effects of immobilization in larvae from two zebrafish strains with muscular dystrophy, the Sapje dystrophin-deficient and the Candyfloss laminin α2-chain-deficient strains. Larvae (4 days post fertilization, dpf of both mutants have significantly lower active force in vitro, alterations in the muscle structure with gaps between muscle fibers and altered birefringence patterns compared to their normal siblings. Complete immobilization (18 hrs to 4 dpf was achieved using a small molecular inhibitor of actin-myosin interaction (BTS, 50 μM. This treatment resulted in a significantly weaker active contraction at 4 dpf in both mutated larvae and normal siblings, most likely reflecting a general effect of immobilization on myofibrillogenesis. The immobilization also significantly reduced the structural damage in the mutated strains, showing that muscle activity is an important pathological mechanism. Following one-day washout of BTS, muscle tension partly recovered in the Candyfloss siblings and caused structural damage in these mutants, indicating activity-induced muscle recovery and damage, respectively.

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

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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.

A review of monoaminergic neuropsychopharmacology in zebrafish.

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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....

Counteracting quasispecies adaptability: extinction of a ribavirin-resistant virus mutant by an alternative mutagenic treatment.

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Celia Perales

Full Text Available BACKGROUND: Lethal mutagenesis, or virus extinction promoted by mutagen-induced elevation of mutation rates of viruses, may meet with the problem of selection of mutagen-resistant variants, as extensively documented for standard, non-mutagenic antiviral inhibitors. Previously, we characterized a mutant of foot-and-mouth disease virus that included in its RNA-dependent RNA polymerase replacement M296I that decreased the sensitivity of the virus to the mutagenic nucleoside analogue ribavirin. METHODOLOGY AND PRINCIPAL FINDINGS: Replacement M296I in the viral polymerase impedes the extinction of the mutant foot-and-mouth disease virus by elevated concentrations of ribavirin. In contrast, wild type virus was extinguished by the same ribavirin treatment and, interestingly, no mutants resistant to ribavirin were selected from the wild type populations. Decreases of infectivity and viral load of the ribavirin-resistant M296I mutant were attained with a combination of the mutagen 5-fluorouracil and the non-mutagenic inhibitor guanidine hydrocloride. However, extinction was achieved with a sequential treatment, first with ribavirin, and then with a minimal dose of 5-fluorouracil in combination with guanidine hydrochloride. Both, wild type and ribavirin-resistant mutant M296I exhibited equal sensitivity to this combination, indicating that replacement M296I in the polymerase did not confer a significant cross-resistance to 5-fluorouracil. We discuss these results in relation to antiviral designs based on lethal mutagenesis. CONCLUSIONS: (i When dominant in the population, a mutation that confers partial resistance to a mutagenic agent can jeopardize virus extinction by elevated doses of the same mutagen. (ii A wild type virus, subjected to identical high mutagenic treatment, need not select a mutagen-resistant variant, and the population can be extinguished. (iii Extinction of the mutagen-resistant variant can be achieved by a sequential treatment of a

Conserved role of unc-79 in ethanol responses in lightweight mutant mice.

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David J Speca

2010-08-01

Full Text Available The mechanisms by which ethanol and inhaled anesthetics influence the nervous system are poorly understood. Here we describe the positional cloning and characterization of a new mouse mutation isolated in an N-ethyl-N-nitrosourea (ENU forward mutagenesis screen for animals with enhanced locomotor activity. This allele, Lightweight (Lwt, disrupts the homolog of the Caenorhabditis elegans (C. elegans unc-79 gene. While Lwt/Lwt homozygotes are perinatal lethal, Lightweight heterozygotes are dramatically hypersensitive to acute ethanol exposure. Experiments in C. elegans demonstrate a conserved hypersensitivity to ethanol in unc-79 mutants and extend this observation to the related unc-80 mutant and nca-1;nca-2 double mutants. Lightweight heterozygotes also exhibit an altered response to the anesthetic isoflurane, reminiscent of unc-79 invertebrate mutant phenotypes. Consistent with our initial mapping results, Lightweight heterozygotes are mildly hyperactive when exposed to a novel environment and are smaller than wild-type animals. In addition, Lightweight heterozygotes exhibit increased food consumption yet have a leaner body composition. Interestingly, Lightweight heterozygotes voluntarily consume more ethanol than wild-type littermates. The acute hypersensitivity to and increased voluntary consumption of ethanol observed in Lightweight heterozygous mice in combination with the observed hypersensitivity to ethanol in C. elegans unc-79, unc-80, and nca-1;nca-2 double mutants suggests a novel conserved pathway that might influence alcohol-related behaviors in humans.

Evaluation of the toxic effects of brominated compounds (BDE-47, 99, 209, TBBPA) and bisphenol A (BPA) using a zebrafish liver cell line, ZFL

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Yang, Jie; Chan, King Ming, E-mail: kingchan@cuhk.edu.hk

2015-02-15

Highlights: • A homologous zebrafish thyroid hormone (TH) receptor (TR) reporter gene system was developed in a zebrafish liver cell-line (ZFL) to study the possible effects of chemicals on TR activities. • BPA was found to have antagonistic effects on T3 induced TR activity, BDE-47, BDE-99, and TBBPA did not show any interference of TR activity. • Down regulation of deiodinases and some sulfation enzymes or phase II enzymes by the tested chemicals indicated their impacts on TH eleiminations. • The up-regulation of tranthyretin by BDE-47 at 96 h long-term exposure gave a link to the CYP family for its role in producing a more toxic and oxidized form. - Abstract: The toxic effects of three polybrominated diphenyl ether (PBDE) congeners (BDE-47, -99, and -209), tetrabromobisphenol A (TBBPA) and bisphenol A (BPA), were evaluated by determining their 24 h and 96 h median lethal concentrations using a zebrafish liver cell line, ZFL. It was found that BDE-47, BDE-99 and TBBPA showed comparative cytotoxicity within the range of 1.2–4.2 μM, and were more toxic than BPA (367.1 μM at 24 h and 357.6 μM at 96 h). However, BDE-209 induced only 15% lethality with exposures up to 25 μM. The molecular stresses of BDE-47, -99, TBBPA and BPA involved in thyroid hormone (TH) homeostasis and hepatic metabolism were also investigated. Using a reporter gene system to detect zebrafish thyroid hormone receptor β (zfTRβ) transcriptional activity, the median effective concentration of triiodothyronine (T3) was determined to be 9.2 × 10{sup −11} M. BDE-47, BDE-99, TBBPA and BPA alone, however, did not exhibit zfTRβ agonistic activity. BPA displayed T3 (0.1 nM) induced zfTRβ antagonistic activity with a median inhibitory concentration of 19.3 μM. BDE-47, BDE-99 and TBBPA displayed no antagonistic effects of T3-induced zfTRβ activity. Target gene expressions were also examined under acute exposures. The significant inhibition of different types of deiodinases by all of

Cellular androgen content influences enzalutamide agonism of F877L mutant androgen receptor

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Coleman, Daniel J.; Van Hook, Kathryn; King, Carly J.; Schwartzman, Jacob; Lisac, Robert; Urrutia, Joshua; Sehrawat, Archana; Woodward, Josha; Wang, Nicholas J.; Gulati, Roman; Thomas, George V.; Beer, Tomasz M.; Gleave, Martin; Korkola, James E.; Gao, Lina; Heiser, Laura M.; Alumkal, Joshi J.

2016-01-01

Prostate cancer is the most commonly diagnosed and second-most lethal cancer among men in the United States. The vast majority of prostate cancer deaths are due to castration-resistant prostate cancer (CRPC) – the lethal form of the disease that has progressed despite therapies that interfere with activation of androgen receptor (AR) signaling. One emergent resistance mechanism to medical castration is synthesis of intratumoral androgens that activate the AR. This insight led to the development of the AR antagonist enzalutamide. However, resistance to enzalutamide invariably develops, and disease progression is nearly universal. One mechanism of resistance to enzalutamide is an F877L mutation in the AR ligand-binding domain that can convert enzalutamide to an agonist of AR activity. However, mechanisms that contribute to the agonist switch had not been fully clarified, and there were no therapies to block AR F877L. Using cell line models of castration-resistant prostate cancer (CRPC), we determined that cellular androgen content influences enzalutamide agonism of mutant F877L AR. Further, enzalutamide treatment of AR F877L-expressing cell lines recapitulated the effects of androgen activation of F877L AR or wild-type AR. Because the BET bromodomain inhibitor JQ-1 was previously shown to block androgen activation of wild-type AR, we tested JQ-1 in AR F877L-expressing CRPC models. We determined that JQ-1 suppressed androgen or enzalutamide activation of mutant F877L AR and suppressed growth of mutant F877L AR CRPC tumors in vivo, demonstrating a new strategy to treat tumors harboring this mutation. PMID:27276681

Functional inhibition of UQCRB suppresses angiogenesis in zebrafish

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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.

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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

Definitive hematopoietic stem/progenitor cells manifest distinct differentiation output in the zebrafish VDA and PBI.

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Jin, Hao; Sood, Raman; Xu, Jin; Zhen, Fenghua; English, Milton A; Liu, P Paul; Wen, Zilong

2009-02-01

One unique feature of vertebrate definitive hematopoiesis is the ontogenic switching of hematopoietic stem cells from one anatomical compartment or niche to another. In mice, hematopoietic stem cells are believed to originate in the aorta-gonad-mesonephros (AGM), subsequently migrate to the fetal liver (FL) and finally colonize the bone marrow (BM). Yet, the differentiation potential of hematopoietic stem cells within early niches such as the AGM and FL remains incompletely defined. Here, we present in vivo analysis to delineate the differentiation potential of definitive hematopoietic stem/progenitor cells (HSPCs) in the zebrafish AGM and FL analogies, namely the ventral wall of dorsal aorta (VDA) and the posterior blood island (PBI), respectively. Cell fate mapping and analysis of zebrafish runx1(w84x) and vlad tepes (vlt(m651)) mutants revealed that HSPCs in the PBI gave rise to both erythroid and myeloid lineages. However, we surprisingly found that HSPCs in the VDA were not quiescent but were uniquely adapted to generate myeloid but not erythroid lineage cells. We further showed that such distinct differentiation output of HSPCs was, at least in part, ascribed to the different micro-environments present in these two niches. Our results highlight the importance of niche in shaping the differentiation output of developing HSPCs.

