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Sample records for directs zebrafish period2

  1. The Zebrafish Period2 Protein Positively Regulates the Circadian Clock through Mediation of Retinoic Acid Receptor (RAR)-related Orphan Receptor α (Rorα)*

    PubMed Central

    Wang, Mingyong; Zhong, Zhaomin; Zhong, Yingbin; Zhang, Wei; Wang, Han

    2015-01-01

    We report the characterization of a null mutant for zebrafish circadian clock gene period2 (per2) generated by transcription activator-like effector nuclease and a positive role of PER2 in vertebrate circadian regulation. Locomotor experiments showed that per2 mutant zebrafish display reduced activities under light-dark and 2-h phase delay under constant darkness, and quantitative real time PCR analyses showed up-regulation of cry1aa, cry1ba, cry1bb, and aanat2 but down-regulation of per1b, per3, and bmal1b in per2 mutant zebrafish, suggesting that Per2 is essential for the zebrafish circadian clock. Luciferase reporter assays demonstrated that Per2 represses aanat2 expression through E-box and enhances bmal1b expression through the Ror/Rev-erb response element, implicating that Per2 plays dual roles in the zebrafish circadian clock. Cell transfection and co-immunoprecipitation assays revealed that Per2 enhances bmal1b expression through binding to orphan nuclear receptor Rorα. The enhancing effect of mouse PER2 on Bmal1 transcription is also mediated by RORα even though it binds to REV-ERBα. Moreover, zebrafish Per2 also appears to have tissue-specific regulatory roles in numerous peripheral organs. These findings help define the essential functions of Per2 in the zebrafish circadian clock and in particular provide strong evidence for a positive role of PER2 in the vertebrate circadian system. PMID:25544291

  2. Direct Visualization of DNA Replication Dynamics in Zebrafish Cells.

    PubMed

    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.

  3. Chemokine guided angiogenesis directs coronary vasculature formation in zebrafish

    PubMed Central

    Harrison, Michael R.M.; Bussmann, Jeroen; Huang, Ying; Zhao, Long; Osorio, Arthela; Burns, C. Geoffrey; Burns, Caroline E.; Sucov, Henry M.; Siekmann, Arndt F.; Lien, Ching-Ling

    2015-01-01

    SUMMARY Interruption of coronary blood supply severely impairs heart function with often-fatal consequences for heart disease patients. However the formation and maturation of these coronary vessels is not fully understood. Here we provide a detailed analysis of coronary vessel development in zebrafish. We observe that coronary vessels form in zebrafish by angiogenic sprouting of arterial cells derived from the endocardium at the atrioventricular canal. Endothelial cells express the CXC-motif chemokine receptor Cxcr4a and migrate to vascularize the ventricle under the guidance of the myocardium-expressed ligand Cxcl12b. cxcr4a mutant zebrafish fail to form a vascular network, whereas ectopic expression of Cxcl12b ligand induces coronary vessel formation. Importantly, cxcr4a mutant zebrafish fail to undergo heart regeneration following injury. Our results suggest that chemokine-signaling has an essential role in coronary vessel formation by directing migration of endocardium-derived endothelial cells. Poorly developed vasculature in cxcr4a mutants likely underlies decreased regenerative potential in adults. PMID:26017769

  4. Direct visualization of replication dynamics in early zebrafish embryos.

    PubMed

    Kuriya, Kenji; Higashiyama, Eriko; Avşar-Ban, Eriko; Okochi, Nanami; Hattori, Kaede; Ogata, Shin; Takebayashi, Shin-Ichiro; Ogata, Masato; Tamaru, Yutaka; Okumura, Katsuzumi

    2016-05-01

    We analyzed DNA replication in early zebrafish embryos. The replicating DNA of whole embryos was labeled with the thymidine analog 5-ethynyl-2'-deoxyuridine (EdU), and spatial regulation of replication sites was visualized in single embryo-derived cells. The results unveiled uncharacterized replication dynamics during zebrafish early embryogenesis.

  5. Strong Static Magnetic Fields Elicit Swimming Behaviors Consistent with Direct Vestibular Stimulation in Adult Zebrafish

    PubMed Central

    Ward, Bryan K.; Tan, Grace X-J; Roberts, Dale C.; Della Santina, Charles C.; Zee, David S.; Carey, John P.

    2014-01-01

    Zebrafish (Danio rerio) offer advantages as model animals for studies of inner ear development, genetics and ototoxicity. However, traditional assessment of vestibular function in this species using the vestibulo-ocular reflex requires agar-immobilization of individual fish and specialized video, which are difficult and labor-intensive. We report that using a static magnetic field to directly stimulate the zebrafish labyrinth results in an efficient, quantitative behavioral assay in free-swimming fish. We recently observed that humans have sustained nystagmus in high strength magnetic fields, and we attributed this observation to magnetohydrodynamic forces acting on the labyrinths. Here, fish were individually introduced into the center of a vertical 11.7T magnetic field bore for 2-minute intervals, and their movements were tracked. To assess for heading preference relative to a magnetic field, fish were also placed in a horizontally oriented 4.7T magnet in infrared (IR) light. A sub-population was tested again in the magnet after gentamicin bath to ablate lateral line hair cell function. Free-swimming adult zebrafish exhibited markedly altered swimming behavior while in strong static magnetic fields, independent of vision or lateral line function. Two-thirds of fish showed increased swimming velocity or consistent looping/rolling behavior throughout exposure to a strong, vertically oriented magnetic field. Fish also demonstrated altered swimming behavior in a strong horizontally oriented field, demonstrating in most cases preferred swimming direction with respect to the field. These findings could be adapted for ‘high-throughput’ investigations of the effects of environmental manipulations as well as for changes that occur during development on vestibular function in zebrafish. PMID:24647586

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

    PubMed Central

    Grama, Abhinav; Engert, Florian

    2012-01-01

    The extraction of the direction of motion is an important computation performed by many sensory systems and in particular, the mechanism by which direction-selective retinal ganglion cells (DS-RGCs) in the retina acquire their selective properties, has been studied extensively. However, whether DS-RGCs simply relay this information to downstream areas or whether additional and potentially de novo processing occurs in these recipient structures is a matter of great interest. Neurons in the larval zebrafish tectum, the largest retino-recipent area in this animal, show direction-selective (DS) responses to moving visual stimuli but how these properties are acquired is still unknown. In order to study this, we first used two-photon calcium imaging to classify the population responses of tectal cells to bars moving at different speeds and in different directions. Subsequently, we performed in vivo whole cell electrophysiology on these DS tectal neurons and we found that their inhibitory inputs were strongly biased toward the null direction of motion, whereas the excitatory inputs showed little selectivity. In addition, we found that excitatory currents evoked by a stimulus moving in the preferred direction occurred before the inhibitory currents whereas a stimulus moving in the null direction evoked currents in the reverse temporal order. The membrane potential modulations resulting from these currents were enhanced by the spike generation mechanism to generate amplified direction selectivity in the spike output. Thus, our results implicate a local inhibitory circuit in generating direction selectivity in tectal neurons. PMID:22969706

  7. The kinematics of directional control in the fast start of zebrafish larvae.

    PubMed

    Nair, Arjun; Azatian, Grigor; McHenry, Matthew J

    2015-12-01

    Larval fish use the 'fast start' escape response to rapidly evade the strike of a predator with a three-dimensional (3D) maneuver. Although this behavior is essential for the survival of fishes, it is not clear how its motion is controlled by the motor system of a larval fish. As a basis for understanding this control, we measured the high-speed kinematics of the body of zebrafish (Danio rerio) larvae when executing the fast start in a variety of directions. We found that the angular excursion in the lateral direction is correlated with the yaw angle in the initial stage of bending (stage 1). In this way, larvae moved in a manner similar to that reported for adult fish. However, larvae also have the ability to control the elevation of a fast start. We found that escapes directed downwards or upwards were achieved by pitching the body throughout the stages of the fast start. Changes in the pitching angle in each stage were significantly correlated with the elevation angle of the trajectory. Therefore, as a larva performs rapid oscillations in yaw that contribute to undulatory motion, the elevation of an escape is generated by more gradual and sustained changes in pitch. These observations are consistent with a model of motor control where elevation is directed through the differential activation of the epaxial and hypaxial musculature. This 3D motion could serve to enhance evasiveness by varying elevation without slowing the escape from a predator.

  8. Effect-directed analysis of Elizabeth River porewater: developmental toxicity in zebrafish (Danio rerio).

    PubMed

    Fang, Mingliang; Getzinger, Gordon J; Cooper, Ellen M; Clark, Bryan W; Garner, Lindsey V T; Di Giulio, Richard T; Ferguson, P Lee; Stapleton, Heather M

    2014-12-01

    In the present study, effect-directed analysis was used to identify teratogenic compounds in porewater collected from a Superfund site along the Elizabeth River estuary (VA, USA). Zebrafish (Danio rerio) exposed to the porewater displayed acute developmental toxicity and cardiac teratogenesis, presumably because of elevated sediment levels of polycyclic aromatic hydrocarbons (PAHs) from historical creosote use. Pretreatment of porewater with several physical and chemical particle removal methods revealed that colloid-bound chemicals constituted the bulk of the observed toxicity. Size-exclusive chromatography and normal-phase high-performance liquid chromatography were used to fractionate Elizabeth River porewater. Acute toxicity of porewater extracts and extract fractions was assessed as the pericardial area in embryonic zebrafish. The most toxic fraction contained several known aryl hydrocarbon receptor (AhR) agonists (e.g., 1,2-benzofluorene and 1,2-benzanthracene) and cytochrome P450 A1 (CPY1A) inhibitors (e.g., dibenzothiophene and fluoranthene). The second most toxic fraction contained known AhR agonists (e.g., benzo[a]pyrene and indeno[1,2,3-cd]pyrene). Addition of a CYP1A inhibitor, fluoranthene, increased toxicity in all active porewater fractions, suggesting synergism between several contaminants present in porewaters. The results indicate that the observed acute toxicity associated with Elizabeth River porewater results from high concentrations of AhR agonistic PAHs and mixture effects related to interactions between compounds co-occurring at the Elizabeth River site. However, even after extensive fractionation and chemical characterization, it remains plausible that some active compounds in Elizabeth River porewater remain unidentified.

  9. EFFECT-DIRECTED ANALYSIS OF ELIZABETH RIVER POREWATER: DEVELOPMENTAL TOXICITY IN ZEBRAFISH (DANIO RERIO)

    PubMed Central

    Fang, Mingliang; Getzinger, Gordon J.; Cooper, Ellen M.; Clark, Bryan W.; Garner, Lindsey V.T.; Di Giulio, Richard T.; Ferguson, P. Lee; Stapleton, Heather M.

    2015-01-01

    In the present study, effect-directed analysis was used to identify teratogenic compounds in porewater collected from a Superfund site along the Elizabeth River estuary (VA, USA). Zebrafish (Danio rerio) exposed to the porewater displayed acute developmental toxicity and cardiac teratogenesis, presumably because of elevated sediment levels of polycyclic aromatic hydrocarbons (PAHs) from historical creosote use. Pretreatment of porewater with several physical and chemical particle removal methods revealed that colloid-bound chemicals constituted the bulk of the observed toxicity. Size-exclusive chromatography and normal-phase high-performance liquid chromatography were used to fractionate Elizabeth River porewater. Acute toxicity of porewater extracts and extract fractions was assessed as the pericardial area in embryonic zebrafish. The most toxic fraction contained several known aryl hydrocarbon receptor (AhR) agonists (e.g., 1,2-benzofluorene and 1,2-benzanthracene) and cytochrome P450 A1 (CPY1A) inhibitors (e.g., dibenzothiophene and fluoranthene). The second most toxic fraction contained known AhR agonists (e.g., benzo[a]pyrene and indeno[1,2,3-cd]pyrene). Addition of a CYP1A inhibitor, fluoranthene, increased toxicity in all active porewater fractions, suggesting synergism between several contaminants present in porewaters. The results indicate that the observed acute toxicity associated with Elizabeth River porewater results from high concentrations of AhR agonistic PAHs and mixture effects related to interactions between compounds co-occurring at the Elizabeth River site. However, even after extensive fractionation and chemical characterization, it remains plausible that some active compounds in Elizabeth River porewater remain unidentified. PMID:25196082

  10. The involvement of Ca2+ and integrins in directional responses of zebrafish keratocytes to electric fields.

    PubMed

    Huang, Ling; Cormie, Peter; Messerli, Mark A; Robinson, Kenneth R

    2009-04-01

    Many cells respond directionally to small DC electrical fields (EFs) by an unknown mechanism, but changes in intracellular Ca(2+) are widely assumed to be involved. We have used zebrafish (Danio rerio) keratocytes in an effort to understand the nature of the EF-cell interaction. We find that the adult zebrafish integument drives substantial currents outward through wounds produced by scale removal, establishing that keratocytes near the wound will experience endogenous EFs. Isolated keratocytes in culture turn toward the cathode in fields as small as 7 mV mm(-1), and the response is independent of cell size. Epidermal sheets are similarly sensitive. The frequency of intracellular Ca(2+) spikes and basal Ca(2+) levels were increased by EFs, but the spikes were not a necessary aspect of migration or EF response. Two-photon imaging failed to detect a pattern of gradients of Ca(2+) across the lamellipodia during normal or EF-induced turning but did detect a sharp, stable Ca(2+) gradient at the junction of the lamellipodium and the cell body. We conclude that gradients of Ca(2+) within the lamellipodium are not required for the EF response. Immunostaining revealed an anode to cathode gradient of integrin beta1 during EF-induced turning, and interference with integrin function attenuated the EF response. Neither electrophoretic redistribution of membrane proteins nor asymmetric perturbations of the membrane potential appear to be involved in the EF response, and we propose a new model in which hydrodynamic forces generated by electro-osmotic water flow mediate EF-cell interactions via effects on focal adhesions.

  11. Direct non-cell autonomous Pax6 activity regulates eye development in the zebrafish

    PubMed Central

    Lesaffre, Brigitte; Joliot, Alain; Prochiantz, Alain; Volovitch, Michel

    2007-01-01

    Background Modifications in Pax6 homeogene expression produce strong eye phenotypes. This suggested to us that eye development might be an appropriate model to verify if homeoprotein intercellular passage has important functions in early development. Similar to other homeoproteins, Pax6 has two domains that enable secretion and internalization by live cells and, thus, intercellular passage. In principle, a straightforward way to test the hypothesis would be to mutate one of the two sequences to produce a 'cell autonomous only' Pax6. However, this was not possible because these sequences are in the homeodomain and their modification would affect Pax6 transcriptional properties. We have thus developed an approach aimed at blocking Pax6 only in the extracellular milieu of developing zebrafish embryos. Results A first strategy was to inject a one-cell embryo with a mRNA encoding a secreted single-chain anti-Pax6 antibody. A second, complementary, strategy was to inject a Pax6 antibody in the blastula extracellular milieu. In both cases, 'dissymmetric eyes', 'one eye only' and 'no eye' phenotypes were produced. In most cases, lens phenotypes paralleled retina malformations. Although eye phenotypes were analyzed 30 hours post-fertilization, there was a strong correlation between early eye field asymmetry, early asymmetry in Pax6 expression and later-occurring eye malformations. Several controls were introduced, demonstrating that the effect is specific to Pax6 and cannot be explained by intracellular antibody activities. Conclusion This study supports the hypothesis that the Pax6 transcription factor is also a signaling molecule with direct non-cell autonomous activity. PMID:17229313

  12. Poised Regeneration of Zebrafish Melanocytes Involves Direct Differentiation and Concurrent Replenishment of Tissue-Resident Progenitor Cells.

    PubMed

    Iyengar, Sharanya; Kasheta, Melissa; Ceol, Craig J

    2015-06-22

    Efficient regeneration following injury is critical for maintaining tissue function and enabling organismal survival. Cells reconstituting damaged tissue are often generated from resident stem or progenitor cells or from cells that have dedifferentiated and become proliferative. While lineage-tracing studies have defined cellular sources of regeneration in many tissues, the process by which these cells execute the regenerative process is largely obscure. Here, we have identified tissue-resident progenitor cells that mediate regeneration of zebrafish stripe melanocytes and defined how these cells reconstitute pigmentation. Nearly all regeneration melanocytes arise through direct differentiation of progenitor cells. Wnt signaling is activated prior to differentiation, and inhibition of Wnt signaling impairs regeneration. Additional progenitors divide symmetrically to sustain the pool of progenitor cells. Combining direct differentiation with symmetric progenitor divisions may serve as a means to rapidly repair injured tissue while preserving the capacity to regenerate.

  13. Rx3 and Shh direct anisotropic growth and specification in the zebrafish tuberal/anterior hypothalamus

    PubMed Central

    Muthu, Victor; Eachus, Helen; Ellis, Pam; Brown, Sarah

    2016-01-01

    In the developing brain, growth and differentiation are intimately linked. Here, we show that in the zebrafish embryo, the homeodomain transcription factor Rx3 coordinates these processes to build the tuberal/anterior hypothalamus. Analysis of rx3 chk mutant/rx3 morphant fish and EdU pulse-chase studies reveal that rx3 is required to select tuberal/anterior hypothalamic progenitors and to orchestrate their anisotropic growth. In the absence of Rx3 function, progenitors accumulate in the third ventricular wall, die or are inappropriately specified, the shh+ anterior recess does not form, and its resident pomc+, ff1b+ and otpb+ Th1+ cells fail to differentiate. Manipulation of Shh signalling shows that Shh coordinates progenitor cell selection and behaviour by acting as an on-off switch for rx3. Together, our studies show that Shh and Rx3 govern formation of a distinct progenitor domain that elaborates patterning through its anisotropic growth and differentiation. PMID:27317806

  14. Zebrafish foxc1a plays a crucial role in early somitogenesis by restricting the expression of aldh1a2 directly.

    PubMed

    Li, Jingyun; Yue, Yunyun; Dong, Xiaohua; Jia, Wenshuang; Li, Kui; Liang, Dong; Dong, Zhangji; Wang, Xiaoxiao; Nan, Xiaoxi; Zhang, Qinxin; Zhao, Qingshun

    2015-04-17

    Foxc1a is a member of the forkhead transcription factors. It plays an essential role in zebrafish somitogenesis. However, little is known about the molecular mechanisms underlying its controlling somitogenesis. To uncover how foxc1a regulates zebrafish somitogenesis, we generated foxc1a knock-out zebrafish using TALEN (transcription activator-like effector nuclease) technology. The foxc1a null embryos exhibited defective somites at early development. Analyses on the expressions of the key genes that control processes of somitogenesis revealed that foxc1a controlled early somitogenesis by regulating the expression of myod1. In the somites of foxc1a knock-out embryos, expressions of fgf8a and deltaC were abolished, whereas the expression of aldh1a2 (responsible for providing retinoic acid signaling) was significantly increased. Once the increased retinoic acid level in the foxc1a null embryos was reduced by knocking down aldh1a2, the reduced expression of myod1 was partially rescued by resuming expressions of fgf8a and deltaC in the somites of the mutant embryos. Moreover, a chromatin immunoprecipitation assay on zebrafish embryos revealed that Foxc1a bound aldh1a2 promoter directly. On the other hand, neither knocking down fgf8a nor inhibiting Notch signaling affected the expression of aldh1a2, although knocking down fgf8a reduced expression of deltaC in the somites of zebrafish embryos at early somitogenesis and vice versa. Taken together, our results demonstrate that foxc1a plays an essential role in early somitogenesis by controlling Fgf and Notch signaling through restricting the expression of aldh1a2 in paraxial mesoderm directly.

  15. Zebrafish hoxd4a Acts Upstream of meis1.1 to Direct Vasculogenesis, Angiogenesis and Hematopoiesis

    PubMed Central

    Amali, Aseervatham Anusha; Sie, Lawrence; Winkler, Christoph; Featherstone, Mark

    2013-01-01

    Mice lacking the 4th-group paralog Hoxd4 display malformations of the anterior vertebral column, but are viable and fertile. Here, we report that zebrafish embryos having decreased function of the orthologous hoxd4a gene manifest striking perturbations in vasculogenesis, angiogenesis and primitive and definitive hematopoiesis. These defects are preceded by reduced expression of the hemangioblast markers scl1, lmo2 and fli1 within the posterior lateral plate mesoderm (PLM) at 13 hours post fertilization (hpf). Epistasis analysis revealed that hoxd4a acts upstream of meis1.1 but downstream of cdx4 as early as the shield stage in ventral-most mesoderm fated to give rise to hemangioblasts, leading us to propose that loss of hoxd4a function disrupts hemangioblast specification. These findings place hoxd4a high in a genetic hierarchy directing hemangioblast formation downstream of cdx1/cdx4 and upstream of meis1.1. An additional consequence of impaired hoxd4a and meis1.1 expression is the deregulation of multiple Hox genes implicated in vasculogenesis and hematopoiesis which may further contribute to the defects described here. Our results add to evidence implicating key roles for Hox genes in their initial phase of expression early in gastrulation. PMID:23554940

  16. Combinatorial activity of Flamingo proteins directs convergence and extension within the early zebrafish embryo via the planar cell polarity pathway.

    PubMed

    Formstone, Caroline J; Mason, Ivor

    2005-06-15

    The seven-transmembrane protocadherin, Flamingo, functions in a number of processes during Drosophila development, including planar cell polarity (PCP). To assess the role(s) of Flamingo1/Celsr1 (Fmi1) during vertebrate embryogenesis we have exploited the zebrafish system, identifying two Fmi1 orthologues (zFmi1a and zFmi1b) and employing morpholinos to induce mis-splicing of zebrafish fmi1 mRNAs, to both imitate mutations identified in Drosophila flamingo and generate novel aberrant Flamingo proteins. We demonstrate that in the zebrafish gastrula, Fmi1 proteins function in concert with each other and with the vertebrate PCP proteins, Wnt11 and Strabismus, to mediate convergence and extension during gastrulation, without altering early dorso-ventral patterning. We show that zebrafish Fmi1a promotes extension of the entire antero-posterior axis of the zebrafish gastrula including prechordal plate and ventral diencephalic precursors. However, while we show that control over axial extension is autonomous, we find that Fmi1a is not required within lateral cells undergoing dorsal convergence.

  17. Microgavage of Zebrafish Larvae

    PubMed Central

    Cocchiaro, Jordan L.; Rawls, John F.

    2013-01-01

    The zebrafish has emerged as a powerful model organism for studying intestinal development1-5, physiology6-11, disease12-16, and host-microbe interactions17-25. Experimental approaches for studying intestinal biology often require the in vivo introduction of selected materials into the lumen of the intestine. In the larval zebrafish model, this is typically accomplished by immersing fish in a solution of the selected material, or by injection through the abdominal wall. Using the immersion method, it is difficult to accurately monitor or control the route or timing of material delivery to the intestine. For this reason, immersion exposure can cause unintended toxicity and other effects on extraintestinal tissues, limiting the potential range of material amounts that can be delivered into the intestine. Also, the amount of material ingested during immersion exposure can vary significantly between individual larvae26. Although these problems are not encountered during direct injection through the abdominal wall, proper injection is difficult and causes tissue damage which could influence experimental results.We introduce a method for microgavage of zebrafish larvae. The goal of this method is to provide a safe, effective, and consistent way to deliver material directly to the lumen of the anterior intestine in larval zebrafish with controlled timing. Microgavage utilizes standard embryo microinjection and stereomicroscopy equipment common to most laboratories that perform zebrafish research. Once fish are properly positioned in methylcellulose, gavage can be performed quickly at a rate of approximately 7-10 fish/ min, and post-gavage survival approaches 100% depending on the gavaged material. We also show that microgavage can permit loading of the intestinal lumen with high concentrations of materials that are lethal to fish when exposed by immersion. To demonstrate the utility of this method, we present a fluorescent dextran microgavage assay that can be used to

  18. Featured organism: Danio rerio, the zebrafish.

    PubMed

    Wixon, J

    2000-09-30

    The zebrafish has long been a favourite model for the study of vertebrate development. Here we provide an overview of the current state of knowledge and resources for the study of this fish, with comments on the future direction of zebrafish genomics from Professor Mark Fishman and Dr Stephen Wilson.

  19. The circadian clock regulates autophagy directly through the nuclear hormone receptor Nr1d1/Rev-erbα and indirectly via Cebpb/(C/ebpβ) in zebrafish

    PubMed Central

    Huang, Guodong; Zhang, Fanmiao; Ye, Qiang; Wang, Han

    2016-01-01

    ABSTRACT Autophagy is a highly conserved intracellular degradation system, and recently was shown to display circadian rhythms in mice. The mechanisms underlying circadian regulation of autophagy, however, are still unclear. Here, we observed that numbers of autophagosomes and autolysosomes exhibit daily rhythms in the zebrafish liver, and cebpb/(c/ebpβ) and various autophagy genes are rhythmically expressed in zebrafish larvae but significantly upregulated in per1b and TALEN-generated nr1d1/rev-erbα mutant fish, indicating that both Per1b and Nr1d1 play critical roles in autophagy rhythms. Luciferase reporter and ChIP assays show that the circadian clock directly regulates autophagy genes through Nr1d1, and also regulates transcription of cebpb through Per1b. We also found that fasting leads to altered expression of both circadian clock genes and autophagy genes in zebrafish adult peripheral organs. Further, transcriptome analysis reveals multiple functions of Nr1d1 in zebrafish. Taken together, these findings provide evidence for how the circadian clock regulates autophagy, imply that nutritional signaling affects both circadian regulation and autophagy activities in peripheral organs, and shed light on how circadian gene mutations act through autophagy to contribute to common metabolic diseases such as obesity. PMID:27171500

  20. The tbx2a/b transcription factors direct pronephros segmentation and corpuscle of Stannius formation in zebrafish.

    PubMed

    Drummond, Bridgette E; Li, Yue; Marra, Amanda N; Cheng, Christina N; Wingert, Rebecca A

    2017-01-01

    The simplified and genetically conserved zebrafish pronephros is an excellent model to examine the cryptic processes of cell fate decisions during the development of nephron segments as well as the origins of associated endocrine cells that comprise the corpuscles of Stannius (CS). Using whole mount in situ hybridization, we found that transcripts of the zebrafish genes t-box 2a (tbx2a) and t-box 2b (tbx2b), which belong to the T-box family of transcription factors, were expressed in the caudal intermediate mesoderm progenitors that give rise to the distal pronephros and CS. Deficiency of tbx2a, tbx2b or both tbx2a/b reduced the size of the distal late (DL) segment, which was accompanied by a proximal convoluted segment (PCT) expansion. Further, tbx2a/b deficiency led to significantly larger CS clusters. These phenotypes were also observed in embryos with the from beyond (fby)(c144) mutation, which encodes a premature stop codon in the tbx2b T-box sequence. Conversely, overexpression of tbx2a and tbx2b in wild-type embryos expanded the DL segment where cells were comingled with the adjacent DE, and also decreased CS cell number, but notably did not alter PCT development-providing independent evidence that tbx2a and tbx2b are each necessary and sufficient to promote DL fate and suppress CS genesis. Epistasis studies indicated that tbx2a acts upstream of tbx2b to regulate the DL and CS fates, and likely has other targets as well. Retinoic acid (RA) addition and inhibition studies revealed that tbx2a and tbx2b are negatively regulated by RA signaling. Interestingly, the CS cell expansion that typifies tbx2a/b deficiency also occurred when blocking Notch signaling with the chemical DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester). Ectopic activation of Notch in Tg(hsp70::Gal4; UAS::NICD)(NICD) embryos led to a reduced CS post heat-shock induction. To further examine the link between the tbx2a/b genes and Notch during CS formation, DAPT

  1. Zebrafish in hematology: sushi or science?

    PubMed Central

    Carradice, Duncan

    2008-01-01

    After a decade of the “modern era” of zebrafish hematology research, what have been their major contributions to hematology and what challenges does the model face? This review argues that, in hematology, zebrafish have demonstrated their suitability, are proving their utility, have supplied timely and novel discoveries, and are poised for further significant contributions. It presents an overview of the anatomy, physiology, and genetics of zebrafish hematopoiesis underpinning their use in hematology research. Whereas reverse genetic techniques enable functional studies of particular genes of interest, forward genetics remains zebrafish's particular strength. Mutants with diverse and interesting hematopoietic defects are emerging from multiple genetic screens. Some mutants model hereditary blood diseases, occasionally leading to disease genes first; others provide insights into developmental hematology. Models of malignant hematologic disorders provide tools for drug-target and pharmaceutics discovery. Numerous transgenic zebrafish with fluorescently marked blood cells enable live-cell imaging of inflammatory responses and host-pathogen interactions previously inaccessible to direct observation in vivo, revealing unexpected aspects of leukocyte behavior. Zebrafish disease models almost uniquely provide a basis for efficient whole animal chemical library screens for new therapeutics. Despite some limitations and challenges, their successes and discovery potential mean that zebrafish are here to stay in hematology research. PMID:18182572

  2. Visually driven chaining of elementary swim patterns into a goal-directed motor sequence: a virtual reality study of zebrafish prey capture.

    PubMed

    Trivedi, Chintan A; Bollmann, Johann H

    2013-01-01

    Prey capture behavior critically depends on rapid processing of sensory input in order to track, approach, and catch the target. When using vision, the nervous system faces the problem of extracting relevant information from a continuous stream of input in order to detect and categorize visible objects as potential prey and to select appropriate motor patterns for approach. For prey capture, many vertebrates exhibit intermittent locomotion, in which discrete motor patterns are chained into a sequence, interrupted by short periods of rest. Here, using high-speed recordings of full-length prey capture sequences performed by freely swimming zebrafish larvae in the presence of a single paramecium, we provide a detailed kinematic analysis of first and subsequent swim bouts during prey capture. Using Fourier analysis, we show that individual swim bouts represent an elementary motor pattern. Changes in orientation are directed toward the target on a graded scale and are implemented by an asymmetric tail bend component superimposed on this basic motor pattern. To further investigate the role of visual feedback on the efficiency and speed of this complex behavior, we developed a closed-loop virtual reality setup in which minimally restrained larvae recapitulated interconnected swim patterns closely resembling those observed during prey capture in freely moving fish. Systematic variation of stimulus properties showed that prey capture is initiated within a narrow range of stimulus size and velocity. Furthermore, variations in the delay and location of swim triggered visual feedback showed that the reaction time of secondary and later swims is shorter for stimuli that appear within a narrow spatio-temporal window following a swim. This suggests that the larva may generate an expectation of stimulus position, which enables accelerated motor sequencing if the expectation is met by appropriate visual feedback.

  3. Sphingosine-1-phosphate receptors regulate individual cell behaviours underlying the directed migration of prechordal plate progenitor cells during zebrafish gastrulation.

    PubMed

    Kai, Masatake; Heisenberg, Carl-Philipp; Tada, Masazumi

    2008-09-01

    During vertebrate gastrulation, cells forming the prechordal plate undergo directed migration as a cohesive cluster. Recent studies revealed that E-cadherin-mediated coherence between these cells plays an important role in effective anterior migration, and that platelet-derived growth factor (Pdgf) appears to act as a guidance cue in this process. However, the mechanisms underlying this process at the individual cell level remain poorly understood. We have identified miles apart (mil) as a suppressor of defective anterior migration of the prospective prechordal plate in silberblick (slb)/wnt11 mutant embryos, in which E-cadherin-mediated coherence of cell movement is reduced. mil encodes Edg5, a sphingosine-1-phosphate (S1P) receptor belonging to a family of five G-protein-coupled receptors (S1PRs). S1P is a lipid signalling molecule that has been implicated in regulating cytoskeletal rearrangements, cell motility and cell adhesion in a variety of cell types. We examined the roles of Mil in anterior migration of prechordal plate progenitor cells and found that, in slb embryos injected with mil-MO, cells migrate with increased motility but decreased directionality, without restoring the coherence of cell migration. This indicates that prechordal plate progenitor cells can migrate effectively as individuals, as well as in a coherent cluster of cells. Moreover, we demonstrate that Mil regulates cell motility and polarisation through Pdgf and its intracellular effecter PI3K, but modulates cell coherence independently of the Pdgf/PI3K pathway, thus co-ordinating cell motility and coherence. These results suggest that the net migration of prechordal plate progenitors is determined by different parameters, including motility, persistence and coherence.

  4. Chemokine-Dependent pH Elevation at the Cell Front Sustains Polarity in Directionally Migrating Zebrafish Germ Cells.

    PubMed

    Tarbashevich, Katsiaryna; Reichman-Fried, Michal; Grimaldi, Cecilia; Raz, Erez

    2015-04-20

    Directional cell migration requires cell polarization with respect to the distribution of the guidance cue. Cell polarization often includes asymmetric distribution of response components as well as elements of the motility machinery. Importantly, the function and regulation of most of these molecules are known to be pH dependent. Intracellular pH gradients were shown to occur in certain cells migrating in vitro, but the functional relevance of such gradients for cell migration and for the response to directional cues, particularly in the intact organism, is currently unknown. In this study, we find that primordial germ cells migrating in the context of the developing embryo respond to the graded distribution of the chemokine Cxcl12 by establishing elevated intracellular pH at the cell front. We provide insight into the mechanisms by which a polar pH distribution contributes to efficient cell migration. Specifically, we show that Carbonic Anhydrase 15b, an enzyme controlling the pH in many cell types, including metastatic cancer cells, is expressed in migrating germ cells and is crucial for establishing and maintaining an asymmetric pH distribution within them. Reducing the level of the protein and thereby erasing the pH elevation at the cell front resulted in abnormal cell migration and impaired arrival at the target. The basis for the disrupted migration is found in the stringent requirement for pH conditions in the cell for regulating contractility, for the polarization of Rac1 activity, and hence for the formation of actin-rich structures at the leading edge of the migrating cells.

  5. Molecular psychiatry of zebrafish

    PubMed Central

    Stewart, Adam Michael; Ullmann, Jeremy F.P.; Norton, William H.J.; Brennan, Caroline H.; Parker, Matthew O.; Gerlai, Robert; Kalueff, Allan V.

    2014-01-01

    Due to their well-characterized neural development and high genetic homology to mammals, zebrafish (Danio rerio) have emerged as a powerful model organism in the field of biological psychiatry. Here, we discuss the molecular psychiatry of zebrafish, and its implications for translational neuroscience research and modeling CNS disorders. In particular, we outline recent genetic and technological developments allowing for in-vivo examinations, high-throughput screening and whole-brain analyses in larval and adult zebrafish. We also summarize the application of these molecular techniques to the understanding of neuropsychiatric disease, outlining the potential of zebrafish for modeling complex brain disorders, including attention-deficit/hyperactivity disorder (ADHD), aggression, post-traumatic stress and substance abuse. Critically evaluating the advantages and limitations of larval and adult fish tests, we suggest that zebrafish models become a rapidly emerging new field in modern biological psychiatry research. PMID:25349164

  6. Electroporation of adult zebrafish.

    PubMed

    Rao, N Madhusudhana; Rambabu, K Murali; Rao, S Harinarayana

    2008-01-01

    We generated transient transgenic zebrafish by applying electrical pulses subsequent to injection of DNA into muscle tissue of 3-6-month old adult zebrafish. Electroporation parameters, such as number of pulses, voltage, and amount of plasmid DNA, were optimized and found that 6 pulses of 40 V/cm at 15 mug/fish increased the luciferase expression by 10-fold compared with those in controls. By measuring the expression of luciferase, in vivo by electroporation in adult zebrafish and in vitro using fish cell line (Xiphophorus xiphidium A2 cells), the strength of three promoters (CMV, human EF-1alpha, and Xenopus EF-1alpha) was compared. Subsequent to electroporation after injecting DNA in the mid region of zebrafish, expression of green fluorescent protein was found far away from the site of injection in the head and the tail sections. Thus, electroporation in adult zebrafish provides a rapid way of testing the behavior of gene sequences in the whole organism.

  7. Detection of Autofluorescent Mycobacterium Chelonae in Living Zebrafish

    PubMed Central

    Whipps, Christopher M.; Moss, Larry G.; Sisk, Dana M.; Murray, Katrina N.; Tobin, David M.

    2014-01-01

    Abstract Mycobacterium chelonae is widespread in aquatic environments and can cause mycobacteriosis with low virulence in zebrafish. The risk of infection in zebrafish is exacerbated in closed-recirculating aquatic systems where rapidly growing mycobacteria can live on biofilms, as well as in zebrafish tissues. We have discovered a method of identifying and visualizing M. chelonae infections in living zebrafish using endogenous autofluorescence. Infected larvae are easily identified and can be excluded from experimental results. Because infection may reduce fertility in zebrafish, the visualization of active infection in contaminated eggs of transparent casper females simplifies screening. Transparent fish are also particularly useful as sentinels that can be examined periodically for the presence of autofluorescence, which can then be tested directly for M. chelonae. PMID:24451037

  8. Analysis of myostatin gene structure, expression and function in zebrafish.

    PubMed

    Xu, Cheng; Wu, Gang; Zohar, Yonathan; Du, Shao-Jun

    2003-11-01

    Myostatin is a member of the TGF-beta family that functions as a negative regulator of skeletal muscle development and growth in mammals. Recently, Myostatin has also been identified in fish; however, its role in fish muscle development and growth remains unknown. We have reported here the isolation and characterization of myostatin genomic gene from zebrafish and analysis of its expression in zebrafish embryos, larvae and adult skeletal muscles. Our data showed that myostatin was weakly expressed in early stage zebrafish embryos, and strongly expressed in swimming larvae, juvenile and skeletal muscles of adult zebrafish. Transient expression analysis revealed that the 1.2 kb zebrafish myostatin 5' flanking sequence could direct green fluorescent protein (GFP) expression predominantly in muscle cells, suggesting that the myostatin 5' flanking sequence contained regulatory elements required for muscle expression. To determine the biological function of Myostatin in fish, we generated a transgenic line that overexpresses the Myostatin prodomain in zebrafish skeletal muscles using a muscle-specific promoter. The Myostatin prodomain could act as a dominant negative and inhibit Myostatin function in skeletal muscles. Transgenic zebrafish expressing the Myostatin prodomain exhibited no significant change in myogenic gene expression and differentiation of slow and fast muscle cells at their embryonic stage. The transgenic fish, however, exhibited an increased number of myofibers in skeletal muscles, but no significant difference in fiber size. Together, these data demonstrate that Myostatin plays an inhibitory role in hyperplastic muscle growth in zebrafish.

  9. Learning from small fry: the zebrafish as a genetic model organism for aquaculture fish species.

    PubMed

    Dahm, Ralf; Geisler, Robert

    2006-01-01

    In recent years, the zebrafish has become one of the most prominent vertebrate model organisms used to study the genetics underlying development, normal body function, and disease. The growing interest in zebrafish research was paralleled by an increase in tools and methods available to study zebrafish. While zebrafish research initially centered on mutagenesis screens (forward genetics), recent years saw the establishment of reverse genetic methods (morpholino knock-down, TILLING). In addition, increasingly sophisticated protocols for generating transgenic zebrafish have been developed and microarrays are now available to characterize gene expression on a near genome-wide scale. The identification of loci underlying specific traits is aided by genetic, physical, and radiation hybrid maps of the zebrafish genome and the zebrafish genome project. As genomic resources for aquacultural species are increasingly being generated, a meaningful interaction between zebrafish and aquacultural research now appears to be possible and beneficial for both sides. In particular, research on nutrition and growth, stress, and disease resistance in the zebrafish can be expected to produce results applicable to aquacultural fish, for example, by improving husbandry and formulated feeds. Forward and reverse genetics approaches in the zebrafish, together with the known conservation of synteny between the species, offer the potential to identify and verify candidate genes for quantitative trait loci (QTLs) to be used in marker-assisted breeding. Moreover, some technologies from the zebrafish field such as TILLING may be directly transferable to aquacultural research and production.

  10. Irf6 directly regulates Klf17 in zebrafish periderm and Klf4 in murine oral epithelium, and dominant-negative KLF4 variants are present in patients with cleft lip and palate

    PubMed Central

    Liu, Huan; Leslie, Elizabeth J.; Jia, Zhonglin; Smith, Tiffany; Eshete, Mekonen; Butali, Azeez; Dunnwald, Martine; Murray, Jeffrey; Cornell, Robert A.

    2016-01-01

    Non-syndromic (NS) cleft lip with or without cleft palate (CL/P) is a common disorder with a strong genetic underpinning. Genome-wide association studies have detected common variants associated with this disorder, but a large portion of the genetic risk for NSCL/P is conferred by unidentified rare sequence variants. Mutations in IRF6 (Interferon Regulatory Factor 6) and GRHL3 (Grainyhead-like 3) cause Van der Woude syndrome, which includes CL/P. Both genes encode members of a regulatory network governing periderm differentiation in model organisms. Here, we report that Krüppel-like factor 17 (Klf17), like Grhl3, acts downstream of Irf6 in this network in zebrafish periderm. Although Klf17 expression is absent from mammalian oral epithelium, a close homologue, Klf4, is expressed in this tissue and is required for the differentiation of epidermis. Chromosome configuration capture and reporter assays indicated that IRF6 directly regulates an oral-epithelium enhancer of KLF4. To test whether rare missense variants of KLF4 contribute risk for NSCL/P, we sequenced KLF4 in approximately 1000 NSCL/P cases and 300 controls. By one statistical test, missense variants of KLF4 as a group were enriched in cases versus controls. Moreover, two patient-derived KLF4 variants disrupted periderm differentiation upon forced expression in zebrafish embryos, suggesting that they have dominant-negative effect. These results indicate that rare NSCL/P risk variants can be found in members of the gene regulatory network governing periderm differentiation. PMID:26692521

  11. Tracking zebrafish larvae in group – Status and perspectives☆

    PubMed Central

    Martineau, Pierre R.; Mourrain, Philippe

    2013-01-01

    Video processing is increasingly becoming a standard procedure in zebrafish behavior investigations as it enables higher research throughput and new or better measures. This trend, fostered by the ever increasing performance-to-price ratio of the required recording and processing equipment, should be expected to continue in the foreseeable future, with video-processing based methods permeating more and more experiments and, as a result, expanding the very role of behavioral studies in zebrafish research. To assess whether the routine video tracking of zebrafish larvae directly in the Petri dish is a capability that can be expected in the near future, the key processing concepts are discussed and illustrated on published zebrafish studies when available or other animals when not. PMID:23707495

  12. Targeted Mutagenesis in Zebrafish Using Customized Zinc Finger Nucleases

    PubMed Central

    Foley, Jonathan E.; Maeder, Morgan L.; Pearlberg, Joseph; Joung, J. Keith; Peterson, Randall T.; Yeh, Jing-Ruey J.

    2009-01-01

    Zebrafish mutants have traditionally been obtained using random mutagenesis or retroviral insertions, methods that cannot be targeted to a specific gene and require laborious gene mapping and sequencing. Recently, we and others have shown that customized zinc finger nucleases (ZFNs) can introduce targeted frame-shift mutations with high efficiency, thereby enabling directed creation of zebrafish gene mutations. Here we describe a detailed protocol for constructing ZFN expression vectors, for generating and introducing ZFN-encoding RNAs into zebrafish embryos, and for identifying ZFN-generated mutations in targeted genomic sites. All of our vectors and methods are compatible with previously described Zinc Finger Consortium reagents for constructing engineered zinc finger arrays. Using these methods, zebrafish founders carrying targeted mutations can be identified within four months. PMID:20010934

  13. Chromatin modification in zebrafish development.

    PubMed

    Cayuso Mas, Jordi; Noël, Emily S; Ober, Elke A

    2011-01-01

    The generation of complex organisms requires that an initial population of cells with identical gene expression profiles can adopt different cell fates during development by progressively diverging transcriptional programs. These programs depend on the binding of transcritional regulators to specific genomic sites, which in turn is controlled by modifications of the chromatin. Chromatin modifications may occur directly upon DNA by methylation of specific nucleotides, or may involve post-translational modification of histones. Local regulation of histone post-translational modifications regionalizes the genome into euchromatic regions, which are more accessible to DNA-binding factors, and condensed heterochromatic regions, inhibiting the binding of such factors. In addition, these modifications may be required in a genome-wide fashion for processes such as DNA replication or chromosome condensation. From an embryologist's point of view chromatin modifications are intensively studied in the context of imprinting and have more recently received increasing attention in understanding the basis of pluripotency and cellular differentiation. Here, we describe recently uncovered roles of chromatin modifications in zebrafish development and regeneration, as well as available resources and commonly used techniques. We provide a general introduction into chromatin modifications and their respective functions with a focus on gene transcription, as well as key aspects of their roles in the early zebrafish embryo, neural development, formation of the digestive system and tissue regeneration.

  14. Mycobacteriosis in Zebrafish Colonies

    PubMed Central

    Whipps, Christopher M.; Lieggi, Christine; Wagner, Robert

    2016-01-01

    Mycobacteriosis, a chronic bacterial infection, has been associated with severe losses in some zebrafish facilities and low-level mortalities and unknown impacts in others. The occurrence of at least six different described species (Mycobacterium abscessus, M. chelonae, M. fortuitum, M. haemophilum, M. marinum, M. peregrinum) from zebrafish complicates diagnosis and control because each species is unique. As a generalization, mycobacteria are often considered opportunists, but M. haemophilum and M. marinum appear to be more virulent. Background genetics of zebrafish and environmental conditions influence the susceptibility of fish and progression of disease, emphasizing the importance of regular monitoring and good husbandry practices. A combined approach to diagnostics is ultimately the most informative, with histology as a first-level screen, polymerase chain reaction for rapid detection and species identification, and culture for strain differentiation. Occurrence of identical strains of Mycobacterium in both fish and biofilms in zebrafish systems suggests transmission can occur when fish feed on infected tissues or tank detritus containing mycobacteria. Within a facility, good husbandry practices and sentinel programs are essential for minimizing the impacts of mycobacteria. In addition, quarantine and screening of animals coming into a facility is important for eliminating the introduction of the more severe pathogens. Elimination of mycobacteria from an aquatic system is likely not feasible because these species readily establish biofilms on surfaces even in extremely low nutrient conditions. Risks associated with each commonly encountered species need to be identified and informed management plans developed. Basic research on the growth characteristics, disinfection, and pathogenesis of zebrafish mycobacteria is critical moving forward. PMID:23382341

  15. An In Vivo Method to Quantify Lymphangiogenesis in Zebrafish

    PubMed Central

    Hoffman, Scott J.; Psaltis, Peter J.; Clark, Karl J.; Spoon, Daniel B.; Chue, Colin D.; Ekker, Stephen C.; Simari, Robert D.

    2012-01-01

    Background Lymphangiogenesis is a highly regulated process involved in the pathogenesis of disease. Current in vivo models to assess lymphangiogenesis are largely unphysiologic. The zebrafish is a powerful model system for studying development, due to its rapid growth and transparency during early stages of life. Identification of a network of trunk lymphatic capillaries in zebrafish provides an opportunity to quantify lymphatic growth in vivo. Methods and Results Late-phase microangiography was used to detect trunk lymphatic capillaries in zebrafish 2- and 3-days post-fertilization. Using this approach, real-time changes in lymphatic capillary development were measured in response to modulators of lymphangiogenesis. Recombinant human vascular endothelial growth factor (VEGF)-C added directly to the zebrafish aqueous environment as well as human endothelial and mouse melanoma cell transplantation resulted in increased lymphatic capillary growth, while morpholino-based knockdown of vegfc and chemical inhibitors of lymphangiogenesis added to the aqueous environment resulted in decreased lymphatic capillary growth. Conclusion Lymphatic capillaries in embryonic and larval zebrafish can be quantified using late-phase microangiography. Human activators and small molecule inhibitors of lymphangiogenesis, as well as transplanted human endothelial and mouse melanoma cells, alter lymphatic capillary development in zebrafish. The ability to rapidly quantify changes in lymphatic growth under physiologic conditions will allow for broad screening of lymphangiogenesis modulators, as well as help define cellular roles and elucidate pathways of lymphatic development. PMID:23028871

  16. Dystrophic muscle improvement in zebrafish via increased heme oxygenase signaling

    PubMed Central

    Kawahara, Genri; Gasperini, Molly J.; Myers, Jennifer A.; Widrick, Jeffrey J.; Eran, Alal; Serafini, Peter R.; Alexander, Matthew S.; Pletcher, Mathew T.; Morris, Carl A.; Kunkel, Louis M.

    2014-01-01

    Duchenne muscular dystrophy (DMD) is caused by a lack of the dystrophin protein and has no effective treatment at present. Zebrafish provide a powerful in vivo tool for high-throughput therapeutic drug screening for the improvement of muscle phenotypes caused by dystrophin deficiency. Using the dystrophin-deficient zebrafish, sapje, we have screened a total of 2640 compounds with known modes of action from three drug libraries to identify modulators of the disease progression. Six compounds that target heme oxygenase signaling were found to rescue the abnormal muscle phenotype in sapje and sapje-like, while upregulating the inducible heme oxygenase 1 (Hmox1) at the protein level. Direct Hmox1 overexpression by injection of zebrafish Hmox1 mRNA into fertilized eggs was found to be sufficient for a dystrophin-independent restoration of normal muscle via an upregulation of cGMP levels. In addition, treatment of mdx5cv mice with the PDE5 inhibitor, sildenafil, which was one of the six drugs impacting the Hmox1 pathway in zebrafish, significantly increased the expression of Hmox1 protein, thus making Hmox1 a novel target for the improvement of dystrophic symptoms. These results demonstrate the translational relevance of our zebrafish model to mammalian models and support the use of zebrafish to screen for new drugs to treat human DMD. The discovery of a small molecule and a specific therapeutic pathway that might mitigate DMD disease progression could lead to significant clinical implications. PMID:24234649

  17. High magnetic field induced otolith fusion in the zebrafish larvae

    PubMed Central

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

    2016-01-01

    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. PMID:27063288

  18. Zebrafish models of cerebrovascular disease.

    PubMed

    Walcott, Brian P; Peterson, Randall T

    2014-04-01

    Perturbations in cerebral blood flow and abnormalities in blood vessel structure are the hallmarks of cerebrovascular disease. While there are many genetic and environmental factors that affect these entities through a heterogeneous group of disease processes, the ultimate final pathologic insult in humans is defined as a stroke, or damage to brain parenchyma. In the case of ischemic stroke, blood fails to reach its target destination whereas in hemorrhagic stroke, extravasation of blood occurs outside of the blood vessel lumen, resulting in direct damage to brain parenchyma. As these acute events can be neurologically devastating, if not fatal, development of novel therapeutics are urgently needed. The zebrafish (Danio rerio) is an attractive model for the study of cerebrovascular disease because of its morphological and physiological similarity to human cerebral vasculature, its ability to be genetically manipulated, and its fecundity allowing for large-scale, phenotype-based screens.

  19. Attraction rules: germ cell migration in zebrafish.

    PubMed

    Raz, Erez; Reichman-Fried, Michal

    2006-08-01

    The migration of zebrafish primordial germ cell towards the region where the gonad develops is guided by the chemokine SDF-1a. Recent studies show that soon after their specification, the cells undergo a series of morphological alterations before they become motile and are able to respond to attractive cues. As migratory cells, primordial germ cells move towards their target while correcting their path upon exiting a cyclic phase in which morphological cell polarity is lost. In the following stages, the cells gather at specific locations and move as cell clusters towards their final target. In all of these stages, zebrafish germ cells respond as individual cells to alterations in the shape of the sdf-1a expression domain, by directed migration towards their target - the position where the gonad develops.

  20. Myristoylation profiling in human cells and zebrafish

    PubMed Central

    Broncel, Malgorzata; Serwa, Remigiusz A; Ciepla, Paulina; Krause, Eberhard; Dallman, Margaret J.; Magee, Anthony I.; Tate, Edward W.

    2015-01-01

    Human cells (HEK 293, HeLa, MCF-7) and zebrafish embryos were metabolically tagged with an alkynyl myristic acid probe, lysed with an SDS buffer and tagged proteomes ligated to multifunctional capture reagents via copper-catalyzed alkyne azide cycloaddition (CuAAC). This allowed for affinity enrichment and high-confidence identification, by delivering direct MS/MS evidence for the modification site, of 87 and 61 co-translationally myristoylated proteins in human cells and zebrafish, respectively. The data have been deposited to ProteomeXchange Consortium (Vizcaíno et al., 2014 Nat. Biotechnol., 32, 223–6) (PXD001863 and PXD001876) and are described in detail in Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic protein lipidation during vertebrate development׳ by Broncel et al., Angew. Chem. Int. Ed. PMID:26217820

  1. Social dominance modulates eavesdropping in zebrafish

    PubMed Central

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

    2015-01-01

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

  2. Zebrafish Models for Dyslipidemia and Atherosclerosis Research

    PubMed Central

    Schlegel, Amnon

    2016-01-01

    Atherosclerotic cardiovascular disease is the leading cause of death. Elevated circulating concentrations of lipids are a central pathogenetic driver of atherosclerosis. While numerous effective therapies for this condition have been developed, there is substantial unmet need for this pandemic illness. Here, I will review nutritional, physiological, genetic, and pathological discoveries in the emerging zebrafish model for studying dyslipidemia and atherosclerosis. The technical and physiological advantages and the pharmacological potential of this organism for discovery and validation of dyslipidemia and atherosclerosis targets are stressed through summary of recent findings. An emerging literature shows that zebrafish, through retention of a cetp ortholog gene and high sensitivity to ingestion of excess cholesterol, rapidly develops hypercholesterolemia, with a pattern of distribution of lipid species in lipoprotein particles similar to humans. Furthermore, recent studies leveraging the optical transparency of zebrafish larvae to monitor the fate of these ingested lipids have provided exciting insights to the development of dyslipidemia and atherosclerosis. Future directions for investigation are considered, with particular attention to the potential for in vivo cell biological study of atherosclerotic plaques. PMID:28018294

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

    PubMed

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

    2014-08-01

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

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

    PubMed

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

    2011-03-15

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

  5. Ozone promotes regeneration by regulating the inflammatory response in zebrafish.

    PubMed

    Hao, Kenan; Li, Yanhao; Feng, Jianyu; Zhang, Wenqing; Zhang, Yiyue; Ma, Ning; Zeng, Qingle; Pang, Huajin; Wang, Chunyan; Xiao, Lijun; He, Xiaofeng

    2015-09-01

    Ozone is thought to advance wound healing by inhibiting inflammation, but the mechanism of this phenomenon has not been determined. Although the zebrafish is often used in regeneration experiments, there has been no report of zebrafish treated with ozonated water. We successfully established a zebrafish model of ozonated water treatment and demonstrate that ozonated water stimulates the regeneration of the zebrafish caudal fin, its mechanism, and time dependence. The growth rate of the caudal fin and the number of neutrophils migrating to the caudal fin wound after resection were higher in the experimental (ozonated) group than in the control group, preliminarily confirming that ozone-promoted regeneration is related to the stimulation of an early inflammatory response by ozone. Ozone modulated the expression of tumor necrosis factor-α (TNF-α) in two ways by regulating interleukin 10 (IL-10) expression. Therefore, ozone promotes tissue regeneration by regulating the inflammatory pathways. This effect of ozone in an experimental zebrafish model is demonstrated for the first time, confirming its promotion of wound healing and the mechanism of its effect in tissue regeneration. These results will open up new directions for ozone and regeneration research.

  6. Nicotinic involvement in memory function in zebrafish.

    PubMed

    Levin, Edward D; Chen, Elaine

    2004-01-01

    Zebrafish are an emerging model for the study of the molecular mechanisms of brain function. To conduct studies of the neural bases of behavior in zebrafish, we must understand the behavioral function of zebrafish and how it is altered by perturbations of brain function. This study determined nicotine actions on memory function in zebrafish. With the methods that we have developed to assess memory in zebrafish using delayed spatial alternation (DSA), we determined the dose effect function of acute nicotine on memory function in zebrafish. As in rodents and primates, low nicotine doses improve memory in zebrafish, while high nicotine doses have diminished effect and can impair memory. This study shows that nicotine affects memory function in zebrafish much like in rats, mice, monkeys and humans. Now, zebrafish can be used to help understand the molecular mechanisms crucial to nicotine effects on memory.

  7. Genome editing using artificial site-specific nucleases in zebrafish.

    PubMed

    Hisano, Yu; Ota, Satoshi; Kawahara, Atsuo

    2014-01-01

    Zebrafish is a model vertebrate suitable for genetic analysis. Forward genetic analysis via chemical mutagenesis screening has established a variety of zebrafish mutants that are defective in various types of organogenesis, and the genes responsible for the individual mutants have been identified from genome mapping. On the other hand, reverse genetic analysis via targeted gene disruption using embryonic stem (ES) cells (e.g., knockout mouse) can uncover gene functions by investigating the phenotypic effects. However, this approach is mostly limited to mice among the vertebrate models because of the difficulty in establishing ES cells. Recently, new gene targeting technologies, such as the transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems, have been developed: that can directly introduce genome modifications at the targeted genomic locus. Here, we summarize these new and powerful genome editing techniques for the study of zebrafish.

  8. Zebrafish: A Model for the Study of Addiction Genetics

    PubMed Central

    Klee, Eric W; Schneider, Henning; Clark, Karl; Cousin, Margot; Ebbert, Jon; Hooten, Michael; Karpyak, Victor; Warner, David; Ekker, Stephen

    2013-01-01

    Drug abuse and dependence are multifaceted disorders with complex genetic underpinnings. Identifying specific genetic correlates is challenging and may be more readily accomplished by defining endophenotypes specific for addictive disorders. Symptoms and syndromes, including acute drug response, consumption, preference, and withdrawal, are potential endophenotypes characterizing addiction that have been investigated using model organisms. We present a review of major genes involved in serotonergic, dopaminergic, GABAergic, and adrenoreceptor signaling that are considered to be directly involved in nicotine, opioid, cannabinoid, and ethanol use and dependence. The zebrafish genome encodes likely homologs of the vast majority of these loci. We also review the known expression patterns of these genes in zebrafish. The information presented in this review provides support for the use of zebrafish as a viable model for studying genetic factors related to drug addiction. Expansion of investigations into drug response using model organisms holds the potential to advance our understanding of drug response and addiction in humans. PMID:22207143

  9. Zebrafish Assays of Ciliopathies

    PubMed Central

    Zaghloul, Norann A.; Katsanis, Nicholas

    2013-01-01

    In light of the growing list of human disorders associated with their dysfunction, primary cilia have recently come to attention as being important regulators of developmental signaling pathways and downstream processes. These organelles, present on nearly every vertebrate cell type, are highly conserved structures allowing for study across a range of species. Zebrafish, in particular, have emerged as useful organisms in which to explore the consequences of ciliary dysfunction and to model human ciliopathies. Here, we present a range of useful techniques that allow for investigation of various aspects of ciliary function. The described assays capitalize on the hallmark gastrulation defects associated with ciliary defects as well as relative ease of visualization of cilia in whole-mount embryos. Further, we describe our recently developed assay for querying functionality of human gene variants in live developing embryos. Finally, a current catalog of known zebrafish ciliary mutant lines is included. The techniques presented here provide a basic toolkit for in vivo investigation of both the biological and genetic mechanisms underlying a growing class of human diseases. PMID:21951534

  10. Analysing regenerative potential in zebrafish models of congenital muscular dystrophy.

    PubMed

    Wood, A J; Currie, P D

    2014-11-01

    fish, a model of MDC1A. Secondly high-throughput small molecule screens not only provide effective therapies, but also an alternative strategy for investigating CMD in zebrafish. In this instance insight into disease mechanism is derived in reverse. Zebrafish models are therefore clearly of critical importance in the advancement of regenerative medicine strategies in CMD. This article is part of a Directed Issue entitled: Regenerative Medicine: The challenge of translation.

  11. Evolutionary conservation of CCND1-ORAOV1-FGF19-FGF4 locus from zebrafish to human.

    PubMed

    Katoh, Masuko; Katoh, Masaru

    2003-07-01

    The CCND1-ORAOV1-FGF19-FGF4-FGF3-FLJ10261-FADD-PPFIA1-EMS1 locus on human chromosome 11q13 is frequently amplified in esophageal cancer, breast cancer, and bladder tumors. FGF19, FGF4 and FGF3 genes are implicated in embryogenesis and carcinogenesis. We proposed in 2002 the hypothesis that mouse Fgf15 might be the ortholog of human FGF19 based on comparative genomics. Here, we identified zebrafish fgf19 and oraov1 genes by using bioinformatics to demonstrate the hypothesis. Zebrafish fgf19 gene, consisting of three exons, was located around nucleotide position 121802-124963 of zebrafish genome draft sequence AL929586.12 in the reverse orientation. Zebrafish fgf19 (209 aa) was more homologous to chicken fgf19 and human FGF19 than to rodent Fgf15. Zebrafish oraov1 gene, consisting of five exons, was located around nucleotide position 112172-115838 of AL929586.12 in the reverse orientation. Zebrafish oraov1 protein (141 aa) was more homologous to human ORAOV1 than to rodent Oraov1. The CCND1-ORAOV1-FGF19-FGF4 locus was well conserved between human and zebrafish genomes in the order of genes, in the direction of genes, and in the exon-intron structure. Rat Ccnd1-Oraov1-Fgf15-Fgf4 locus was synthenic to mouse Ccnd1-Oraov1 (also known as 2210010N10Rik)-Fgf15-Fgf4 locus. Fgf15, homologous to human FGF19 and zebrafish fgf19, was located on the synthenic locus of human FGF19 and zebrafish fgf19 within rodent genomes. Based on the evolutionary conservation of the CCND1-ORAOV1-FGF19-FGF4 locus from zebrafish to human, it was concluded that Fgf15 gene is the rodent ortholog of human FGF19 gene.

  12. Thrombin Generation in Zebrafish Blood

    PubMed Central

    Hemker, Coenraad; Lindhout, Theo; Kelchtermans, Hilde; de Laat, Bas

    2016-01-01

    To better understand hypercoagulability as an underlying cause for thrombosis, the leading cause of death in the Western world, new assays to study ex vivo coagulation are essential. The zebrafish is generally accepted as a good model for human hemostasis and thrombosis, as the hemostatic system proved to be similar to that in man. Their small size however, has been a hurdle for more widespread use in hemostasis related research. In this study we developed a method that enables the measurement of thrombin generation in a single drop of non-anticoagulated zebrafish blood. Pre-treatment of the fish with inhibitors of FXa and thrombin, resulted in a dose dependent diminishing of thrombin generation, demonstrating the validity of the assay. In order to establish the relationship between whole blood thrombin generation and fibrin formation, we visualized the resulting fibrin network by scanning electron microscopy. Taken together, in this study we developed a fast and reliable method to measure thrombin generation in whole blood collected from a single zebrafish. Given the similarities between coagulation pathways of zebrafish and mammals, zebrafish may be an ideal animal model to determine the effect of novel therapeutics on thrombin generation. Additionally, because of the ease with which gene functions can be silenced, zebrafish may serve as a model organism for mechanistical research in thrombosis and hemostasis. PMID:26872266

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

    PubMed Central

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

    2015-01-01

    The evolutionary conservation of genomic, biochemical and developmental features between zebrafish and humans is gradually coming into focus with the end result that the zebrafish embryo model has emerged as a powerful tool for uncovering the effects of environmental exposures on a multitude of biological processes with direct relevance to human health. In this review, we highlight advances in automation, high-throughput (HT) screening, and analysis that leverage the power of the zebrafish embryo model for unparalleled advances in our understanding of how chemicals in our environment affect our health and wellbeing. PMID:25678986

  14. Proteomic analysis of zebrafish caudal fin regeneration.

    PubMed

    Saxena, Sandeep; Singh, Sachin K; Lakshmi, Mula G Meena; Meghah, Vuppalapaty; Bhatti, Bhawna; Swamy, Cherukuvada V Brahmendra; Sundaram, Curam S; Idris, Mohammed M

    2012-06-01

    The epimorphic regeneration of zebrafish caudal fin is rapid and complete. We have analyzed the biomechanism of zebrafish caudal fin regeneration at various time points based on differential proteomics approaches. The spectrum of proteome changes caused by regeneration were analyzed among controls (0 h) and 1, 12, 24, 48, and 72 h postamputation involving quantitative differential proteomics analysis based on two-dimensional gel electrophoresis matrix-assisted laser desorption/ionization and differential in-gel electrophoresis Orbitrap analysis. A total of 96 proteins were found differentially regulated between the control nonregenerating and regenerating tissues of different time points for having at least 1.5-fold changes. 90 proteins were identified as differentially regulated for regeneration based on differential in-gel electrophoresis analysis between the control and regenerating tissues. 35 proteins were characterized for its expression in all of the five regenerating time points against the control samples. The proteins identified and associated with regeneration were found to be directly allied with various molecular, biological, and cellular functions. Based on network pathway analysis, the identified proteome data set for regeneration was majorly associated in maintaining cellular structure and architecture. Also the proteins were found associated for the cytoskeleton remodeling pathway and cellular immune defense mechanism. The major proteins that were found differentially regulated during zebrafish caudal fin regeneration includes keratin and its 10 isoforms, cofilin 2, annexin a1, skeletal α1 actin, and structural proteins. Annexin A1 was found to be exclusively undergoing phosphorylation during regeneration. The obtained differential proteome and the direct association of the various proteins might lead to a new understanding of the regeneration mechanism.

  15. INDUCED AND SPONTANEOUS NEOPLASIA IN ZEBRAFISH.

    EPA Science Inventory

    To address the potential of zebrafish as a cancer model, it is important to determine the susceptibility of zebrafish to tumors, and to compare zebrafish tumors with human tumors. To determine whether the commonly-used germ line mutagen, ethylnitrosourea (ENU) induces tumors, we ...

  16. Period-2: a tumor suppressor gene in breast cancer

    PubMed Central

    Xiang, Shulin; Coffelt, Seth B; Mao, Lulu; Yuan, Lin; Cheng, Qi; Hill, Steven M

    2008-01-01

    Previous reports have suggested that the ablation of the Period 2 gene (Per 2) leads to enhanced development of lymphoma and leukemia in mice. Employing immunoblot analyses, we have demonstrated that PER 2 is endogenously expressed in human breast epithelial cell lines but is not expressed or is expressed at significantly reduced level in human breast cancer cell lines. Expression of PER 2 in MCF-7 breast cancer cells significantly inhibited the growth of MCF-7 human breast cancer cells, and, when PER 2 was co-expressed with the Crytochrome 2 (Cry 2) gene, an even greater growth-inhibitory effect was observed. The inhibitory effect of PER 2 on breast cancer cells was also demonstrated by its suppression of the anchorage-independent growth of MCF-7 cells as evidenced by the reduced number and size of colonies. A corresponding blockade of MCF-7 cells in the G1 phase of the cell cycle was also observed in response to the expression of PER 2 alone or in combination with CRY 2. Expression of PER 2 also induced apoptosis of MCF-7 breast cancer cells as demonstrated by an increase in PARP [poly (ADP-ribose) polymerase] cleavage. Finally, our studies demonstrate that PER 2 expression in MCF-7 breast cancer cells is associated with a significant decrease in the expression of cyclin D1 and an up-regulation of p53 levels. PMID:18334030

  17. Period-2: a tumor suppressor gene in breast cancer.

    PubMed

    Xiang, Shulin; Coffelt, Seth B; Mao, Lulu; Yuan, Lin; Cheng, Qi; Hill, Steven M

    2008-03-11

    Previous reports have suggested that the ablation of the Period 2 gene (Per 2) leads to enhanced development of lymphoma and leukemia in mice. Employing immunoblot analyses, we have demonstrated that PER 2 is endogenously expressed in human breast epithelial cell lines but is not expressed or is expressed at significantly reduced level in human breast cancer cell lines. Expression of PER 2 in MCF-7 breast cancer cells significantly inhibited the growth of MCF-7 human breast cancer cells, and, when PER 2 was co-expressed with the Crytochrome 2 (Cry 2) gene, an even greater growth-inhibitory effect was observed. The inhibitory effect of PER 2 on breast cancer cells was also demonstrated by its suppression of the anchorage-independent growth of MCF-7 cells as evidenced by the reduced number and size of colonies. A corresponding blockade of MCF-7 cells in the G1 phase of the cell cycle was also observed in response to the expression of PER 2 alone or in combination with CRY 2. Expression of PER 2 also induced apoptosis of MCF-7 breast cancer cells as demonstrated by an increase in PARP [poly (ADP-ribose) polymerase] cleavage. Finally, our studies demonstrate that PER 2 expression in MCF-7 breast cancer cells is associated with a significant decrease in the expression of cyclin D1 and an up-regulation of p53 levels.

  18. Budget Period 2 Summary Report Part 2: Hywind Maine Project

    SciTech Connect

    Driscoll, Frederick; Platt, Andrew; Sirnivas, Senu

    2015-08-15

    This project was performed under the Work for Others—Funds in Agreement FIA-14-1793 between Statoil and the Alliance for Sustainable Energy, manager and operator of the National Renewable Energy Laboratory (NREL). To support the development of a 6-MW spar-mounted offshore wind turbine, Statoil funded NREL to perform tasks in the following three categories: 1. Design and analysis 2. Wake modeling 3. Concept resource assessment. This study expands upon the work conducted in Budget Period 1 (BP1) to investigate the influence of the wake generated from an upstream turbine on a downstream turbine using Computational Fluid Dynamics (CFD) high-fidelity modeling tool. Simulator fOr Wind Farms Application (SOWFA) [1] is an NREL high fidelity modeling tool that couples OpenFOAM [2] CFD and NREL’s Aero-Elastic code Fatigue, Aerodynamics, Structures, and Turbulence (FAST)[3]. In BP1 the configuration was based on Hywind-3MW at 140 m water depth in the Gulf of Maine; however this study for Budget Period 2 (BP2) the configuration investigated is based on Hywind-6MW at 220 m water depth off the coast of Boston. The objectives were to perform two-turbines One-Way Coupling (OWC), three-turbines Two-Way Coupling (TWC), and to investigate wind power plant optimization.

  19. Maintenance of Zebrafish Lines at the European Zebrafish Resource Center

    PubMed Central

    Borel, Nadine; Ferg, Marco; Maier, Jana Viktoria; Strähle, Uwe

    2016-01-01

    Abstract We have established a European Zebrafish Resource Center (EZRC) at the KIT. This center not only maintains and distributes a large number of existing mutant and transgenic zebrafish lines but also gives zebrafish researchers access to screening services and technologies such as imaging and high-throughput sequencing, provided by the Institute of Toxicology and Genetics (ITG). The EZRC maintains and distributes the stock collection of the Nüsslein-Volhard laboratory, comprising over 2000 publicly released mutations, as frozen sperm samples. Within the framework of the ZF-HEALTH EU project, the EZRC distributes over 10,000 knockout mutations from the Sanger Institute (United Kingdom), as well as over 100 mutant and transgenic lines from other sources. In this article, we detail the measures we have taken to ensure the health of our fish, including hygiene, quarantine, and veterinary inspections. PMID:27351617

  20. Zebrafish Sensitivity to Botulinum Neurotoxins

    PubMed Central

    Chatla, Kamalakar; Gaunt, Patricia S.; Petrie-Hanson, Lora; Ford, Lorelei; Hanson, Larry A.