Molecular and Chemical Genetic Approaches to Developmental Origins of Aging and Disease in Zebrafish

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Sasaki, Tomoyuki; Kishi, Shuji

2013-01-01

The incidence of diseases increases rapidly with age, accompanied by progressive deteriorations of physiological functions in organisms. Aging-associated diseases are sporadic but mostly inevitable complications arising from senescence. Senescence is often considered the antithesis of early development, but yet there may be factors and mechanisms in common between these two phenomena over the dynamic process of aging. The association between early development and late-onset disease with advancing age is thought to come from a consequence of developmental plasticity, the phenomenon by which one genotype can give rise to a range of physiologically and/or morphologically adaptive states in response to different environmental or genetic perturbations. On the one hand, we hypothesized that the future aging process can be predictive based on adaptivity during the early developmental period. Modulating the thresholds of adaptive plasticity by chemical genetic approaches, we have been investigating whether any relationship exists between the regulatory mechanisms that function in early development and in senescence using the zebrafish (Danio rerio), a small freshwater fish and a useful model animal for genetic studies. We have successfully conducted experiments to isolate zebrafish mutants expressing apparently altered senescence phenotypes during embryogenesis (“embryonic senescence”), subsequently showing shortened lifespan in adulthoods. We anticipate that previously uncharacterized developmental genes may mediate the aging process and play a pivotal role in senescence. On the other hand, unexpected senescence-related genes might also be involved in the early developmental process and regulation. The ease of manipulation using the zebrafish system allows us to conduct an exhaustive exploration of novel genes and small molecular compounds that can be linked to the senescence phenotype, and thereby facilitates searching for the evolutionary and developmental origins

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

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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

Social learning of an associative foraging task in zebrafish

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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.

Plexin A3 and turnout regulate motor axonal branch morphogenesis in zebrafish.

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Rajiv Sainath

Full Text Available During embryogenesis motor axons navigate to their target muscles, where individual motor axons develop complex branch morphologies. The mechanisms that control axonal branching morphogenesis have been studied intensively, yet it still remains unclear when branches begin to form or how branch locations are determined. Live cell imaging of individual zebrafish motor axons reveals that the first axonal branches are generated at the ventral extent of the myotome via bifurcation of the growth cone. Subsequent branches are generated by collateral branching restricted to their synaptic target field along the distal portion of the axon. This precisely timed and spatially restricted branching process is disrupted in turnout mutants we identified in a forward genetic screen. Molecular genetic mapping positioned the turnout mutation within a 300 kb region encompassing eight annotated genes, however sequence analysis of all eight open reading frames failed to unambiguously identify the turnout mutation. Chimeric analysis and single cell labeling reveal that turnout function is required cell non-autonomously for intraspinal motor axon guidance and peripheral branch formation. turnout mutant motor axons form the first branch on time via growth cone bifurcation, but unlike wild-type they form collateral branches precociously, when the growth cone is still navigating towards the ventral myotome. These precocious collateral branches emerge along the proximal region of the axon shaft typically devoid of branches, and they develop into stable, permanent branches. Furthermore, we find that null mutants of the guidance receptor plexin A3 display identical motor axon branching defects, and time lapse analysis reveals that precocious branch formation in turnout and plexin A3 mutants is due to increased stability of otherwise short-lived axonal protrusions. Thus, plexin A3 dependent intrinsic and turnout dependent extrinsic mechanisms suppress collateral branch

MicroCT-based phenomics in the zebrafish skeleton reveals virtues of deep phenotyping in a distributed organ system.

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Hur, Matthew; Gistelinck, Charlotte A; Huber, Philippe; Lee, Jane; Thompson, Marjorie H; Monstad-Rios, Adrian T; Watson, Claire J; McMenamin, Sarah K; Willaert, Andy; Parichy, David M; Coucke, Paul; Kwon, Ronald Y

2017-09-08

Phenomics, which ideally involves in-depth phenotyping at the whole-organism scale, may enhance our functional understanding of genetic variation. Here, we demonstrate methods to profile hundreds of phenotypic measures comprised of morphological and densitometric traits at a large number of sites within the axial skeleton of adult zebrafish. We show the potential for vertebral patterns to confer heightened sensitivity, with similar specificity, in discriminating mutant populations compared to analyzing individual vertebrae in isolation. We identify phenotypes associated with human brittle bone disease and thyroid stimulating hormone receptor hyperactivity. Finally, we develop allometric models and show their potential to aid in the discrimination of mutant phenotypes masked by alterations in growth. Our studies demonstrate virtues of deep phenotyping in a spatially distributed organ system. Analyzing phenotypic patterns may increase productivity in genetic screens, and facilitate the study of genetic variants associated with smaller effect sizes, such as those that underlie complex diseases.

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

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

2018-05-07

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

Exome sequencing for gene discovery in lethal fetal disorders--harnessing the value of extreme phenotypes.

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Filges, Isabel; Friedman, Jan M

2015-10-01

Massively parallel sequencing has revolutionized our understanding of Mendelian disorders, and many novel genes have been discovered to cause disease phenotypes when mutant. At the same time, next-generation sequencing approaches have enabled non-invasive prenatal testing of free fetal DNA in maternal blood. However, little attention has been paid to using whole exome and genome sequencing strategies for gene identification in fetal disorders that are lethal in utero, because they can appear to be sporadic and Mendelian inheritance may be missed. We present challenges and advantages of applying next-generation sequencing approaches to gene discovery in fetal malformation phenotypes and review recent successful discovery approaches. We discuss the implication and significance of recessive inheritance and cross-species phenotyping in fetal lethal conditions. Whole exome sequencing can be used in individual families with undiagnosed lethal congenital anomaly syndromes to discover causal mutations, provided that prior to data analysis, the fetal phenotype can be correlated to a particular developmental pathway in embryogenesis. Cross-species phenotyping allows providing further evidence for causality of discovered variants in genes involved in those extremely rare phenotypes and will increase our knowledge about normal and abnormal human developmental processes. Ultimately, families will benefit from the option of early prenatal diagnosis. © 2014 John Wiley & Sons, Ltd.

Deciphering the Mechanisms of Developmental Disorders (DMDD: a new programme for phenotyping embryonic lethal mice

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Timothy Mohun

2013-05-01

International efforts to test gene function in the mouse by the systematic knockout of each gene are creating many lines in which embryonic development is compromised. These homozygous lethal mutants represent a potential treasure trove for the biomedical community. Developmental biologists could exploit them in their studies of tissue differentiation and organogenesis; for clinical researchers they offer a powerful resource for investigating the origins of developmental diseases that affect newborns. Here, we outline a new programme of research in the UK aiming to kick-start research with embryonic lethal mouse lines. The ‘Deciphering the Mechanisms of Developmental Disorders’ (DMDD programme has the ambitious goal of identifying all embryonic lethal knockout lines made in the UK over the next 5 years, and will use a combination of comprehensive imaging and transcriptomics to identify abnormalities in embryo structure and development. All data will be made freely available, enabling individual researchers to identify lines relevant to their research. The DMDD programme will coordinate its work with similar international efforts through the umbrella of the International Mouse Phenotyping Consortium [see accompanying Special Article (Adams et al., 2013] and, together, these programmes will provide a novel database for embryonic development, linking gene identity with molecular profiles and morphology phenotypes.

Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

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

2015-12-01

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

Comparative proteomics of a tor inducible Aspergillus fumigatus mutant reveals involvement of the Tor kinase in iron regulation.

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Baldin, Clara; Valiante, Vito; Krüger, Thomas; Schafferer, Lukas; Haas, Hubertus; Kniemeyer, Olaf; Brakhage, Axel A

2015-07-01

The Tor (target of rapamycin) kinase is one of the major regulatory nodes in eukaryotes. Here, we analyzed the Tor kinase in Aspergillus fumigatus, which is the most important airborne fungal pathogen of humans. Because deletion of the single tor gene was apparently lethal, we generated a conditional lethal tor mutant by replacing the endogenous tor gene by the inducible xylp-tor gene cassette. By both 2DE and gel-free LC-MS/MS, we found that Tor controls a variety of proteins involved in nutrient sensing, stress response, cell cycle progression, protein biosynthesis and degradation, but also processes in mitochondria, such as respiration and ornithine metabolism, which is required for siderophore formation. qRT-PCR analyses indicated that mRNA levels of ornithine biosynthesis genes were increased under iron limitation. When tor was repressed, iron regulation was lost. In a deletion mutant of the iron regulator HapX also carrying the xylp-tor cassette, the regulation upon iron deprivation was similar to that of the single tor inducible mutant strain. In line, hapX expression was significantly reduced when tor was repressed. Thus, Tor acts either upstream of HapX or independently of HapX as a repressor of the ornithine biosynthesis genes and thereby regulates the production of siderophores. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Ionic channels underlying the ventricular action potential in zebrafish embryo.

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

2014-06-01

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

High magnetic field induced otolith fusion in the zebrafish larvae.

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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.

Excision-repair in mutants of Escherichia coli deficient in DNA polymerase I and/or its associated 5'. -->. 3' exonuclease

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Cooper, P [Stanford Univ., Calif. (USA). Dept. of Biological Sciences

1977-01-01

The UV sensitivity of E.coli mutants deficient in the 5'..-->..3' exonuclease activity of DNA polymerase I is intermediate between that of pol/sup +/ strains and mutants which are deficient in the polymerizing activity of pol I (polA1). Like polA1 mutants, the 5'-econuclease deficient mutants exhibit increased UV-induced DNA degradation and increased repair synthesis compared to a pol/sup +/ strain, although the increase is not as great as in polA1 or in the conditionally lethal mutant BT4113ts deficient in both polymerase I activities. When dimer excision was measured at UV doses low enough to avoid interference from extensive DNA degradation, all three classes of polymerase I deficient mutants were found to remove dimers efficiently from their DNA. We conclude that enzymes alternative to polymerase I can operate in both the excision and resynthesis steps of excision repair and that substitution for either of the polymerase I functions results in longer patches of repair. A model is proposed detailing the possible events in the alternative pathways.