    2016-01-01

    Botulinum neurotoxins (BoNT) are the most potent known toxins. The mouse LD50 assay is the gold standard for testing BoNT potency, but is not sensitive enough to detect the extremely low levels of neurotoxin that may be present in the serum of sensitive animal species that are showing the effects of BoNT toxicity, such as channel catfish affected by visceral toxicosis of catfish. Since zebrafish are an important animal model for diverse biomedical and basic research, they are readily available and have defined genetic lines that facilitate reproducibility. This makes them attractive for use as an alternative bioassay organism. The utility of zebrafish as a bioassay model organism for BoNT was investigated. The 96 h median immobilizing doses of BoNT/A, BoNT/C, BoNT/E, and BoNT/F for adult male Tübingen strain zebrafish (0.32 g mean weight) at 25 °C were 16.31, 124.6, 4.7, and 0.61 picograms (pg)/fish, respectively. These findings support the use of the zebrafish-based bioassays for evaluating the presence of BoNT/A, BoNT/E, and BoNT/F. Evaluating the basis of the relatively high resistance of zebrafish to BoNT/C and the extreme sensitivity to BoNT/F may reveal unique functional patterns to the action of these neurotoxins. PMID:27153088

  1. Neurochemical measurements in the zebrafish brain

    PubMed Central

    Jones, Lauren J.; McCutcheon, James E.; Young, Andrew M. J.; Norton, William H. J.

    2015-01-01

    The zebrafish is an ideal model organism for behavioral genetics and neuroscience. The high conservation of genes and neurotransmitter pathways between zebrafish and other vertebrates permits the translation of research between species. Zebrafish behavior can be studied at both larval and adult stages and recent research has begun to establish zebrafish models for human disease. Fast scan cyclic voltammetry (FSCV) is an electrochemical technique that permits the detection of neurotransmitter release and reuptake. In this study we have used in vitro FSCV to measure the release of analytes in the adult zebrafish telencephalon. We compare different stimulation methods and present a characterization of neurochemical changes in the wild-type zebrafish brain. This study represents the first FSCV recordings in zebrafish, thus paving the way for neurochemical analysis of the fish brain. PMID:26441575

  2. Quantifying Aggressive Behavior in Zebrafish.

    PubMed

    Teles, Magda C; Oliveira, Rui F

    2016-01-01

    Aggression is a complex behavior that influences social relationships and can be seen as adaptive or maladaptive depending on the context and intensity of expression. A model organism suitable for genetic dissection of the underlying neural mechanisms of aggressive behavior is still needed. Zebrafish has already proven to be a powerful vertebrate model organism for the study of normal and pathological brain function. Despite the fact that zebrafish is a gregarious species that forms shoals, when allowed to interact in pairs, both males and females express aggressive behavior and establish dominance hierarchies. Here, we describe two protocols that can be used to quantify aggressive behavior in zebrafish, using two different paradigms: (1) staged fights between real opponents and (2) mirror-elicited fights. We also discuss the methodology for the behavior analysis, the expected results for both paradigms, and the advantages and disadvantages of each paradigm in face of the specific goals of the study.

  3. Coupled mutagenesis screens and genetic mapping in zebrafish.

    PubMed Central

    Rawls, John F; Frieda, Matthew R; McAdow, Anthony R; Gross, Jason P; Clayton, Chad M; Heyen, Candy K; Johnson, Stephen L

    2003-01-01

    Forward genetic analysis is one of the principal advantages of the zebrafish model system. However, managing zebrafish mutant lines derived from mutagenesis screens and mapping the corresponding mutations and integrating them into the larger collection of mutations remain arduous tasks. To simplify and focus these endeavors, we developed an approach that facilitates the rapid mapping of new zebrafish mutations as they are generated through mutagenesis screens. We selected a minimal panel of 149 simple sequence length polymorphism markers for a first-pass genome scan in crosses involving C32 and SJD inbred lines. We also conducted a small chemical mutagenesis screen that identified several new mutations affecting zebrafish embryonic melanocyte development. Using our first-pass marker panel in bulked-segregant analysis, we were able to identify the genetic map positions of these mutations as they were isolated in our screen. Rapid mapping of the mutations facilitated stock management, helped direct allelism tests, and should accelerate identification of the affected genes. These results demonstrate the efficacy of coupling mutagenesis screens with genetic mapping. PMID:12663538

  4. Whole-body multispectral photoacoustic imaging of adult zebrafish

    PubMed Central

    Huang, Na; Guo, Heng; Qi, Weizhi; Zhang, Zhiwei; Rong, Jian; Yuan, Zhen; Ge, Wei; Jiang, Huabei; Xi, Lei

    2016-01-01

    The zebrafish, an ideal vertebrate for studying developmental biology and genetics, is increasingly being used to understand human diseases, due to its high similarity to the human genome and its optical transparency during embryonic stages. Once the zebrafish has fully developed, especially wild-type breeds, conventional optical imaging techniques have difficulty in imaging the internal organs and structures with sufficient resolution and penetration depth. Even with established mutant lines that remain transparent throughout their life cycle, it is still challenging for purely optical imaging modalities to visualize the organs of juvenile and adult zebrafish at a micro-scale spatial resolution. In this work, we developed a non-invasive three-dimensional photoacoustic imaging platform with an optimized illumination pattern and a cylindrical-scanning-based data collection system to image entire zebrafish with micro-scale resolutions of 80 μm and 600 μm in the lateral and axial directions, respectively. In addition, we employed a multispectral strategy that utilized excitation wavelengths from 690 nm to 930 nm to statistically quantify the relative optical absorption spectrum of major organs. PMID:27699119

  5. Object recognition memory in zebrafish.

    PubMed

    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.

  6. Through the Looking Glass: Visualizing Leukemia Growth, Migration, and Engraftment Using Fluorescent Transgenic Zebrafish

    PubMed Central

    Moore, Finola E.; Langenau, David M.

    2012-01-01

    Zebrafish have emerged as a powerful model of development and cancer. Human, mouse, and zebrafish malignancies exhibit striking histopathologic and molecular similarities, underscoring the remarkable conservation of genetic pathways required to induce cancer. Zebrafish are uniquely suited for large-scale studies in which hundreds of animals can be used to investigate cancer processes. Moreover, zebrafish are small in size, optically clear during development, and amenable to genetic manipulation. Facile transgenic approaches and new technologies in gene inactivation have provided much needed genomic resources to interrogate the function of specific oncogenic and tumor suppressor pathways in cancer. This manuscript focuses on the unique attribute of labeling leukemia cells with fluorescent proteins and directly visualizing cancer processes in vivo including tumor growth, dissemination, and intravasation into the vasculature. We will also discuss the use of fluorescent transgenic approaches and cell transplantation to assess leukemia-propagating cell frequency and response to chemotherapy. PMID:22829834

  7. Effect of acute ethanol administration on zebrafish tail-beat motion.

    PubMed

    Bartolini, Tiziana; Mwaffo, Violet; Butail, Sachit; Porfiri, Maurizio

    2015-11-01

    Zebrafish is becoming a species of choice in neurobiological and behavioral studies of alcohol-related disorders. In these efforts, the activity of adult zebrafish is typically quantified using indirect activity measures that are either scored manually or identified automatically from the fish trajectory. The analysis of such activity measures has produced important insight into the effect of acute ethanol exposure on individual and social behavior of this vertebrate species. Here, we leverage a recently developed tracking algorithm that reconstructs fish body shape to investigate the effect of acute ethanol administration on zebrafish tail-beat motion in terms of amplitude and frequency. Our results demonstrate a significant effect of ethanol on the tail-beat amplitude as well as the tail-beat frequency, both of which were found to robustly decrease for high ethanol concentrations. Such a direct measurement of zebrafish motor functions is in agreement with evidence based on indirect activity measures, offering a complementary perspective in behavioral screening.

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

    PubMed Central

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

    2009-01-01

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

  9. A student team in a University of Michigan biomedical engineering design course constructs a microfluidic bioreactor for studies of zebrafish development.

    PubMed

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

    2009-06-01

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

  10. Carbon Quantum Dots for Zebrafish Fluorescence Imaging

    NASA Astrophysics Data System (ADS)

    Kang, Yan-Fei; Li, Yu-Hao; Fang, Yang-Wu; Xu, Yang; Wei, Xiao-Mi; Yin, Xue-Bo

    2015-07-01

    Carbon quantum dots (C-QDs) are becoming a desirable alternative to metal-based QDs and dye probes owing to their high biocompatibility, low toxicity, ease of preparation, and unique photophysical properties. Herein, we describe fluorescence bioimaging of zebrafish using C-QDs as probe in terms of the preparation of C-QDs, zebrafish husbandry, embryo harvesting, and introduction of C-QDs into embryos and larvae by soaking and microinjection. The multicolor of C-QDs was validated with their imaging for zebrafish embryo. The distribution of C-QDs in zebrafish embryos and larvae were successfully observed from their fluorescence emission. the bio-toxicity of C-QDs was tested with zebrafish as model and C-QDs do not interfere to the development of zebrafish embryo. All of the results confirmed the high biocompatibility and low toxicity of C-QDs as imaging probe. The absorption, distribution, metabolism and excretion route (ADME) of C-QDs in zebrafish was revealed by their distribution. Our work provides the useful information for the researchers interested in studying with zebrafish as a model and the applications of C-QDs. The operations related zebrafish are suitable for the study of the toxicity, adverse effects, transport, and biocompatibility of nanomaterials as well as for drug screening with zebrafish as model.

  11. Carbon Quantum Dots for Zebrafish Fluorescence Imaging

    PubMed Central

    Kang, Yan-Fei; Li, Yu-Hao; Fang, Yang-Wu; Xu, Yang; Wei, Xiao-Mi; Yin, Xue-Bo

    2015-01-01

    Carbon quantum dots (C-QDs) are becoming a desirable alternative to metal-based QDs and dye probes owing to their high biocompatibility, low toxicity, ease of preparation, and unique photophysical properties. Herein, we describe fluorescence bioimaging of zebrafish using C-QDs as probe in terms of the preparation of C-QDs, zebrafish husbandry, embryo harvesting, and introduction of C-QDs into embryos and larvae by soaking and microinjection. The multicolor of C-QDs was validated with their imaging for zebrafish embryo. The distribution of C-QDs in zebrafish embryos and larvae were successfully observed from their fluorescence emission. the bio-toxicity of C-QDs was tested with zebrafish as model and C-QDs do not interfere to the development of zebrafish embryo. All of the results confirmed the high biocompatibility and low toxicity of C-QDs as imaging probe. The absorption, distribution, metabolism and excretion route (ADME) of C-QDs in zebrafish was revealed by their distribution. Our work provides the useful information for the researchers interested in studying with zebrafish as a model and the applications of C-QDs. The operations related zebrafish are suitable for the study of the toxicity, adverse effects, transport, and biocompatibility of nanomaterials as well as for drug screening with zebrafish as model. PMID:26135470

  12. Functional aging and gradual senescence in zebrafish.

    PubMed

    Kishi, Shuji

    2004-06-01

    Zebrafish (Danio rerio) has been recognized as a powerful model for genetic studies in developmental biology. Recently, the zebrafish system also has given insights into several human diseases such as neurodegenerative, hematopoietic, and cardiovascular disease, and cancer. Because aging processes affect these and various other human disorders, it is important to compare zebrafish and mammalian senescence. However, the aging process of zebrafish remains largely unexplored, and little is known about functional aging and senescence in zebrafish. In our initial studies to assess aging phenotypes in zebrafish, we have identified several potential aging biomarkers in an ongoing search for suitable ones on zebrafish aging. In aging zebrafish, we detected senescence-associated beta-galactosidase activity in skin and oxidized protein accumulation in muscle. On the other hand, we did not observe lipofuscin granules (aging pigments), which accumulate in postmitotic cells, in muscle of zebrafish with advancing age. Consistently, there were continuously proliferating myocytes that incorporated BrdU in muscle tissues of the aged fish. Moreover, we demonstrated that zebrafish have constitutively abundant telomerase activity in adult somatic tissues implicating unlimited replicative ability of cells throughout their lives. Although some stress-associated markers are upregulated and minor histological changes are observed during the aging process of zebrafish, our studies together with other evidence of remarkable reproductive and regenerative abilities suggest that zebrafish show very gradual senescence. By using those biological and biochemical aging markers already characterized in normal zebrafish, transgenic fish analyses and genetic mutant fish screens can be readily performed. These efforts will help to elucidate the role and molecular mechanisms of common or different pathways of aging among vertebrates from fish to humans and also will contribute to the discovery of

  13. Zebrafish sex: a complicated affair

    PubMed Central

    Liew, Woei Chang

    2014-01-01

    In this review, we provide a detailed overview of studies on the elusive sex determination (SD) and gonad differentiation mechanisms of zebrafish (Danio rerio). We show that the data obtained from most studies are compatible with polygenic sex determination (PSD), where the decision is made by the allelic combinations of several loci. These loci are typically dispersed throughout the genome, but in some teleost species a few of them might be located on a preferential pair of (sex) chromosomes. The PSD system has a much higher level of variation of SD genotypes both at the level of gametes and the sexual genotype of individuals, than that of the chromosomal sex determination systems. The early sexual development of zebrafish males is a complicated process, as they first develop a ‘juvenile ovary’, that later undergoes a transformation to give way to a testis. To date, three major developmental pathways were shown to be involved with gonad differentiation through the modulation of programmed cell death. In our opinion, there are more pathways participating in the regulation of zebrafish gonad differentiation/transformation. Introduction of additional powerful large-scale genomic approaches into the analysis of zebrafish reproduction will result in further deepening of our knowledge as well as identification of additional pathways and genes associated with these processes in the near future. PMID:24148942

  14. Behavioural fever in zebrafish larvae.

    PubMed

    Rey, Sonia; Moiche, Visila; Boltaña, Sebastian; Teles, Mariana; MacKenzie, Simon

    2017-02-01

    Behavioural fever has been reported in different species of mobile ectotherms including the zebrafish, Danio rerio, in response to exogenous pyrogens. In this study we report, to our knowledge for the first time, upon the ontogenic onset of behavioural fever in zebrafish (Danio rerio) larvae. For this, zebrafish larvae (from first feeding to juveniles) were placed in a continuous thermal gradient providing the opportunity to select their preferred temperature. The novel thermal preference aquarium was based upon a continuous vertical column system and allows for non-invasive observation of larvae vertical distribution under isothermal (TR at 28 °C) and thermal gradient conditions (TCH: 28-32 °C). Larval thermal preference was assessed under both conditions with or without an immersion challenge, in order to detect the onset of the behavioural fever response. Our results defined the onset of the dsRNA induced behavioural fever at 18-20 days post fertilization (dpf). Significant differences were observed in dsRNA challenged larvae, which prefer higher temperatures (1-4 °C increase) throughout the experimental period as compared to non-challenged larvae. In parallel we measured the abundance of antiviral transcripts; viperin, gig2, irf7, trim25 and Mxb mRNAs in dsRNA challenged larvae under both thermal regimes: TR and TCh. Significant increases in the abundance of all measured transcripts were recorded under thermal choice conditions signifying that thermo-coupling and the resultant enhancement of the immune response to dsRNA challenge occurs from 18 dpf onwards in the zebrafish. The results are of importance as they identify a key developmental stage where the neuro-immune interface matures in the zebrafish likely providing increased resistance to viral infection.

  15. Development of social behavior in young zebrafish

    PubMed Central

    Dreosti, Elena; Lopes, Gonçalo; Kampff, Adam R.; Wilson, Stephen W.

    2015-01-01

    Adult zebrafish are robustly social animals whereas larva is not. We designed an assay to determine at what stage of development zebrafish begin to interact with and prefer other fish. One week old zebrafish do not show significant social preference whereas most 3 weeks old zebrafish strongly prefer to remain in a compartment where they can view conspecifics. However, for some individuals, the presence of conspecifics drives avoidance instead of attraction. Social preference is dependent on vision and requires viewing fish of a similar age/size. In addition, over the same 1–3 weeks period larval zebrafish increasingly tend to coordinate their movements, a simple form of social interaction. Finally, social preference and coupled interactions are differentially modified by an NMDAR antagonist and acute exposure to ethanol, both of which are known to alter social behavior in adult zebrafish. PMID:26347614

  16. Glial Cell Development and Function in Zebrafish

    PubMed Central

    Lyons, David A.; Talbot, William S.

    2015-01-01

    The zebrafish is a premier vertebrate model system that offers many experimental advantages for in vivo imaging and genetic studies. This review provides an overview of glial cell types in the central and peripheral nervous system of zebrafish. We highlight some recent work that exploited the strengths of the zebrafish system to increase the understanding of the role of Gpr126 in Schwann cell myelination and illuminate the mechanisms controlling oligodendrocyte development and myelination. We also summarize similarities and differences between zebrafish radial glia and mammalian astrocytes and consider the possibility that their distinct characteristics may represent extremes in a continuum of cell identity. Finally, we focus on the emergence of zebrafish as a model for elucidating the development and function of microglia. These recent studies have highlighted the power of the zebrafish system for analyzing important aspects of glial development and function. PMID:25395296

  17. Functional development of the circadian clock in the zebrafish pineal gland.

    PubMed

    Ben-Moshe, Zohar; Foulkes, Nicholas S; Gothilf, Yoav

    2014-01-01

    The zebrafish constitutes a powerful model organism with unique advantages for investigating the vertebrate circadian timing system and its regulation by light. In particular, the remarkably early and rapid development of the zebrafish circadian system has facilitated exploring the factors that control the onset of circadian clock function during embryogenesis. Here, we review our understanding of the molecular basis underlying functional development of the central clock in the zebrafish pineal gland. Furthermore, we examine how the directly light-entrainable clocks in zebrafish cell lines have facilitated unravelling the general mechanisms underlying light-induced clock gene expression. Finally, we summarize how analysis of the light-induced transcriptome and miRNome of the zebrafish pineal gland has provided insight into the regulation of the circadian system by light, including the involvement of microRNAs in shaping the kinetics of light- and clock-regulated mRNA expression. The relative contributions of the pineal gland central clock and the distributed peripheral oscillators to the synchronization of circadian rhythms at the whole animal level are a crucial question that still remains to be elucidated in the zebrafish model.

  18. Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

    PubMed Central

    Williams, Caitlin; Bryson-Richardson, Robert J.

    2016-01-01

    Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays. Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction. In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function. PMID:27842370

  19. Zebrafish Social Behavior in the Wild.

    PubMed

    Suriyampola, Piyumika S; Shelton, Delia S; Shukla, Rohitashva; Roy, Tamal; Bhat, Anuradha; Martins, Emília P

    2016-02-01

    Wild zebrafish exhibit a wide range of behavior. We found abundant wild zebrafish in flowing rivers and still water, in large, tightly-knit groups of hundreds of individuals, as well as in small, loose shoals. In two still-water populations, zebrafish were quite small in body size, common, and in tight groups of up to 22 fish. As in earlier laboratory studies, these zebrafish exhibited very low levels of aggression. In slowly flowing water in central India, zebrafish were relatively rare and gathered in small shoals (4-12 fish), often with other small fish, such as Rasbora daniconius. These stream zebrafish were larger in body size (27 mm TL) and much more aggressive than those in still water. In a second river population with much faster flowing water, zebrafish were abundant and again relatively large (21 mm TL). These zebrafish occurred in very large (up to 300 individuals) and tightly-knit (nearest-neighbor distances up to 21 mm) groups that exhibited collective rheotaxis and almost no aggression. This remarkable variation in social behavior of wild zebrafish offers an opportunity for future studies of behavioral genetics, development, and neuroscience.

  20. Zebrafish respond to the geomagnetic field by bimodal and group-dependent orientation

    PubMed Central

    Takebe, Akira; Furutani, Toshiki; Wada, Tatsunori; Koinuma, Masami; Kubo, Yoko; Okano, Keiko; Okano, Toshiyuki

    2012-01-01

    A variety of animals use Earth's magnetic field as a reference for their orientation behaviour. Although distinctive magnetoreception mechanisms have been postulated for many migrating or homing animals, the molecular mechanisms are still undefined. In this study, we found that zebrafish, a model organism suitable for genetic manipulation, responded to a magnetic field as weak as the geomagnetic field. Without any training, zebrafish were individually released into a circular arena that was placed in an artificial geomagnetic field, and their preferred magnetic directions were recorded. Individuals from five out of the seven zebrafish groups studied, groups mostly comprised of the offspring of predetermined pairs, showed bidirectional orientation with group-specific preferences regardless of close kinships. The preferred directions did not seem to depend on gender, age or surrounding environmental factors, implying that directional preference was genetically defined. The present findings may facilitate future study on the molecular mechanisms underlying magnetoreception. PMID:23061010

  1. Zebrafish respond to the geomagnetic field by bimodal and group-dependent orientation.

    PubMed

    Takebe, Akira; Furutani, Toshiki; Wada, Tatsunori; Koinuma, Masami; Kubo, Yoko; Okano, Keiko; Okano, Toshiyuki

    2012-01-01

    A variety of animals use Earth's magnetic field as a reference for their orientation behaviour. Although distinctive magnetoreception mechanisms have been postulated for many migrating or homing animals, the molecular mechanisms are still undefined. In this study, we found that zebrafish, a model organism suitable for genetic manipulation, responded to a magnetic field as weak as the geomagnetic field. Without any training, zebrafish were individually released into a circular arena that was placed in an artificial geomagnetic field, and their preferred magnetic directions were recorded. Individuals from five out of the seven zebrafish groups studied, groups mostly comprised of the offspring of predetermined pairs, showed bidirectional orientation with group-specific preferences regardless of close kinships. The preferred directions did not seem to depend on gender, age or surrounding environmental factors, implying that directional preference was genetically defined. The present findings may facilitate future study on the molecular mechanisms underlying magnetoreception.

  2. Tracking tagged molecules in single neurons in intact zebrafish.

    PubMed

    Armisen, Ricardo; Gleason, Michelle R; Fetcho, Joseph R; Mandel, Gail

    2007-08-01

    INTRODUCTIONThis protocol describes an approach for monitoring the movement of tagged molecules in single neurons in intact embryonic and larval zebrafish. The intact preparation provides a meaningful context for the physiological event being studied. Other advantages offered by the young zebrafish include direct in vivo imaging, the ability to produce large numbers of labeled embryos easily using microinjection, and the existence of identified sensory circuits that can be exploited to activate a particular cell type. One limitation of this system is the fragility of 2- to 3-d-old embryos, which demands delicate physical manipulation of the fish during all stages preceding and during the experiment. In contrast to brain slices or isolated cells, nearly all original neural connections and sensory components are maintained in the intact preparation, so the occurrence of a downstream event may be precluded (or its manifestation enhanced) by some complex interplay of biological processes that are not fully understood.

  3. Diversity of zebrafish peripheral oscillators revealed by luciferase reporting.

    PubMed

    Kaneko, Maki; Hernandez-Borsetti, Nancy; Cahill, Gregory M

    2006-09-26

    In various multicellular organisms, circadian clocks are present not only in the central nervous system, but also in peripheral organs and tissues. In mammals peripheral oscillators are not directly responsive to light, but are entrained by the central oscillator in the suprachiasmatic nucleus. These individual oscillators are diverse in their free-running periods and phases. In contrast, cultured peripheral tissues and cell lines from zebrafish are not only rhythmic, but can also be directly entrained by light. Because of the convenience of studying rhythms in cultured cells, however, little has been known about properties of individual oscillators in intact zebrafish. Here, we show the remarkable diversity and consistency of oscillator properties in various peripheral organs and tissues from the period3-luciferase (per3-luc) transgenic zebrafish. Tissue-dependent differences were found in free-running period, phase, response to light, and temperature compensation. Furthermore, cycling amplitudes were reduced at lower temperatures in some, but not all, of the organs tested. Finally, we found that per3-luc rhythms can free run in both constant dark and constant light with remarkably similar amplitudes, phases, and periods, despite the fact that the mRNA of per2 and per1 has been shown not to oscillate in constant light.

  4. DNA repair capacity of zebrafish.

    PubMed

    Sussman, Raquel

    2007-08-14

    Damage to the genome is unavoidable in living creatures, because of sunlight exposure as well as environmental chemicals present in food and drinking water. There is a need to monitor and purify the drinking water; therefore, several methods of detection have been developed. A very promising model system for this purpose is the zebrafish (Danio rerio), which is endowed with special qualities for detecting external as well as internal abnormalities. Grossman and Wei's assay [Grossman L, Wei Q (1995) Clin Chem 12:1854-1863], which measures the expression level of a nonreplicating recombinant plasmid DNA containing a UV-damaged luciferase reporter gene, shows that zebrafish can repair chromosomal lesions to a much greater extent than the human population. This vertebrate model is still very promising after possible down-regulation of the DNA repair enzymes.

  5. Zebrafish Behavior: Opportunities and Challenges.

    PubMed

    Orger, Michael B; de Polavieja, Gonzalo G

    2017-04-03

    A great challenge in neuroscience is understanding how activity in the brain gives rise to behavior. The zebrafish is an ideal vertebrate model to address this challenge, thanks to the capacity, at the larval stage, for precise behavioral measurements, genetic manipulations, and recording and manipulation of neural activity noninvasively and at single-neuron resolution throughout the whole brain. These techniques are being further developed for application in freely moving animals and juvenile stages to study more complex behaviors including learning, decision making, and social interactions. We review some of the approaches that have been used to study the behavior of zebrafish and point to opportunities and challenges that lie ahead. Expected final online publication date for the Annual Review of Neuroscience Volume 40 is July 8, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  6. Zebrafish genomics comes of age.

    PubMed

    Tan, Haihan; Zsigmond, Aron

    2013-09-01

    The ZF-HEALTH/EuFishBiomed workshop on "Genomics and High-throughput Sequencing Technologies with the Zebrafish Model" took place in December 2012 in Cambridge, United Kingdom. The organisers, Fiona Wardle and Ferenc Müller, brought together developmental biologists, geneticists, and bioinformaticians from Europe and the rest of the world to share findings and insights about the latest genomic capabilities and applications in this popular model organism.

  7. The Zebrafish Model Organism Database: new support for human disease models, mutation details, gene expression phenotypes and searching.

    PubMed

    Howe, Douglas G; Bradford, Yvonne M; Eagle, Anne; Fashena, David; Frazer, Ken; Kalita, Patrick; Mani, Prita; Martin, Ryan; Moxon, Sierra Taylor; Paddock, Holly; Pich, Christian; Ramachandran, Sridhar; Ruzicka, Leyla; Schaper, Kevin; Shao, Xiang; Singer, Amy; Toro, Sabrina; Van Slyke, Ceri; Westerfield, Monte

    2017-01-04

    The Zebrafish Model Organism Database (ZFIN; http://zfin.org) is the central resource for zebrafish (Danio rerio) genetic, genomic, phenotypic and developmental data. ZFIN curators provide expert manual curation and integration of comprehensive data involving zebrafish genes, mutants, transgenic constructs and lines, phenotypes, genotypes, gene expressions, morpholinos, TALENs, CRISPRs, antibodies, anatomical structures, models of human disease and publications. We integrate curated, directly submitted, and collaboratively generated data, making these available to zebrafish research community. Among the vertebrate model organisms, zebrafish are superbly suited for rapid generation of sequence-targeted mutant lines, characterization of phenotypes including gene expression patterns, and generation of human disease models. The recent rapid adoption of zebrafish as human disease models is making management of these data particularly important to both the research and clinical communities. Here, we describe recent enhancements to ZFIN including use of the zebrafish experimental conditions ontology, 'Fish' records in the ZFIN database, support for gene expression phenotypes, models of human disease, mutation details at the DNA, RNA and protein levels, and updates to the ZFIN single box search.

  8. The Zebrafish Model Organism Database: new support for human disease models, mutation details, gene expression phenotypes and searching

    PubMed Central

    Howe, Douglas G.; Bradford, Yvonne M.; Eagle, Anne; Fashena, David; Frazer, Ken; Kalita, Patrick; Mani, Prita; Martin, Ryan; Moxon, Sierra Taylor; Paddock, Holly; Pich, Christian; Ramachandran, Sridhar; Ruzicka, Leyla; Schaper, Kevin; Shao, Xiang; Singer, Amy; Toro, Sabrina; Van Slyke, Ceri; Westerfield, Monte

    2017-01-01

    The Zebrafish Model Organism Database (ZFIN; http://zfin.org) is the central resource for zebrafish (Danio rerio) genetic, genomic, phenotypic and developmental data. ZFIN curators provide expert manual curation and integration of comprehensive data involving zebrafish genes, mutants, transgenic constructs and lines, phenotypes, genotypes, gene expressions, morpholinos, TALENs, CRISPRs, antibodies, anatomical structures, models of human disease and publications. We integrate curated, directly submitted, and collaboratively generated data, making these available to zebrafish research community. Among the vertebrate model organisms, zebrafish are superbly suited for rapid generation of sequence-targeted mutant lines, characterization of phenotypes including gene expression patterns, and generation of human disease models. The recent rapid adoption of zebrafish as human disease models is making management of these data particularly important to both the research and clinical communities. Here, we describe recent enhancements to ZFIN including use of the zebrafish experimental conditions ontology, ‘Fish’ records in the ZFIN database, support for gene expression phenotypes, models of human disease, mutation details at the DNA, RNA and protein levels, and updates to the ZFIN single box search. PMID:27899582

  9. Zebrafish Discoveries in Cancer Epigenetics

    PubMed Central

    Chernyavskaya, Yelena; Kent, Brandon

    2017-01-01

    The cancer epigenome is fundamentally different than that of normal cells. How these differences arise in and contribute to carcinogenesis is not known, and studies using model organisms such as zebrafish provide an opportunity to address these important questions. Modifications of histones and DNA comprise the complex epigenome, and these influence chromatin structure, genome stability and gene expression, all of which are fundamental to the cellular changes that cause cancer. The cancer genome atlas covers the wide spectrum of genetic changes associated with nearly every cancer type, however, this catalog is currently unidimensional. As the pattern of epigenetic marks and chromatin structure in cancer cells is described and overlaid on the mutational landscape, the map of the cancer genome becomes multi-dimensional and highly complex. Two major questions remain in the field: (1) how the epigenome becomes repatterned in cancer and (2) which of these changes are cancer-causing. Zebrafish provide a tractable in vivo system to monitor the epigenome during transformation and to identify epigenetic drivers of cancer. In this chapter, we review principles of cancer epigenetics and discuss recent work using zebrafish whereby epigenetic modifiers were established as cancer driver genes, thus providing novel insights into the mechanisms of epigenetic reprogramming in cancer. PMID:27165354

  10. Stressing Zebrafish for Behavioral Genetics

    PubMed Central

    Clark, Karl J.; Boczek, Nicole J.; Ekker, Stephen C.

    2012-01-01

    Synopsis The stress response is a normal reaction to a real or perceived threat. However, stress response systems that are overwhelmed or out of balance can increase both the incidence and severity of diseases including addiction and mood and anxiety disorders. Using an animal model with both genetic diversity and large family size can help discover the specific genetic and environmental contributions to these behavioral diseases. The stress response has been studied extensively in teleosts because of their importance in food production. The zebrafish (Danio rerio) is a major model organism with a strong record for use in developmental biology, genetic screening, and genomic studies. More recently, the stress response of larval and adult zebrafish has been documented. High-throughput automated tracking systems make possible behavioral readouts of the stress response in zebrafish. This non-invasive measure of the stress response can be combined with mutagenesis methods to dissect the genes involved in complex stress response behaviors in vertebrates. Understanding the genetic and epigenetic basis for the stress response in vertebrates will help to develop advanced screening and therapies for stress-aggravated diseases like addiction and mood and anxiety disorders. PMID:21615261

  11. Inflammatory diseases modelling in zebrafish

    PubMed Central

    Morales Fénero, Camila Idelí; Colombo Flores, Alicia Angelina; Câmara, Niels Olsen Saraiva

    2016-01-01

    The ingest of diets with high content of fats and carbohydrates, low or no physical exercise and a stressful routine are part of the everyday lifestyle of most people in the western world. These conditions are triggers for different diseases with complex interactions between the host genetics, the metabolism, the immune system and the microbiota, including inflammatory bowel diseases (IBD), obesity and diabetes. The incidence of these disorders is growing worldwide; therefore, new strategies for its study are needed. Nowadays, the majority of researches are in use of murine models for understand the genetics, physiopathology and interaction between cells and signaling pathways to find therapeutic solutions to these diseases. The zebrafish, a little tropical water fish, shares 70% of our genes and conserves anatomic and physiological characteristics, as well as metabolical pathways, with mammals, and is rising as a new complementary model for the study of metabolic and inflammatory diseases. Its high fecundity, fast development, transparency, versatility and low cost of maintenance makes the zebrafish an interesting option for new researches. In this review, we offer a discussion of the existing genetic and induced zebrafish models of two important Western diseases that have a strong inflammatory component, the IBD and the obesity. PMID:26929916

  12. Vitrification of zebrafish embryo blastomeres in microvolumes.

    PubMed

    Cardona-Costa, J; García-Ximénez, F

    2007-01-01

    Cryopreservation of fish embryos may play an important role in biodiversity preservation and in aquaculture, but it is very difficult. In addition, the cryopreservation of fish embryo blastomeres makes conservation strategies feasible when they are used in germ-line chimaerism, including interspecific chimaerism. Fish embryo blastomere cryopreservation has been achieved by equilibrium procedures, but to our knowledge, no data on vitrification procedures are available. In the present work, zebrafish embryo blastomeres were successfully vitrified in microvolumes: a number of 0.25 microl drops, sufficient to contain all the blastomeres of an embryo at blastula stage (from 1000-cell stage to oblong stage), were placed over a 2.5 cm loop of nylon filament. In this procedure, where intracellular cryoprotectant permeation is not required, blastomeres were exposed to cryoprotectants for a maximum of 25 sec prior vitrification. The assayed cryoprotectants (ethylene glycol, propylene glycol, dimethyl sulphoxide, glycerol and methanol) are all frequently used in fish embryo and blastomere cryopreservation. Methanol was finally rejected because of the excessive concentration required for the vitrification (15M). All other cryoprotectants were prepared (individually) at 5 M in Hanks' buffered salt solution (sigma) plus 20% FBS (vitrification solutions: vs). After direct thawing in Hanks' buffered salt solution plus 20% FBS, acceptable survival rates were obtained with ethylene glycol: 82.8%, propylene glycol: 87.7%, dimethyl sulphoxide: 93.4%, and glycerol: 73.9% (p < 0.05). Dimethyl sulphoxide showed the highest blastomere survival rate and allowed the rescue of as much as 20% of the total blastomeres from each zebrafish blastula embryo.

  13. A Comparative Map of the Zebrafish Genome

    PubMed Central

    Woods, Ian G.; Kelly, Peter D.; Chu, Felicia; Ngo-Hazelett, Phuong; Yan, Yi-Lin; Huang, Hui; Postlethwait, John H.; Talbot, William S.