Neutrophil Reverse Migration Becomes Transparent with Zebrafish

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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.

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

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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.

Induction and isolation of DNA transformation mutants in the yeast Saccharomyces cerevisiae

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Hegerich, P.A.; Bruschi, C.V.

1987-01-01

The objective of this research was to induce and isolate mutants of the yeast Saccharomyces cerevisiae which have become transformable by purified plasmid DNA. Non-transformable yeast cells were mutagenized by ultraviolet light using a 65% lethal dose (480 ergs/mm 2 ). After a period of overnight liquid holding recovery, the irradiated cells were subjected to DNA transformation using our CaCl 2 protocol with the multi-marker shuttle plasmid pBB carrying the LEU 2 leucine gene. Following transformation the colonies that grew on selective leucineless medium were identified and subjected to further genetic analysis. From a total of 1 x 10 9 cells the authors have isolated 7 colonies deriving from putative mutants that have acquired the capability to uptake plasmid DNA. The transformants were cured from the plasmid by its mitotic loss on non-selective medium, then re-transformed to verify their genetic competence to give rise to a number of transformants comparable to transformable strains. We have identified and isolated one mutant, coded trs-1, which is able to reproduce a frequency of transformation comparable with the tranformable control. They, therefore, conclude that this mutant is specific for plasmid DNA transformation and that the mutation is mitotically stable

Zebrafish in Toxicology and Environmental Health.

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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

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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

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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

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

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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.

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

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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.

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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

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

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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

Effects of metallothionein on zinc metabolism in lethal-milk mutant mice

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Grider, A. Jr.

1986-01-01

The lethal-milk mice (C57BL/6J-Im) exhibit various pleiotropic effects, including a congenital otolith defect, production of zinc-deficient milk, and clinical signs of a systemic Zn deficiency by one year of age. The clinical signs include alopecia, dermatitis, and skin lesions. The systemic zinc deficiency may be due to increased levels of metallothionein (MT) in the intestine and/or liver of Im mice. The untreated Im mice contain twice as much intestinal MT as do C57BL/6J-(+/sup im//+ /sup Im/) (B6) controls. This was determined by a sulfhydryl assay, by the 109 Cd-saturation/hemolysate method, and by the 65 Zn-binding assay. Various concentrations of Cd or Zn were added to the drinking water three days before assaying for MT. Compared to B6 mice, the Im mice exhibited more MT in their liver by the 65 Zn-MT binding assay (3-fold) and by the 109 Cd-saturation/hemolysate method (18-fold). The effects of the two zinc treatments did not differ significantly between Im and B6 mice. The retention and excretion of 65 Zn (administered intraperitoneally) were determined over a 14-day period, but the results did not different between the Im and B6 mice. The increased concentrations of MT within the Im mice was not significantly different for the intestine and liver. Based on these data and other studies, the Im mice may exhibit alterations in zinc homeostasis due to some deregulation of MT metabolism, including the inner ear of the fetus, the lactating mammary gland, and the intestine and liver of adults by one year of age

Effects of metallothionein on zinc metabolism in lethal-milk mutant mice

Energy Technology Data Exchange (ETDEWEB)

Grider, A. Jr.

1986-01-01

The lethal-milk mice (C57BL/6J-Im) exhibit various pleiotropic effects, including a congenital otolith defect, production of zinc-deficient milk, and clinical signs of a systemic Zn deficiency by one year of age. The clinical signs include alopecia, dermatitis, and skin lesions. The systemic zinc deficiency may be due to increased levels of metallothionein (MT) in the intestine and/or liver of Im mice. The untreated Im mice contain twice as much intestinal MT as do C57BL/6J-(+/sup im//+ /sup Im/) (B6) controls. This was determined by a sulfhydryl assay, by the /sup 109/Cd-saturation/hemolysate method, and by the /sup 65/Zn-binding assay. Various concentrations of Cd or Zn were added to the drinking water three days before assaying for MT. Compared to B6 mice, the Im mice exhibited more MT in their liver by the /sup 65/Zn-MT binding assay (3-fold) and by the /sup 109/Cd-saturation/hemolysate method (18-fold). The effects of the two zinc treatments did not differ significantly between Im and B6 mice. The retention and excretion of /sup 65/Zn (administered intraperitoneally) were determined over a 14-day period, but the results did not different between the Im and B6 mice. The increased concentrations of MT within the Im mice was not significantly different for the intestine and liver. Based on these data and other studies, the Im mice may exhibit alterations in zinc homeostasis due to some deregulation of MT metabolism, including the inner ear of the fetus, the lactating mammary gland, and the intestine and liver of adults by one year of age.

Pharmacological Modulation of Hemodynamics in Adult Zebrafish In Vivo.

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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.

Calreticulin mutants in mice induce an MPL-dependent thrombocytosis with frequent progression to myelofibrosis.

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Marty, Caroline; Pecquet, Christian; Nivarthi, Harini; El-Khoury, Mira; Chachoua, Ilyas; Tulliez, Micheline; Villeval, Jean-Luc; Raslova, Hana; Kralovics, Robert; Constantinescu, Stefan N; Plo, Isabelle; Vainchenker, William

2016-03-10

Frameshift mutations in the calreticulin (CALR) gene are seen in about 30% of essential thrombocythemia and myelofibrosis patients. To address the contribution of the CALR mutants to the pathogenesis of myeloproliferative neoplasms, we engrafted lethally irradiated recipient mice with bone marrow cells transduced with retroviruses expressing these mutants. In contrast to wild-type CALR, CALRdel52 (type I) and, to a lesser extent, CALRins5 (type II) induced thrombocytosis due to a megakaryocyte (MK) hyperplasia. Disease was transplantable into secondary recipients. After 6 months, CALRdel52-, in contrast to rare CALRins5-, transduced mice developed a myelofibrosis associated with a splenomegaly and a marked osteosclerosis. Monitoring of virus-transduced populations indicated that CALRdel52 leads to expansion at earlier stages of hematopoiesis than CALRins5. However, both mutants still specifically amplified the MK lineage and platelet production. Moreover, a mutant deleted of the entire exon 9 (CALRdelex9) did not induce a disease, suggesting that the oncogenic property of CALR mutants was related to the new C-terminus peptide. To understand how the CALR mutants target the MK lineage, we used a cell-line model and demonstrated that the CALR mutants, but not CALRdelex9, specifically activate the thrombopoietin (TPO) receptor (MPL) to induce constitutive activation of Janus kinase 2 and signal transducer and activator of transcription 5/3/1. We confirmed in c-mpl- and tpo-deficient mice that expression of Mpl, but not of Tpo, was essential for the CALR mutants to induce thrombocytosis in vivo, although Tpo contributes to disease penetrance. Thus, CALR mutants are sufficient to induce thrombocytosis through MPL activation. © 2016 by The American Society of Hematology.

The use of mrp1-deficient (Danio rerio) zebrafish embryos to investigate the role of Mrp1 in the toxicity of cadmium chloride and benzo[a]pyrene

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Tian, Jingjing [CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu 215163 (China); Hu, Jia [School of Biology & Basic Medical Sciences, Medical College, Soochow University, Suzhou 215123, Jiangsu (China); Chen, Mingli [CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu 215163 (China); Yin, Huancai [CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu 215163 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Miao, Peng; Bai, Pengli [CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu 215163 (China); Yin, Jian, E-mail: yinj@sibet.ac.cn [CAS Key Lab of Bio-Medical Diagnostics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, Jiangsu 215163 (China)

2017-05-15

Previous studies in our lab have revealed that both P-glycoprotein (Pgp) and multi-resistance associated protein (Mrp) 1 played important roles in the detoxification of heavy metals and polycyclic aromatic hydrocarbon (PAH) in zebrafish embryos. This paper aims to extend this research by using mrp1-deficient model to illustrate the individual function of Mrp1. In this respect, CRISPR/Cas9 system was employed to generate a frameshift mutation in zebrafish mrp1 causing premature translational stops in Mrp1. Significant reduction on the efflux function of Mrps was found in mutant zebrafish embryos, which correlated well with the significantly enhanced accumulation and toxicity of cadmium chloride (CdCl{sub 2}) and benzo[a]pyrene (BαP), indicating the protective role of the corresponding protein. The different alteration on the accumulation and toxicity of Cd{sup 2+} and BαP could be attributed to the fact that Cd{sup 2+} and its metabolites were mainly excreted by Mrp1, while BαP was primarily pumped out by Pgp. More importantly, the compensation mechanism for the absence of Mrp1, including elevated glutathione (GSH) level and up-regulated expression of pgp and mrp2 were also found. Thus, mrp1-deficient zebrafish embryo could be a useful tool in the investigation of Mrp1 functions in the early life stages of aquatic organisms. However, compensation mechanism should be taken into consideration in the interpretation of results obtained with mrp1-deficient fish.

The use of mrp1-deficient (Danio rerio) zebrafish embryos to investigate the role of Mrp1 in the toxicity of cadmium chloride and benzo[a]pyrene

International Nuclear Information System (INIS)

Tian, Jingjing; Hu, Jia; Chen, Mingli; Yin, Huancai; Miao, Peng; Bai, Pengli; Yin, Jian

2017-01-01

Previous studies in our lab have revealed that both P-glycoprotein (Pgp) and multi-resistance associated protein (Mrp) 1 played important roles in the detoxification of heavy metals and polycyclic aromatic hydrocarbon (PAH) in zebrafish embryos. This paper aims to extend this research by using mrp1-deficient model to illustrate the individual function of Mrp1. In this respect, CRISPR/Cas9 system was employed to generate a frameshift mutation in zebrafish mrp1 causing premature translational stops in Mrp1. Significant reduction on the efflux function of Mrps was found in mutant zebrafish embryos, which correlated well with the significantly enhanced accumulation and toxicity of cadmium chloride (CdCl_2) and benzo[a]pyrene (BαP), indicating the protective role of the corresponding protein. The different alteration on the accumulation and toxicity of Cd"2"+ and BαP could be attributed to the fact that Cd"2"+ and its metabolites were mainly excreted by Mrp1, while BαP was primarily pumped out by Pgp. More importantly, the compensation mechanism for the absence of Mrp1, including elevated glutathione (GSH) level and up-regulated expression of pgp and mrp2 were also found. Thus, mrp1-deficient zebrafish embryo could be a useful tool in the investigation of Mrp1 functions in the early life stages of aquatic organisms. However, compensation mechanism should be taken into consideration in the interpretation of results obtained with mrp1-deficient fish.