    2000-01-01

    Zebrafish mutations define the functions of hundreds of essential genes in the vertebrate genome. To accelerate the molecular analysis of zebrafish mutations and to facilitate comparisons among the genomes of zebrafish and other vertebrates, we used a homozygous diploid meiotic mapping panel to localize polymorphisms in 691 previously unmapped genes and expressed sequence tags (ESTs). Together with earlier efforts, this work raises the total number of markers scored in the mapping panel to 2119, including 1503 genes and ESTs and 616 previously characterized simple-sequence length polymorphisms. Sequence analysis of zebrafish genes mapped in this study and in prior work identified putative human orthologs for 804 zebrafish genes and ESTs. Map comparisons revealed 139 new conserved syntenies, in which two or more genes are on the same chromosome in zebrafish and human. Although some conserved syntenies are quite large, there were changes in gene order within conserved groups, apparently reflecting the relatively frequent occurrence of inversions and other intrachromosomal rearrangements since the divergence of teleost and tetrapod ancestors. Comparative mapping also shows that there is not a one-to-one correspondence between zebrafish and human chromosomes. Mapping of duplicate gene pairs identified segments of 20 linkage groups that may have arisen during a genome duplication that occurred early in the evolution of teleosts after the divergence of teleost and mammalian ancestors. This comparative map will accelerate the molecular analysis of zebrafish mutations and enhance the understanding of the evolution of the vertebrate genome. PMID:11116086

  14. Zebrafish as a model for human osteosarcoma.

    PubMed

    Mohseny, A B; Hogendoorn, P C W

    2014-01-01

    For various reasons involving biological comparativeness, expansive technological possibilities, accelerated experimental speed, and competitive costs, zebrafish has become a comprehensive model for cancer research. Hence, zebrafish embryos and full-grown fish have been instrumental for studies of leukemia, melanoma, pancreatic cancer, bone tumors, and other malignancies. Although because of its similarities to human osteogenesis zebrafish appears to be an appealing model to investigate osteosarcoma, only a few osteosarcoma specific studies have been accomplished yet. Here, we review interesting related and unrelated reports of which the findings might be extrapolated to osteosarcoma. More importantly, rational but yet unexplored applications of zebrafish are debated to expand the window of opportunities for future establishment of osteosarcoma models. Accordingly technological advances of zebrafish based cancer research, such as robotic high-throughput multicolor injection systems and advanced imaging methods are discussed. Furthermore, various use of zebrafish embryos for screening drug regimens by combinations of chemotherapy, novel drug deliverers, and immune system modulators are suggested. Concerning the etiology, the high degree of genetic similarity between zebrafish and human cancers indicates that affected regions are evolutionarily conserved. Therefore, zebrafish as a swift model system that allows for the investigation of multiple candidate gene-defects is presented.

  15. Identification and functional characterization of zebrafish solute carrier Slc16a2 (Mct8) as a thyroid hormone membrane transporter.

    PubMed

    Arjona, Francisco J; de Vrieze, Erik; Visser, Theo J; Flik, Gert; Klaren, Peter H M

    2011-12-01

    Most components of the thyroid system in bony fish have been described and characterized, with the notable exception of thyroid hormone membrane transporters. We have cloned, sequenced, and expressed the zebrafish solute carrier Slc16a2 (also named monocarboxylate transporter Mct8) cDNA and established its role as a thyroid hormone transport protein. The cloned cDNA shares 56-57% homology with its mammalian orthologs. The 526-amino-acid sequence contains 12 predicted transmembrane domains. An intracellular N-terminal PEST domain, thought to be involved in proteolytic processing of the protein, is present in the zebrafish sequence. Measured at initial rate and at the body/rearing temperature of zebrafish (26 C), T(3) uptake by zebrafish Slc16a2 is a saturable process with a calculated Michaelis-Menten constant of 0.8 μM T(3). The rate of T(3) uptake is temperature dependent and Na(+) independent. Interestingly, at 26 C, zebrafish Slc16a2 does not transport T(4). This implies that at a normal body temperature in zebrafish, Slc16a2 protein is predominantly involved in T(3) uptake. When measured at 37 C, zebrafish Slc16a2 transports T(4) in a Na(+)-independent manner. In adult zebrafish, the Slc16a2 gene is highly expressed in brain, gills, pancreas, liver, pituitary, heart, kidney, and gut. Beginning from the midblastula stage, Slc16a2 is also expressed during zebrafish early development, the highest expression levels occurring 48 h after fertilization. This is the first direct evidence for thyroid hormone membrane transporters in fish. We suggest that Slc16a2 plays a key role in the local availability of T(3) in adult tissues as well as during the completion of morphogenesis of primary organ systems.

  16. Optimized cell transplantation using adult rag2 mutant zebrafish

    PubMed Central

    Tang, Qin; Abdelfattah, Nouran S.; Blackburn, Jessica S.; Moore, John C.; Martinez, Sarah A.; Moore, Finola E.; Lobbardi, Riadh; Tenente, Inês M.; Ignatius, Myron S.; Berman, Jason N.; Liwski, Robert S.; Houvras, Yariv; Langenau, David M.

    2014-01-01

    Cell transplantation into adult zebrafish has lagged behind mouse due to the lack of immune compromised models. Here, we have created homozygous rag2E450fs mutant zebrafish that have reduced numbers of functional T and B cells but are viable and fecund. Mutant fish engraft zebrafish muscle, blood stem cells, and cancers. rag2E450fs mutant zebrafish are the first immune compromised zebrafish model that permits robust, long-term engraftment of multiple tissues and cancer. PMID:25042784

  17. Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior.

    PubMed

    Ben-Moshe Livne, Zohar; Alon, Shahar; Vallone, Daniela; Bayleyen, Yared; Tovin, Adi; Shainer, Inbal; Nisembaum, Laura G; Aviram, Idit; Smadja-Storz, Sima; Fuentes, Michael; Falcón, Jack; Eisenberg, Eli; Klein, David C; Burgess, Harold A; Foulkes, Nicholas S; Gothilf, Yoav

    2016-11-01

    The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior.

  18. Genetically Blocking the Zebrafish Pineal Clock Affects Circadian Behavior

    PubMed Central

    Alon, Shahar; Vallone, Daniela; Tovin, Adi; Shainer, Inbal; Nisembaum, Laura G.; Aviram, Idit; Smadja-Storz, Sima; Fuentes, Michael; Falcón, Jack; Eisenberg, Eli; Klein, David C.; Burgess, Harold A.; Foulkes, Nicholas S.; Gothilf, Yoav

    2016-01-01

    The master circadian clock in fish has been considered to reside in the pineal gland. This dogma is challenged, however, by the finding that most zebrafish tissues contain molecular clocks that are directly reset by light. To further examine the role of the pineal gland oscillator in the zebrafish circadian system, we generated a transgenic line in which the molecular clock is selectively blocked in the melatonin-producing cells of the pineal gland by a dominant-negative strategy. As a result, clock-controlled rhythms of melatonin production in the adult pineal gland were disrupted. Moreover, transcriptome analysis revealed that the circadian expression pattern of the majority of clock-controlled genes in the adult pineal gland is abolished. Importantly, circadian rhythms of behavior in zebrafish larvae were affected: rhythms of place preference under constant darkness were eliminated, and rhythms of locomotor activity under constant dark and constant dim light conditions were markedly attenuated. On the other hand, global peripheral molecular oscillators, as measured in whole larvae, were unaffected in this model. In conclusion, characterization of this novel transgenic model provides evidence that the molecular clock in the melatonin-producing cells of the pineal gland plays a key role, possibly as part of a multiple pacemaker system, in modulating circadian rhythms of behavior. PMID:27870848

  19. Developmental effects of simulated microgravity on zebrafish, (Danio rerio)

    NASA Astrophysics Data System (ADS)

    Stoyek, Matthew; Edsall, Sara; Franz-Odendaal, Tamara; Smith, Frank; Croll, Roger

    Zebrafish are widely used model vertebrates in research and recently this species has been used to study the effects of microgravity on fundamental biological processes. In this study we used a NASA-designed rotating wall vessel (RWV) to investigate the effects of simulated microgravity (SMG) on zebrafish development up to 14 days post fertilization (dpf). At developmental stages beyond the 3-4 somite stage we found SMG-exposed embryos reached key developmental stag-ing points more rapidly than fish raised within a non-rotating vessel. By the 21 somite stage, both groups were again synchronized in their developmental staging. However, SMG-exposed embryos eventually exhibited a delay in hatching time compared to controls. Otolith and to-tal body size were observed to be greater in larvae raised in SMG. In addition, pigmentation patterns in SMG exposed fish differed, with larger and differentially aggregated melanocytes . Heart development was slowed in SMG exposed fish, but no change in nervous system de-velopment was detected. Ongoing research will focus on differences in heart and respiration rates. Finally, by developing a method to extend the duration of SMG exposure, we found the swimming behaviour of SMG-exposed animals was altered with time in the RWV. Initially SMG-exposed animals swam in the direction of RWV rotation (5-9dpf) but older (9+dpf) fish swam against rotation and demonstrated righting behaviour with each rotation. These results suggest that vestibular reflexes may develop normally and be maintained in animals exposed to SMG. Together, our data provide insights into how zebrafish may develop when flown in space, permitting better formulation of experiments to test mechanisms by which microgravity may affect ontogeny of this model organism. Keywords: microgravity, zebrafish, growth, development

  20. Streptococcus-Zebrafish Model of Bacterial Pathogenesis

    PubMed Central

    Neely, Melody N.; Pfeifer, John D.; Caparon, Michael

    2002-01-01

    Due to its small size, rapid generation time, powerful genetic systems, and genomic resources, the zebrafish has emerged as an important model of vertebrate development and human disease. Its well-developed adaptive and innate cellular immune systems make the zebrafish an ideal model for the study of infectious diseases. With a natural and important pathogen of fish, Streptococcus iniae, we have established a streptococcus- zebrafish model of bacterial pathogenesis. Following injection into the dorsal muscle, zebrafish developed a lethal infection, with a 50% lethal dose of 103 CFU, and died within 2 to 3 days. The pathogenesis of infection resembled that of S. iniae in farmed fish populations and that of several important human streptococcal diseases and was characterized by an initial focal necrotic lesion that rapidly progressed to invasion of the pathogen into all major organ systems, including the brain. Zebrafish were also susceptible to infection by the human pathogen Streptococcus pyogenes. However, disease was characterized by a marked absence of inflammation, large numbers of extracellular streptococci in the dorsal muscle, and extensive myonecrosis that occurred far in advance of any systemic invasion. The genetic systems available for streptococci, including a novel method of mutagenesis which targets genes whose products are exported, were used to identify several mutants attenuated for virulence in zebrafish. This combination of a genetically amenable pathogen with a well-defined vertebrate host makes the streptococcus-zebrafish model of bacterial pathogenesis a powerful model for analysis of infectious disease. PMID:12065534

  1. Animal Models of Tuberculosis: Zebrafish

    PubMed Central

    van Leeuwen, Lisanne M.; van der Sar, Astrid M.; Bitter, Wilbert

    2015-01-01

    Over the past decade the zebrafish (Danio rerio) has become an attractive new vertebrate model organism for studying mycobacterial pathogenesis. The combination of medium-throughput screening and real-time in vivo visualization has allowed new ways to dissect host pathogenic interaction in a vertebrate host. Furthermore, genetic screens on the host and bacterial sides have elucidated new mechanisms involved in the initiation of granuloma formation and the importance of a balanced immune response for control of mycobacterial pathogens. This article will highlight the unique features of the zebrafish–Mycobacterium marinum infection model and its added value for tuberculosis research. PMID:25414379

  2. Proteomic analysis of the Rett syndrome experimental model mecp2(Q63X) mutant zebrafish.

    PubMed

    Cortelazzo, Alessio; Pietri, Thomas; De Felice, Claudio; Leoncini, Silvia; Guerranti, Roberto; Signorini, Cinzia; Timperio, Anna Maria; Zolla, Lello; Ciccoli, Lucia; Hayek, Joussef

    2017-02-10

    Rett syndrome (RTT) is a severe genetic disorder resulting from mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene. Recently, a zebrafish carrying a mecp2-null mutation has been developed with the resulting phenotypes exhibiting defective sensory and thigmotactic responses, and abnormal motor behavior reminiscent of the human disease. Here, we performed a proteomic analysis to examine protein expression changes in mecp2-null vs. wild-type larvae and adult zebrafish. We found a total of 20 proteins differentially expressed between wild-type and mutant zebrafish, suggesting skeletal and cardiac muscle functional defects, a stunted glycolysis and depleted energy availability. This molecular evidence is directly linked to the mecp2-null zebrafish observed phenotype. In addition, we identified changes in expression of proteins critical for a proper redox balance, suggesting an enhanced oxidative stress, a phenomenon also documented in human patients and RTT murine models. The molecular alterations observed in the mecp2-null zebrafish expand our knowledge on the molecular cascade of events that lead to the RTT phenotype.

  3. The influences of parental diet and vitamin E intake on the embryonic zebrafish transcriptome

    PubMed Central

    Miller, Galen W.; Truong, Lisa; Barton, Carrie L.; Labut, Edwin M.; Lebold, Katie M.; Traber, Maret G.; Tanguay, Robert L.

    2014-01-01

    The composition of the typical commercial diet fed to zebrafish can dramatically vary. By utilizing defined diets we sought to answer two questions: 1) How does the embryonic zebrafish transcriptome change when the parental adults are fed a commercial lab diet compared with a sufficient, defined diet (E+)? 2) Does a vitamin Edeficient parental diet (E−) further change the embryonic transcriptome? We conducted a global gene expression study using embryos from zebrafish fed a commercial (Lab), an E+ or an E− diet. To capture differentially expressed transcripts prior to onset of overt malformations observed in E− embryos at 48 h post-fertilization (hpf), embryos were collected from each group at 36 hpf. Lab embryos differentially expressed (p < 0.01) 946 transcripts compared with the E+ embryos, and 2656 transcripts compared with the E− embryos. The differences in protein, fat and micronutrient intakes in zebrafish fed the Lab compared with the E + diet demonstrate that despite overt morphologic consistency, significant differences in gene expression occurred. Moreover, functional analysis of the significant transcripts in the E−embryos suggested perturbed energy metabolism, leading to overt malformations and mortality. Thus, these findings demonstrate that parental zebrafish diet has a direct impact on the embryonic transcriptome. PMID:24657723

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

    PubMed Central

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

    2016-01-01

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

  5. Association of Early Atherosclerosis with Vascular Wall Shear Stress in Hypercholesterolemic Zebrafish

    PubMed Central

    Lee, Sang Joon; Choi, Woorak; Seo, Eunseok; Yeom, Eunseop

    2015-01-01

    Although atherosclerosis is a multifactorial disease, the role of hemodynamic information has become more important. Low and oscillating wall shear stress (WSS) that changes its direction is associated with the early stage of atherosclerosis. Several in vitro and in vivo models were proposed to reveal the relation between the WSS and the early atherosclerosis. However, these models possess technical limitations in mimicking real physiological conditions and monitoring the developmental course of the early atherosclerosis. In this study, a hypercholesterolaemic zebrafish model is proposed as a novel experimental model to resolve these limitations. Zebrafish larvae are optically transparent, which enables temporal observation of pathological variations under in vivo condition. WSS in blood vessels of 15 days post-fertilisation zebrafish was measured using a micro particle image velocimetry (PIV) technique, and spatial distribution of lipid deposition inside the model was quantitatively investigated after feeding high cholesterol diet for 10 days. Lipids were mainly deposited in blood vessel of low WSS. The oscillating WSS was not induced by the blood flows in zebrafish models. The present hypercholesterolaemic zebrafish would be used as a potentially useful model for in vivo study about the effects of low WSS in the early atherosclerosis. PMID:26561854

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

    PubMed

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

    2016-06-11

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

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

    PubMed

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

    2013-06-01

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

  8. Nonhatching Decapsulated Artemia Cysts As a Replacement to Artemia Nauplii in Juvenile and Adult Zebrafish Culture.

    PubMed

    Tye, Marc; Rider, Dana; Duffy, Elizabeth A; Seubert, Adam; Lothert, Brogen; Schimmenti, Lisa A

    2015-12-01

    Feeding Artemia nauplii as the main nutrition source for zebrafish is a common practice for many research facilities. Culturing live feed can be time-consuming and requires additional equipment to be purchased, maintained, and cleaned. Nonhatching decapsulated Artemia cysts (decaps) are a commercially available product that can be fed directly to fish. Several other ornamental fish species have been successfully cultured using decaps. Replacing Artemia nauplii with decaps could reduce the overall time and costs associated with the operation of a zebrafish facility. The objective of this study was to determine if decaps could be a suitable replacement to Artemia nauplii in juvenile and adult zebrafish culture. Wild-type zebrafish were fed one of three dietary treatments: decaps only, nauplii only, or a standard consisting of nauplii plus a commercially prepared pellet food. Survival, growth (length and weight), and embryo production were analyzed between the treatments. Fish receiving the decap diet demonstrated a significantly higher growth and embryo production when compared to the fish receiving the nauplii-only diet. When comparing the decap fish to the standard fish, no significant difference was found in mean survival, mean weight at 90 days postfertilization, or mean embryo production. It was determined that nonhatching decapsulated Artemia cysts can be used as a suitable replacement to Artemia nauplii in juvenile and adult zebrafish culture.

  9. Mesoderm is required for coordinated cell movements within zebrafish neural plate in vivo

    PubMed Central

    2014-01-01

    Background Morphogenesis of the zebrafish neural tube requires the coordinated movement of many cells in both time and space. A good example of this is the movement of the cells in the zebrafish neural plate as they converge towards the dorsal midline before internalizing to form a neural keel. How these cells are regulated to ensure that they move together as a coherent tissue is unknown. Previous work in other systems has suggested that the underlying mesoderm may play a role in this process but this has not been shown directly in vivo. Results Here we analyze the roles of subjacent mesoderm in the coordination of neural cell movements during convergence of the zebrafish neural plate and neural keel formation. Live imaging demonstrates that the normal highly coordinated movements of neural plate cells are lost in the absence of underlying mesoderm and the movements of internalization and neural tube formation are severely disrupted. Despite this, neuroepithelial polarity develops in the abnormal neural primordium but the resulting tissue architecture is very disorganized. Conclusions We show that the movements of cells in the zebrafish neural plate are highly coordinated during the convergence and internalization movements of neurulation. Our results demonstrate that the underlying mesoderm is required for these coordinated cell movements in the zebrafish neural plate in vivo. PMID:24755297

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

    PubMed

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

    2016-04-01

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

  11. Learning and memory in zebrafish larvae.

    PubMed

    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.

  12. Waterborne Risperidone Decreases Stress Response in Zebrafish.

    PubMed

    Idalencio, Renan; Kalichak, Fabiana; Rosa, João Gabriel Santos; de Oliveira, Tiago Acosta; Koakoski, Gessi; Gusso, Darlan; Abreu, Murilo Sander de; Giacomini, Ana Cristina Varrone; Barcellos, Heloísa Helena de Alcântara; Piato, Angelo L; Barcellos, Leonardo José Gil

    2015-01-01

    The presence of drugs and their metabolites in surface waters and municipal effluents has been reported in several studies, but its impacts on aquatic organisms are not yet well understood. This study investigated the effects of acute exposure to the antipsychotic risperidone on the stress and behavioral responses in zebrafish. It became clear that intermediate concentration of risperidone inhibited the hypothalamic-pituitary-interrenal axis and displayed anxiolytic-like effects in zebrafish. The data presented here suggest that the presence of this antipsychotic in aquatic environments can alter neuroendocrine and behavior profiles in zebrafish.

  13. Learning and memory in zebrafish larvae

    PubMed Central

    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

  14. Zebrafish tracking using convolutional neural networks

    PubMed Central

    XU, Zhiping; Cheng, Xi En

    2017-01-01

    Keeping identity for a long term after occlusion is still an open problem in the video tracking of zebrafish-like model animals, and accurate animal trajectories are the foundation of behaviour analysis. We utilize the highly accurate object recognition capability of a convolutional neural network (CNN) to distinguish fish of the same congener, even though these animals are indistinguishable to the human eye. We used data augmentation and an iterative CNN training method to optimize the accuracy for our classification task, achieving surprisingly accurate trajectories of zebrafish of different size and age zebrafish groups over different time spans. This work will make further behaviour analysis more reliable. PMID:28211462

  15. Zebrafish tracking using convolutional neural networks

    NASA Astrophysics Data System (ADS)

    Xu, Zhiping; Cheng, Xi En

    2017-02-01

    Keeping identity for a long term after occlusion is still an open problem in the video tracking of zebrafish-like model animals, and accurate animal trajectories are the foundation of behaviour analysis. We utilize the highly accurate object recognition capability of a convolutional neural network (CNN) to distinguish fish of the same congener, even though these animals are indistinguishable to the human eye. We used data augmentation and an iterative CNN training method to optimize the accuracy for our classification task, achieving surprisingly accurate trajectories of zebrafish of different size and age zebrafish groups over different time spans. This work will make further behaviour analysis more reliable.

  16. Waterborne Risperidone Decreases Stress Response in Zebrafish

    PubMed Central

    Kalichak, Fabiana; Rosa, João Gabriel Santos; de Oliveira, Tiago Acosta; Koakoski, Gessi; Gusso, Darlan; de Abreu, Murilo Sander; Giacomini, Ana Cristina Varrone; Barcellos, Heloísa Helena de Alcântara

    2015-01-01

    The presence of drugs and their metabolites in surface waters and municipal effluents has been reported in several studies, but its impacts on aquatic organisms are not yet well understood. This study investigated the effects of acute exposure to the antipsychotic risperidone on the stress and behavioral responses in zebrafish. It became clear that intermediate concentration of risperidone inhibited the hypothalamic-pituitary-interrenal axis and displayed anxiolytic-like effects in zebrafish. The data presented here suggest that the presence of this antipsychotic in aquatic environments can alter neuroendocrine and behavior profiles in zebrafish. PMID:26473477

  17. Identifying proteins in zebrafish embryos using spectral libraries generated from dissected adult organs and tissues.

    PubMed

    van der Plas-Duivesteijn, Suzanne J; Mohammed, Yassene; Dalebout, Hans; Meijer, Annemarie; Botermans, Anouk; Hoogendijk, Jordy L; Henneman, Alex A; Deelder, André M; Spaink, Herman P; Palmblad, Magnus

    2014-03-07

    Spectral libraries provide a sensitive and accurate method for identifying peptides from tandem mass spectra, complementary to searching genome-derived databases or sequencing de novo. Their application requires comprehensive libraries including peptides from low-abundant proteins. Here we describe a method for constructing such libraries using biological differentiation to "fractionate" the proteome by harvesting adult organs and tissues and build comprehensive libraries for identifying proteins in zebrafish (Danio rerio) embryos and larvae (an important and widely used model system). Hierarchical clustering using direct comparison of spectra was used to prioritize organ selection. The resulting and publicly available library covers 14,164 proteins, significantly improved the number of peptide-spectrum matches in zebrafish developmental stages, and can be used on data from different instruments and laboratories. The library contains information on tissue and organ expression of these proteins and is also applicable for adult experiments. The approach itself is not limited to zebrafish but would work for any model system.

  18. Benzo[a]pyrene decreases global and gene specific DNA methylation during zebrafish development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA methylation is important for gene regulation and is vulnerable to early-life exposure to environmental contaminants. We found that direct waterborne benzo[a]pyrene (BaP) exposure at 24 'g/L from 2.5 to 96 hours post fertilization (hpf) to zebrafish embryos significantly decreased global cytosine...

  19. Heme Binding to the Mammalian Circadian Clock Protein Period 2 is Non-Specific†

    PubMed Central

    Airola, Michael V.; Du, Jing; Dawson, John H.; Crane, Brian R.

    2010-01-01

    The mammalian circadian clock synchronizes physical and metabolic activity with the diurnal cycle through a transcriptional-posttranslational feedback loop. An additional feedback mechanism regulating clock timing has been proposed to involve oscillation in heme availability. Period 2 (PER2), an integral component in the negative feedback loop that establishes circadian rhythms in mammals, has been identified as a heme binding protein. However, the majority of evidence for heme binding is based upon in vitro heme binding assays. We sought to ascertain if these largely spectral assays could distinguish between specific and non-specific heme interactions. Heme binding properties by a number of other well-characterized proteins, all with no known biological role involving heme interaction, corresponded to those displayed by PER2. Site-directed mutants of putative heme-binding residues identified by MCD were unable to locate a specific heme-binding site on PER2. Protein film electrochemistry also indicates that heme binds PER2 non-specifically on the protein surface. Our results establish the inability of typical in vitro assays to easily distinguish between specific and non-specific heme binding. We conclude that heme binding to PER2 is likely to be non-specific and does not involve the hydrophobic pocket within the PER2 PAS domains that in other PAS proteins commonly recognizes cofactors. These findings also question the significance of in vivo studies that implicate heme interactions with the clock proteins PER2 and nPAS2 in biological function. PMID:20411915

  20. Zebrafish Models for Human Acute Organophosphorus Poisoning.

    PubMed

    Faria, Melissa; Garcia-Reyero, Natàlia; Padrós, Francesc; Babin, Patrick J; Sebastián, David; Cachot, Jérôme; Prats, Eva; Arick Ii, Mark; Rial, Eduardo; Knoll-Gellida, Anja; Mathieu, Guilaine; Le Bihanic, Florane; Escalon, B Lynn; Zorzano, Antonio; Soares, Amadeu M V M; Raldúa, Demetrio

    2015-10-22

    Terrorist use of organophosphorus-based nerve agents and toxic industrial chemicals against civilian populations constitutes a real threat, as demonstrated by the terrorist attacks in Japan in the 1990 s or, even more recently, in the Syrian civil war. Thus, development of more effective countermeasures against acute organophosphorus poisoning is urgently needed. Here, we have generated and validated zebrafish models for mild, moderate and severe acute organophosphorus poisoning by exposing zebrafish larvae to different concentrations of the prototypic organophosphorus compound chlorpyrifos-oxon. Our results show that zebrafish models mimic most of the pathophysiological mechanisms behind this toxidrome in humans, including acetylcholinesterase inhibition, N-methyl-D-aspartate receptor activation, and calcium dysregulation as well as inflammatory and immune responses. The suitability of the zebrafish larvae to in vivo high-throughput screenings of small molecule libraries makes these models a valuable tool for identifying new drugs for multifunctional drug therapy against acute organophosphorus poisoning.

  1. Modeling Syndromic Congenital Heart Defects in Zebrafish.

    PubMed

    Grant, Meagan G; Patterson, Victoria L; Grimes, Daniel T; Burdine, Rebecca D

    2017-01-01

    Cardiac development is a dynamic process regulated by spatial and temporal cues that are integrated to effect molecular, cellular, and tissue-level events that form the adult heart. Disruption of these highly orchestrated events can be devastating for cardiac form and function. Aberrations in heart development result in congenital heart defects (CHDs), which affect 1 in 100 infants in the United States each year. Zebrafish have proven informative as a model organism to understand both heart development and the mechanisms associated with CHDs due to the similarities in heart morphogenesis among vertebrates, as well as their genetic tractability and amenability to live imaging. In this review, we discuss the mechanisms of zebrafish heart development and the utility of zebrafish for understanding syndromic CHDs, those cardiac abnormalities that occur in the context of multisystem disorders. We conclude with avenues of zebrafish research that will potentially inform future therapeutic approaches for the treatment of CHDs.

  2. Zebrafish Models for Human Acute Organophosphorus Poisoning

    PubMed Central

    Faria, Melissa; Garcia-Reyero, Natàlia; Padrós, Francesc; Babin, Patrick J.; Sebastián, David; Cachot, Jérôme; Prats, Eva; Arick II, Mark; Rial, Eduardo; Knoll-Gellida, Anja; Mathieu, Guilaine; Le Bihanic, Florane; Escalon, B. Lynn; Zorzano, Antonio; Soares, Amadeu M.V.M; Raldúa, Demetrio

    2015-01-01

    Terrorist use of organophosphorus-based nerve agents and toxic industrial chemicals against civilian populations constitutes a real threat, as demonstrated by the terrorist attacks in Japan in the 1990 s or, even more recently, in the Syrian civil war. Thus, development of more effective countermeasures against acute organophosphorus poisoning is urgently needed. Here, we have generated and validated zebrafish models for mild, moderate and severe acute organophosphorus poisoning by exposing zebrafish larvae to different concentrations of the prototypic organophosphorus compound chlorpyrifos-oxon. Our results show that zebrafish models mimic most of the pathophysiological mechanisms behind this toxidrome in humans, including acetylcholinesterase inhibition, N-methyl-D-aspartate receptor activation, and calcium dysregulation as well as inflammatory and immune responses. The suitability of the zebrafish larvae to in vivo high-throughput screenings of small molecule libraries makes these models a valuable tool for identifying new drugs for multifunctional drug therapy against acute organophosphorus poisoning. PMID:26489395

  3. Zebrafish olfactory receptor ORA1 recognizes a putative reproductive pheromone

    PubMed Central

    Ahuja, Gaurav; Korsching, Sigrun

    2014-01-01

    Teleost v1r-related ora genes constitute a small and highly conserved olfactory receptor gene family, and their direct orthologs are present in lineages as distant as cartilaginous fishes. Recently, the first member of the ora gene family was deorphanized. ORA1 detects p-hydroxyphenylacetic acid with high sensitivity and specificity. This compound elicits olfactory-mediated oviposition behavior in adult zebrafish mating pairs, suggesting a potential function as a reproductive pheromone for pHPAA itself or a related substance. This association of an odor and its cognate receptor with an oviposition response may provide a molecular basis for studying neural circuits involved in fish reproduction. PMID:26842458

  4. How mitochondrial dysfunction affects zebrafish development and cardiovascular function: an in vivo model for testing mitochondria-targeted drugs

    PubMed Central

    Pinho, Brígida R; Santos, Miguel M; Fonseca-Silva, Anabela; Valentão, Patrícia; Andrade, Paula B; Oliveira, Jorge M A

    2013-01-01

    Background and Purpose Mitochondria are a drug target in mitochondrial dysfunction diseases and in antiparasitic chemotherapy. While zebrafish is increasingly used as a biomedical model, its potential for mitochondrial research remains relatively unexplored. Here, we perform the first systematic analysis of how mitochondrial respiratory chain inhibitors affect zebrafish development and cardiovascular function, and assess multiple quinones, including ubiquinone mimetics idebenone and decylubiquinone, and the antimalarial atovaquone. Experimental Approach Zebrafish (Danio rerio) embryos were chronically and acutely exposed to mitochondrial inhibitors and quinone analogues. Concentration-response curves, developmental and cardiovascular phenotyping were performed together with sequence analysis of inhibitor-binding mitochondrial subunits in zebrafish versus mouse, human and parasites. Phenotype rescuing was assessed in co-exposure assays. Key Results Complex I and II inhibitors induced developmental abnormalities, but their submaximal toxicity was not additive, suggesting active alternative pathways for complex III feeding. Complex III inhibitors evoked a direct normal-to-dead transition. ATP synthase inhibition arrested gastrulation. Menadione induced hypochromic anaemia when transiently present following primitive erythropoiesis. Atovaquone was over 1000-fold less lethal in zebrafish than reported for Plasmodium falciparum, and its toxicity partly rescued by the ubiquinone precursor 4-hydroxybenzoate. Idebenone and decylubiquinone delayed rotenone- but not myxothiazol- or antimycin-evoked cardiac dysfunction. Conclusion and Implications This study characterizes pharmacologically induced mitochondrial dysfunction phenotypes in zebrafish, laying the foundation for comparison with future studies addressing mitochondrial dysfunction in this model organism. It has relevant implications for interpreting zebrafish disease models linked to complex I/II inhibition. Further

  5. Polygenic Sex Determination System in Zebrafish

    PubMed Central

    Liew, Woei Chang; Bartfai, Richard; Lim, Zijie; Sreenivasan, Rajini; Siegfried, Kellee R.; Orban, Laszlo

    2012-01-01

    Background Despite the popularity of zebrafish as a research model, its sex determination (SD) mechanism is still unknown. Most cytogenetic studies failed to find dimorphic sex chromosomes and no primary sex determining switch has been identified even though the assembly of zebrafish genome sequence is near to completion and a high resolution genetic map is available. Recent publications suggest that environmental factors within the natural range have minimal impact on sex ratios of zebrafish populations. The primary aim of this study is to find out more about how sex is determined in zebrafish. Methodology/Principal Findings Using classical breeding experiments, we found that sex ratios across families were wide ranging (4.8% to 97.3% males). On the other hand, repeated single pair crossings produced broods of very similar sex ratios, indicating that parental genotypes have a role in the sex ratio of the offspring. Variation among family sex ratios was reduced after selection for breeding pairs with predominantly male or female offspring, another indication that zebrafish sex is regulated genetically. Further examinations by a PCR-based “blind assay" and array comparative genomic hybridization both failed to find universal sex-linked differences between the male and female genomes. Together with the ability to increase the sex bias of lines by selective breeding, these data suggest that zebrafish is unlikely to utilize a chromosomal sex determination (CSD) system. Conclusions/Significance Taken together, our study suggests that zebrafish sex is genetically determined with limited, secondary influences from the environment. As we have not found any sign for CSD in the species, we propose that the zebrafish has a polygenic sex determination system. PMID:22506019

  6. Spinal cord transection in the larval zebrafish.

    PubMed

    Briona, Lisa K; Dorsky, Richard I

    2014-05-21

    Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6 weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2 days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

  7. Patterning of angiogenesis in the zebrafish embryo.

    PubMed

    Childs, Sarah; Chen, Jau-Nian; Garrity, Deborah M; Fishman, Mark C

    2002-02-01

    -type endothelial cells to assume the anomalous ISV pattern of obd embryos. Thus, the launching position of the new sprout and its initial trajectory are directed by inhibitory signals from ventral somites. Zebrafish ISVs are a tractable system for defining the origins and fates of vessels, and for dissecting elements that govern patterns of vessel growth.

  8. Neutron induced bystander effect among zebrafish embryos

    NASA Astrophysics Data System (ADS)

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

    2015-12-01

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

  9. Active microrheology of fluids inside developing zebrafish

    NASA Astrophysics Data System (ADS)

    Taormina, Mike; Parthasarathy, Raghuveer

    2014-03-01

    Biological fluids are a source of diverse and interesting behavior for the soft matter physicist. Since their mechanical properties must be tuned to fulfill functional roles important to the development and health of living things, they often display complex behavior on length and time scales spanning many orders of magnitude. For microbes colonizing an animal host, for example, the mechanical properties of the host environment are of great importance, affecting mobility and hence the ability to establish a stable population. Indeed, some species possess the ability to affect the fluidity of their environment, both directly by chemically modifying it, and indirectly by influencing the host cells' secretion of mucus. Driving magnetically doped micron-scale probes which have been orally micro-gavaged into the intestinal bulb of a larval zebrafish allows the rheology of the mucosal layer within the fish to be measured over three decades of frequency, complementing ecological data on microbial colonization with physical information about the gut environment. Here, we describe the technique, provide the first measurement of mucosal viscosity in a developing animal, and explore the technique's applicability to other small-volume or spatially inhomogeneous fluid samples.

  10. Zebrafish heart as a model for human cardiac electrophysiology.

    PubMed

    Vornanen, Matti; Hassinen, Minna

    2016-01-01

    The zebrafish (Danio rerio) has become a popular model for human cardiac diseases and pharmacology including cardiac arrhythmias and its electrophysiological basis. Notably, the phenotype of zebrafish cardiac action potential is similar to the human cardiac action potential in that both have a long plateau phase. Also the major inward and outward current systems are qualitatively similar in zebrafish and human hearts. However, there are also significant differences in ionic current composition between human and zebrafish hearts, and the molecular basis and pharmacological properties of human and zebrafish cardiac ionic currents differ in several ways. Cardiac ionic currents may be produced by non-orthologous genes in zebrafish and humans, and paralogous gene products of some ion channels are expressed in the zebrafish heart. More research on molecular basis of cardiac ion channels, and regulation and drug sensitivity of the cardiac ionic currents are needed to enable rational use of the zebrafish heart as an electrophysiological model for the human heart.

  11. Can Zebrafish be used to Identify Developmentally Neurotoxic Chemicals

    EPA Science Inventory

    Can Zebrafish be Used to Identify Developmentally Neurotoxic Chemicals? The U.S. Environmental Protection Agency is evaluating methods to screen and prioritize large numbers of chemicals for developmental neurotoxicity. We are exploring behavioral methods using zebrafish by desig...