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

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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.

Effects of particle size and coating on nanoscale Ag and TiO₂ exposure in zebrafish (Danio rerio) embryos.

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Osborne, Olivia J; Johnston, Blair D; Moger, Julian; Balousha, Mohammed; Lead, Jamie R; Kudoh, Tetsuhiro; Tyler, Charles R

2013-12-01

Manufactured metal (oxide) nanoparticles are entering the aquatic environment with little understanding on their potential health impacts for exposed organisms. Adopting an integrative approach, we investigated effects of particle size and coating on biological responses for two of the most commonly used metal (oxide) nanoscale particles, silver (Ag) and titanium dioxide (TiO₂) in zebrafish embryos. Titanium dioxide nanoparticles (nominally, 4 nm, 10 nm, 30 nm and 134 nm) had little or no toxicity on the endpoints measured. Ag both in nano form (10 nm and 35 nm) and its larger counterpart (600-1600 nm) induced dose-dependent lethality and morphological defects, occurring predominantly during gastrula stage. Of the silver material tested 10 nm nanoparticles appeared to be the most toxic. Coating Ag nanoparticles with citrate or fulvic acid decreased toxicity significantly. In situ hybridisation analysis identified the yolk syncytial layer (YSL) as a target tissue for Ag-nano toxicity where there was a significant induction of the heavy metal stress response gene, metallothionein 2 (Mt2) at sub-lethal exposures. Coherent Anti-stroke Raman Scattering (CARS) microscopy provided no evidence for silver particles crossing the chorionic membrane in exposed embryos. Collectively, our data suggest that silver ions play a major role in the toxicity of Ag nanoparticles.

N-Ethylmaleimide–Sensitive Factor b (nsfb) Is Required for Normal Pigmentation of the Zebrafish Retinal Pigment Epithelium

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Hanovice, Nicholas J.; Daly, Christina M. S.; Gross, Jeffrey M.

2015-01-01

Purpose Despite the number of albinism-causing mutations identified in human patients and animal models, there remain a significant number of cases for which no mutation has been identified, suggesting that our understanding of melanogenesis is incomplete. Previously, we identified two oculocutaneous albinism mutations in zebrafish, au13 and au18. Here, we sought to identify the mutated loci and determine how the affected proteins contribute to normal pigmentation of the retinal pigment epithelium (RPE). Methods Complementation analyses revealed that au13 and au18 belonged to a single complementation group, suggesting that they affected the same locus. Whole-genome sequencing and single nucleotide polymorphism (SNP) analysis was performed to identify putative mutations, which were confirmed by cDNA sequencing and mRNA rescue. Transmission electron microscopy (TEM) and image quantification were used to identify the cellular basis of hypopigmentation. Results Whole-genome sequencing and SNP mapping identified a nonsense mutation in the N-ethylmaleimide–sensitive factor b (nsfb) gene in au18 mutants. Complementary DNA sequencing confirmed the presence of the mutation (C893T), which truncates the nsfb protein by roughly two-thirds (Y297X). No coding sequence mutations were identified in au13, but quantitative PCR revealed a significant decrease in nsfb expression, and nsfb mRNA injection rescued the hypopigmentation phenotype, suggesting a regulatory mutation. In situ hybridization revealed that nsfb is broadly expressed during embryonic development, including in the RPE. Transmission electron microscopy analyses indicated that average melanosome density and maturity were significantly decreased in nsfb mutants. Conclusions au18 and au13 contain mutations in nsfb, which encodes a protein that is required for the maturation of melanosomes in zebrafish RPE. PMID:26618645

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.

Knocking Down Snrnp200 Initiates Demorphogenesis of Rod Photoreceptors in Zebrafish

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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.

OECD validation study to assess intra- and inter-laboratory reproducibility of the zebrafish embryo toxicity test for acute aquatic toxicity testing.

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Busquet, François; Strecker, Ruben; Rawlings, Jane M; Belanger, Scott E; Braunbeck, Thomas; Carr, Gregory J; Cenijn, Peter; Fochtman, Przemyslaw; Gourmelon, Anne; Hübler, Nicole; Kleensang, André; Knöbel, Melanie; Kussatz, Carola; Legler, Juliette; Lillicrap, Adam; Martínez-Jerónimo, Fernando; Polleichtner, Christian; Rzodeczko, Helena; Salinas, Edward; Schneider, Katharina E; Scholz, Stefan; van den Brandhof, Evert-Jan; van der Ven, Leo T M; Walter-Rohde, Susanne; Weigt, Stefan; Witters, Hilda; Halder, Marlies

2014-08-01

The OECD validation study of the zebrafish embryo acute toxicity test (ZFET) for acute aquatic toxicity testing evaluated the ZFET reproducibility by testing 20 chemicals at 5 different concentrations in 3 independent runs in at least 3 laboratories. Stock solutions and test concentrations were analytically confirmed for 11 chemicals. Newly fertilised zebrafish eggs (20/concentration and control) were exposed for 96h to chemicals. Four apical endpoints were recorded daily as indicators of acute lethality: coagulation of the embryo, lack of somite formation, non-detachment of the tail bud from the yolk sac and lack of heartbeat. Results (LC50 values for 48/96h exposure) show that the ZFET is a robust method with a good intra- and inter-laboratory reproducibility (CV30%) for some very toxic or volatile chemicals, and chemicals tested close to their limit of solubility. The ZFET is now available as OECD Test Guideline 236. Considering the high predictive capacity of the ZFET demonstrated by Belanger et al. (2013) in their retrospective analysis of acute fish toxicity and fish embryo acute toxicity data, the ZFET is ready to be considered for acute fish toxicity for regulatory purposes. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Staphylococcus aureus β-Toxin Mutants Are Defective in Biofilm Ligase and Sphingomyelinase Activity, and Causation of Infective Endocarditis and Sepsis.

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Herrera, Alfa; Vu, Bao G; Stach, Christopher S; Merriman, Joseph A; Horswill, Alexander R; Salgado-Pabón, Wilmara; Schlievert, Patrick M

2016-05-03

β-Toxin is an important virulence factor of Staphylococcus aureus, contributing to colonization and development of disease [Salgado-Pabon, W., et al. (2014) J. Infect. Dis. 210, 784-792; Huseby, M. J., et al. (2010) Proc. Natl. Acad. Sci. U.S.A. 107, 14407-14412; Katayama, Y., et al. (2013) J. Bacteriol. 195, 1194-1203]. This cytotoxin has two distinct mechanisms of action: sphingomyelinase activity and DNA biofilm ligase activity. However, the distinct mechanism that is most important for its role in infective endocarditis is unknown. We characterized the active site of β-toxin DNA biofilm ligase activity by examining deficiencies in site-directed mutants through in vitro DNA precipitation and biofilm formation assays. Possible conformational changes in mutant structure compared to that of wild-type toxin were assessed preliminarily by trypsin digestion analysis, retention of sphingomyelinase activity, and predicted structures based on the native toxin structure. We addressed the contribution of each mechanism of action to producing infective endocarditis and sepsis in vivo in a rabbit model. The H289N β-toxin mutant, lacking sphingomyelinase activity, exhibited lower sepsis lethality and infective endocarditis vegetation formation compared to those of the wild-type toxin. β-Toxin mutants with disrupted biofilm ligase activity did not exhibit decreased sepsis lethality but were deficient in infective endocarditis vegetation formation compared to the wild-type protein. Our study begins to characterize the DNA biofilm ligase active site of β-toxin and suggests β-toxin functions importantly in infective endocarditis through both of its mechanisms of action.

Disruption of murine mp29/Syf2/Ntc31 gene results in embryonic lethality with aberrant checkpoint response.

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Chia-Hsin Chen

Full Text Available Human p29 is a putative component of spliceosomes, but its role in pre-mRNA is elusive. By siRNA knockdown and stable overexpression, we demonstrated that human p29 is involved in DNA damage response and Fanconi anemia pathway in cultured cells. In this study, we generated p29 knockout mice (mp29(GT/GT using the mp29 gene trap embryonic stem cells to study the role of mp29 in DNA damage response in vivo. Interruption of mp29 at both alleles resulted in embryonic lethality. Embryonic abnormality occurred as early as E6.5 in mp29(GT/GT mice accompanied with decreased mRNA levels of α-tubulin and Chk1. The reduction of α-tubulin and Chk1 mRNAs is likely due to an impaired post-transcriptional event. An aberrant G2/M checkpoint was found in mp29 gene trap embryos when exposed to aphidicolin and UV light. This embryonic lethality was rescued by crossing with mp29 transgenic mice. Additionally, the knockdown of zfp29 in zebrafish resulted in embryonic death at 72 hours of development postfertilization (hpf. A lower level of acetylated α-tubulin was also observed in zfp29 morphants. Together, these results illustrate an indispensable role of mp29 in DNA checkpoint response during embryonic development.

Evaluation of MWNT toxic effects on daphnia and zebrafish embryos

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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).

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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.

Expression of prostaglandin synthases (pgds and pges) during zebrafish gonadal differentiation

DEFF Research Database (Denmark)

Jørgensen, Anne; Nielsen, John E; Nielsen, Betina Frydenlund

2010-01-01

The present study aimed at elucidating whether the expression pattern of the membrane bound form of prostaglandin E2 synthase (pges) and especially the lipocalin-type prostaglandin D2 synthase (pgds) indicates involvement in gonadal sex differentiation in zebrafish as has previously been found....... In this study, a sexually dimorphic expression of pgds was found in gonads of adult zebrafish with expression in testis but not in ovaries. To determine whether the sex-specific expression pattern of pgds was present in gonads of juvenile zebrafish and therefore could be an early marker of sex in zebrafish, we...... microdissected gonads from four randomly selected individual zebrafish for every second day in the period 2-20 days post hatch (dph) and 0-1 dph. The temporal expression of pgds and pges was investigated in the microdissected gonads, however, no differential expression that could indicate sex-specific difference...