  12. Neuroblastoma and Its Zebrafish Model.

    PubMed

    Zhu, Shizhen; Thomas Look, A

    2016-01-01

    Neuroblastoma, an important developmental tumor arising in the peripheral sympathetic nervous system (PSNS), accounts for approximately 10 % of all cancer-related deaths in children. Recent genomic analyses have identified a spectrum of genetic alterations in this tumor. Amplification of the MYCN oncogene is found in 20 % of cases and is often accompanied by mutational activation of the ALK (anaplastic lymphoma kinase) gene, suggesting their cooperation in tumor initiation and spread. Understanding how complex genetic changes function together in oncogenesis has been a continuing and daunting task in cancer research. This challenge was addressed in neuroblastoma by generating a transgenic zebrafish model that overexpresses human MYCN and activated ALK in the PSNS, leading to tumors that closely resemble human neuroblastoma and new opportunities to probe the mechanisms that underlie the pathogenesis of this tumor. For example, coexpression of activated ALK with MYCN in this model triples the penetrance of neuroblastoma and markedly accelerates tumor onset, demonstrating the interaction of these modified genes in tumor development. Further, MYCN overexpression induces adrenal sympathetic neuroblast hyperplasia, blocks chromaffin cell differentiation, and ultimately triggers a developmentally-timed apoptotic response in the hyperplastic sympathoadrenal cells. In the context of MYCN overexpression, activated ALK provides prosurvival signals that block this apoptotic response, allowing continued expansion and oncogenic transformation of hyperplastic neuroblasts, thus promoting progression to neuroblastoma. This application of the zebrafish model illustrates its value in rational assessment of the multigenic changes that define neuroblastoma pathogenesis and points the way to future studies to identify novel targets for therapeutic intervention.

  13. Viral diseases in zebrafish: what is known and unknown.

    PubMed

    Crim, Marcus J; Riley, Lela K

    2012-01-01

    Naturally occurring viral infections have the potential to introduce confounding variability that leads to invalid and misinterpreted data. Whereas the viral diseases of research rodents are well characterized and closely monitored, no naturally occurring viral infections have been characterized for the laboratory zebrafish (Danio rerio), an increasingly important biomedical research model. Despite the ignorance about naturally occurring zebrafish viruses, zebrafish models are rapidly expanding in areas of biomedical research where the confounding effects of unknown infectious agents present a serious concern. In addition, many zebrafish research colonies remain linked to the ornamental (pet) zebrafish trade, which can contribute to the introduction of new pathogens into research colonies, whereas mice used for research are purpose bred, with no introduction of new mice from the pet industry. Identification, characterization, and monitoring of naturally occurring viruses in zebrafish are crucial to the improvement of zebrafish health, the reduction of unwanted variability, and the continued development of the zebrafish as a model organism. This article addresses the importance of identifying and characterizing the viral diseases of zebrafish as the scope of zebrafish models expands into new research areas and also briefly addresses zebrafish susceptibility to experimental viral infection and the utility of the zebrafish as an infection and immunology model.

  14. Viral Diseases in Zebrafish: What Is Known and Unknown

    PubMed Central

    Crim, Marcus J.; Riley, Lela K.

    2013-01-01

    Naturally occurring viral infections have the potential to introduce confounding variability that leads to invalid and misinterpreted data. Whereas the viral diseases of research rodents are well characterized and closely monitored, no naturally occurring viral infections have been characterized for the laboratory zebrafish (Danio rerio), an increasingly important biomedical research model. Despite the ignorance about naturally occurring zebrafish viruses, zebrafish models are rapidly expanding in areas of biomedical research where the confounding effects of unknown infectious agents present a serious concern. In addition, many zebrafish research colonies remain linked to the ornamental (pet) zebrafish trade, which can contribute to the introduction of new pathogens into research colonies, whereas mice used for research are purpose bred, with no introduction of new mice from the pet industry. Identification, characterization, and monitoring of naturally occurring viruses in zebrafish are crucial to the improvement of zebrafish health, the reduction of unwanted variability, and the continued development of the zebrafish as a model organism. This article addresses the importance of identifying and characterizing the viral diseases of zebrafish as the scope of zebrafish models expands into new research areas and also briefly addresses zebrafish susceptibility to experimental viral infection and the utility of the zebrafish as an infection and immunology model. PMID:23382345

  15. Disruption of Epithalamic Left-Right Asymmetry Increases Anxiety in Zebrafish.

    PubMed

    Facchin, Lucilla; Duboué, Erik R; Halpern, Marnie E

    2015-12-02

    Differences between the left and right sides of the brain are found throughout the animal kingdom, but the consequences of altered neural asymmetry are not well understood. In the zebrafish epithalamus, the parapineal is located on the left side of the brain where it influences development of the adjacent dorsal habenular (dHb) nucleus, causing the left and right dHb to differ in their organization, gene expression, and connectivity. Left-right (L-R) reversal of parapineal position and dHb asymmetry occurs spontaneously in a small percentage of the population, whereas the dHb develop symmetrically following experimental ablation of the parapineal. The habenular region was previously implicated in modulating fear in both mice and zebrafish, but the relevance of its L-R asymmetry is unclear. We now demonstrate that disrupting directionality of the zebrafish epithalamus causes reduced exploratory behavior and increased cortisol levels, indicative of enhanced anxiety. Accordingly, exposure to buspirone, an anxiolytic agent, significantly suppresses atypical behavior. Axonal projections from the parapineal to the dHb are more variable when it is located on the right side of the brain, revealing that L-R reversals do not necessarily represent a neuroanatomical mirror image. The results highlight the importance of directional asymmetry of the epithalamus in the regulation of stress responses in zebrafish.

  16. Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

    PubMed Central

    Sidhaye, Jaydeep; Tomancak, Pavel; Preibisch, Stephan; Norden, Caren

    2016-01-01

    Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments. PMID:27167079

  17. Methods for generating and colonizing gnotobiotic zebrafish

    PubMed Central

    Pham, Linh N.; Kanther, Michelle; Semova, Ivana; Rawls, John F.

    2008-01-01

    Vertebrates are colonized at birth by complex and dynamic communities of microorganisms that can contribute significantly to host health and disease. The ability to raise animals in the absence of microorganisms has been a powerful tool for elucidating the relationships between animal hosts and their microbial residents. The optical transparency of the developing zebrafish and relative ease of generating germ-free zebrafish makes it an attractive model organism for gnotobiotic research. Here we provide a protocol for: generating zebrafish embryos; deriving and rearing germ-free zebrafish; and colonizing zebrafish with microorganisms. Using these methods, we typically obtain 80–90% sterility rates in our germ-free derivations with 90% survival in germ-free animals and 50–90% survival in colonized animals through larval stages. Obtaining embryos for derivation requires approximately 1–2 hours with a 3–8 hour incubation period prior to derivation. Derivation of germ-free animals takes 1–1.5 hours, and daily maintenance requires 1–2 hours. PMID:19008873

  18. Defects of the Glycinergic Synapse in Zebrafish.

    PubMed

    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.

  19. Production of Androgenetic Zebrafish (Danio Rerio)

    PubMed Central

    Corley-Smith, G. E.; Lim, C. J.; Brandhorst, B. P.

    1996-01-01

    To help investigate the evolutionary origin of the imprinting (parent-of-origin mono-allelic expression) of paternal genes observed in mammals, we constructed haploid and diploid androgenetic zebrafish (Danio rerio). Haploid androgenotes were produced by fertilizing eggs that had been X-ray irradiated to eliminate the maternal genome. Subsequent inhibition of the first mitotic division of haploid androgenotes by heat shock produced diploid androgenotes. The lack of inheritance of maternal-specific DNA markers (RAPD and SSR) by putative diploid and haploid androgenotes confirmed the androgenetic origin of their genomes. Marker analysis was performed on 18 putative androgenotes (five diploids and 13 haploids) from six families. None of 157 maternal-specific RAPD markers analyzed, some of which were apparently homozygous, were passed on to any of these putative androgenotes. A mean of 7.7 maternal-specific markers were assessed per family. The survival of androgenetic zebrafish suggests that if paternal imprinting occurs in zebrafish, it does not result in essential genes being inactivated when their expression is required for development. Production of haploid androgenotes can be used to determine the meiotic recombination rate in male zebrafish. Androgenesis may also provide useful information about the mechanism of sex determination in zebrafish. PMID:8846903

  20. Latent learning in zebrafish (Danio rerio).

    PubMed

    Gómez-Laplaza, Luis M; Gerlai, Robert

    2010-04-02

    The zebrafish may represent an excellent compromise between system complexity and practical simplicity for behavioral brain research. It may be particularly appropriate for large scale screening studies whose aim is to identify mutants with altered phenotypes or novel compounds with particular efficacy. For example, the zebrafish may have utility in the analysis of the biological mechanisms of learning and memory. Although learning and memory have been extensively studied and hundreds of underlying molecular mechanisms have been identified, this number may represent only the fraction of genes involved in these complex brain functions. Thus large scale mutagenesis screens may have utility. In order for such screens to succeed, appropriate screening paradigms must be developed. The first step in this research is the characterization of learning and memory capabilities of zebrafish and the development of automatable tasks. Here we show that zebrafish is capable of latent learning, i.e. can acquire memory of their environment after being allowed to explore it. For example, we found experimental zebrafish that experienced an open left tunnel or an open right tunnel of a maze during the unrewarded exploration phase of the test to show the appropriate side bias during a probe trial when they had to swim to a group of conspecifics (the reward). Given that exploration of the maze does not require the presence of the experimenter and the probe trial, during which the subjects are video-recorded and their memory is tested, is short, we argue that the paradigm has utility in high-throughput screening.

  1. Regeneration of the Pancreas in Adult Zebrafish

    PubMed Central

    Moss, Jennifer B.; Koustubhan, Punita; Greenman, Melanie; Parsons, Michael J.; Walter, Ingrid; Moss, Larry G.

    2009-01-01

    OBJECTIVE Regenerating organs in diverse biological systems have provided clues to processes that can be harnessed to repair damaged tissue. Adult mammalian β-cells have a limited capacity to regenerate, resulting in diabetes and lifelong reliance on insulin. Zebrafish have been used as a model for the regeneration of many organs. We demonstrate the regeneration of adult zebrafish pancreatic β-cells. This nonmammalian model can be used to define pathways for islet-cell regeneration in humans. RESEARCH DESIGN AND METHODS Adult transgenic zebrafish were injected with a single high dose of streptozotocin or metronidazole and anesthetized at 3, 7, or 14 days or pancreatectomized. Blood glucose measurements were determined and gut sections were analyzed using specific endocrine, exocrine, and duct cell markers as well as markers for dividing cells. RESULTS Zebrafish recovered rapidly without the need for insulin injections, and normoglycemia was attained within 2 weeks. Although few proliferating cells were present in vehicles, ablation caused islet destruction and a striking increase of proliferating cells, some of which were Pdx1 positive. Dividing cells were primarily associated with affected islets and ducts but, with the exception of surgical partial pancreatectomy, were not extensively β-cells. CONCLUSIONS The ability of the zebrafish to regenerate a functional pancreas using chemical, genetic, and surgical approaches enabled us to identify patterns of cell proliferation in islets and ducts. Further study of the origin and contribution of proliferating cells in reestablishing islet function could provide strategies for treating human diseases. PMID:19491207

  2. Transcriptome Analysis of Zebrafish Embryogenesis Using Microarrays

    PubMed Central

    Mathavan, Sinnakaruppan; Lee, Serene G. P; Mak, Alicia; Miller, Lance D; Murthy, Karuturi Radha Krishna; Govindarajan, Kunde R; Tong, Yan; Wu, Yi Lian; Lam, Siew Hong; Yang, Henry; Ruan, Yijun; Korzh, Vladimir; Gong, Zhiyuan; Liu, Edison T; Lufkin, Thomas

    2005-01-01

    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). PMID:16132083

  3. Zebrafish as an emerging model for studying complex brain disorders

    PubMed Central

    Kalueff, Allan V.; Stewart, Adam Michael; Gerlai, Robert

    2014-01-01

    The zebrafish (Danio rerio) is rapidly becoming a popular model organism in pharmacogenetics and neuropharmacology. Both larval and adult zebrafish are currently used to increase our understanding of brain function, dysfunction, and their genetic and pharmacological modulation. Here we review the developing utility of zebrafish in the analysis of complex brain disorders (including, for example, depression, autism, psychoses, drug abuse and cognitive disorders), also covering zebrafish applications towards the goal of modeling major human neuropsychiatric and drug-induced syndromes. We argue that zebrafish models of complex brain disorders and drug-induced conditions have become a rapidly emerging critical field in translational neuropharmacology research. PMID:24412421

  4. Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

    PubMed Central

    Lopez-Ramirez, Miguel A.; Calvo, Charles-Félix; Ristori, Emma; Thomas, Jean-Léon; Nicoli, Stefania

    2016-01-01

    The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish. PMID:26967835

  5. Developing 'integrative' zebrafish models of behavioral and metabolic disorders.

    PubMed

    Nguyen, Michael; Yang, Ester; Neelkantan, Nikhil; Mikhaylova, Alina; Arnold, Raymond; Poudel, Manoj K; Stewart, Adam Michael; Kalueff, Allan V

    2013-11-01

    Recently, the pathophysiological overlap between metabolic and mental disorders has received increased recognition. Zebrafish (Danio rerio) are rapidly becoming a popular model organism for translational biomedical research due to their genetic tractability, low cost, quick reproductive cycle, and ease of behavioral, pharmacological or genetic manipulation. High homology to mammalian physiology and the availability of well-developed assays also make the zebrafish an attractive organism for studying human disorders. Zebrafish neurobehavioral and endocrine phenotypes show promise for the use of zebrafish in studies of stress, obesity and related behavioral and metabolic disorders. Here, we discuss the parallels between zebrafish and other model species in stress and obesity physiology, as well as outline the available zebrafish models of weight gain, metabolic deficits, feeding, stress, anxiety and related behavioral disorders. Overall, zebrafish demonstrate a strong potential for modeling human behavioral and metabolic disorders, and their comorbidity.

  6. In vitro development of zebrafish vascular networks.

    PubMed

    Ibrahim, Muhammad; Richardson, Michael K

    2017-02-09

    A major limitation to culturing tissues and organs is the lack of a functional vascular network in vitro. The zebrafish possess many useful properties which makes it a promising model for such studies. Unfortunately, methods of culturing endothelial cells from this species are not well characterised. Here, we tried two methods (embryoid body culture and organ explants from transgenic zebrafish kdrl:GFP embryos) to develop in vitro vascular networks. In the kdrl:GFP line, endothelial cells expresses green fluorescent protein, which allows to track the vascular development in live cultures. We found that embryoid bodies showed significantly longer and wider branches of connected endothelial cells when grown in a microfluidic system than in static culture. Similarly, sprouting of kdrl:GFP(+) cells from the tissue explants was observed in a 3D hydrogel matrix. This study is a step towards the development of zebrafish vascular networks in vitro.

  7. Conditional gene-trap mutagenesis in zebrafish.

    PubMed

    Maddison, Lisette A; Li, Mingyu; Chen, Wenbiao

    2014-01-01

    Zebrafish has become a widely used model for analysis of gene function. Several methods have been used to create mutations in this organism and thousands of mutant lines are available. However, all the conventional zebrafish mutations affect the gene in all cells at all time, making it difficult to determine tissue-specific functions. We have adopted a FlEx Trap approach to generate conditional mutations in zebrafish by gene-trap mutagenesis. Combined with appropriate Cre or Flp lines, the insertional mutants not only allow spatial- and temporal-specific gene inactivation but also permit spatial- and temporal-specific rescue of the disrupted gene. We provide experimental details on how to generate and use such mutations.

  8. 15 years of zebrafish chemical screening

    PubMed Central

    Rennekamp, Andrew J.; Peterson, Randall T.

    2015-01-01

    In 2000, the first chemical screen using living zebrafish in a multi-well plate was reported. Since then, more than 60 additional screens have been published describing whole-organism drug and pathway discovery projects in zebrafish. To investigate the scope of the work reported in the last 14 years and to identify trends in the field, we analyzed the discovery strategies of 64 primary research articles from the literature. We found that zebrafish screens have expanded beyond the use of developmental phenotypes to include behavioral, cardiac, metabolic, proliferative and regenerative endpoints. Additionally, many creative strategies have been used to uncover the mechanisms of action of new small molecules including chemical phenocopy, genetic phenocopy, mutant rescue, and spatial localization strategies. PMID:25461724

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

    PubMed

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

    2017-01-20

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

  10. Fishing for Fetal Alcohol Spectrum Disorders: Zebrafish as a Model for Ethanol Teratogenesis.

    PubMed

    Lovely, Charles Ben; Fernandes, Yohaan; Eberhart, Johann K

    2016-10-01

    Fetal Alcohol Spectrum Disorders (FASD) describes a wide array of ethanol-induced developmental defects, including craniofacial dysmorphology and cognitive impairments. It affects ∼1 in 100 children born in the United States each year. Due to the pleiotropic effects of ethanol, animal models have proven critical in characterizing the mechanisms of ethanol teratogenesis. In this review, we focus on the utility of zebrafish in characterizing ethanol-induced developmental defects. A growing number of laboratories have focused on using zebrafish to examine ethanol-induced defects in craniofacial, cardiac, ocular, and neural development, as well as cognitive and behavioral impairments. Growing evidence supports that genetic predisposition plays a role in these ethanol-induced defects, yet little is understood about these gene-ethanol interactions. With a high degree of genetic amenability, zebrafish is at the forefront of identifying and characterizing the gene-ethanol interactions that underlie FASD. Because of the conservation of gene function between zebrafish and humans, these studies will directly translate to studies of candidate genes in human populations and allow for better diagnosis and treatment of FASD.

  11. Development of high-content assays for kidney progenitor cell expansion in transgenic zebrafish.

    PubMed

    Sanker, Subramaniam; Cirio, Maria Cecilia; Vollmer, Laura L; Goldberg, Natasha D; McDermott, Lee A; Hukriede, Neil A; Vogt, Andreas

    2013-12-01

    Reactivation of genes normally expressed during organogenesis is a characteristic of kidney regeneration. Enhancing this reactivation could potentially be a therapeutic target to augment kidney regeneration. The inductive events that drive kidney organogenesis in zebrafish are similar to the initial steps in mammalian kidney organogenesis. Therefore, quantifying embryonic signals that drive zebrafish kidney development is an attractive strategy for the discovery of potential novel therapeutic modalities that accelerate kidney regeneration. The Lim1 homeobox protein, Lhx1, is a marker of kidney development that is also expressed in the regenerating kidneys after injury. Using a fluorescent Lhx1a-EGFP transgene whose phenotype faithfully recapitulates that of the endogenous protein, we developed a high-content assay for Lhx1a-EGFP expression in transgenic zebrafish embryos employing an artificial intelligence-based image analysis method termed cognition network technology (CNT). Implementation of the CNT assay on high-content readers enabled automated real-time in vivo time-course, dose-response, and variability studies in the developing embryo. The Lhx1a assay was complemented with a kidney-specific secondary CNT assay that enables direct measurements of the embryonic renal tubule cell population. The integration of fluorescent transgenic zebrafish embryos with automated imaging and artificial intelligence-based image analysis provides an in vivo analysis system for structure-activity relationship studies and de novo discovery of novel agents that augment innate regenerative processes.

  12. Mmp23b promotes liver development and hepatocyte proliferation through the TNF pathway in zebrafish

    PubMed Central

    Qi, Fei; Song, Jianbo; Yang, Hanshuo; Gao, Wei; Liu, Ning-ai; Zhang, Bo; Lin, Shuo

    2012-01-01

    The matrix metalloproteinase (MMP) family of proteins degrades extracellular matrix (ECM) components as well as processes cytokines and growth factors. MMPs are involved in regulating ECM homeostasis in both normal physiology and disease pathophysiology. Here, we report the critical roles of mmp23b in normal zebrafish liver development. Mmp23b was initially identified as a gene linked to the genomic locus of an enhancer trap transgenic zebrafish line in which GFP expression was restricted to the developing liver. Follow-up analysis of mmp23b mRNA expression confirmed its liver-specific expression pattern. Morpholino (MO) knockdown of mmp23b resulted in defective hepatocyte proliferation, causing a reduction in liver size while maintaining relatively normal pancreas and gut development. Genetically, we showed that mmp23b functions through the tumor necrosis factor (TNF) signaling pathway. Antisense knockdown of tnfa or tnfb in zebrafish caused similar reductions of liver size whereas overexpression of tnfa or tnfb rescued liver defects in mmp23b morphants but not vice versa. Biochemically, MMP23B, the human ortholog of Mmp23b, directly interacts with TNF and mediates its release from the cell membrane in a cell culture system. Since mmp23b/MMP23B is highly conserved, our findings in zebrafish warrant further investigation of its role in regulating liver development in mammals. PMID:21064033

  13. Dynamic microtubules at the vegetal cortex predict the embryonic axis in zebrafish.

    PubMed

    Tran, Long Duc; Hino, Hiromu; Quach, Helen; Lim, Shimin; Shindo, Asako; Mimori-Kiyosue, Yuko; Mione, Marina; Ueno, Naoto; Winkler, Christoph; Hibi, Masahiko; Sampath, Karuna

    2012-10-01

    In zebrafish, as in many animals, maternal dorsal determinants are vegetally localized in the egg and are transported after fertilization in a microtubule-dependent manner. However, the organization of early microtubules, their dynamics and their contribution to axis formation are not fully understood. Using live imaging, we identified two populations of microtubules, perpendicular bundles and parallel arrays, which are directionally oriented and detected exclusively at the vegetal cortex before the first cell division. Perpendicular bundles emanate from the vegetal cortex, extend towards the blastoderm, and orient along the animal-vegetal axis. Parallel arrays become asymmetric on the vegetal cortex, and orient towards dorsal. We show that the orientation of microtubules at 20 minutes post-fertilization can predict where the embryonic dorsal structures in zebrafish will form. Furthermore, we find that parallel microtubule arrays colocalize with wnt8a RNA, the candidate maternal dorsal factor. Vegetal cytoplasmic granules are displaced with parallel arrays by ~20°, providing in vivo evidence of a cortical rotation-like process in zebrafish. Cortical displacement requires parallel microtubule arrays, and probably contributes to asymmetric transport of maternal determinants. Formation of parallel arrays depends on Ca(2+) signaling. Thus, microtubule polarity and organization predicts the zebrafish embryonic axis. In addition, our results suggest that cortical rotation-like processes might be more common in early development than previously thought.

  14. Efficient homologous recombination-mediated genome engineering in zebrafish using TALE nucleases.

    PubMed

    Shin, Jimann; Chen, Jiakun; Solnica-Krezel, Lilianna

    2014-10-01

    Custom-designed nucleases afford a powerful reverse genetic tool for direct gene disruption and genome modification in vivo. Among various applications of the nucleases, homologous recombination (HR)-mediated genome editing is particularly useful for inserting heterologous DNA fragments, such as GFP, into a specific genomic locus in a sequence-specific fashion. However, precise HR-mediated genome editing is still technically challenging in zebrafish. Here, we establish a GFP reporter system for measuring the frequency of HR events in live zebrafish embryos. By co-injecting a TALE nuclease and GFP reporter targeting constructs with homology arms of different size, we defined the length of homology arms that increases the recombination efficiency. In addition, we found that the configuration of the targeting construct can be a crucial parameter in determining the efficiency of HR-mediated genome engineering. Implementing these modifications improved the efficiency of zebrafish knock-in generation, with over 10% of the injected F0 animals transmitting gene-targeting events through their germline. We generated two HR-mediated insertion alleles of sox2 and gfap loci that express either superfolder GFP (sfGFP) or tandem dimeric Tomato (tdTomato) in a spatiotemporal pattern that mirrors the endogenous loci. This efficient strategy provides new opportunities not only to monitor expression of endogenous genes and proteins and follow specific cell types in vivo, but it also paves the way for other sophisticated genetic manipulations of the zebrafish genome.

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

    PubMed

    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.

  16. Immunohistochemical Characterization of Intestinal Neoplasia in Zebrafish (Danio rerio) Indicates Epithelial Origin

    PubMed Central

    Paquette, Colleen E.; Kent, Michael L.; Peterson, Tracy S.; Wang, Rong; Dashwood, Roderick H.; Löhr, Christiane V.

    2015-01-01

    Spontaneous neoplasia of the intestinal tract in sentinel and moribund zebrafish (Danio rerio) is common in some zebrafish facilities. We previously classified these tumors as adenocarcinoma, small-cell carcinoma, or carcinoma otherwise unspecified based on histomorphologic characteristics. Based on histological presentation, the primary differential diagnosis for the intestinal carcinomas was tumor of neuroendocrine cells (e.g., carcinoids). To further characterize the phenotype of the neoplastic cells, select tissue sections were stained with a panel of antibodies directed toward human epithelial (Cytokeratin Wide Spectrum Screening [WSS], AE1/AE3) or neuroendocrine (S100, chromogranin A) markers. We also investigated antibody specificity by Western blot analysis, using a human cell line and zebrafish tissues. Nine of the intestinal neoplasms (64%) stained for AE1/AE3, seven (50%) also stained for WSS. None of the intestinal neoplastic cells were stained for chromogranin A or S100. Endocrine cells of the pituitary gland and neurons and axons of peripheral nerves and ganglia stained for Chromogranin A, whereas perineural and periaxonal cells of peripheral intestinal ganglia, and glial and ependymal cells of the brain stained for S100. Immunohistochemistry for cytokeratins confirmed the majority of intestinal neoplasms in this cohort of zebrafish as carcinomas. PMID:26503773

  17. Nicotine alters the expression of molecular markers of endocrine disruption in zebrafish.

    PubMed

    Kanungo, Jyotshna; Cuevas, Elvis; Guo, Xiaoqing; Lopez, Aida G; Ramirez-Lee, Manuel A; Trickler, William; Paule, Merle G; Ali, Syed F

    2012-09-27

    Nicotine, a drug of abuse, has been reported to have many adverse effects on the developing nervous system. In rodents, chronic nicotine exposure inhibits estrogen-mediated neuroprotection against cerebral ischemia in females suggesting that nicotine could disrupt endocrine targets. Zebrafish have been used as a model system for examining mechanisms underlying nicotinic effects on neuronal development. Here, using zebrafish embryos, we demonstrate that nicotine alters the expression of the validated endocrine disruption (ED) biomarkers, vitellogenin (vtg 1 and vtg 2) and cytochrome p450 aromatase (cyp19a1a and cyp19a1b) at the transcriptional level. Increased expression of three of these molecular markers (vtg 1, vtg 2 and cyp19a1b) in response to 17β-estradiol (E2) was more pronounced in 48hpf (hours post-fertilization) embryos than in the 24hpf embryos. While 24hpf embryos were non-responsive in this regard to 25μM nicotine, a similar exposure of the 48hpf embryos for 24h significantly down-regulated the expression of all four ED biomarker genes indicating that nicotine's anti-estrogenic effects are detectable in the 48hpf zebrafish embryos. These results provide direct molecular evidence that nicotine is an endocrine disruptor in zebrafish.

  18. Characterization of zebrafish dysferlin by morpholino knockdown

    SciTech Connect

    Kawahara, Genri; Serafini, Peter R.; Myers, Jennifer A.; Alexander, Matthew S.; Kunkel, Louis M.

    2011-09-23

    Highlights: {yields} cDNAs of zebrafish dysferlin were cloned (6.3 kb). {yields} The dysferlin expression was detected in skeletal muscle, heart and eye. {yields} Injection of antisense morpholinos to dysferlin caused marked muscle disorganization. {yields} Zebrafish dysferlin expression may be involved in stabilizing muscle structures. -- Abstract: Mutations in the gene encoding dysferlin cause two distinct muscular dystrophy phenotypes: limb-girdle muscular dystrophy type 2B (LGMD-2B) and Miyoshi myopathy (MM). Dysferlin is a large transmembrane protein involved in myoblast fusion and membrane resealing. Zebrafish represent an ideal animal model to use for studying muscle disease including abnormalities of dysferlin. cDNAs of zebrafish dysferlin were cloned (6.3 kb) and the predicted amino acid sequences, showed 68% similarity to predicted amino acid sequences of mammalian dysferlin. The expression of dysferlin was mainly in skeletal muscle, heart and eye, and the expression could be detected as early as 11 h post fertilization (hpf). Three different antisense oligonucleotide morpholinos were targeted to inhibit translation of this dysferlin mRNA and the morpholino-injected fish showed marked muscle disorganization which could be detected by birefringence assay. Western blot analysis using dysferlin antibodies showed that the expression of dysferlin was reduced in each of the three morphants. Dysferlin expression was shown to be reduced at the myosepta of zebrafish muscle using immunohistochemistry, although the expression of other muscle membrane components, dystrophin, laminin, {beta}-dystroglycan were detected normally. Our data suggest that zebrafish dysferlin expression is involved in stabilizing muscle structures and its downregulation causes muscle disorganization.

  19. Oxidative stress in zebrafish (Danio rerio) sperm.

    PubMed

    Hagedorn, Mary; McCarthy, Megan; Carter, Virginia L; Meyers, Stuart A

    2012-01-01

    Laboratories around the world have produced tens of thousands of mutant and transgenic zebrafish lines. As with mice, maintaining all of these valuable zebrafish genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Because reducing oxidative stress has become an important aspect of reducing the variability in mouse sperm cryopreservation, we examined whether antioxidants might improve cryopreservation of zebrafish sperm. Four experiments were conducted in this study. First, we used the xanthine-xanthine oxidase (X-XO) system to generate reactive oxygen species (ROS). The X-XO system was capable of producing a stress reaction in zebrafish sperm reducing its sperm motility in a concentration dependent manner (P<0.05). Second, we examined X-XO and the impact of antioxidants on sperm viability, ROS and motility. Catalase (CAT) mitigated stress and maintained viability and sperm motility (P>0.05), whereas superoxide dismutase (SOD) and vitamin E did not (P<0.05). Third, we evaluated ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility. Methanol (8%) reduced viability and sperm motility (P<0.05), but the addition of CAT mitigated these effects (P>0.05), producing a mean 2.0 to 2.9-fold increase in post-thaw motility. Fourth, we examined the effect of additional cryoprotectants and CAT on fresh sperm motility. Cryoprotectants, 8% methanol and 10% dimethylacetamide (DMA), reduced the motility over the control value (P<0.5), whereas 10% dimethylformamide (DMF) with or without CAT did not (P>0.05). Zebrafish sperm protocols should be modified to improve the reliability of the cryopreservation process, perhaps using a different cryoprotectant. Regardless, the simple addition of CAT to present-day procedures will significantly improve this process, assuring increased and less variable fertilization success and allowing resource managers to dependably plan how many straws are needed to safely

  20. 77 FR 2022 - Fisheries of the Northeastern United States; Spiny Dogfish Fishery; Commercial Period 2 Quota...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-01-13

    ... Northeastern United States; Spiny Dogfish Fishery; Commercial Period 2 Quota Harvested AGENCY: National Marine...: Temporary rule; closure of spiny dogfish fishery. SUMMARY: NMFS announces that the spiny dogfish commercial..., federally permitted spiny dogfish vessels may not fish for, possess, transfer, or land spiny dogfish...

  1. 75 FR 4004 - Fisheries of the Northeastern United States; Spiny Dogfish Fishery; Commercial Period 2 Quota...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-26

    ... United States; Spiny Dogfish Fishery; Commercial Period 2 Quota Harvested AGENCY: National Marine...: Temporary rule; Closure of spiny dogfish fishery. SUMMARY: NMFS announces that the spiny dogfish commercial..., federally permitted spiny dogfish vessels may not fish for, possess, transfer, or land spiny dogfish...

  2. Glyphosate induces neurotoxicity in zebrafish.

    PubMed

    Roy, Nicole M; Carneiro, Bruno; Ochs, Jeremy

    2016-03-01

    Glyphosate based herbicides (GBH) like Roundup(®) are used extensively in agriculture as well as in urban and rural settings as a broad spectrum herbicide. Its mechanism of action was thought to be specific only to plants and thus considered safe and non-toxic. However, mounting evidence suggests that GBHs may not be as safe as once thought as initial studies in frogs suggest that GBHs may be teratogenic. Here we utilize the zebrafish vertebrate model system to study early effects of glyphosate exposure using technical grade glyphosate and the Roundup(®) Classic formulation. We find morphological abnormalities including cephalic and eye reductions and a loss of delineated brain ventricles. Concomitant with structural changes in the developing brain, using in situ hybridization analysis, we detect decreases in genes expressed in the eye, fore and midbrain regions of the brain including pax2, pax6, otx2 and ephA4. However, we do not detect changes in hindbrain expression domains of ephA4 nor exclusive hindbrain markers krox-20 and hoxb1a. Additionally, using a Retinoic Acid (RA) mediated reporter transgenic, we detect no alterations in the RA expression domains in the hindbrain and spinal cord, but do detect a loss of expression in the retina. We conclude that glyphosate and the Roundup(®) formulation is developmentally toxic to the forebrain and midbrain but does not affect the hindbrain after 24 h exposure.

  3. The zebrafish early arrest mutants.

    PubMed

    Kane, D A; Maischein, H M; Brand, M; van Eeden, F J; Furutani-Seiki, M; Granato, M; Haffter, P; Hammerschmidt, M; Heisenberg, C P; Jiang, Y J; Kelsh, R N; Mullins, M C; Odenthal, J; Warga, R M; Nüsslein-Volhard, C

    1996-12-01

    This report describes mutants of the zebrafish having phenotypes causing a general arrest in early morphogenesis. These mutants identify a group of loci making up about 20% of the loci identified by mutants with visible morphological phenotypes within the first day of development. There are 12 Class I mutants, which fall into 5 complementation groups and have cells that lyse before morphological defects are observed. Mutants at three loci, speed bump, ogre and zombie, display abnormal nuclei. The 8 Class II mutants, which fall into 6 complementation groups, arrest development before cell lysis is observed. These mutants seemingly stop development in the late segmentation stages, and maintain a body shape similar to a 20 hour embryo. Mutations in speed bump, ogre, zombie, specter, poltergeist and troll were tested for cell lethality by transplanting mutant cells into wild-type hosts. With poltergeist, transplanted mutant cells all survive. The remainder of the mutants tested were autonomously but conditionally lethal: mutant cells, most of which lyse, sometimes survive to become notochord, muscles, or, in rare cases, large neurons, all cell types which become postmitotic in the gastrula. Some of the genes of the early arrest group may be necessary for progression though the cell cycle; if so, the survival of early differentiating cells may be based on having their terminal mitosis before the zygotic requirement for these genes.

  4. Dynamic focusing in the zebrafish beating heart

    NASA Astrophysics Data System (ADS)

    Andrés-Delgado, L.; Peralta, M.; Mercader, N.; Ripoll, J.

    2016-03-01

    Of the large amount of the animal models available for cardiac research, the zebrafish is extremely valuable due to its transparency during early stages of development. In this work a dual illumination laser sheet microscope with simultaneous dual camera imaging is used to image the beating heart at 200 fps, dynamically and selectively focusing inside the beating heart through the use of a tunable lens. This dual color dynamic focusing enables imaging with cellular resolution at unprecedented high frame rates, allowing 3D imaging of the whole beating heart of embryonic zebrafish.

  5. Crypt cells are involved in kin recognition in larval zebrafish.

    PubMed

    Biechl, Daniela; Tietje, Kristin; Gerlach, Gabriele; Wullimann, Mario F

    2016-04-18

    Zebrafish larvae imprint on visual and olfactory kin cues at day 5 and 6 postfertilization, respectively, resulting in kin recognition later in life. Exposure to non-kin cues prevents imprinting and kin recognition. Imprinting depends on MHC class II related signals and only larvae sharing MHC class II alleles can imprint on each other. Here, we analyzed which type of olfactory sensory neuron (OSN) detects kin odor. The single teleost olfactory epithelium harbors ciliated OSNs carrying OR and TAAR gene family receptors (mammals: main olfactory epithelium) and microvillous OSNs with V1R and V2R gene family receptors (mammals: vomeronasal organ). Additionally, teleosts exhibit crypt cells which possess microvilli and cilia. We used the activity marker pERK (phosphorylated extracellular signal regulated kinase) after stimulating 9 day old zebrafish larvae with either non-kin conspecific or food odor. While food odor activated both ciliated and microvillous OSNs, only the latter were activated by conspecific odor, crypt cells showed no activation to both stimuli. Then, we tested imprinted and non-imprinted larvae (full siblings) for kin odor detection. We provide the first direct evidence that crypt cells, and likely a subpopulation of microvillous OSNs, but not ciliated OSNs, play a role in detecting a kin odor related signal.