TOXICITY EVALUATION OF NEW ENGINEERED NANOMATERIALS IN ZEBRAFISH

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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.

Single and joint toxicity assessment of four currently used pesticides to zebrafish (Danio rerio) using traditional and molecular endpoints.

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Wang, Yanhua; Wu, Shenggan; Chen, Jine; Zhang, Changpeng; Xu, Zhenlan; Li, Gang; Cai, Leiming; Shen, Weifeng; Wang, Qiang

2018-02-01

Pesticides usually present in mixtures in surface waters, although they are traditionally regulated on an individual basis in aquatic ecosystems. In this study, we aimed to investigate the lethal and transcriptional responses of individual and combined pesticides (iprodione, pyrimethanil, pyraclostrobin and acetamiprid) on zebrafish (Danio rerio). Semi-static toxicity test indicated that the greatest toxicity to the four life stages (embryonic, larval, juvenile and adult stages) of D. rerio was detected from pyraclostrobin, followed by iprodione and pyrimethanil. In contrast, the lowest toxicity to the organisms was found from acetamiprid. Most of the selected pesticides exerted greater toxicities to D. rerio of embryonic stage compared with other life stages. Synergistic responses were observed from all binary mixtures of iprodione in combination with pyrimethanil or acetamiprid and ternary mixtures of iprodione+pyraclostrobin in combination with pyrimethanil or acetamiprid. The expressions of 16 genes related to cell apoptosis pathway, oxidative stress response, innate immunity and endocrine disruption at the mRNA level showed that zebrafish embryos were affected by the individual or combined pesticides. The expressions of P53, Tnf, TRβ, Tsh and Cyp19a exhibited greater changes upon exposure to combined pesticides compared with individual pesticides. Taken together, increased toxicity might be triggered by the simultaneous presence of several pesticides in the aquatic environment, which seriously damaged the non-target organisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

A bioenergetic model for zebrafish Danio rerio (Hamilton)

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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.

Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.

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Seiler, Christoph; Gebhart, Nichole; Zhang, Yong; Shinton, Susan A; Li, Yue-sheng; Ross, Nicola L; Liu, Xingjun; Li, Qin; Bilbee, Alison N; Varshney, Gaurav K; LaFave, Matthew C; Burgess, Shawn M; Balciuniene, Jorune; Balciunas, Darius; Hardy, Richard R; Kappes, Dietmar J; Wiest, David L; Rhodes, Jennifer

2015-01-01

Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain) genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP) during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.

Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.

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Christoph Seiler

Full Text Available Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.

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

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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

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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.

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

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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.

The Zebrafish Models to Explore Genetic and Epigenetic Impacts on Evolutionary Developmental Origins of Aging

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Kishi, Shuji

2014-01-01

Can we reset, reprogram, rejuvenate or reverse the organismal aging process? Certain genetic manipulations could at least reset and reprogram epigenetic dynamics beyond phenotypic plasticity and elasticity in cells, which can be further manipulated into organisms. However, in a whole complex aging organism, how can we rejuvenate intrinsic resources and infrastructures in an intact/noninvasive manner? The incidence of diseases increases exponentially with age, accompanied by progressive deteriorations of physiological functions in organisms. Aging-associated diseases are sporadic but essentially inevitable complications arising from senescence. Senescence is often considered the antithesis of early development, but yet there may be factors and mechanisms in common between these two phenomena to rejuvenate over the dynamic process of aging. The association between early development and late-onset disease with advancing age is thought to come from a consequence of developmental plasticity, the phenomenon by which one genotype can give rise to a range of physiologically and/or morphologically adaptive states based on diverse epigenotypes, in response to intrinsic or extrinsic environmental cues and genetic perturbations. We hypothesized that the future aging process can be predictive based on adaptivity during the early developmental period. Modulating the thresholds and windows of plasticity and its robustness by molecular genetic and chemical epigenetic approaches, we have successfully conducted experiments to isolate zebrafish mutants expressing apparently altered senescence phenotypes during their embryonic and/or larval stages (“embryonic/larval senescence”). Subsequently, at least some of these mutant animals were found to show shortened lifespan, while some others would be expected to live longer in adulthoods. We anticipate that previously uncharacterized developmental genes may mediate the aging process and play a pivotal role in senescence. On the other

The Hypocretin/Orexin Neuronal Networks in Zebrafish.

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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.

Graph theoretical model of a sensorimotor connectome in zebrafish.

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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.

Expression of sall4 in taste buds of zebrafish.

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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.

Zebrafish as a Model for the Study of Human Myeloid Malignancies

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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.

Egfl6 is involved in zebrafish notochord development.

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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.

Cyp1a reporter zebrafish reveals target tissues for dioxin

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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.

Depdc5 knockdown causes mTOR-dependent motor hyperactivity in zebrafish.

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de Calbiac, Hortense; Dabacan, Adriana; Marsan, Elise; Tostivint, Hervé; Devienne, Gabrielle; Ishida, Saeko; Leguern, Eric; Baulac, Stéphanie; Muresan, Raul C; Kabashi, Edor; Ciura, Sorana

2018-05-01

DEPDC5 was identified as a major genetic cause of focal epilepsy with deleterious mutations found in a wide range of inherited forms of focal epilepsy, associated with malformation of cortical development in certain cases. Identification of frameshift, truncation, and deletion mutations implicates haploinsufficiency of DEPDC5 in the etiology of focal epilepsy. DEPDC5 is a component of the GATOR1 complex, acting as a negative regulator of mTOR signaling. Zebrafish represents a vertebrate model suitable for genetic analysis and drug screening in epilepsy-related disorders. In this study, we defined the expression of depdc5 during development and established an epilepsy model with reduced Depdc5 expression. Here we report a zebrafish model of Depdc5 loss-of-function that displays a measurable behavioral phenotype, including hyperkinesia, circular swimming, and increased neuronal activity. These phenotypic features persisted throughout embryonic development and were significantly reduced upon treatment with the mTORC1 inhibitor, rapamycin, as well as overexpression of human WT DEPDC5 transcript. No phenotypic rescue was obtained upon expression of epilepsy-associated DEPDC5 mutations (p.Arg487* and p.Arg485Gln), indicating that these mutations cause a loss of function of the protein. This study demonstrates that Depdc5 knockdown leads to early-onset phenotypic features related to motor and neuronal hyperactivity. Restoration of phenotypic features by WT but not epilepsy-associated Depdc5 mutants, as well as by mTORC1 inhibition confirm the role of Depdc5 in the mTORC1-dependent molecular cascades, defining this pathway as a potential therapeutic target for DEPDC5 -inherited forms of focal epilepsy.

Influence of carbon nanotube length on toxicity to zebrafish embryos

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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

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

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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.

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

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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/.

Multimodality optical coherence tomography and fluorescence confocal scanning laser ophthalmoscopy in a zebrafish model of retinal vascular occlusion and remodeling

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Li, Xiaoyue; Spitz, Kathleen; Bozic, Ivan; Tao, Yuankai K.

2018-02-01

Neovascularization in diabetic retinopathy (DR) and age-related macular degeneration (AMD) result in severe vision-loss and are two of the leading causes of blindness. The structural, metabolic, and vascular changes underlying retinal neovascularization are unknown and, thus, there is an unmet need to identify mechanisms of pathogenesis and novel anti-angiogenic therapies. Zebrafish is a robust ophthalmological model because its retina has comparable structure to the human retina and its fecundity and life-cycle enable development of mutant phenotypes of human pathologies. Here, we perform multimodal imaging with OCT and fluorescence confocal scanning laser ophthalmoscopy (cSLO) to identify changes in retinal structure and function in a zebrafish model of vascular leakage. Transgenic zebrafish with EGFP tagged plasma protein were imaged longitudinally at six time points over two weeks to visualize vascular perfusion changes from diethylaminobenzaldehyde (DEAB) treatment. Complementary contrast from OCT-A perfusion maps and cSLO imaging of plasma protein EGFP shows vascular occlusions posttreatment. cSLO images confirm presence of vessels despite loss of OCT-A signal. Plasma protein EGFP contrast also shows significant changes in vessel structure as compared to baseline images. OCT structural volumes show empty vessel cross-sections confirming non-perfusion. In addition, we present algorithms for automated biometric identification of OCT datasets using OCT-A vascular patterns in the presence of significant vascular perfusion changes. These results establish a framework for large-scale in vivo assays to identify novel anti-angiogenic compounds and understand the mechanisms ofneovascularization associated with retinal ocular pathologies.

Identification of a Bacillus subtilis secretion mutant using a ß-galactosidase screening procedure

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Jacobs, Myra F.; Andersen, Jens Bo; Borchert, Torben V.

1995-01-01

High-level synthesis of exportable beta-galactosidase (LacZ) fusion proteins in Bacillus subtilis results in a lethal phenotype, and has been suggested as a tool for the selection of secretion mutants. We tested a plasmid-based, inducible lacZ fusion gene system for this purpose, but frequent...... mutations in cis, which reduced expression of the fusion gene, forced abandonment of the induction-selection strategy. Instead, after modification of the indicator plasmid, a screening procedure for increased basal LacZ activity levels was adopted. This led to the identification of a conditional B. subtilis...

Identification of a Bacillus subtilis secretion mutant using a beta-galactosidase screening procedure

DEFF Research Database (Denmark)

Jacobs, M F; Borchert, T V; Kontinen, V P

1995-01-01

High-level synthesis of exportable beta-galactosidase (LacZ) fusion proteins in Bacillus subtilis results in a lethal phenotype, and has been suggested as a tool for the selection of secretion mutants. We tested a plasmid-based, inducible lacZ fusion gene system for this purpose, but frequent...... mutations in cis, which reduced expression of the fusion gene, forced abandonment of the induction-selection strategy. Instead, after modification of the indicator plasmid, a screening procedure for increased basal LacZ activity levels was adopted. This led to the identification of a conditional B. subtilis...