  6. Crypt cells are involved in kin recognition in larval zebrafish

    PubMed Central

    Biechl, Daniela; Tietje, Kristin; Gerlach, Gabriele; Wullimann, Mario F.

    2016-01-01

    Zebrafish larvae imprint on visual and olfactory kin cues at day 5 and 6 postfertilization, respectively, resulting in kin recognition later in life. Exposure to non-kin cues prevents imprinting and kin recognition. Imprinting depends on MHC class II related signals and only larvae sharing MHC class II alleles can imprint on each other. Here, we analyzed which type of olfactory sensory neuron (OSN) detects kin odor. The single teleost olfactory epithelium harbors ciliated OSNs carrying OR and TAAR gene family receptors (mammals: main olfactory epithelium) and microvillous OSNs with V1R and V2R gene family receptors (mammals: vomeronasal organ). Additionally, teleosts exhibit crypt cells which possess microvilli and cilia. We used the activity marker pERK (phosphorylated extracellular signal regulated kinase) after stimulating 9 day old zebrafish larvae with either non-kin conspecific or food odor. While food odor activated both ciliated and microvillous OSNs, only the latter were activated by conspecific odor, crypt cells showed no activation to both stimuli. Then, we tested imprinted and non-imprinted larvae (full siblings) for kin odor detection. We provide the first direct evidence that crypt cells, and likely a subpopulation of microvillous OSNs, but not ciliated OSNs, play a role in detecting a kin odor related signal. PMID:27087508

  7. Sparc Protein Is Required for Normal Growth of Zebrafish Otoliths

    PubMed Central

    Kang, Young-Jin; Stevenson, Amy K.; Yau, Peter M.

    2008-01-01

    Otoliths and the homologous otoconia in the inner ear are essential for balance. Their morphogenesis is less understood than that of other biominerals, such as bone, and only a small number of their constituent proteins have been characterized. As a novel approach to identify unknown otolith proteins, we employed shotgun proteomics to analyze crude extracts from trout and catfish otoliths. We found three proteins that had not been associated previously with otolith or otoconia formation: ‘Secreted acidic cysteine rich glycoprotein’ (Sparc), an important bone protein that binds collagen and Ca2+; precerebellin-like protein, which contains a C1q domain and may associate with the collagenous otolin-1 during its assembly into a framework; and neuroserpin, a serine protease inhibitor that may regulate local protease activity during framework assembly. We then used the zebrafish to investigate whether Sparc plays a role in otolith morphogenesis. Immunodetection demonstrated that Sparc is a true constituent of otoliths. Knockdown of Sparc expression in morphant zebrafish resulted in four principal types of defective otoliths: smaller, extra and ectopic, missing and fused, or completely absent. Smaller size was the predominant phenotype and independent of the severity of otic-vesicle defects. These results suggested that Sparc is directly required for normal otolith growth. PMID:18784957

  8. Startle response memory and hippocampal changes in adult zebrafish pharmacologically-induced to exhibit anxiety/depression-like behaviors.

    PubMed

    Pittman, Julian T; Lott, Chad S

    2014-01-17

    Zebrafish (Danio rerio) are rapidly becoming a popular animal model for neurobehavioral and psychopharmacological research. While startle testing is a well-established assay to investigate anxiety-like behaviors in different species, screening of the startle response and its habituation in zebrafish is a new direction of translational biomedical research. This study focuses on a novel behavioral protocol to assess a tapping-induced startle response and its habituation in adult zebrafish that have been pharmacologically-induced to exhibit anxiety/depression-like behaviors. We demonstrated that zebrafish exhibit robust learning performance in a task adapted from the mammalian literature, a modified plus maze, and showed that ethanol and fluoxetine impair memory performance in this maze when administered after training at a dose that does not impair motor function, however, leads to significant upregulation of hippocampal serotoninergic neurons. These results suggest that the maze associative learning paradigm has face and construct validity and that zebrafish may become a translationally relevant study species for the analysis of the mechanisms of learning and memory changes associated with psychopharmacological treatment of anxiety/depression.

  9. High-Content and Semi-Automated Quantification of Responses to Estrogenic Chemicals Using a Novel Translucent Transgenic Zebrafish.

    PubMed

    Green, Jon M; Metz, Jeremy; Lee, Okhyun; Trznadel, Maciej; Takesono, Aya; Brown, A Ross; Owen, Stewart F; Kudoh, Tetsuhiro; Tyler, Charles R

    2016-06-21

    Rapid embryogenesis, together with genetic similarities with mammals, and the desire to reduce mammalian testing, are major incentives for using the zebrafish model in chemical screening and testing. Transgenic zebrafish, engineered for identifying target gene expression through expression of fluorophores, have considerable potential for both high-content and high-throughput testing of chemicals for endocrine activity. Here we generated an estrogen responsive transgenic zebrafish model in a pigment-free "Casper" phenotype, facilitating identification of target tissues and quantification of these responses in whole intact fish. Using the ERE-GFP-Casper model we show chemical type and concentration dependence for green fluorescent protein (GFP) induction and both spatial and temporal responses for different environmental estrogens tested. We also developed a semiautomated (ArrayScan) imaging and image analysis system that we applied to quantify whole body fluorescence responses for a range of different estrogenic chemicals in the new transgenic zebrafish model. The zebrafish model developed provides a sensitive and highly integrative system for identifying estrogenic chemicals, their target tissues and effect concentrations for exposures in real time and across different life stages. It thus has application for chemical screening to better direct health effects analysis of environmental estrogens and for investigating the functional roles of estrogens in vertebrates.

  10. Expression Pattern and Biochemical Properties of Zebrafish N-Acetylglutamate Synthase

    PubMed Central

    Caldovic, Ljubica; Haskins, Nantaporn; Mumo, Amy; Majumdar, Himani; Pinter, Mary; Tuchman, Mendel; Krufka, Alison

    2014-01-01

    The urea cycle converts ammonia, a waste product of protein catabolism, into urea. Because fish dispose ammonia directly into water, the role of the urea cycle in fish remains unknown. Six enzymes, N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase III, ornithine transcarbamylase, argininosuccinate synthase, argininosuccinate lyase and arginase 1, and two membrane transporters, ornithine transporter and aralar, comprise the urea cycle. The genes for all six enzymes and both transporters are present in the zebrafish genome. NAGS (EC 2.3.1.1) catalyzes the formation of N-acetylglutamate from glutamate and acetyl coenzyme A and in zebrafish is partially inhibited by L-arginine. NAGS and other urea cycle genes are highly expressed during the first four days of zebrafish development. Sequence alignment of NAGS proteins from six fish species revealed three regions of sequence conservation: the mitochondrial targeting signal (MTS) at the N-terminus, followed by the variable and conserved segments. Removal of the MTS yields mature zebrafish NAGS (zfNAGS-M) while removal of the variable segment from zfNAGS-M results in conserved NAGS (zfNAGS-C). Both zfNAGS-M and zfNAGS-C are tetramers in the absence of L-arginine; addition of L-arginine decreased partition coefficients of both proteins. The zfNAGS-C unfolds over a broader temperature range and has higher specific activity than zfNAGS-M. In the presence of L-arginine the apparent Vmax of zfNAGS-M and zfNAGS-C decreased, their Kmapp for acetyl coenzyme A increased while the Kmapp for glutamate remained unchanged. The expression pattern of NAGS and other urea cycle genes in developing zebrafish suggests that they may have a role in citrulline and/or arginine biosynthesis during the first day of development and in ammonia detoxification thereafter. Biophysical and biochemical properties of zebrafish NAGS suggest that the variable segment may stabilize a tetrameric state of zfNAGS-M and that under physiological

  11. Gene-specific differential response to anti-apoptotic therapies in zebrafish models of ocular coloboma

    PubMed Central

    Moosajee, Mariya; Shan, Xianghong; Gregory-Evans, Kevin

    2011-01-01

    Purpose We recently demonstrated that molecular therapy using aminoglycosides can overcome the underlying genetic defect in two zebrafish models of ocular coloboma and showed abnormal cell death to be a key feature associated with the optic fissure closure defects. In further studies to identify molecular therapies for this common congenital malformation, we now examine the effects of anti-apoptotic compounds in zebrafish models of ocular coloboma in vivo. Methods Two ocular coloboma zebrafish lines (pax2.1/noitu29a and lamb1/gupm189) were exposed to diferuloylmethane (curcumin) or benzyloxycarbonyl-Val-Ala-Asp(Ome)-fluoromethylketone (zVAD-fmk; a pan-caspase inhibitor) for up to 8 days post-fertilization. The effects of these compounds were assessed by morphology, histology, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and western blot analysis. Results The size of the coloboma in gup zebrafish mutants treated with diferuloylmethane was greatly reduced. In treated mutants a reduction in TUNEL staining and a 67% decrease in activated caspase-3 protein were observed. The release of cytochrome c from the mitochondria into the cytosol was reduced fourfold by in vivo diferuloylmethane treatment, suggesting that the drug was acting to inhibit the intrinsic apoptotic pathway. Inhibition of caspases directly with zVAD-fmk also resulted in a similar reduction in coloboma phenotype. Treatment with either diferuloylmethane or zVAD-fmk resulted in a statistically significant 1.4 fold increase in length of survival of these mutant zebrafish (p<0.001), which normally succumb to the lethal genetic mutation. In contrast, the coloboma phenotype in noi zebrafish mutants did not respond to either diferuloylmethane or zVAD-fmk exposure, even though inhibition of apoptotic cell death was observed by a reduction in TUNEL staining. Conclusions The differential sensitivity to anti-apoptotic agents in lamb1-deficient and pax2.1-deficient zebrafish models

  12. Primary neuron culture for nerve growth and axon guidance studies in zebrafish (Danio rerio).

    PubMed

    Chen, Zheyan; Lee, Han; Henle, Steven J; Cheever, Thomas R; Ekker, Stephen C; Henley, John R

    2013-01-01

    Zebrafish (Danio rerio) is a widely used model organism in genetics and developmental biology research. Genetic screens have proven useful for studying embryonic development of the nervous system in vivo, but in vitro studies utilizing zebrafish have been limited. Here, we introduce a robust zebrafish primary neuron culture system for functional nerve growth and guidance assays. Distinct classes of central nervous system neurons from the spinal cord, hindbrain, forebrain, and retina from wild type zebrafish, and fluorescent motor neurons from transgenic reporter zebrafish lines, were dissociated and plated onto various biological and synthetic substrates to optimize conditions for axon outgrowth. Time-lapse microscopy revealed dynamically moving growth cones at the tips of extending axons. The mean rate of axon extension in vitro was 21.4±1.2 µm hr(-1) s.e.m. for spinal cord neurons, which corresponds to the typical ∼0.5 mm day(-1) growth rate of nerves in vivo. Fluorescence labeling and confocal microscopy demonstrated that bundled microtubules project along axons to the growth cone central domain, with filamentous actin enriched in the growth cone peripheral domain. Importantly, the growth cone surface membrane expresses receptors for chemotropic factors, as detected by immunofluorescence microscopy. Live-cell functional assays of axon extension and directional guidance demonstrated mammalian brain-derived neurotrophic factor (BDNF)-dependent stimulation of outgrowth and growth cone chemoattraction, whereas mammalian myelin-associated glycoprotein inhibited outgrowth. High-resolution live-cell Ca(2+)-imaging revealed local elevation of cytoplasmic Ca(2+) concentration in the growth cone induced by BDNF application. Moreover, BDNF-induced axon outgrowth, but not basal outgrowth, was blocked by treatments to suppress cytoplasmic Ca(2+) signals. Thus, this primary neuron culture model system may be useful for studies of neuronal development, chemotropic axon

  13. Nanomaterial Toxicity Screening in Developing Zebrafish Embryos

    EPA Science Inventory

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

  14. Protocadherin-17 Function in Zebrafish Retinal Development

    PubMed Central

    Chen, Yun; Londraville, Richard; Brickner, Sarah; El-Shaar, Lana; Fankhauser, Kelsee; Dearth, Cassandra; Fulton, Leah; Sochacka, Alicja; Bhattarai, Sunil; Marrs, James A.; Liu, Qin

    2012-01-01

    Cadherin cell adhesion molecules play crucial roles in vertebrate development including the development of the retina. Most studies have focused on examining functions of classic cadherins (e.g. N-cadherin) in retinal development. There is little information on the function of protocadherins in the development of the vertebrate visual system. We previously showed that protocadherin-17 mRNA was expressed in developing zebrafish retina during critical stages of the retinal development. To gain insight into protocadherin-17 function in the formation of the retina, we analyzed eye development and differentiation of retinal cells in zebrafish embryos injected with protocadherin-17 specific antisense morpholino oligonucleotides (MOs). Protocadherin-17 knockdown embryos (pcdh17 morphants) had significantly reduced eyes due mainly to decreased cell proliferation. Differentiation of several retinal cell types (e.g. retinal ganglion cells) was also disrupted in the pcdh17 morphants. Phenotypic rescue was achieved by injection of protocadherin-17 mRNA. Injection of a vivo-protocadherin-17 MO into one eye of embryonic zebrafish resulted in similar eye defects. Our results suggest that protocadherin-17 plays an important role in the normal formation of the zebrafish retina. PMID:22927092

  15. Zebrafish phenotypic screen identifies novel Notch antagonists.

    PubMed

    Velaithan, Vithya; Okuda, Kazuhide Shaun; Ng, Mei Fong; Samat, Norazwana; Leong, Sze Wei; Faudzi, Siti Munirah Mohd; Abas, Faridah; Shaari, Khozirah; Cheong, Sok Ching; Tan, Pei Jean; Patel, Vyomesh

    2017-04-01

    Zebrafish represents a powerful in vivo model for phenotype-based drug discovery to identify clinically relevant small molecules. By utilizing this model, we evaluated natural product derived compounds that could potentially modulate Notch signaling that is important in both zebrafish embryogenesis and pathogenic in human cancers. A total of 234 compounds were screened using zebrafish embryos and 3 were identified to be conferring phenotypic alterations similar to embryos treated with known Notch inhibitors. Subsequent secondary screens using HEK293T cells overexpressing truncated Notch1 (HEK293TΔE) identified 2 compounds, EDD3 and 3H4MB, to be potential Notch antagonists. Both compounds reduced protein expression of NOTCH1, Notch intracellular domain (NICD) and hairy and enhancer of split-1 (HES1) in HEK293TΔE and downregulated Notch target genes. Importantly, EDD3 treatment of human oral cancer cell lines demonstrated reduction of Notch target proteins and genes. EDD3 also inhibited proliferation and induced G0/G1 cell cycle arrest of ORL-150 cells through inducing p27(KIP1). Our data demonstrates the utility of the zebrafish phenotypic screen and identifying EDD3 as a promising Notch antagonist for further development as a novel therapeutic agent.

  16. Behavorial assessments of larval zebrafish neurotoxicology

    EPA Science Inventory

    Fishes have long been a popular organism in ecotoxicology research, and are increasingly used in human health research as an alternative animal model for chemical screening. Our laboratory incorporates a zebrafish (Danio rerio) embryo/larval assay to screen chemicals for developm...

  17. Regeneration of Zebrafish CNS: Adult Neurogenesis

    PubMed Central

    Ghosh, Sukla; Hui, Subhra Prakash

    2016-01-01

    Regeneration in the animal kingdom is one of the most fascinating problems that have allowed scientists to address many issues of fundamental importance in basic biology. However, we came to know that the regenerative capability may vary across different species. Among vertebrates, fish and amphibians are capable of regenerating a variety of complex organs through epimorphosis. Zebrafish is an excellent animal model, which can repair several organs like damaged retina, severed spinal cord, injured brain and heart, and amputated fins. The focus of the present paper is on spinal cord regeneration in adult zebrafish. We intend to discuss our current understanding of the cellular and molecular mechanism(s) that allows formation of proliferating progenitors and controls neurogenesis, which involve changes in epigenetic and transcription programs. Unlike mammals, zebrafish retains radial glia, a nonneuronal cell type in their adult central nervous system. Injury induced proliferation involves radial glia which proliferate, transcribe embryonic genes, and can give rise to new neurons. Recent technological development of exquisite molecular tools in zebrafish, such as cell ablation, lineage analysis, and novel and substantial microarray, together with advancement in stem cell biology, allowed us to investigate how progenitor cells contribute to the generation of appropriate structures and various underlying mechanisms like reprogramming. PMID:27382491

  18. Teaching Stress Physiology Using Zebrafish ("Danio Rerio")

    ERIC Educational Resources Information Center

    Cooper, Michael; Dhawale, Shree; Mustafa, Ahmed

    2009-01-01

    A straightforward and inexpensive laboratory experiment is presented that investigates the physiological stress response of zebrafish after a 5 degree C increase in water temperature. This experiment is designed for an undergraduate physiology lab and allows students to learn the scientific method and relevant laboratory techniques without causing…

  19. An outbreak of Plesimonus Shigelloides in Zebrafish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plesiomonas shigelloides is a flagellated, gram-negative rod that is an emergent pathogen associated with human gastroenteritis. Recently, we experienced a disease outbreak in zebrafish that were obtained from a commercial source. Fourteen days after being held at 27°C in our flow-through quarantine...

  20. Detecting Developmental Neurotoxicants Using Zebrafish Embryos

    EPA Science Inventory

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

  1. Thyroid development in zebrafish lacking Taz.

    PubMed

    Pappalardo, Andrea; Porreca, Immacolata; Caputi, Luigi; De Felice, Elena; Schulte-Merker, Stephan; Zannini, Mariastella; Sordino, Paolo

    2015-11-01

    Taz is a signal-responsive transcriptional coregulator implicated in several biological functions, from chondrogenesis to regulation of organ size. Less well studied, however, is its role in thyroid formation. Here, we explored the in vivo effects on thyroid development of morpholino (MO)-mediated knockdown of wwtr1, the gene encoding zebrafish Taz. The wwtr1 gene is expressed in the thyroid primordium and pharyngeal tissue of developing zebrafish. Compared to mammalian cells, in which Taz promotes expression of thyroid transcription factors and thyroid differentiation genes, wwtr1 MO injection in zebrafish had little or no effect on the expression of thyroid transcription factors, and differentially altered the expression of thyroid differentiation genes. Analysis of wwtr1 morphants at later stages of development revealed that the number and the lumen of thyroid follicles, and the number of thyroid follicle cells, were significantly smaller. In addition, Taz-depleted larvae displayed patterning defects in ventral cranial vessels that correlate with lateral displacement of thyroid follicles. These findings indicate that the zebrafish Taz protein is needed for the normal differentiation of the thyroid and are the first to suggest that Taz confers growth advantage to the endocrine gland.

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

    NASA Astrophysics Data System (ADS)

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

    2016-10-01

    Ambient mass spectrometry imaging has become an increasingly powerful technique for the direct analysis of biological tissues in the open environment with minimal sample preparation and fast analysis times. In this study, we introduce desorption electrospray ionization mass spectrometry imaging (DESI-MSI) as a novel, rapid, and sensitive approach to localize the accumulation of a mildly toxic ionic liquid (IL), AMMOENG 130 in zebrafish (Danio rerio). The work demonstrates that DESI-MSI has the potential to rapidly monitor the accumulation of IL pollutants in aquatic organisms. AMMOENG 130 is a quaternary ammonium-based IL reported to be broadly used as a surfactant in commercialized detergents. It is known to exhibit acute toxicity to zebrafish causing extensive damage to gill secondary lamellae and increasing membrane permeability. Zebrafish were exposed to the IL in a static 96-h exposure study in concentrations near the LC50 of 1.25, 2.5, and 5.0 mg/L. DESI-MS analysis of zebrafish gills demonstrated the appearance of a dealkylated AMMOENG 130 metabolite in the lowest concentration of exposure identified by a high resolution hybrid LTQ-Orbitrap mass spectrometer as the trimethylstearylammonium ion, [C21H46N]+. With DESI-MSI, the accumulation of AMMOENG 130 and its dealkylated metabolite in zebrafish tissue was found in the nervous and respiratory systems. AMMOENG 130 and the metabolite were capable of penetrating the blood brain barrier of the fish with significant accumulation in the brain. Hence, we report for the first time the simultaneous characterization, distribution, and metabolism of a toxic IL in whole body zebrafish analyzed by DESI-MSI. This ambient mass spectrometry imaging technique shows great promise for the direct analysis of biological tissues to qualitatively monitor foreign, toxic, and persistent compounds in aquatic organisms from the environment.

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

    PubMed

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

    2016-10-24

    Ambient mass spectrometry imaging has become an increasingly powerful technique for the direct analysis of biological tissues in the open environment with minimal sample preparation and fast analysis times. In this study, we introduce desorption electrospray ionization mass spectrometry imaging (DESI-MSI) as a novel, rapid, and sensitive approach to localize the accumulation of a mildly toxic ionic liquid (IL), AMMOENG 130 in zebrafish (Danio rerio). The work demonstrates that DESI-MSI has the potential to rapidly monitor the accumulation of IL pollutants in aquatic organisms. AMMOENG 130 is a quaternary ammonium-based IL reported to be broadly used as a surfactant in commercialized detergents. It is known to exhibit acute toxicity to zebrafish causing extensive damage to gill secondary lamellae and increasing membrane permeability. Zebrafish were exposed to the IL in a static 96-h exposure study in concentrations near the LC50 of 1.25, 2.5, and 5.0 mg/L. DESI-MS analysis of zebrafish gills demonstrated the appearance of a dealkylated AMMOENG 130 metabolite in the lowest concentration of exposure identified by a high resolution hybrid LTQ-Orbitrap mass spectrometer as the trimethylstearylammonium ion, [C21H46N](+). With DESI-MSI, the accumulation of AMMOENG 130 and its dealkylated metabolite in zebrafish tissue was found in the nervous and respiratory systems. AMMOENG 130 and the metabolite were capable of penetrating the blood brain barrier of the fish with significant accumulation in the brain. Hence, we report for the first time the simultaneous characterization, distribution, and metabolism of a toxic IL in whole body zebrafish analyzed by DESI-MSI. This ambient mass spectrometry imaging technique shows great promise for the direct analysis of biological tissues to qualitatively monitor foreign, toxic, and persistent compounds in aquatic organisms from the environment. Graphical Abstract ᅟ.

  4. Defects of the Glycinergic Synapse in Zebrafish

    PubMed Central

    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

  5. Expression and functions of ASIC1 in the zebrafish retina.

    PubMed

    Liu, Sha; Wang, Mei-Xia; Mao, Cheng-Jie; Cheng, Xiao-Yu; Wang, Chen-Tao; Huang, Jian; Zhong, Zhao-Min; Hu, Wei-Dong; Wang, Fen; Hu, Li-Fang; Wang, Han; Liu, Chun-Feng

    2014-12-12

    It has been demonstrated that acid sensing ionic channels (ASICs) are present in the central and peripheral nervous system of mammals, including the retina. However, it remains unclear whether the zebrafish retina also expresses ASICs. In the present study, the expression and distribution of zasic1 were examined in the retina of zebrafish. Both zasic1 mRNA and protein expressions were detected in the adult zebrafish retina. A wide distribution of ASIC1 in zebrafish retina was confirmed using whole mount in situ hybridization and immunohistochemistry study. Acidosis-induced currents in the isolated retinal ganglion cells (RGCs) were also recorded using whole cell patch clamping. Moreover, blockade of ASICs channel significantly reduced the locomotion of larval zebrafish in response to light exposure. In sum, our data demonstrate the presence of ASIC1 and its possible functional relevance in the retina of zebrafish.

  6. Conserved gene regulation during acute inflammation between zebrafish and mammals.

    PubMed

    Forn-Cuní, G; Varela, M; Pereiro, P; Novoa, B; Figueras, A

    2017-02-03

    Zebrafish (Danio rerio), largely used as a model for studying developmental processes, has also emerged as a valuable system for modelling human inflammatory diseases. However, in a context where even mice have been questioned as a valid model for these analysis, a systematic study evaluating the reproducibility of human and mammalian inflammatory diseases in zebrafish is still lacking. In this report, we characterize the transcriptomic regulation to lipopolysaccharide in adult zebrafish kidney, liver, and muscle tissues using microarrays and demonstrate how the zebrafish genomic responses can effectively reproduce the mammalian inflammatory process induced by acute endotoxin stress. We provide evidence that immune signaling pathways and single gene expression is well conserved throughout evolution and that the zebrafish and mammal acute genomic responses after lipopolysaccharide stimulation are highly correlated despite the differential susceptibility between species to that compound. Therefore, we formally confirm that zebrafish inflammatory models are suited to study the basic mechanisms of inflammation in human inflammatory diseases, with great translational impact potential.

  7. Behavioral analysis of zebrafish larvae swimming in three dimensions

    NASA Astrophysics Data System (ADS)

    Feng, Ruopei; Girdhar, Kiran; Chemla, Yann; Gruebele, Martin

    2015-03-01

    Behavioral biologists have a strong interest in studying the behavior of larval zebrafish because the limited number of locomotor neurons in larval zebrafish couples with the rich repertoire of movements as a vertebrate animal. Current research uses a priori-selected parameters to describe their movements. Most research also only considers the 2D movements of zebrafish, leaving out the vertical component of their locomotion. Our lab has developed a method to reduce the dimensionality of the locomotion of zebrafish and determine the behavioral space of 2D swimming. We are extending this work to capture 3D locomotion of zebrafish larvae. Here we present our preliminary analysis of the 3D locomotion of zebrafish.

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

    SciTech Connect

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

    2010-04-15

    Dithiocarbamates (DTCs) are a class of compounds that are extensively used in agriculture as pesticides. As such, humans and wildlife are undoubtedly exposed to these chemicals. Although DTCs are thought to be relatively safe due to their short half lives, it is well established that they are teratogenic to vertebrates, especially to fish. In zebrafish, these teratogenic effects are characterized by distorted notochord development and shortened anterior to posterior axis. DTCs are known copper (Cu) chelators but this does not fully explain the observed teratogenic effects. We show here that DTCs cause malformations in zebrafish that highly resemble teratogenic effects observed by direct inhibition of a group of cuproenzymes termed lysyl oxidases (LOX). Additionally, we demonstrate that partial knockdown of three LOX genes, lox, loxl1 and loxl5b, sensitizes the developing embryo to DTC exposure. Finally, we show that DTCs directly inhibit zebrafish LOX activity in an ex vivo amine oxidase assay. Taken together, these results provide the first evidence that DTC induced teratogenic effects are, at least in part, caused by direct inhibition of LOX activity.

  9. Dimentionality and behavior of swimming Zebrafish: ``The EigenFish''

    NASA Astrophysics Data System (ADS)

    Girdhar, Kiran; Gruebele, Martin; Chemla, Yann

    2013-03-01

    How simple is the underlying control mechanism for the complex locomotion of vertebrates? To answer this question, we study the swimming behavior of zebrafish larvae. A dimensionality reduction method (singular value decomposition), in analogy to previous studies of worms, is used to analyze swimming movies of fish. That way, the animals can directly provide us with a minimal set of shapes to describe their motion, rather than us imposing arbitrary coordinates. We show that two low imensional attractors (an ellipse and a distorted ellipse) embedded in a threedimensional space of motion coordinates are sufficient to describe > 95% of the locomotion. We also show that scoots and R-turns, previously thought to be independent behaviors based on qualitative studies, are in fact just extremes of a continuous family of motions bounded by the two attractors.

  10. Optical micromanipulation of nanoparticles and cells inside living zebrafish

    PubMed Central

    Johansen, Patrick Lie; Fenaroli, Federico; Evensen, Lasse; Griffiths, Gareth; Koster, Gerbrand

    2016-01-01

    Regulation of biological processes is often based on physical interactions between cells and their microenvironment. To unravel how and where interactions occur, micromanipulation methods can be used that offer high-precision control over the duration, position and magnitude of interactions. However, lacking an in vivo system, micromanipulation has generally been done with cells in vitro, which may not reflect the complex in vivo situation inside multicellular organisms. Here using optical tweezers we demonstrate micromanipulation throughout the transparent zebrafish embryo. We show that different cells, as well as injected nanoparticles and bacteria can be trapped and that adhesion properties and membrane deformation of endothelium and macrophages can be analysed. This non-invasive micromanipulation inside a whole-organism gives direct insights into cell interactions that are not accessible using existing approaches. Potential applications include screening of nanoparticle-cell interactions for cancer therapy or tissue invasion studies in cancer and infection biology. PMID:26996121

  11. Optical micromanipulation of nanoparticles and cells inside living zebrafish

    NASA Astrophysics Data System (ADS)

    Johansen, Patrick Lie; Fenaroli, Federico; Evensen, Lasse; Griffiths, Gareth; Koster, Gerbrand

    2016-03-01

    Regulation of biological processes is often based on physical interactions between cells and their microenvironment. To unravel how and where interactions occur, micromanipulation methods can be used that offer high-precision control over the duration, position and magnitude of interactions. However, lacking an in vivo system, micromanipulation has generally been done with cells in vitro, which may not reflect the complex in vivo situation inside multicellular organisms. Here using optical tweezers we demonstrate micromanipulation throughout the transparent zebrafish embryo. We show that different cells, as well as injected nanoparticles and bacteria can be trapped and that adhesion properties and membrane deformation of endothelium and macrophages can be analysed. This non-invasive micromanipulation inside a whole-organism gives direct insights into cell interactions that are not accessible using existing approaches. Potential applications include screening of nanoparticle-cell interactions for cancer therapy or tissue invasion studies in cancer and infection biology.

  12. Hearing Assessment in Zebrafish During the First Week Postfertilization

    PubMed Central

    Yao, Qi; DeSmidt, Alexandra A.; Tekin, Mustafa; Liu, Xuezhong

    2016-01-01

    Abstract The zebrafish (Danio rerio) is a valuable vertebrate model for human hearing disorders because of many advantages in genetics, embryology, and in vivo visualization. In this study, we investigated auditory function of zebrafish during the first week postfertilization using microphonic potential recording. Extracellular microphonic potentials were recorded from hair cells in the inner ear of wild-type AB and transgenic Et(krt4:GFP)sqet4 zebrafish at 3, 5, and 7 days postfertilization in response to 20, 50, 100, 200, 300, and 400-Hz acoustic stimulation. We found that microphonic threshold significantly decreased with age in zebrafish. However, there was no significant difference of microphonic responses between wild-type and transgenic zebrafish, indicating that the transgenic zebrafish have normal hearing like wild-type zebrafish. In addition, we observed that microphonic threshold did not change with the recording electrode location. Furthermore, microphonic threshold increased significantly at all tested stimulus frequencies after displacement of the saccular otolith but only increased at low frequencies after displacement of the utricular otolith, showing that the saccule rather than the utricle plays the major role in larval zebrafish hearing. These results enhance our knowledge of early development of auditory function in zebrafish and the factors affecting hearing assessment with microphonic potential recording. PMID:26982161

  13. Biologically inspired robots elicit a robust fear response in zebrafish

    NASA Astrophysics Data System (ADS)

    Ladu, Fabrizio; Bartolini, Tiziana; Panitz, Sarah G.; Butail, Sachit; Macrı, Simone; Porfiri, Maurizio

    2015-03-01

    We investigate the behavioral response of zebrafish to three fear-evoking stimuli. In a binary choice test, zebrafish are exposed to a live allopatric predator, a biologically-inspired robot, and a computer-animated image of the live predator. A target tracking algorithm is developed to score zebrafish behavior. Unlike computer-animated images, the robotic and live predator elicit a robust avoidance response. Importantly, the robotic stimulus elicits more consistent inter-individual responses than the live predator. Results from this effort are expected to aid in hypothesis-driven studies on zebrafish fear response, by offering a valuable approach to maximize data-throughput and minimize animal subjects.

  14. Visualizing Human Hematopoietic Stem Cell Trafficking In Vivo Using a Zebrafish Xenograft Model.

    PubMed

    Staal, Frank J T; Spaink, Herman P; Fibbe, Willem E

    2016-02-15

    Zebrafish is gaining increased popularity as a model organism to study stem cell biology. It also is widely used as model system to visualize human leukemic stem cells. However, xenotransplantation of primary human stem/progenitor cells has not been described. Here, we use casper pigmentation mutant fish that are transparent crossed to fli-GFP transgenic fish as recipients of red labeled human CD34(+) cells. We have investigated various conditions and protocols with the aim to monitor and visualize the fate of transplanted human CD34(+) cells. We here report successful use of casper mutant zebrafish embryos for the direct monitoring of human hematopoietic stem cell transplantation, differentiation, and trafficking in vivo.

  15. The role of Rho kinase (Rock) in re-epithelialization of adult zebrafish skin wounds.

    PubMed

    Richardson, Rebecca; Hammerschmidt, Matthias

    2016-08-03

    Complete re-epithelialization of full-thickness skin wounds in adult mammals takes days to complete and relies on numerous signaling cues and multiple overlapping cellular processes that take place both within the epidermis itself and in other participating tissues. We have previously shown that re-epithelialization of full-thickness skin wounds of adult zebrafish, however, is extremely rapid and largely independent of the other processes of wound healing allowing for the dissection of specific processes that occur in, or have a direct effect on, re-epithelializing keratinocytes. Recently, we have shown that, in addition to lamellipodial crawling at the leading edge, re-epithelialization of zebrafish partial- and full-thickness wounds requires long-range epithelial rearrangements including radial intercalations, flattening and directed elongation and that each of these processes involves Rho kinase (Rock) signaling. Our studies demonstrate how these coordinated signaling events allow for the rapid collective cell migration observed in adult zebrafish wound healing. Here we discuss the particular contribution of Rock to each of these processes.