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

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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

Tributyltin and Zebrafish: Swimming in Dangerous Water

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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

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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.

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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

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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

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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.

Polarization and migration in the zebrafish posterior lateral line system.

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Hildur Knutsdottir

2017-04-01

Full Text Available Collective cell migration plays an important role in development. Here, we study the posterior lateral line primordium (PLLP a group of about 100 cells, destined to form sensory structures, that migrates from head to tail in the zebrafish embryo. We model mutually inhibitory FGF-Wnt signalling network in the PLLP and link tissue subdivision (Wnt receptor and FGF receptor activity domains to receptor-ligand parameters. We then use a 3D cell-based simulation with realistic cell-cell adhesion, interaction forces, and chemotaxis. Our model is able to reproduce experimentally observed motility with leading cells migrating up a gradient of CXCL12a, and trailing (FGF receptor active cells moving actively by chemotaxis towards FGF ligand secreted by the leading cells. The 3D simulation framework, combined with experiments, allows an investigation of the role of cell division, chemotaxis, adhesion, and other parameters on the shape and speed of the PLLP. The 3D model demonstrates reasonable behaviour of control as well as mutant phenotypes.

Glyphosate and Roundup® alter morphology and behavior in zebrafish.

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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

Graph theoretical model of a sensorimotor connectome in zebrafish.

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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.

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

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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.

Zebrafish neurofibromatosis type 1 genes have redundant functions in tumorigenesis and embryonic development

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Jimann Shin

2012-11-01

Neurofibromatosis type 1 (NF1 is a common, dominantly inherited genetic disorder that results from mutations in the neurofibromin 1 (NF1 gene. Affected individuals demonstrate abnormalities in neural-crest-derived tissues that include hyperpigmented skin lesions and benign peripheral nerve sheath tumors. NF1 patients also have a predisposition to malignancies including juvenile myelomonocytic leukemia (JMML, optic glioma, glioblastoma, schwannoma and malignant peripheral nerve sheath tumors (MPNSTs. In an effort to better define the molecular and cellular determinants of NF1 disease pathogenesis in vivo, we employed targeted mutagenesis strategies to generate zebrafish harboring stable germline mutations in nf1a and nf1b, orthologues of NF1. Animals homozygous for loss-of-function alleles of nf1a or nf1b alone are phenotypically normal and viable. Homozygous loss of both alleles in combination generates larval phenotypes that resemble aspects of the human disease and results in larval lethality between 7 and 10 days post fertilization. nf1-null larvae demonstrate significant central and peripheral nervous system defects. These include aberrant proliferation and differentiation of oligodendrocyte progenitor cells (OPCs, dysmorphic myelin sheaths and hyperplasia of Schwann cells. Loss of nf1 contributes to tumorigenesis as demonstrated by an accelerated onset and increased penetrance of high-grade gliomas and MPNSTs in adult nf1a+/−; nf1b−/−; p53e7/e7 animals. nf1-null larvae also demonstrate significant motor and learning defects. Importantly, we identify and quantitatively analyze a novel melanophore phenotype in nf1-null larvae, providing the first animal model of the pathognomonic pigmentation lesions of NF1. Together, these findings support a role for nf1a and nf1b as potent tumor suppressor genes that also function in the development of both central and peripheral glial cells as well as melanophores in zebrafish.

Knockdown of Pnpla6 protein results in motor neuron defects in zebrafish

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Yang Song

2013-03-01

Mutations in patatin-like phospholipase domain containing 6 (PNPLA6, also known as neuropathy target esterase (NTE or SPG39, cause hereditary spastic paraplegia (HSP. Although studies on animal models, including mice and Drosophila, have extended our understanding of PNPLA6, its roles in neural development and in HSP are not clearly understood. Here, we describe the generation of a vertebrate model of PNPLA6 insufficiency using morpholino oligonucleotide knockdown in zebrafish (Danio rerio. Pnpla6 knockdown resulted in developmental abnormalities and motor neuron defects, including axon truncation and branching. The phenotypes in pnpla6 knockdown morphants were rescued by the introduction of wild-type, but not mutant, human PNPLA6 mRNA. Our results also revealed the involvement of BMP signaling in pnpla6 knockdown phenotypes. Taken together, these results demonstrate an important role of PNPLA6 in motor neuron development and implicate overexpression of BMP signaling as a possible mechanism underlying the developmental defects in pnpla6 morphants.

Human cathepsin L rescues the neurodegeneration and lethality incathepsin B/L double deficient mice

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Sevenich, Lisa; Pennacchio, Len A.; Peters, Christoph; Reinheckel, Thomas

2006-01-09

Cathepsin B (CTSB) and cathepsin L (CTSL) are two widelyexpressed cysteine proteases thought to predominantly reside withinlysosomes. Functional analysis of CTSL in humans is complicated by theexistence of two CTSL-like homologues (CTSL and CTSL2), in contrast tomice which contain only one CTSL enzyme. Thus transgenic expression ofhuman CTSL in CTSL deficient mice provides an opportunity to study the invivo functions of this human protease without interference by its highlyrelated homologue. While mice with single gene deficiencies for murineCTSB or CTSL survive without apparent neuromuscular impairment, murineCTSB/CTSL double deficient mice display degeneration of cerebellarPurkinje cells and neurons of the cerebral cortex, resulting in severehypotrophy, motility defects, and lethality during their third to fourthweek of life. Here we show that expression of human CTSL through agenomic transgene results in widespread expression of human CTSL in themouse which is capable of rescuing the lethality found in CTSB/CTSLdouble-deficient animals. Human CTSL is expressed in the brain of thesecompound mutants predominantly in neurons of the cerebral cortex and inPurkinje cells of the cerebellum, where it appears to prevent neuronalcell death.

Analyses in zebrafish embryos reveal that nanotoxicity profiles are dependent on surface-functionalization controlled penetrance of biological membranes.

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Paatero, Ilkka; Casals, Eudald; Niemi, Rasmus; Özliseli, Ezgi; Rosenholm, Jessica M; Sahlgren, Cecilia

2017-08-21

Mesoporous silica nanoparticles (MSNs) are extensively explored as drug delivery systems, but in depth understanding of design-toxicity relationships is still scarce. We used zebrafish (Danio rerio) embryos to study toxicity profiles of differently surface functionalized MSNs. Embryos with the chorion membrane intact, or dechoroniated embryos, were incubated or microinjected with amino (NH 2 -MSNs), polyethyleneimine (PEI-MSNs), succinic acid (SUCC-MSNs) or polyethyleneglycol (PEG-MSNs) functionalized MSNs. Toxicity was assessed by viability and cardiovascular function. NH 2 -MSNs, SUCC-MSNs and PEG-MSNs were well tolerated, 50 µg/ml PEI-MSNs induced 100% lethality 48 hours post fertilization (hpf). Dechoroniated embryos were more sensitive and 10 µg/ml PEI-MSNs reduced viability to 5% at 96hpf. Sensitivity to PEG- and SUCC-, but not NH 2 -MSNs, was also enhanced. Typically cardiovascular toxicity was evident prior to lethality. Confocal microscopy revealed that PEI-MSNs penetrated into the embryos whereas PEG-, NH2- and SUCC-MSNs remained aggregated on the skin surface. Direct exposure of inner organs by microinjecting NH 2 -MSNs and PEI-MSNs demonstrated that the particles displayed similar toxicity indicating that functionalization affects the toxicity profile by influencing penetrance through biological barriers. The data emphasize the need for careful analyses of toxicity mechanisms in relevant models and constitute an important knowledge step towards the development of safer and sustainable nanotherapies.

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

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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.

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

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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.

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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.

Jeast (Saccharomyces cerevisial) mutants with enhanced induced mutagenesis

International Nuclear Information System (INIS)

Ivanov, E.L.; Koval'tsova, S.V.; Korolev, V.G.

1987-01-01

The influence of him1-1, him2-1, him3-1 and himX mutations on induction frequency and specificity of UV-induced adenine-dependent mutations in the yeast Saccharomyces cerevisiae has been. Him mutations do not render haploid cells more sensitive to the lethal action of UV-light; however, in him strains adeine-dependent mutations (ade, ade2) were induced more frequently (1.5-2-fold), as compared to the HIM strain. An analysis of the molecular nature of ade2 mutants revealed than him1-1, him2-1, and himX mutations increase specifically the yield of transitions (AT-GC and GC→AT), whereas in the him3-1, strain the yield of transversions was enhanced as well. We suggest him mutations analysed to affect specific repair pathway for mismatch correction

Factors influencing circadian rhythms in acetaminophen lethality.

Science.gov (United States)

Schnell, R C; Bozigian, H P; Davies, M H; Merrick, B A; Park, K S; McMillan, D A

1984-01-01

Experiments were conducted to examine the effects of changes in lighting schedules and food consumption on circadian rhythms in acetaminophen lethality and hepatic glutathione levels in male mice. Under a normal lighting schedule (light: 06.00-18.00 h), male mice exhibited a circadian rhythm in acetaminophen lethality (peak: 18.00 h; nadir: 06.00, 10.00 h) and an inverse rhythm in hepatic glutathione concentrations (peak: 06.00, 10.00 h; nadir: 18.00 h). Under a reversed lighting schedule (light: 18.00-06.00 h) the glutathione rhythm was reversed and the rhythm in acetaminophen lethality was altered showing greater sensitivity to the drug. Under continuous light, there was a shift in the acetaminophen lethality and the hepatic glutathione rhythms. Under continuous dark, both rhythms were abolished. Under a normal lighting regimen, hepatic glutathione levels were closely correlated with food consumption; i.e., both were increased during the dark phase and decreased during the light phase. Fasting the mice for 12 h abolished the rhythms in acetaminophen lethality and hepatic glutathione levels; moreover, the lethality was increased and the hepatic glutathione levels were decreased. These experiments show that both lighting schedules and feeding can alter the circadian rhythms in acetaminophen lethality and hepatic glutathione levels in male mice.

Connexin 39.9 Protein Is Necessary for Coordinated Activation of Slow-twitch Muscle and Normal Behavior in Zebrafish*

Science.gov (United States)

Hirata, Hiromi; Wen, Hua; Kawakami, Yu; Naganawa, Yuriko; Ogino, Kazutoyo; Yamada, Kenta; Saint-Amant, Louis; Low, Sean E.; Cui, Wilson W.; Zhou, Weibin; Sprague, Shawn M.; Asakawa, Kazuhide; Muto, Akira; Kawakami, Koichi; Kuwada, John Y.

2012-01-01

In many tissues and organs, connexin proteins assemble between neighboring cells to form gap junctions. These gap junctions facilitate direct intercellular communication between adjoining cells, allowing for the transmission of both chemical and electrical signals. In rodents, gap junctions are found in differentiating myoblasts and are important for myogenesis. Although gap junctions were once believed to be absent from differentiated skeletal muscle in mammals, recent studies in teleosts revealed that differentiated muscle does express connexins and is electrically coupled, at least at the larval stage. These findings raised questions regarding the functional significance of gap junctions in differentiated muscle. Our analysis of gap junctions in muscle began with the isolation of a zebrafish motor mutant that displayed weak coiling at day 1 of development, a behavior known to be driven by slow-twitch muscle (slow muscle). We identified a missense mutation in the gene encoding Connexin 39.9. In situ hybridization found connexin 39.9 to be expressed by slow muscle. Paired muscle recordings uncovered that wild-type slow muscles are electrically coupled, whereas mutant slow muscles are not. The further examination of cellular activity revealed aberrant, arrhythmic touch-evoked Ca2+ transients in mutant slow muscle and a reduction in the number of muscle fibers contracting in response to touch in mutants. These results indicate that Connexin 39.9 facilitates the spreading of neuronal inputs, which is irregular during motor development, beyond the muscle cells and that gap junctions play an essential role in the efficient recruitment of slow muscle fibers. PMID:22075003

Distinct functional and temporal requirements for zebrafish Hdac1 during neural crest-derived craniofacial and peripheral neuron development.

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Myron S Ignatius

Full Text Available The regulation of gene expression is accomplished by both genetic and epigenetic means and is required for the precise control of the development of the neural crest. In hdac1(b382 mutants, craniofacial cartilage development is defective in two distinct ways. First, fewer hoxb3a, dlx2 and dlx3-expressing posterior branchial arch precursors are specified and many of those that are consequently undergo apoptosis. Second, in contrast, normal numbers of progenitors are present in the anterior mandibular and hyoid arches, but chondrocyte precursors fail to terminally differentiate. In the peripheral nervous system, there is a disruption of enteric, DRG and sympathetic neuron differentiation in hdac1(b382 mutants compared to wildtype embryos. Specifically, enteric and DRG-precursors differentiate into neurons in the anterior gut and trunk respectively, while enteric and DRG neurons are rarely present in the posterior gut and tail. Sympathetic neuron precursors are specified in hdac1(b382 mutants and they undergo generic neuronal differentiation but fail to undergo noradrenergic differentiation. Using the HDAC inhibitor TSA, we isolated enzyme activity and temporal requirements for HDAC function that reproduce hdac1(b382 defects in craniofacial and sympathetic neuron development. Our study reveals distinct functional and temporal requirements for zebrafish hdac1 during neural crest-derived craniofacial and peripheral neuron development.

Distinct functional and temporal requirements for zebrafish Hdac1 during neural crest-derived craniofacial and peripheral neuron development.

Science.gov (United States)

Ignatius, Myron S; Unal Eroglu, Arife; Malireddy, Smitha; Gallagher, Glen; Nambiar, Roopa M; Henion, Paul D

2013-01-01

The regulation of gene expression is accomplished by both genetic and epigenetic means and is required for the precise control of the development of the neural crest. In hdac1(b382) mutants, craniofacial cartilage development is defective in two distinct ways. First, fewer hoxb3a, dlx2 and dlx3-expressing posterior branchial arch precursors are specified and many of those that are consequently undergo apoptosis. Second, in contrast, normal numbers of progenitors are present in the anterior mandibular and hyoid arches, but chondrocyte precursors fail to terminally differentiate. In the peripheral nervous system, there is a disruption of enteric, DRG and sympathetic neuron differentiation in hdac1(b382) mutants compared to wildtype embryos. Specifically, enteric and DRG-precursors differentiate into neurons in the anterior gut and trunk respectively, while enteric and DRG neurons are rarely present in the posterior gut and tail. Sympathetic neuron precursors are specified in hdac1(b382) mutants and they undergo generic neuronal differentiation but fail to undergo noradrenergic differentiation. Using the HDAC inhibitor TSA, we isolated enzyme activity and temporal requirements for HDAC function that reproduce hdac1(b382) defects in craniofacial and sympathetic neuron development. Our study reveals distinct functional and temporal requirements for zebrafish hdac1 during neural crest-derived craniofacial and peripheral neuron development.

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

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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.

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...

Directed Differentiation of Zebrafish Pluripotent Embryonic Cells to Functional Cardiomyocytes

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Yao Xiao

2016-09-01

Full Text Available A cardiomyocyte differentiation in vitro system from zebrafish embryos remains to be established. Here, we have determined pluripotency window of zebrafish embryos by analyzing their gene-expression patterns of pluripotency factors together with markers of three germ layers, and have found that zebrafish undergoes a very narrow period of pluripotency maintenance from zygotic genome activation to a brief moment after oblong stage. Based on the pluripotency and a combination of appropriate conditions, we established a rapid and efficient method for cardiomyocyte generation in vitro from primary embryonic cells. The induced cardiomyocytes differentiated into functional and specific cardiomyocyte subtypes. Notably, these in vitro generated cardiomyocytes exhibited typical contractile kinetics and electrophysiological features. The system provides a new paradigm of cardiomyocyte differentiation from primary embryonic cells in zebrafish. The technology provides a new platform for the study of heart development and regeneration, in addition to drug discovery, disease modeling, and assessment of cardiotoxic agents.

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

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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.

Effects of Ru(CO)3Cl-glycinate on the developmental toxicities induced by X-ray and carbon-ion irradiation in zebrafish embryos

Energy Technology Data Exchange (ETDEWEB)

Zhou, Rong [Department of Radiation Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Basic Research on Heavy Ion Radiation Application in Medicine, Gansu Province, Lanzhou 730000 (China); Song, Jing’e [School/Hospital of stomatology, Lanzhou University, Lanzhou 730000 (China); Si, Jing [Department of Radiation Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Basic Research on Heavy Ion Radiation Application in Medicine, Gansu Province, Lanzhou 730000 (China); Zhang, Hong, E-mail: zhangh@impcas.ac.cn [Department of Radiation Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Basic Research on Heavy Ion Radiation Application in Medicine, Gansu Province, Lanzhou 730000 (China); Liu, Bin [School/Hospital of stomatology, Lanzhou University, Lanzhou 730000 (China); Gan, Lu; Zhou, Xin [Department of Radiation Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Basic Research on Heavy Ion Radiation Application in Medicine, Gansu Province, Lanzhou 730000 (China); and others

2016-11-15

Highlights: • CORM-3 pretreatment could significantly inhibit the X-ray irradiation-induced developmental toxicity and apoptosis with ROS generation. • CORM-3 pretreatment showed little effect on carbon-ion irradiation-induced developmental toxicity and apoptosis without ROS generation. • CORM-3 could inhibit apoptosis induced by ionizing radiation with low-LET as an effective ROS scavenger. • CORM-3 could suppress apoptosis and DNA damage by inhibiting the activation of P53 and the mitochondrial apoptotic pathway. - Abstract: The inhibitory effects of carbon monoxide (CO), generated by Ru(CO){sub 3}Cl-glycinate [CO-releasing molecule (CORM-3)], on developmental toxicity in zebrafish embryos induced by ionizing radiation with different linear energy transfer (LET) were studied. Zebrafish embryos at 5 h post-fertilization were irradiated with X-ray (low-LET) and carbon-ion (high-LET) with or without pretreatment of CORM-3 1 h before irradiation. CORM-3 pre-treatment showed a significant inhibitory effect on X-ray irradiation-induced developmental toxicity, but had little effect on carbon-ion irradiation-induced developmental toxicity. X-ray irradiation-induced significant increase in ROS levels and cell apoptosis could be modified by CORM-3 pretreatment. However, embryos exposed to carbon-ion irradiation showed significantly increase of cell apoptosis without obvious ROS generation, which could not be attenuated by CORM-3 pretreatment. CORM-3 could inhibit apoptosis induced by ionizing radiation with low-LET as an effective ROS scavenger. The expression of pro-apoptotic genes increased significantly after X-ray irradiation, but increased expression was reduced markedly when CORM-3 was applied before irradiation. Moreover, the protein levels of P53 and γ-H2AX increased markedly after X-ray irradiation, which could be modified by the presence of CORM-3. The protective effect of CORM-3 on X-ray irradiation occurred mainly by suppressing ROS generation and DNA

Acute toxicity and sublethal effects of gallic and pelargonic acids on the zebrafish Danio rerio.

Science.gov (United States)

Techer, Didier; Milla, Sylvain; Fontaine, Pascal; Viot, Sandrine; Thomas, Marielle

2015-04-01

Gallic and pelargonic acids are naturally found in a variety of plants and food products. Despite their extensive use in man-made applications, little is known regarding their potential risks to aquatic vertebrates. The aim of this work was to assess the acute toxicity of these polyphenolic and fatty acid compounds to the zebrafish. In order to get insights into sublethal effects, the enzyme activity of usual biomarkers related to oxidative stress and biotransformation were also assessed in fish. These latter included total superoxide dismutase, catalase as well as total glutathione peroxidase for antioxidant defence mechanisms and glutathione S-transferase for biotransformation related enzyme. Gallic acid was practically non-toxic (96-h lethal concentration (LC50) > 100 mg/L) whereas pelargonic acid was slightly toxic (96-h LC50 of 81.2 mg/L). Moreover, biomarker analyses indicated enhanced superoxide dismutase activity in fish exposed to 20, 40 and 100 mg/L of gallic acid compared to control. A dose-dependent induction of glutathione peroxidase and glutathione S-transferase was reported following gallic acid exposure at the tested concentrations of 10, 20 and 40 mg/L, with the exception of 100 mg/L of substance where basal activity levels were reported. In the case of pelargonic acid, there was no change in antioxidant enzyme activity while an inhibition of glutathione S-transferase was observed from organisms exposed to 45, 58 and 76 mg/L of test solution. The results concerning sublethal effects on biological parameters of zebrafish highlighted thereby the need for further investigations following chronic exposure to both organic acids.