  16. Osteogenic programs during zebrafish fin regeneration

    PubMed Central

    Watson, Claire J; Kwon, Ronald Y

    2015-01-01

    Recent advances in genomic, screening and imaging technologies have provided new opportunities to examine the molecular and cellular landscape underlying human physiology and disease. In the context of skeletal research, technologies for systems genetics, high-throughput screening and high-content imaging can aid an unbiased approach when searching for new biological, pathological or therapeutic pathways. However, these approaches necessitate the use of specialized model systems that rapidly produce a phenotype, are easy to manipulate, and amenable to optical study, all while representing mammalian bone physiologies at the molecular and cellular levels. The emerging use of zebrafish (Danio rerio) for modeling human disease highlights its potential to accelerate therapeutic and pathway discovery in the mammalian skeleton. In this review, we consider the potential value of zebrafish fin ray regeneration (a rapid, genetically tractable and optically transparent model of intramembranous ossification) as a translational model for such studies. PMID:26421148

  17. Characterization of rag1 mutant zebrafish leukocytes

    PubMed Central

    Petrie-Hanson, Lora; Hohn, Claudia; Hanson, Larry

    2009-01-01

    Background Zebrafish may prove to be one of the best vertebrate models for innate immunology. These fish have sophisticated immune components, yet rely heavily on innate immune mechanisms. Thus, the development and characterization of mutant and/or knock out zebrafish are critical to help define immune cell and immune gene functions in the zebrafish model. The use of Severe Combined Immunodeficient (SCID) and recombination activation gene 1 and 2 mutant mice has allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Similar zebrafish mutants are now being used in biomedical and fish immunology related research. This report describes the leukocyte populations in a unique model, recombination activation gene 1-/- mutant zebrafish (rag1 mutants). Results Differential counts of peripheral blood leukocytes (PBL) showed that rag1 mutants had significantly decreased lymphocyte-like cell populations (34.7%) compared to wild-types (70.5%), and significantly increased granulocyte populations (52.7%) compared to wild-types (17.6%). Monocyte/macrophage populations were similar between mutants and wild-types, 12.6% and 11.3%, respectively. Differential leukocyte counts of rag1 mutant kidney hematopoietic tissue showed a significantly reduced lymphocyte-like cell population (8%), a significantly increased myelomonocyte population (57%), 34.8% precursor cells, and 0.2% thrombocytes, while wild-type hematopoietic kidney tissue showed 29.4% lymphocytes/lymphocyte-like cells, 36.4% myelomonocytes, 33.8% precursors and 0.5% thrombocytes. Flow cytometric analyses of kidney hematopoietic tissue revealed three leukocyte populations. Population A was monocytes and granulocytes and comprised 34.7% of the gated cells in rag1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for rag1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte

  18. Understanding cardiac sarcomere assembly with zebrafish genetics.

    PubMed

    Yang, Jingchun; Shih, Yu-Huan; Xu, Xiaolei

    2014-09-01

    Mutations in sarcomere genes have been found in many inheritable human diseases, including hypertrophic cardiomyopathy. Elucidating the molecular mechanisms of sarcomere assembly shall facilitate understanding of the pathogenesis of sarcomere-based cardiac disease. Recently, biochemical and genomic studies have identified many new genes encoding proteins that localize to the sarcomere. However, their precise functions in sarcomere assembly and sarcomere-based cardiac disease are unknown. Here, we review zebrafish as an emerging vertebrate model for these studies. We summarize the techniques offered by this animal model to manipulate genes of interest, annotate gene expression, and describe the resulting phenotypes. We survey the sarcomere genes that have been investigated in zebrafish and discuss the potential of applying this in vivo model for larger-scale genetic studies.

  19. What optimization principle explains the zebrafish vasculature?

    NASA Astrophysics Data System (ADS)

    Chang, Shyr-Shea; Baek, Kyung In; Hsiai, Tzung; Roper, Marcus

    2016-11-01

    Many multicellular organisms depend on biological transport networks; from the veins of leaves to the animal circulatory system, to redistribute nutrients internally. Since natural selection rewards efficiency, those networks are thought to minimize the cost of maintaining the flow inside. But optimizing these costs creates tradeoffs with other functions, e.g. mixing or uniform distribution of nutrients. We develop an extended Lagrange multiplier approach that allows the optimization of general network functionals. We also follow the real zebrafish vasculature and blood flows during organism development. Taken together, our work shows that the challenge of uniform oxygen perfusion, and not transport efficiency, explain zebrafish vascular organization. Ruth L. Kirschstein National Research Service Award (T32-GM008185).

  20. BDE 49 and developmental toxicity in zebrafish

    PubMed Central

    McClain, Valerie; Stapleton, Heather M.; Gallagher, Evan

    2011-01-01

    The polybrominated diphenyl ethers (PBDEs) are a group of brominated flame retardants. Human health concerns of these agents have largely centered upon their potential to elicit reproductive and developmental effects. Of the various congeners, BDE 49 (2,2’,4,5’-tetrabromodiphenyl ether) has been poorly studied, despite the fact that it is often detected in the tissues of fish and wildlife species. Furthermore, we have previously shown that BDE 49 is a metabolic debromination product of BDE 99 hepatic metabolism in salmon, carp and trout, underscoring the need for a better understanding of biological effects. In the current study, we investigated the developmental toxicity of BDE 49 using the zebrafish (Danio rerio) embryo larval model. Embryo and larval zebrafish were exposed to BDE 49 at either 5 hours post fertilization (hpf) or 24 hpf and monitored for developmental and neurotoxicity. Exposure to BDE 49 at concentrations of 4 µM- 32 µM caused a dose-dependent loss in survivorship at 6 days post fertilization (dpf). Morphological impairments were observed prior to the onset of mortality, the most striking of which included severe dorsal curvatures of the tail. The incidence of dorsal tail curvatures was dose and time dependent. Exposure to BDE 49 caused cardiac toxicity as evidenced by a significant reduction in zebrafish heart rates at 6 dpf but not earlier, suggesting that cardiac toxicity was non-specific and associated with physiological stress. Neurobehavioral injury from BDE 49 was evidenced by an impairment of touch-escape responses observed at 5 dpf. Our results indicate that BDE 49 is a developmental toxicant in larval zebrafish that can cause morphological abnormalities and adversely affect neurobehavior. The observed toxicities from BDE 49 were similar in scope to those previously reported for the more common tetrabrominated congener, BDE 47, and also for other lower brominated PBDEs, suggest that these compounds may share similarities in risk to

  1. The Morphogenesis of Cranial Sutures in Zebrafish

    PubMed Central

    Topczewska, Jolanta M.; Shoela, Ramy A.; Tomaszewski, Joanna P.; Mirmira, Rupa B.; Gosain, Arun K.

    2016-01-01

    Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births. PMID:27829009

  2. Phenylthiourea disrupts thyroid function in developing zebrafish.

    PubMed

    Elsalini, Osama A; Rohr, Klaus B

    2003-01-01

    Thyroid hormone (T4) can be detected in thyroid follicles in wild-type zebrafish larvae from 3 days of development, when the thyroid has differentiated. In contrast, embryos or larvae treated with goitrogens (substances such as methimazole, potassium percholorate, and 6-n-propyl-2-thiouracil) are devoid of thyroid hormone immunoreactivity. Phenythiourea (PTurea; also commonly known as PTU) is widely used in zebrafish research to suppress pigmentation in developing embryos/fry. PTurea contains a thiocarbamide group that is responsible for goitrogenic activity in methimazole and 6-n-propyl-2-thiouracil. In the present study, we show that commonly used doses of 0.003% PTurea abolish T4 immunoreactivity of the thyroid follicles of zebrafish larvae. As development of the thyroid gland is not affected, these data suggest that PTurea blocks thyroid hormone production. Like other goitrogens, PTurea causes delayed hatching, retardation and malformation of embryos or larvae with increasing doses. At doses of 0.003% PTurea, however, toxic side effects seem to be at a minimum, and the maternal contribution of the hormone might compensate for compromised thyroid function during the first days of development.

  3. The zebrafish infraorbital bones: a descriptive study.

    PubMed

    Chang, Carolyn; Franz-Odendaal, Tamara Anne

    2014-02-01

    The infraorbital (IO) bone series, a component of the circumorbital series, makes up five of the eight dermal bones found in the orbital region of the zebrafish skull. Ossifying in a set sequence, the IOs are closely associated with the cranial lateral line system as they house neuromast sensory receptors in bony canals. We conducted a detailed analysis of the condensation to mineralization phases of development of these bones. Our analyses involved both bone and osteoblast staining of zebrafish at 20 different time points. IO bone condensations are shaped as templates for the final bone shape, and they mineralize at one or more centers of ossification. Initially, mineralization is closely associated with the lateral line canals and/or foramen, and the onset of mineralization is temporally variable. Canal wall mineralization is a process that continues into adulthood and completely mineralized canal roofs were not found. Our comprehensive growth series detailing the ossification of each IO bone provides important insight into the growth and development of this series of neural crest-derived flat bones in the zebrafish craniofacial skeleton.

  4. Single stimulus learning in zebrafish larvae

    PubMed Central

    O’Neale, Ashley; Ellis, Joseph; Creton, Robbert; Colwill, Ruth M.

    2014-01-01

    Learning about a moving visual stimulus was examined in zebrafish larvae using an automated imaging system and a t1-t2 design. In three experiments, zebrafish larvae were exposed to one of two inputs at t1 (either a gray bouncing disk or an identical but stationary disk) followed by a common test at t2 (the gray bouncing disk). Using 7 days post-fertilization (dpf) larvae and 12 stimulus exposures, Experiment 1 established that these different treatments produced differential responding to the moving disk during testing. Larvae familiar with the moving test stimulus were significantly less likely to be still in its presence than larvae that had been exposed to the identical but stationary stimulus. Experiment 2 confirmed this result in 7 dpf larvae and extended the finding to 5 and 6 dpf larvae. Experiment 3 found differential responding to the moving test stimulus with 4 or 8 stimulus exposures but not with just one exposure in 7 dpf larvae. These results provide evidence for learning in very young zebrafish larvae. The merits and challenges of the t1-t2 framework to study learning are discussed. PMID:24012906

  5. Repressor Dimerization in the Zebrafish Somitogenesis Clock

    PubMed Central

    Cinquin, Olivier

    2007-01-01

    The oscillations of the somitogenesis clock are linked to the fundamental process of vertebrate embryo segmentation, yet little is known about their generation. In zebrafish, it has been proposed that Her proteins repress the transcription of their own mRNA. However, in its simplest form, this model is incompatible with the fact that morpholino knockdown of Her proteins can impair expression of their mRNA. Simple self-repression models also do not account for the spatiotemporal pattern of gene expression, with waves of gene expression shrinking as they propagate. Here we study computationally the networks generated by the wealth of dimerization possibilities amongst transcriptional repressors in the zebrafish somitogenesis clock. These networks can reproduce knockdown phenotypes, and strongly suggest the existence of a Her1–Her7 heterodimer, so far untested experimentally. The networks are the first reported to reproduce the spatiotemporal pattern of the zebrafish somitogenesis clock; they shed new light on the role of Her13.2, the only known link between the somitogenesis clock and positional information in the paraxial mesoderm. The networks can also account for perturbations of the clock by manipulation of FGF signaling. Achieving an understanding of the interplay between clock oscillations and positional information is a crucial first step in the investigation of the segmentation mechanism. PMID:17305423

  6. Standardized Welfare Terms for the Zebrafish Community

    PubMed Central

    Karp, Natasha A.; Blackledge, Samuel; Clark, Bradley; Keeble, Rosemary; Kovacs, Ceri; Murray, Katrina N.; Price, Michael; Thompson, Peter; Bussell, James

    2016-01-01

    Abstract Managing the welfare of laboratory animals is critical to animal health, vital in the understanding of phenotypes created by treatment or genetic alteration and ensures compliance of regulations. Part of an animal welfare assessment is the requirement to record observations, ensuring all those responsible for the animals are aware of their health status and can act accordingly. Although the use of zebrafish in research continues to increase, guidelines for conducting welfare assessments and the reporting of observations are considered unclear compared to mammalian species. To support the movement of zebrafish between facilities, significant improvement would be achieved through the use of standardized terms to ensure clarity and consistency between facilities. Improving the clarity of terminology around welfare not only addresses our ethical obligation but also supports the research goals and provides a searchable description of the phenotypes. A Collaboration between the Wellcome Trust Sanger Institute and Cambridge University (Department of Medicine-Laboratory of Molecular Biology) has led to the creation of the zebrafish welfare terms from which standardization of terminology can be achieved. PMID:27096380

  7. Characterization of the Enigma family in zebrafish.

    PubMed

    Ott, Elisabeth B; Sakalis, Philippe A; Marques, Ines J; Bagowski, Christoph P

    2007-11-01

    The three Enigma subfamily proteins, Enigma, Enigma homologue, and Cypher/ZASP belong to the PDZ and LIM encoding protein family, which is characterized by the presence of a PDZ- and one or more LIM domains. PDZ/LIM proteins play important biological roles, and all members have been shown to associate with the actin cytoskeleton. We describe here the splice form specific expression patterns for the three Enigma subfamily members during zebrafish embryogenesis. Whole-mount in situ hybridization revealed common and distinct expression patterns for the different PDZ or LIM domain encoding splice variants. We further studied the role of enigma in zebrafish development. Enigma knockdown appeared to be embryonic lethal shortly after the end of gastrulation and in few surviving embryos led to elongation defects and disorganized somites. In summary, we show here the temporal and spatial expression patterns of the three Enigma family members and their PDZ and LIM domain encoding splice forms during zebrafish embryogenesis. Our results suggest that enigma is important for the formation and organization of somites and might play an important role for actin cytoskeleton organization during development.

  8. Short Stories on Zebrafish Long Noncoding RNAs

    PubMed Central

    Haque, Shadabul; Kaushik, Kriti; Leonard, Vincent Elvin; Kapoor, Shruti; Sivadas, Ambily; Joshi, Adita

    2014-01-01

    Abstract The recent re-annotation of the transcriptome of human and other model organisms, using next-generation sequencing approaches, has unravelled a hitherto unknown repertoire of transcripts that do not have a potential to code for proteins. These transcripts have been largely classified into an amorphous class popularly known as long noncoding RNAs (lncRNA). This discovery of lncRNAs in human and other model systems have added a new layer to the understanding of gene regulation at the transcriptional and post-transcriptional levels. In recent years, three independent studies have discovered a number of lncRNAs expressed in different stages of zebrafish development and adult tissues using a high-throughput RNA sequencing approach, significantly adding to the repertoire of genes known in zebrafish. A subset of these transcripts also shows distinct and specific spatiotemporal patterns of gene expression, pointing to a tight regulatory control and potential functional roles in development, organogenesis, and/ or homeostasis. This review provides an overview of the lncRNAs in zebrafish and discusses how their discovery could provide new insights into understanding biology, explaining mutant phenotypes, and helping in potentially modeling disease processes. PMID:25110965

  9. Facilitating drug discovery: an automated high-content inflammation assay in zebrafish.

    PubMed

    Wittmann, Christine; Reischl, Markus; Shah, Asmi H; Mikut, Ralf; Liebel, Urban; Grabher, Clemens

    2012-07-16

    Zebrafish larvae are particularly amenable to whole animal small molecule screens due to their small size and relative ease of manipulation and observation, as well as the fact that compounds can simply be added to the bathing water and are readily absorbed when administered in a <1% DMSO solution. Due to the optical clarity of zebrafish larvae and the availability of transgenic lines expressing fluorescent proteins in leukocytes, zebrafish offer the unique advantage of monitoring an acute inflammatory response in vivo. Consequently, utilizing the zebrafish for high-content small molecule screens aiming at the identification of immune-modulatory compounds with high throughput has been proposed, suggesting inflammation induction scenarios e.g. localized nicks in fin tissue, laser damage directed to the yolk surface of embryos or tailfin amputation. The major drawback of these methods however was the requirement of manual larva manipulation to induce wounding, thus preventing high-throughput screening. Introduction of the chemically induced inflammation (ChIn) assay eliminated these obstacles. Since wounding is inflicted chemically the number of embryos that can be treated simultaneously is virtually unlimited. Temporary treatment of zebrafish larvae with copper sulfate selectively induces cell death in hair cells of the lateral line system and results in rapid granulocyte recruitment to injured neuromasts. The inflammatory response can be followed in real-time by using compound transgenic cldnB::GFP/lysC::DsRED2 zebrafish larvae that express a green fluorescent protein in neuromast cells, as well as a red fluorescent protein labeling granulocytes. In order to devise a screening strategy that would allow both high-content and high-throughput analyses we introduced robotic liquid handling and combined automated microscopy with a custom developed software script. This script enables automated quantification of the inflammatory response by scoring the percent area

  10. Developmental lead exposure causes startle response deficits in zebrafish.

    PubMed

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

    2011-10-01

    Lead (Pb(2+)) exposure continues to be an important concern for fish populations. Research is required to assess the long-term behavioral effects of low-level concentrations of Pb(2+) and the physiological mechanisms that control those behaviors. Newly fertilized zebrafish embryos (<2h post fertilization; hpf) were exposed to one of three concentrations of lead (as PbCl(2)): 0, 10, or 30 nM until 24 hpf. (1) Response to a mechanosensory stimulus: Individual larvae (168 hpf) were tested for response to a directional, mechanical stimulus. The tap frequency was adjusted to either 1 or 4 taps/s. Startle response was recorded at 1000 fps. Larvae responded in a concentration-dependent pattern for latency to reaction, maximum turn velocity, time to reach V(max) and escape time. With increasing exposure concentrations, a larger number of larvae failed to respond to even the initial tap and, for those that did respond, ceased responding earlier than control larvae. These differences were more pronounced at a frequency of 4 taps/s. (2) Response to a visual stimulus: Fish, exposed as embryos (2-24 hpf) to Pb(2+) (0-10 μM) were tested as adults under low light conditions (≈ 60 μW/m(2)) for visual responses to a rotating black bar. Visual responses were significantly degraded at Pb(2+) concentrations of 30 nM. These data suggest that zebrafish are viable models for short- and long-term sensorimotor deficits induced by acute, low-level developmental Pb(2+) exposures.

  11. Identifying Structural Alerts Based on Zebrafish Developmental Morphological Toxicity (TDS)

    EPA Science Inventory

    Zebrafish constitute a powerful alternative animal model for chemical hazard evaluation. To provide an in vivo complement to high-throughput screening data from the ToxCast program, zebrafish developmental toxicity screens were conducted on the ToxCast Phase I (Padilla et al., 20...

  12. Host-Pathogen Interactions Made Transparent with the Zebrafish Model

    PubMed Central

    Meijer, Annemarie H; Spaink, Herman P

    2011-01-01

    The zebrafish holds much promise as a high-throughput drug screening model for immune-related diseases, including inflammatory and infectious diseases and cancer. This is due to the excellent possibilities for in vivo imaging in combination with advanced tools for genomic and large scale mutant analysis. The context of the embryo’s developing immune system makes it possible to study the contribution of different immune cell types to disease progression. Furthermore, due to the temporal separation of innate immunity from adaptive responses, zebrafish embryos and larvae are particularly useful for dissecting the innate host factors involved in pathology. Recent studies have underscored the remarkable similarity of the zebrafish and human immune systems, which is important for biomedical applications. This review is focused on the use of zebrafish as a model for infectious diseases, with emphasis on bacterial pathogens. Following a brief overview of the zebrafish immune system and the tools and methods used to study host-pathogen interactions in zebrafish, we discuss the current knowledge on receptors and downstream signaling components that are involved in the zebrafish embryo’s innate immune response. We summarize recent insights gained from the use of bacterial infection models, particularly the Mycobacterium marinum model, that illustrate the potential of the zebrafish model for high-throughput antimicrobial drug screening. PMID:21366518

  13. Characterization of behavioral and endocrine effects of LSD on zebrafish.

    PubMed

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

    2010-12-25

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

  14. DRUG EFFECTS ON THE LOCOMOTOR ACTIVITY OF LARVAL ZEBRAFISH.

    EPA Science Inventory

    As part of an effort to develop a rapid in vivo screen for EPA’s prioritization of toxic chemicals, we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae and the effects of prototype drugs. Zebrafish larvae (6-7 days post-fertilization) were indiv...

  15. Pleistophora hyphessobryconis (Microsporidia) infecting zebrafish (Danio rerio) in research facilities

    PubMed Central

    Sanders, Justin L; Lawrence, Christian; Nichols, Donald K; Brubaker, Jeffrey F.; Peterson, Tracy S; Murray, Katrina N.; Kent, Michael L

    2014-01-01

    Zebrafish (Danio rerio) are important models for biomedical research, and thus there is an increased concern about diseases afflicting them. Here we describe infections by Pleistophora hyphessobryconis (Microsporidia) in zebrafish from three laboratories. As reported in other aquarium fishes, affected zebrafish exhibited massive infections in the skeletal muscle, with no involvement of smooth or cardiac muscle. In addition, numerous spores within macrophages were observed in the visceral organs, including the ovaries. Transmission studies and ribosomal RNA (rRNA) gene sequence comparisons confirmed that the parasite from zebrafish was P. hyphessobryconis as described from neon tetra Paracheirodon innesi. Ten 15-day-old zebrafish were exposed to P. hyphessobryconis collected from one infected neon tetra, and 7 of 10 fish became infected. Comparison of P. hyphessobryconis small subunit rRNA gene sequence from neon tetra with that obtained from zebrafish was nearly identical, with < 1% difference. Given the severity of infections, P. hyphessobryconis should be added to the list of pathogens that should be avoided in zebrafish research facilities, and it would be prudent to not mix zebrafish used in research with other aquarium fishes. PMID:20853741

  16. Zebrafish heart as a model for human cardiac electrophysiology

    PubMed Central

    Vornanen, Matti; Hassinen, Minna

    2016-01-01

    ABSTRACT The zebrafish (Danio rerio) has become a popular model for human cardiac diseases and pharmacology including cardiac arrhythmias and its electrophysiological basis. Notably, the phenotype of zebrafish cardiac action potential is similar to the human cardiac action potential in that both have a long plateau phase. Also the major inward and outward current systems are qualitatively similar in zebrafish and human hearts. However, there are also significant differences in ionic current composition between human and zebrafish hearts, and the molecular basis and pharmacological properties of human and zebrafish cardiac ionic currents differ in several ways. Cardiac ionic currents may be produced by non-orthologous genes in zebrafish and humans, and paralogous gene products of some ion channels are expressed in the zebrafish heart. More research on molecular basis of cardiac ion channels, and regulation and drug sensitivity of the cardiac ionic currents are needed to enable rational use of the zebrafish heart as an electrophysiological model for the human heart. PMID:26671745

  17. Scale development in zebrafish (Danio rerio)

    PubMed Central

    SIRE, JEAN-YVES; ALLIZARD, FRANCOISE; BABIAR, OLIVIER; BOURGUIGNON, JACQUELINE; QUILHAC, ALEXANDRA

    1997-01-01

    In the course of an extensive comparative, structural and developmental study of the cranial and postcranial dermal skeleton (teeth and scales) in osteichthyan fishes, we have undertaken investigations on scale development in zebrafish (Danio (Brachydanio) rerio) using alizarin red staining, and light and transmission electron microscopy. The main goal was to know whether zebrafish scales can be used as a model for further research on the processes controlling the development of the dermal skeleton in general, especially epithelial–mesenchymal interactions. Growth series of laboratory bred specimens were used to study in detail: (1) the relationship of scale appearance with size and age; (2) the squamation pattern; and (3) the events taking place in the epidermis and in the dermis, before and during scale initiation and formation, with the aim of searching for morphological indications of epithelial-mesenchymal interactions. Scales form late in ontogeny, generally when zebrafish are more than 8.0 mm in standard length. Within a population of zebrafish of the same age scale appearance is related to standard length, but when comparing populations of different age the size of the fish at scale appearance is also related to age. Scales always appear first in the posterior region of the body and the squamation then extends anteriorly. Scales develop in the dermis but closely apposed to the epidermal–dermal boundary. Cellular modifications occurring in the basal layer of the epidermis and in the dermis before scale formation clearly indicate that the basal epidermal cells differentiate first, before any evidence of differentiation of the progenitors of the scale-forming cells in the dermis. This strongly suggests that scale differentiation could be initiated by the epidermal basal layer cells which probably produce a molecular signal towards the dermis below. Subsequently dermal cells accumulate close to the epidermis, and differentiate to form scale papillae. The

  18. Robust circadian rhythms in organoid cultures from PERIOD2::LUCIFERASE mouse small intestine.

    PubMed

    Moore, Sean R; Pruszka, Jill; Vallance, Jefferson; Aihara, Eitaro; Matsuura, Toru; Montrose, Marshall H; Shroyer, Noah F; Hong, Christian I

    2014-09-01

    Disruption of circadian rhythms is a risk factor for several human gastrointestinal (GI) diseases, ranging from diarrhea to ulcers to cancer. Four-dimensional tissue culture models that faithfully mimic the circadian clock of the GI epithelium would provide an invaluable tool to understand circadian regulation of GI health and disease. We hypothesized that rhythmicity of a key circadian component, PERIOD2 (PER2), would diminish along a continuum from ex vivo intestinal organoids (epithelial 'miniguts'), nontransformed mouse small intestinal epithelial (MSIE) cells and transformed human colorectal adenocarcinoma (Caco-2) cells. Here, we show that bioluminescent jejunal explants from PERIOD2::LUCIFERASE (PER2::LUC) mice displayed robust circadian rhythms for >72 hours post-excision. Circadian rhythms in primary or passaged PER2::LUC jejunal organoids were similarly robust; they also synchronized upon serum shock and persisted beyond 2 weeks in culture. Remarkably, unshocked organoids autonomously synchronized rhythms within 12 hours of recording. The onset of this autonomous synchronization was slowed by >2 hours in the presence of the glucocorticoid receptor antagonist RU486 (20 μM). Doubling standard concentrations of the organoid growth factors EGF, Noggin and R-spondin enhanced PER2 oscillations, whereas subtraction of these factors individually at 24 hours following serum shock produced no detectable effects on PER2 oscillations. Growth factor pulses induced modest phase delays in unshocked, but not serum-shocked, organoids. Circadian oscillations of PER2::LUC bioluminescence aligned with Per2 mRNA expression upon analysis using quantitative PCR. Concordant findings of robust circadian rhythms in bioluminescent jejunal explants and organoids provide further evidence for a peripheral clock that is intrinsic to the intestinal epithelium. The rhythmic and organotypic features of organoids should offer unprecedented advantages as a resource for elucidating the role

  19. Studying the immune response to human viral infections using zebrafish.

    PubMed

    Goody, Michelle F; Sullivan, Con; Kim, Carol H

    2014-09-01

    Humans and viruses have a long co-evolutionary history. Viral illnesses have and will continue to shape human history: from smallpox, to influenza, to HIV, and beyond. Animal models of human viral illnesses are needed in order to generate safe and effective antiviral medicines, adjuvant therapies, and vaccines. These animal models must support the replication of human viruses, recapitulate aspects of human viral illnesses, and respond with conserved immune signaling cascades. The zebrafish is perhaps the simplest, most commonly used laboratory model organism in which innate and/or adaptive immunity can be studied. Herein, we will discuss the current zebrafish models of human viral illnesses and the insights they have provided. We will highlight advantages of early life stage zebrafish and the importance of innate immunity in human viral illnesses. We will also discuss viral characteristics to consider before infecting zebrafish with human viruses as well as predict other human viruses that may be able to infect zebrafish.

  20. Host-microbe interactions in the developing zebrafish

    PubMed Central

    Kanther, Michelle; Rawls, John F.

    2010-01-01

    Summary of recent advances The amenability of the zebrafish to in vivo imaging and genetic analysis has fueled expanded use of this vertebrate model to investigate the molecular and cellular foundations of host-microbe relationships. Study of microbial encounters in zebrafish hosts has concentrated on developing embryonic and larval stages, when the advantages of the zebrafish model are maximized. A comprehensive understanding of these host-microbe interactions requires appreciation of the developmental context into which a microbe is introduced, as well as the effects of that microbial challenge on host ontogeny. In this review, we discuss how in vivo imaging and genetic analysis in zebrafish has advanced our knowledge of host-microbe interactions in the context of a developing vertebrate host. We focus on recent insights into immune cell ontogeny and function, commensal microbial relationships in the intestine, and microbial pathogenesis in zebrafish hosts. PMID:20153622

  1. Neutrophils in host defense: new insights from zebrafish

    PubMed Central

    Harvie, Elizabeth A.; Huttenlocher, Anna

    2015-01-01

    Neutrophils are highly motile phagocytic cells that play a critical role in the immune response to infection. Zebrafish (Danio rerio) are increasingly used to study neutrophil function and host-pathogen interactions. The generation of transgenic zebrafish lines with fluorescently labeled leukocytes has made it possible to visualize the neutrophil response to infection in real time by use of optically transparent zebrafish larvae. In addition, the genetic tractability of zebrafish has allowed for the generation of models of inherited neutrophil disorders. In this review, we discuss several zebrafish models of infectious disease, both in the context of immunocompetent, as well as neutrophil-deficient hosts and how these models have shed light on neutrophil behavior during infection. PMID:25717145

  2. Zebrafish Models of Human Liver Development and Disease

    PubMed Central

    Wilkins, Benjamin J.; Pack, Michael

    2016-01-01

    The liver performs a large number of essential synthetic and regulatory functions that are acquired during fetal development and persist throughout life. Their disruption underlies a diverse group of heritable and acquired diseases that affect both pediatric and adult patients. Although experimental analyses used to study liver development and disease are typically performed in cell culture models or rodents, the zebrafish is increasingly used to complement discoveries made in these systems. Forward and reverse genetic analyses over the past two decades have shown that the molecular program for liver development is largely conserved between zebrafish and mammals, and that the zebrafish can be used to model heritable human liver disorders. Recent work has demonstrated that zebrafish can also be used to study the mechanistic basis of acquired liver diseases. Here, we provide a comprehensive summary of how the zebrafish has contributed to our understanding of human liver development and disease. PMID:23897685

  3. Acute caffeine administration affects zebrafish response to a robotic stimulus.

    PubMed

    Ladu, Fabrizio; Mwaffo, Violet; Li, Jasmine; Macrì, Simone; Porfiri, Maurizio

    2015-08-01

    Zebrafish has been recently proposed as a valid animal model to investigate the fundamental mechanisms regulating emotional behavior and evaluate the modulatory effects exerted by psychoactive compounds. In this study, we propose a novel methodological framework based on robotics and information theory to investigate the behavioral response of zebrafish exposed to acute caffeine treatment. In a binary preference test, we studied the response of caffeine-treated zebrafish to a replica of a shoal of conspecifics moving in the tank. A purely data-driven information theoretic approach was used to infer the influence of the replica on zebrafish behavior as a function of caffeine concentration. Our results demonstrate that acute caffeine administration modulates both the average speed and the interaction with the replica. Specifically, zebrafish exposed to elevated doses of caffeine show reduced locomotion and increased sensitivity to the motion of the replica. The methodology developed in this study may complement traditional experimental paradigms developed in the field of behavioral pharmacology.

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

    NASA Technical Reports Server (NTRS)

    Moorman, S. J.

    2001-01-01

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

  5. [Potential of the zebrafish model to study congenital muscular dystrophies].

    PubMed

    Ryckebüsch, Lucile

    2015-10-01

    In order to better understand the complexity of congenital muscular dystrophies (CMD) and develop new strategies to cure them, it is important to establish new disease models. Due to its numerous helpful attributes, the zebrafish has recently become a very powerful animal model for the study of CMD. For some CMD, this vertebrate model is phenotypically closer to human pathology than the murine model. Over the last few years, researchers have developed innovative techniques to screen rapidly and on a large scale for muscle defects in zebrafish. Furthermore, new genome editing techniques in zebrafish make possible the identification of new disease models. In this review, the major attributes of zebrafish for CMD studies are discussed and the principal models of CMD in zebrafish are highlighted.

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

    PubMed

    Butail, Sachit; Mwaffo, Violet; Porfiri, Maurizio

    2016-04-01

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

  7. Identification of distal cis-regulatory elements at mouse mitoferrin loci using zebrafish transgenesis.

    PubMed

    Amigo, Julio D; Yu, Ming; Troadec, Marie-Berengere; Gwynn, Babette; Cooney, Jeffrey D; Lambert, Amy J; Chi, Neil C; Weiss, Mitchell J; Peters, Luanne L; Kaplan, Jerry; Cantor, Alan B; Paw, Barry H

    2011-04-01

    Mitoferrin 1 (Mfrn1; Slc25a37) and mitoferrin 2 (Mfrn2; Slc25a28) function as essential mitochondrial iron importers for heme and Fe/S cluster biogenesis. A genetic deficiency of Mfrn1 results in a profound hypochromic anemia in vertebrate species. To map the cis-regulatory modules (CRMs) that control expression of the Mfrn genes, we utilized genome-wide chromatin immunoprecipitation (ChIP) datasets for the major erythroid transcription factor GATA-1. We identified the CRMs that faithfully drive the expression of Mfrn1 during blood and heart development and Mfrn2 ubiquitously. Through in vivo analyses of the Mfrn-CRMs in zebrafish and mouse, we demonstrate their functional and evolutionary conservation. Using knockdowns with morpholinos and cell sorting analysis in transgenic zebrafish embryos, we show that GATA-1 directly regulates the expression of Mfrn1. Mutagenesis of individual GATA-1 binding cis elements (GBE) demonstrated that at least two of the three GBE within this CRM are functionally required for GATA-mediated transcription of Mfrn1. Furthermore, ChIP assays demonstrate switching from GATA-2 to GATA-1 at these elements during erythroid maturation. Our results provide new insights into the genetic regulation of mitochondrial function and iron homeostasis and, more generally, illustrate the utility of genome-wide ChIP analysis combined with zebrafish transgenesis for identifying long-range transcriptional enhancers that regulate tissue development.

  8. In vivo imaging of zebrafish digestive organ function using multiple quenched fluorescent reporters.

    PubMed

    Hama, Kotaro; Provost, Elayne; Baranowski, Timothy C; Rubinstein, Amy L; Anderson, Jennifer L; Leach, Steven D; Farber, Steven A

    2009-02-01

    Optical clarity of larvae makes the zebrafish ideal for real-time analyses of vertebrate organ function through the use of fluorescent reporters of enzymatic activities. A key function of digestive organs is to couple the generation of enzymes with mechanical processes that enable nutrient availability and absorption. However, it has been extremely difficult, and in many cases not possible, to directly observe digestive processes in a live vertebrate. Here we describe a new method to visualize intestinal protein and lipid processing simultaneously in live zebrafish larvae using a quenched fluorescent protein (EnzChek) and phospholipid (PED6). By employing these reagents, we found that wild-type larvae exhibit significant variation in intestinal phospholipase and protease activities within a group but display a strong correlation between the activities within individuals. Furthermore, we found that pancreas function is essential for larval digestive protease activity but not for larval intestinal phospholipase activity. Although fat-free (ffr) mutant larvae were previously described to exhibit impaired lipid processes, we found they also had significantly reduced protease activity. Finally, we selected and evaluated compounds that were previously suggested to have altered phospholipase activity and are known or suspected to have inflammatory effects in the intestinal tract including nonsteroidal anti-inflammatory drugs, and identified a compound that significantly increases intestinal phospholipid processing. Thus the multiple fluorescent reporter-based methodology facilitates the rapid analysis of digestive organ function in live zebrafish larvae.

  9. Target of rapamycin (TOR)-based therapy for cardiomyopathy: evidence from zebrafish and human studies.

    PubMed

    Kushwaha, Sudhir; Xu, Xiaolei

    2012-02-01

    Rapamycin is a U.S. Food and Drug Administration-approved drug for the prevention of immunorejection following organ transplantation. Pharmacological studies suggest a potential new application of rapamycin in attenuating cardiomyopathy, but the potential for this application is not yet supported by genetic studies of genes in target of rapamycin (TOR) signaling in rodents. Recently, supporting genetic evidence was presented in zebrafish using two adult cardiomyopathy models. By characterizing a heterozygous zebrafish target of rapamycin (ztor) mutant, the therapeutic effect of long-term TOR signaling inhibition was demonstrated. Dose- and stage-dependent functions of TOR signaling provide an explanation for the seemingly contradictory results obtained in genetic studies of TOR components in rodents. The results from the zebrafish studies, together with the supporting preliminary clinical studies, suggested that TOR signaling inhibition should be further pursued as a novel therapeutic strategy for cardiomyopathy. Future directions for developing TOR-based therapy include assessing the long-term benefits of rapamycin as a candidate drug for heart failure patients, defining the dynamic activity of TOR, exploring the impacts of TOR signaling manipulation in different models of cardiomyopathies, and elucidating the downstream signaling branches that confer the therapeutic effects of TOR signaling inhibition.