Cofilin-1 inactivation leads to proteinuria--studies in zebrafish, mice and humans.

Directory of Open Access Journals (Sweden)

Sharon Ashworth

Full Text Available BACKGROUND: Podocytes are highly specialized epithelial cells on the visceral side of the glomerulus. Their interdigitating primary and secondary foot processes contain an actin based contractile apparatus that can adjust to changes in the glomerular perfusion pressure. Thus, the dynamic regulation of actin bundles in the foot processes is critical for maintenance of a well functioning glomerular filtration barrier. Since the actin binding protein, cofilin-1, plays a significant role in the regulation of actin dynamics, we examined its role in podocytes to determine the impact of cofilin-1 dysfunction on glomerular filtration. METHODS AND FINDINGS: We evaluated zebrafish pronephros function by dextran clearance and structure by TEM in cofilin-1 morphant and mutant zebrafish and we found that cofilin-1 deficiency led to foot process effacement and proteinuria. In vitro studies in murine and human podocytes revealed that PMA stimulation induced activation of cofilin-1, whereas treatment with TGF-β resulted in cofilin-1 inactivation. Silencing of cofilin-1 led to an accumulation of F-actin fibers and significantly decreased podocyte migration ability. When we analyzed normal and diseased murine and human glomerular tissues to determine cofilin-1 localization and activity in podocytes, we found that in normal kidney tissues unphosphorylated, active cofilin-1 was distributed throughout the cell. However, in glomerular diseases that affect podocytes, cofilin-1 was inactivated by phosphorylation and observed in the nucleus. CONCLUSIONS: Based on these in vitro and in vivo studies we concluded cofilin-1 is an essential regulator for actin filament recycling that is required for the dynamic nature of podocyte foot processes. Therefore, we describe a novel pathomechanism of proteinuria development.

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.

Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter

International Nuclear Information System (INIS)

Wan Haiyan; Korzh, Svitlana; Li Zhen; Mudumana, Sudha Puttur; Korzh, Vladimir; Jiang Yunjin; Lin Shuo; Gong Zhiyuan

2006-01-01

In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development

Contribution of single-strand breaks and alkali-labile bonds to the loss of infectivity of γ-irradiated phiX174 RF-DNA in E. coli cells mutant in various repair functions

International Nuclear Information System (INIS)

McKee, R.H.

1975-01-01

Twenty-one radiation sensitive mutants have been examined for their capacity to support gamma-irradiated phiX174 RF-DNA. The survival of phiX174 RF-DNA was reduced in essentially all of the sensitive mutants. The irradiated phiX174 RF-DNA was then separated into populations containing either single-strand breaks or alkali-labile bonds to examine the capacity of the mutants to repair each of the classes of lesions. It was found that all E. coli strains are unable to repair 22 percent of the single-strand breaks and all sensitive mutants are unable to repair an additional 10 percent of the breaks. All the repair functions examined are involved in single-strand break repair and none are more or less necessary than any of the others. PhiX174 RF-DNA is also inactivated by alkali-labile bonds. In the normal strains the inactivation efficiency is 0.16 lethal events per lesion with a threshold dose of 15 to 20 krads. The mutants are divided into two classes by their sensitivity to alkali-labile bonds. Both classes of mutants are also inactivated by alkali-labile bonds with efficiencies of about 0.17 and 0.29 lethal events per lesion, respectively. It is proposed that the differences seen in survival curves of phiX174 measured in the sensitive mutants is due to this difference. Although in normal cells the efficiency of inactivation of phiX174 by single-strand breaks is 50 percent greater than by alkali-labile bonds, alkali-labile bonds are produced at approximately twice the rate of single-strand breaks so alkali-labile bonds account for about 61 percent of the overall inactivation. In the mutants of least sensitivity alkali-labile bonds account for about 54 percent of the inactivating events and in the most sensitive about 67 percent

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

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

Science.gov (United States)

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

2018-03-01

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

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.

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

International Nuclear Information System (INIS)

Pan, Keyao; Deem, Michael W

2011-01-01

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

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

Science.gov (United States)

Pan, Keyao; Deem, Michael W.

2011-10-01

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

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...

Repairing quite swimmingly: advances in regenerative medicine using zebrafish.

Science.gov (United States)

Goessling, Wolfram; North, Trista E

2014-07-01

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

Analysis of the albino-locus region of the mouse. II. Mosaic mutants

International Nuclear Information System (INIS)

Russell, L.B.

1979-01-01

Among 119 mutations involving the c locus that were recovered in the course of mouse specific-locus experiments with external radiations, 16 were found in mosaic, or fractional, mutants. The number of additional c-locus fractionals that could have occurred in these experiments and, for a variety of reasons, might not have been clearly identified, probably does not exceed the present number. There was no evidence for radiation induction of the fractionals, and even those occurring in the irradiated groups may thus be assumed to be of spontaneous origin. Since only two mutations in the control groups were found in whole-body mutants, it appears that the bulk of spontaneous c-locus mutations are fractionals. None of the mutations recovered in fractional mutants was homozygous lethal; 25% were viable intermediate alleles, and the remainder were albino-like mutants, all viable except for one subvital and one not tested. Genetic tests of the fractionals indicated no major selection against the new mutations, either gametically or in the progeny. For the group of fractionals as a whole, about one-half of the germinal tissue carried the mutation, indicating that the fractionals came from an overall blastomere population that was one-half mutant. Such a population could result from mutation in one strand of the gamete DNA, in a daughter chromosome derived from pronuclear DNA synthesis of the zygote, or in one of the first two blastomeres prior to replication. Since the mouse embryo does not stem from all of the cleavage products of the zygote, the frequency of fractionals observeed underestimates the frequency of mutational events that result in two types of blastomeres

Auxin-dependent microtubule responses and seedling development are affected in a rice mutant resistant to EPC

International Nuclear Information System (INIS)

Nick, P.; Yatou, O.; Furuya, M.; Lambert, A.M.

1994-01-01

Mutants in rice (Oryza sativa L. cv. japonica) were used to study the role of the cytoskeleton in signal-dependent morphogenesis. Mutants obtained by gamma ray irradiation were selected that failed to show inhibition of coleoptile elongation by the anti microtubular drug ethyl-N-phenylcarbamate (EPC). The mutation EPC-Resistant 31 (ER31), isolated from such a screen, caused lethality in putatively homozygous embryos. Heterozygotes exhibited drug resistance, impaired development of crown roots, and characteristic changes in the pattern of cell elongation: cell elongation was enhanced in mesocotyls and leaf sheaths, but inhibited in coleoptiles. The orientation of cortical microtubules changed correspondingly: for etiolated seedlings, compared with the wild-type, they were more transverse with respect to the long cell axis in mesocotyls and leaf sheaths, but more longitudinal in coleoptiles. In mutant coleoptiles, in contrast to wild-type, microtubules did not reorient in response to auxin, and their response to microtubule-eliminating and microtubule-stabilizing drugs was conspicuously reduced. In contrast, they responded normally to other stimuli such as gibberellins or red light. Auxin sensitivity as assayed by the dose-response for callus induction did not show any significant differences between wild-type and mutant. The mutant phenotype is interpreted in terms of an interrupted link between auxin-triggered signal transduction and microtubule reorientation. (author)

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.

Developmental toxicity of thyroid-active compounds in a zebrafish embryotoxicity test

NARCIS (Netherlands)

Jomaa, B.; Hermsen, S.A.B.; Kessels, M.Y.; Berg, van den J.H.J.; Peijenburg, A.C.M.; Aarts, J.M.M.J.G.; Piersma, A.H.; Rietjens, I.

2014-01-01

Zebrafish embryos were exposed to concentration ranges of selected thyroid-active model compounds in order to assess the applicability of zebrafish-based developmental scoring systems within an alternative testing strategy to detect the developmental toxicity of thyroid-active compounds. Model

Sex specific response in cholesterol level in zebrafish (Danio rerio) after long-term exposure of difenoconazole

International Nuclear Information System (INIS)

Mu, Xiyan; Wang, Kai; Chai, Tingting; Zhu, Lizhen; Yang, Yang; Zhang, Jie; Pang, Sen; Wang, Chengju; Li, Xuefeng

2015-01-01

Difenoconazole is a widely used triazole fungicide, its extensive application may potentially cause toxic effects on non-target organisms. To investigate the effect of difenoconazole on cholesterol content and related mechanism, adult zebrafish were exposed to environmental related dosage (0.1, 10 and 500 μg/L) difenoconazole. The body weight and hepatic total cholesterol (TCHO) level was tested at 7, 15 and 30 days post exposure (dpe). The expressions of eight cholesterol synthesis genes and one cholesterol metabolism gene were assessed via Quantitative PCR method. The significant decrease of TCHO level in male zebrafish liver was observed at 15 and 30 dpe, which was accompanied by apparent hepatic cholesterol-genesis genes expression decline. In comparison with males, female zebrafish showed different transcription modification of tested genes, and the cholesterol content maintain normal level during the whole exposure. - Highlights: • Difenoconazle could reduce TCHO level in male zebrafish liver. • Difenoconazole could inhibit sterol-genesis genes expression in male zebrafish. • Female zebrafish didn't show obvious change of TCHO level after exposure. • Difenoconazole could inhibit body weight of both male and female zebrafish. - Difenoconazle could reduce cholesterol level and sterol-genesis genes expression in male zebrafish. While female zebrafish showed no obvious cholesterol content change during exposure

Automated processing of zebrafish imaging data: a survey.

Science.gov (United States)

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

2013-09-01

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

Automated Processing of Zebrafish Imaging Data: A Survey

Science.gov (United States)