  10. Reversible loss of reproductive fitness in zebrafish on chronic alcohol exposure.

    PubMed

    Dewari, Pooran Singh; Ajani, Funmilola; Kushawah, Gopal; Kumar, Damera Santhosh; Mishra, Rakesh K

    2016-02-01

    Alcoholism is one of the most prevalent diseases in society and causes significant health and social problems. Alcohol consumption by pregnant women is reported to cause adverse effects on the physical and psychological growth of the fetus. However, the direct effect of chronic alcohol consumption on reproductive fitness has not been tested. In recent years, the zebrafish (Danio rerio) has emerged as a versatile model system to study the effects of alcohol on behavior and embryonic development. We utilized the zebrafish model system to address the effect of chronic alcohol exposure (0.5% alcohol in the holding tank for 9 weeks) on reproductive capacity. We found a dramatic decrease in fecundity, measured by counting the number of eggs laid, when at least one of the parents is subject to chronic alcohol exposure. Interestingly, a 9-week alcohol withdrawal program completely restored the reproductive capacity of the treated subjects. In agreement with observations on fecundity, the chronic alcohol exposure leads to increased anxiety, as measured by the novel-tank diving assay. Conversely, the withdrawal program diminished heightened anxiety in alcohol-exposed subjects. Our results highlight the adverse effects of chronic alcohol exposure on the reproductive capacity of both males and females, and underscore the utility of the zebrafish model system to understand the biology of chronic alcoholism.

  11. OCCURRENCE OF A MYXOZOAN PARASITE MYXIDIUM STREISINGERI N. SP. IN LABORATORY ZEBRAFISH DANIO RERIO

    PubMed Central

    Whipps, Christopher M.; Murray, Katrina N.; Kent, Michael L.

    2015-01-01

    Over several years of screening diagnostic cases, the Zebrafish International Resource Center Health Services have encountered a myxozoan parasite of the ducts associated with the kidney in zebrafish Danio rerio from and average of 21% of facilities submitting specimens over 5 yr. The parasite is coelozoic and is associated with no appreciable histological changes. Plasmodia bear ovoid spores with 3 sutural ridges. Spores are consistent with the genus Myxidium, but are distinct from any known species, and are thus described as Myxidium streisingeri n. sp. Phylogenetically, this parasite is a member of the polyphyletic urinary bladder clade, which is consistent with the site of infection. The common occurrence of a myxozoan in this closed husbandry system is unexpected because these parasites are known to have complex life cycles, alternating between a vertebrate and invertebrate host. It may be that biofilters provide habitat for suitable invertebrate hosts or perhaps M. streisingeri n. sp. can be transmitted directly. Future control of this parasite in zebrafish research laboratories depends on a better understanding of this life cycle. PMID:25277837

  12. Lactobacillus rhamnosus GG Effect on Behavior of Zebrafish During Chronic Ethanol Exposure.

    PubMed

    Schneider, Ana Claudia Reis; Rico, Eduardo Pacheco; de Oliveira, Diogo Losch; Rosemberg, Denis Broock; Guizzo, Ranieli; Meurer, Fábio; da Silveira, Themis Reverbel

    2016-01-01

    Ethanol is a widely consumed drug, which acts on the central nervous system to induce behavioral alterations ranging from disinhibition to sedation. Recent studies have produced accumulating evidence for the therapeutic role of probiotic bacteria in behavior. We aimed to investigate the effect of Lactobacillus rhamnosus GG (LGG) on the behavior of adult zebrafish chronically exposed to ethanol. Adult wild-type zebrafish were randomly divided into four groups, each containing 15 fish. The following groups were formed: Control (C), received unsupplemented feed during the trial period; Probiotic (P), fed with feed supplemented with LGG; Ethanol (E), received unsupplemented feed and 0.5% of ethanol directly added to the tank water; and Probiotic+Ethanol (P+E), group under ethanol exposure (0.5%) and fed with LGG supplemented feed. After 2 weeks of exposure, the novel tank test was used to evaluate fish behavior, which was analyzed using computer-aided video tracking. LGG alone did not alter swimming behavior of the fish. Ethanol exposure led to robust behavioral effects in the form of reduced anxiety levels, as indicated by increased vertical exploration and more time spent in the upper region of the novel tank. The group exposed to ethanol and treated with LGG behaved similarly to animals exposed to ethanol alone. Taken together, these results show that zebrafish behavior was not altered by LGG per se, as seen in murine models. This was the first study to investigate the effects of a probiotic diet on behavior after a chronic ethanol exposure.

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

    NASA Astrophysics Data System (ADS)

    Butail, Sachit; Mwaffo, Violet; Porfiri, Maurizio

    2016-04-01

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

  14. miR-19b Regulates Ventricular Action Potential Duration in Zebrafish

    PubMed Central

    Benz, Alexander; Kossack, Mandy; Auth, Dominik; Seyler, Claudia; Zitron, Edgar; Juergensen, Lonny; Katus, Hugo A.; Hassel, David

    2016-01-01

    Sudden cardiac death due to ventricular arrhythmias often caused by action potential duration (APD) prolongation is a common mode of death in heart failure (HF). microRNAs, noncoding RNAs that fine tune gene expression, are frequently dysregulated during HF, suggesting a potential involvement in the electrical remodeling process accompanying HF progression. Here, we identified miR-19b as an important regulator of heart function. Zebrafish lacking miR-19b developed severe bradycardia and reduced cardiac contractility. miR-19b deficient fish displayed increased sensitivity to AV-block, a characteristic feature of long QT syndrome in zebrafish. Patch clamp experiments from whole hearts showed that miR-19b deficient zebrafish exhibit significantly prolonged ventricular APD caused by impaired repolarization. We found that miR-19b directly and indirectly regulates the expression of crucial modulatory subunits of cardiac ion channels, and thereby modulates AP duration and shape. Interestingly, miR-19b knockdown mediated APD prolongation can rescue a genetically induced short QT phenotype. Thus, miR-19b might represent a crucial modifier of the cardiac electrical activity, and our work establishes miR-19b as a potential candidate for human long QT syndrome. PMID:27805004

  15. Occurrence of a myxozoan parasite Myxidium streisingeri n. sp. in laboratory zebrafish Danio rerio.

    PubMed

    Whipps, Christopher M; Murray, Katrina N; Kent, Michael L

    2015-02-01

    Over several years of screening diagnostic cases, the Zebrafish International Resource Center Health Services has encountered a myxozoan parasite of the ducts associated with the kidney in zebrafish, Danio rerio , from an average of 21% of facilities submitting specimens over 5 yr. The parasite is coelozoic and is associated with no appreciable histological changes. Plasmodia bear ovoid spores with 3 sutural ridges. Spores are consistent with the genus Myxidium, but they are distinct from any known species and are thus described as Myxidium streisingeri n. sp. Phylogenetically, this parasite is a member of the polyphyletic urinary bladder clade, which is consistent with the site of infection. The common occurrence of a myxozoan in this closed husbandry system is unexpected because these parasites are known to have complex life cycles, alternating between a vertebrate and invertebrate host. It may be that biofilters provide habitat for suitable invertebrate hosts or perhaps M. streisingeri n. sp. can be transmitted directly. Future control of this parasite in zebrafish research laboratories depends on a better understanding of this life cycle.

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

    SciTech Connect

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

    2009-05-15

    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.

  17. Protein‐Functionalized DNA Nanostructures as Tools to Control Transcription in Zebrafish Embryos

    PubMed Central

    Angelin, Alessandro; Kassel, Olivier; Rastegar, Sepand; Strähle, Uwe

    2016-01-01

    Abstract The unique structure‐directing properties of DNA origami nanostructures (DONs) show great potential to specifically manipulate intracellular processes. We report an innovative concept to selectively activate the transcription of a single gene in the developing zebrafish embryo. We reason that engineering a designer transcription factor in which a rigid DON imposes a fixed distance between the DNA‐binding domain (DBD) and the transactivation domain (TAD) would allow the selective activation of a gene harboring the same distance between the corresponding transcription factor binding site and the core promoter. As a test case, a rigid tubular DON was designed to separate the DBD of the GAL4 transcription factor and the VP16 viral protein as a TAD. This construct was microinjected in the yolk of one‐cell‐stage zebrafish embryos, together with a reporter plasmid to assess its functionality. The large DON was efficiently distributed to cells of the developing embryo and showed no signs of toxicity. However, because the DON showed only a cytosolic localization, it did not activate transcription of the reporter gene. Although this work clearly demonstrates that DON microinjection enables the intracellular distribution of multi‐protein architectures in most of the cells of the developing zebrafish embryo, further refinements are necessary to enable selective gene activation in vivo. PMID:28168148

  18. A Dominant Negative Zebrafish Ahr2 Partially Protects Developing Zebrafish from Dioxin Toxicity

    PubMed Central

    Lanham, Kevin A.; Prasch, Amy L.; Weina, Kasia M.; Peterson, Richard E.; Heideman, Warren

    2011-01-01

    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. PMID:22194803

  19. Chronic exposure to ethanol causes steatosis and inflammation in zebrafish liver

    PubMed Central

    Schneider, Ana Claudia Reis; Gregório, Cleandra; Uribe-Cruz, Carolina; Guizzo, Ranieli; Malysz, Tais; Faccioni-Heuser, Maria Cristina; Longo, Larisse; da Silveira, Themis Reverbel

    2017-01-01

    AIM To evaluate the effects of chronic exposure to ethanol in the liver and the expression of inflammatory genes in zebrafish. METHODS Zebrafish (n = 104), wild type, adult, male and female, were divided into two groups: Control and ethanol (0.05 v/v). The ethanol was directly added into water; tanks water were changed every two days and the ethanol replaced. The animals were fed twice a day with fish food until satiety. After two and four weeks of trial, livers were dissected, histological analysis (hematoxilin-eosin and Oil Red staining) and gene expression assessment of adiponectin, adiponectin receptor 2 (adipor2), sirtuin-1 (sirt-1), tumor necrosis factor-alpha (tnf-a), interleukin-1b (il-1b) and interleukin-10 (il-10) were performed. Ultrastructural evaluations were conducted at fourth week. RESULTS Exposing zebrafish to 0.5% ethanol developed intense liver steatosis after four weeks, as demonstrated by oil red staining. In ethanol-treated animals, the main ultrastructural changes were related to cytoplasmic lipid particles and droplets, increased number of rough endoplasmic reticulum cisterns and glycogen particles. Between two and four weeks, hepatic mRNA expression of il-1b, sirt-1 and adipor2 were upregulated, indicating that ethanol triggered signaling molecules which are key elements in both hepatic inflammatory and protective responses. Adiponectin was not detected in the liver of animals exposed and not exposed to ethanol, and il-10 did not show significant difference. CONCLUSION Data suggest that inflammatory signaling and ultrastructural alterations play a significant role during hepatic steatosis in zebrafish chronically exposed to ethanol. PMID:28357029

  20. Early doors (Edo) mutant mouse reveals the importance of period 2 (PER2) PAS domain structure for circadian pacemaking

    PubMed Central

    Militi, Stefania; Maywood, Elizabeth S.; Sandate, Colby R.; Chesham, Johanna E.; Parsons, Michael J.; Vibert, Jennifer L.; Joynson, Greg M.; Partch, Carrie L.; Hastings, Michael H.; Nolan, Patrick M.

    2016-01-01

    The suprachiasmatic nucleus (SCN) defines 24 h of time via a transcriptional/posttranslational feedback loop in which transactivation of Per (period) and Cry (cryptochrome) genes by BMAL1–CLOCK complexes is suppressed by PER–CRY complexes. The molecular/structural basis of how circadian protein complexes function is poorly understood. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutation, early doors (Edo), in the PER-ARNT-SIM (PAS) domain dimerization region of period 2 (PER2) (I324N) that accelerates the circadian clock of Per2Edo/Edo mice by 1.5 h. Structural and biophysical analyses revealed that Edo alters the packing of the highly conserved interdomain linker of the PER2 PAS core such that, although PER2Edo complexes with clock proteins, its vulnerability to degradation mediated by casein kinase 1ε (CSNK1E) is increased. The functional relevance of this mutation is revealed by the ultrashort (<19 h) but robust circadian rhythms in Per2Edo/Edo; Csnk1eTau/Tau mice and the SCN. These periods are unprecedented in mice. Thus, Per2Edo reveals a direct causal link between the molecular structure of the PER2 PAS core and the pace of SCN circadian timekeeping. PMID:26903623

  1. Effect of Mefloquine, a Gap Junction Blocker, on Circadian Period2 Gene Oscillation in the Mouse Suprachiasmatic Nucleus Ex Vivo

    PubMed Central

    Koo, Jinmi; Choe, Han Kyoung; Kim, Hee-Dae; Chun, Sung Kook; Son, Gi Hoon

    2015-01-01

    Background In mammals, the master circadian pacemaker is localized in an area of the ventral hypothalamus known as the suprachiasmatic nucleus (SCN). Previous studies have shown that pacemaker neurons in the SCN are highly coupled to one another, and this coupling is crucial for intrinsic self-sustainability of the SCN central clock, which is distinguished from peripheral oscillators. One plausible mechanism underlying the intercellular communication may involve direct electrical connections mediated by gap junctions. Methods We examined the effect of mefloquine, a neuronal gap junction blocker, on circadian Period 2 (Per2) gene oscillation in SCN slice cultures prepared from Per2::luciferase (PER2::LUC) knock-in mice using a real-time bioluminescence measurement system. Results Administration of mefloquine causes instability in the pulse period and a slight reduction of amplitude in cyclic PER2::LUC expression. Blockade of gap junctions uncouples PER2::LUC-expressing cells, in terms of phase transition, which weakens synchrony among individual cellular rhythms. Conclusion These findings suggest that neuronal gap junctions play an important role in synchronizing the central pacemaker neurons and contribute to the distinct self-sustainability of the SCN master clock. PMID:25491783

  2. Loss of the small heat shock protein αA-crystallin does not lead to detectable defects in early zebrafish lens development.

    PubMed

    Posner, Mason; Skiba, Jackie; Brown, Mary; Liang, Jennifer O; Nussbaum, Justin; Prior, Heather

    2013-11-01

    Alpha crystallins are small heat shock proteins essential to normal ocular lens function. They also help maintain homeostasis in many non-ocular vertebrate tissues and their expression levels change in multiple diseases of the nervous and cardiovascular system and during cancer. The specific roles that α-crystallins may play in eye development are unclear. Studies with knockout mice suggested that only one of the two mammalian α-crystallins is required for normal early lens development. However, studies in two fish species suggested that reduction of αA-crystallin alone could inhibit normal fiber cell differentiation, cause cataract and contribute to lens degeneration. In this study we used synthetic antisense morpholino oligomers to suppress the expression of zebrafish αA-crystallin to directly test the hypothesis that, unlike mammals, the zebrafish requires αA-crystallin for normal early lens development. Despite the reduction of zebrafish αA-crystallin protein to undetectable levels by western analysis through 4 days of development we found no changes in fiber cell differentiation, lens morphology or transparency. In contrast, suppression of AQP0a expression, previously shown to cause lens cataract, produced irregularly shaped lenses, delay in fiber cell differentiation and lens opacities detectable by confocal microscopy. The normal development observed in αA-crystallin deficient zebrafish embryos may reflect similarly non-essential roles for this protein in the early stages of both zebrafish and mammalian lens development. This finding has ramifications for a growing number of researchers taking advantage of the zebrafish's transparent external embryos to study vertebrate eye development. Our demonstration that lens cataracts can be visualized in three-dimensions by confocal microscopy in a living zebrafish provides a new tool for studying the causes, development and prevention of lens opacities.

  3. Immobilization of zebrafish larvae on a chip-based device for environmental scanning electron microscopy (ESEM) imaging

    NASA Astrophysics Data System (ADS)

    Akagi, Jin; Hall, Chris J.; Crosier, Kathryn E.; Crosier, Philip S.; Wlodkowic, Donald

    2013-12-01

    Small vertebrate model organisms have recently gained popularity as attractive experimental models that enhance our understanding of human tissue and organ development. Laser microsurgery on zebrafish larvae combined with Scanning Electron Microscopy (SEM) imaging can in particular provide accelerated insights into the tissue regeneration phenomena. Conventional SEM exposes, however, specimens to high vacuum environments, and often requires laborintensive and time-consuming pretreatments and manual positioning. Moreover, there are virtually no technologies available that can quickly immobilize the zebrafish larvae for high definition SEM imaging. This work describes the proof-of-concept design and validation of a microfluidic chip-based system for immobilizing zebrafish larvae and it's interfacing with Environmental Scanning Electron Microscope (ESEM) imaging. The Lab-on-a-Chip (LOC) device was fabricated using a high-speed infrared laser micromachining and consists of a reservoir with multiple semispherical microwells, which hold the yolk of zebrafish larvae, and drain channels that allow removing excess of medium during SEM imaging. Paper filter is used to actuate the chip and immobilization of the larvae by gentle suction that occurs during water drainage. The trapping region allows multiple specimens to be positioned on the chip. The device is then inserted directly inside the ESEM and imaged in a near 100% humidity atmosphere. This facilitates ESEM imaging of untreated biological samples.

  4. Zebrafish: an animal model for research in veterinary medicine.

    PubMed

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

    2015-01-01

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

  5. Adaptive locomotor behavior in larval zebrafish.

    PubMed

    Portugues, Ruben; Engert, Florian

    2011-01-01

    In this study we report that larval zebrafish display adaptive locomotor output that can be driven by unexpected visual feedback. We develop a new assay that addresses visuomotor integration in restrained larval zebrafish. The assay involves a closed-loop environment in which the visual feedback a larva receives depends on its own motor output in a way that resembles freely swimming conditions. The experimenter can control the gain of this closed feedback loop, so that following a given motor output the larva experiences more or less visual feedback depending on whether the gain is high or low. We show that increases and decreases in this gain setting result in adaptive changes in behavior that lead to a generalized decrease or increase of motor output, respectively. Our behavioral analysis shows that both the duration and tail beat frequency of individual swim bouts can be modified, as well as the frequency with which bouts are elicited. These changes can be implemented rapidly, following an exposure to a new gain of just 175 ms. In addition, modifications in some behavioral parameters accumulate over tens of seconds and effects last for at least 30 s from trial to trial. These results suggest that larvae establish an internal representation of the visual feedback expected from a given motor output and that the behavioral modifications are driven by an error signal that arises from the discrepancy between this expectation and the actual visual feedback. The assay we develop presents a unique possibility for studying visuomotor integration using imaging techniques available in the larval zebrafish.

  6. Adaptive Locomotor Behavior in Larval Zebrafish

    PubMed Central

    Portugues, Ruben; Engert, Florian

    2011-01-01

    In this study we report that larval zebrafish display adaptive locomotor output that can be driven by unexpected visual feedback. We develop a new assay that addresses visuomotor integration in restrained larval zebrafish. The assay involves a closed-loop environment in which the visual feedback a larva receives depends on its own motor output in a way that resembles freely swimming conditions. The experimenter can control the gain of this closed feedback loop, so that following a given motor output the larva experiences more or less visual feedback depending on whether the gain is high or low. We show that increases and decreases in this gain setting result in adaptive changes in behavior that lead to a generalized decrease or increase of motor output, respectively. Our behavioral analysis shows that both the duration and tail beat frequency of individual swim bouts can be modified, as well as the frequency with which bouts are elicited. These changes can be implemented rapidly, following an exposure to a new gain of just 175 ms. In addition, modifications in some behavioral parameters accumulate over tens of seconds and effects last for at least 30 s from trial to trial. These results suggest that larvae establish an internal representation of the visual feedback expected from a given motor output and that the behavioral modifications are driven by an error signal that arises from the discrepancy between this expectation and the actual visual feedback. The assay we develop presents a unique possibility for studying visuomotor integration using imaging techniques available in the larval zebrafish. PMID:21909325

  7. montalcino, a Zebrafish Model for Variegate Porphyria

    PubMed Central

    Dooley, Kimberly A.; Fraenkel, Paula G.; Langer, Nathaniel B.; Schmid, Bettina; Davidson, Alan J.; Weber, Gerhard; Chiang, Ken; Foott, Helen; Dwyer, Caitlin; Wingert, Rebecca A.; Zhou, Yi; Paw, Barry H.; Zon, Leonard I.

    2008-01-01

    Objective Inherited or acquired mutations in the heme biosynthetic pathway lead to a debilitating class of diseases collectively known as porphyrias, with symptoms that can include anemia, cutaneous photosensitivity, and neurovisceral dysfunction. In a genetic screen for hematopoietic mutants, we isolated a zebrafish mutant, montalcino (mno), which displays hypochromic anemia and porphyria. The objective of this study was to identify the defective gene and characterize the phenotype of the zebrafish mutant. Methods Genetic linkage analysis was utilized to identify the region harboring the mno mutation. Candidate gene analysis together with RT-PCR was utilized to identify the genetic mutation, which was confirmed via allele specific oligo hybridizations. Whole mount in situ hybridizations and 0-dianisidine staining were used to characterize the phenotype of the mno mutant. mRNA and morpholino microinjections were performed to phenocopy and/or rescue the mutant phenotype. Results Homozygous mno mutant embryos have a defect in the protoporphyrinogen oxidase (ppox) gene, which encodes the enzyme that catalyzes the oxidation of protoporphyrinogen. Homozygous mutant embryos are deficient in hemoglobin, and by 36 hpf are visibly anemic and porphyric. The hypochromic anemia of mno embryos was partially rescued by human ppox, providing evidence for the conservation of function between human and zebrafish ppox. Conclusion In humans, mutations in ppox result in variegate porphyria. At present, effective treatment for acute attacks requires the administration intravenous hemin and/or glucose. Thus, mno represents a powerful model for investigation, and a tool for future screens aimed at identifying chemical modifiers of variegate porphyria. PMID:18550261

  8. GROWTH AND BEHAVIOR OF LARVAL ZEBRAFISH Danio ...

    EPA Pesticide Factsheets

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

  9. Novel biomarkers of perchlorate exposure in zebrafish

    USGS Publications Warehouse

    Mukhi, S.; Carr, J.A.; Anderson, T.A.; Patino, R.

    2005-01-01

    Perchlorate inhibits iodide uptake by thyroid follicles and lowers thyroid hormone production. Although several effects of perchlorate on the thyroid system have been reported, the utility of these pathologies as markers of environmental perchlorate exposures has not been adequately assessed. The present study examined time-course and concentration-dependent effects of perchlorate on thyroid follicle hypertrophy, colloid depletion, and angiogenesis; alterations in whole-body thyroxine (T4) levels; and somatic growth and condition factor of subadult and adult zebrafish. Changes in the intensity of the colloidal T4 ring previously observed in zebrafish also were examined immunohistochemically. Three-month-old zebrafish were exposed to ammonium perchlorate at measured perchlorate concentrations of 0, 11, 90, 1,131, and 11,480 ppb for 12 weeks and allowed to recover in clean water for 12 weeks. At two weeks of exposure, the lowest-observed-effective concentrations (LOECs) of perchlorate that induced angiogenesis and depressed the intensity of colloidal T4 ring were 90 and 1,131 ppb, respectively; other parameters were not affected (whole-body T4 was not determined at this time). At 12 weeks of exposure, LOECs for colloid depletion, hypertrophy, angiogenesis, and colloidal T4 ring were 11,480, 1,131, 90, and 11 ppb, respectively. All changes were reversible, but residual effects on angiogenesis and colloidal T4 ring intensity were still present after 12 weeks of recovery (LOEC, 11,480 ppb). Whole-body T 4 concentration, body growth (length and weight), and condition factor were not affected by perchlorate. The sensitivity and longevity of changes in colloidal T4 ring intensity and angiogenesis suggest their usefulness as novel markers of perchlorate exposure. The 12-week LOEC for colloidal T4 ring is the lowest reported for any perchlorate biomarker in aquatic vertebrates. ?? 2005 SETAC.

  10. Acclimation of zebrafish to transport stress.

    PubMed

    Dhanasiri, Anusha K S; Fernandes, Jorge M O; Kiron, Viswanath

    2013-03-01

    Welfare of fish is commonly neglected when they are transported. This study examines the effect of a 72-h mock transport on certain aspects of the stress physiology of two groups of zebrafish-the first transported in water enriched with a nitrifying bacterial consortium and the second in water without the enrichment. Zebrafish were examined at different time points-before packing (BP), immediately after packing them in transport bags (AP), at the end of transport (AT), and 72 h thereafter (PT)-to assess the primary (cortisol) and secondary (glucose) stress responses. In addition, the relevant genes in hypothalamic-pituitary-interrenal (HPI) axis (crf in brain, mc2r, star, cyp11c1, and hsd11b2 in kidney), including that of mineralocorticoid receptor (mr in kidney), were studied. Procedures during packing caused an increase in whole body cortisol levels of both fish groups. Only in the fish transported without the bacterial consortium, an increase in the levels of whole body cortisol as well as blood glucose was observed at the end of the transport. At the same time point and in the same fish group, the transcripts of mr and hsd11b2 were enhanced, probably to cope with the stress and to maintain homeostasis. The mRNA levels of the other genes in the HPI stress axis (crf, mc2r, star, and cyp11c1) were not significantly altered. Zebrafish transported in water enriched with the bacterial consortium exhibited a speedier stress acclimation. Nevertheless, only through in-depth studies the beneficial effect of the consortium can be confirmed.

  11. Heme reversibly damps PERIOD2 rhythms in mouse suprachiasmatic nucleus explants.

    PubMed

    Guenthner, C J; Bickar, D; Harrington, M E

    2009-12-01

    The hypothalamic suprachiasmatic nucleus (SCN), which in mammals serves as the master circadian pacemaker by synchronizing autonomous clocks in peripheral tissues, is composed of coupled single-cell oscillators that are driven by interlocking positive/negative transcriptional/translational feedback loops. Several studies have suggested that heme, a common prosthetic group that is synthesized and degraded in a circadian manner in the SCN, may modulate the function of several feedback loop components, including the REV-ERB nuclear receptors and PERIOD2 (PER2). We found that ferric heme (hemin, 3-100 microM) dose-dependently and reversibly damped luminescence rhythms in SCN explants from mice expressing a PER2::LUCIFERASE (PER2::LUC) fusion protein. Inhibitors of heme oxygenases (HOs, which degrade heme to biliverdin, carbon monoxide, and iron) mimicked heme's effects on PER2 rhythms. In contrast, heme and HO inhibition did not damp luminescence rhythms in thymus and esophagus explants and had only a small effect on PER2::LUC damping in spleen explants, suggesting that heme's effects are tissue-specific. Analysis of the effects of heme's degradation products on SCN PER2::LUC rhythms indicated that they probably were not responsible for heme's effects on rhythms. The heme synthesis inhibitor N-methylprotoporphyrinIX (NMP) lengthened the circadian period of SCN PER2::LUC rhythms by about an hour. These data are consistent with an important role for heme in the circadian system.

  12. Glucocorticoid-mediated Period2 induction delays the phase of circadian rhythm.

    PubMed

    Cheon, Solmi; Park, Noheon; Cho, Sehyung; Kim, Kyungjin

    2013-07-01

    Glucocorticoid (GC) signaling synchronizes the circadian rhythm of individual peripheral cells and induces the expression of circadian genes, including Period1 (Per1) and Period2 (Per2). However, no GC response element (GRE) has been reported in the Per2 promoter region. Here we report the molecular mechanisms of Per2 induction by GC signaling and its relevance to the regulation of circadian timing. We found that GC prominently induced Per2 expression and delayed the circadian phase. The overlapping GRE and E-box (GE2) region in the proximal Per2 promoter was responsible for GC-mediated Per2 induction. The GRE in the Per2 promoter was unique in that brain and muscle ARNT-like protein-1 (BMAL1) was essential for GC-induced Per2 expression, whereas other GRE-containing promoters, such as Per1 and mouse mammary tumor virus, responded to dexamethasone in the absence of BMAL1. This specialized regulatory mechanism was mediated by BMAL1-dependent binding of the GC receptor to GRE in Per2 promoter. When Per2 induction was abrogated by the mutation of the GRE or E-box, the circadian oscillation phase failed to be delayed compared with that of the wild-type. Therefore, the current study demonstrates that the rapid Per2 induction mediated by GC is crucial for delaying the circadian rhythm.

  13. Glucocorticoid-mediated Period2 induction delays the phase of circadian rhythm

    PubMed Central

    Cheon, Solmi; Park, Noheon; Cho, Sehyung; Kim, Kyungjin

    2013-01-01

    Glucocorticoid (GC) signaling synchronizes the circadian rhythm of individual peripheral cells and induces the expression of circadian genes, including Period1 (Per1) and Period2 (Per2). However, no GC response element (GRE) has been reported in the Per2 promoter region. Here we report the molecular mechanisms of Per2 induction by GC signaling and its relevance to the regulation of circadian timing. We found that GC prominently induced Per2 expression and delayed the circadian phase. The overlapping GRE and E-box (GE2) region in the proximal Per2 promoter was responsible for GC-mediated Per2 induction. The GRE in the Per2 promoter was unique in that brain and muscle ARNT-like protein-1 (BMAL1) was essential for GC-induced Per2 expression, whereas other GRE-containing promoters, such as Per1 and mouse mammary tumor virus, responded to dexamethasone in the absence of BMAL1. This specialized regulatory mechanism was mediated by BMAL1-dependent binding of the GC receptor to GRE in Per2 promoter. When Per2 induction was abrogated by the mutation of the GRE or E-box, the circadian oscillation phase failed to be delayed compared with that of the wild-type. Therefore, the current study demonstrates that the rapid Per2 induction mediated by GC is crucial for delaying the circadian rhythm. PMID:23620290

  14. The circadian clock component PERIOD2: from molecular to cerebral functions.

    PubMed

    Ripperger, Jürgen A; Albrecht, Urs

    2012-01-01

    The circadian clock is based on a molecular oscillator, which simulates the external day within nearly all of a body's cells. This "internalized" day then defines activity and rest phases for the cells and the organism by generating precise rhythms in the metabolism, physiology, and behavior. In its perfect state, this timing system allows for the synchronization of an organism to its environment and this may optimize energy handling and responses to daily recurring challenges. However, nowadays, we believe that desynchronization of an organism due to its lifestyle or problems with its circadian clock not only causes discomfort but also may aggravate conditions such as depression, metabolic syndrome, addiction, or cancer. In this review, we focus on one simple cogwheel of the mammalian circadian clock, the PERIOD2 (PER2) protein. Originally identified as an integral part of the molecular mechanism that yields overt rhythms of about 24h, more recently multiple other functions have been identified. In essence, the PER proteins, in addition to their important function within the molecular oscillator, can be seen not only as integrators on the input side of the circadian clock but also as mediators of clock output. This diversity in their function is possible, because the PER proteins can interact with a multitude of other proteins transferring oscillator timing information to the latter. In this fashion, the circadian clock synchronizes many rhythmic processes.

  15. Three-Dimensional Neurophenotyping of Adult Zebrafish Behavior

    PubMed Central

    Cachat, Jonathan; Stewart, Adam; Utterback, Eli; Hart, Peter; Gaikwad, Siddharth; Wong, Keith; Kyzar, Evan; Wu, Nadine; Kalueff, Allan V.

    2011-01-01

    The use of adult zebrafish (Danio rerio) in neurobehavioral research is rapidly expanding. The present large-scale study applied the newest video-tracking and data-mining technologies to further examine zebrafish anxiety-like phenotypes. Here, we generated temporal and spatial three-dimensional (3D) reconstructions of zebrafish locomotion, globally assessed behavioral profiles evoked by several anxiogenic and anxiolytic manipulations, mapped individual endpoints to 3D reconstructions, and performed cluster analysis to reconfirm behavioral correlates of high- and low-anxiety states. The application of 3D swim path reconstructions consolidates behavioral data (while increasing data density) and provides a novel way to examine and represent zebrafish behavior. It also enables rapid optimization of video tracking settings to improve quantification of automated parameters, and suggests that spatiotemporal organization of zebrafish swimming activity can be affected by various experimental manipulations in a manner predicted by their anxiolytic or anxiogenic nature. Our approach markedly enhances the power of zebrafish behavioral analyses, providing innovative framework for high-throughput 3D phenotyping of adult zebrafish behavior. PMID:21408171

  16. Zebrafish: A Versatile Animal Model for Fertility Research.

    PubMed

    Hoo, Jing Ying; Kumari, Yatinesh; Shaikh, Mohd Farooq; Hue, Seow Mun; Goh, Bey Hing

    2016-01-01

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

  17. Zebrafish: A Versatile Animal Model for Fertility Research

    PubMed Central

    Hoo, Jing Ying; Kumari, Yatinesh; Shaikh, Mohd Farooq; Hue, Seow Mun

    2016-01-01

    The utilization of zebrafish in biomedical research is very common in the research world nowadays. Today, it has emerged as a favored vertebrate organism for the research in science of reproduction. There is a significant growth in amount numbers of scientific literature pertaining to research discoveries in reproductive sciences in zebrafish. It has implied the importance of zebrafish in this particular field of research. In essence, the current available literature has covered from the very specific brain region or neurons of zebrafish, which are responsible for reproductive regulation, until the gonadal level of the animal. The discoveries and findings have proven that this small animal is sharing a very close/similar reproductive system with mammals. More interestingly, the behavioral characteristics and along with the establishment of animal courtship behavior categorization in zebrafish have laid an even stronger foundation and firmer reason on the suitability of zebrafish utilization in research of reproductive sciences. In view of the immense importance of this small animal for the development of reproductive sciences, this review aimed at compiling and describing the proximate close similarity of reproductive regulation on zebrafish and human along with factors contributing to the infertility, showing its versatility and its potential usage for fertility research. PMID:27556045

  18. Genetic determinants of hyaloid and retinal vasculature in zebrafish

    PubMed Central

    Alvarez, Yolanda; Cederlund, Maria L; Cottell, David C; Bill, Brent R; Ekker, Stephen C; Torres-Vazquez, Jesus; Weinstein, Brant M; Hyde, David R; Vihtelic, Thomas S; Kennedy, Breandan N

    2007-01-01

    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. PMID:17937808

  19. Functionally conserved effects of rapamycin exposure on zebrafish.

    PubMed

    Sucularli, Ceren; Shehwana, Huma; Kuscu, Cem; Dungul, Dilay Ciglidag; Ozdag, Hilal; Konu, Ozlen

    2016-05-01

    Mechanistic target of rapamycin (mTOR) is a conserved serine/threonine kinase important in cell proliferation, growth and protein translation. Rapamycin, a well‑known anti‑cancer agent and immunosuppressant drug, inhibits mTOR activity in different taxa including zebrafish. In the present study, the effect of rapamycin exposure on the transcriptome of a zebrafish fibroblast cell line, ZF4, was investigated. Microarray analysis demonstrated that rapamycin treatment modulated a large set of genes with varying functions including protein synthesis, assembly of mitochondrial and proteasomal machinery, cell cycle, metabolism and oxidative phosphorylation in ZF4 cells. A mild however, coordinated reduction in the expression of proteasomal and mitochondrial ribosomal subunits was detected, while the expression of numerous ribosomal subunits increased. Meta‑analysis of heterogeneous mouse rapamycin microarray datasets enabled the comparison of zebrafish and mouse pathways modulated by rapamycin, using Kyoto Encyclopedia of Genes and Genomes and Gene Ontology pathway analysis. The analyses demonstrated a high degree of functional conservation between zebrafish and mice in response to rapamycin. In addition, rapamycin treatment resulted in a marked dose‑dependent reduction in body size and pigmentation in zebrafish embryos. The present study is the first, to the best of our knowledge, to evaluate the conservation of rapamycin‑modulated functional pathways between zebrafish and mice, in addition to the dose‑dependent growth curves of zebrafish embryos upon rapamycin exposure.

  20. Designing and Testing of Self-Cleaning Recirculating Zebrafish Tanks.

    PubMed

    Nema, Shubham; Bhargava, Yogesh

    2016-08-01

    Maintenance of large number of zebrafish in captive conditions is a daunting task. This can be eased by the use of recirculating racks with self-cleaning zebrafish tanks. Commercially available systems are costly, and compatibility of intercompany products has never been investigated. Although various cost-effective designs and methods of construction of custom-made recirculating zebrafish racks are available in literature, the design of self-cleaning zebrafish tanks is still not available. In this study, we report the design and method of construction of the self-cleaning unit, which can be fitted in any zebrafish tank. We validated the design by investigating sediment cleaning process in rectangular and cylindrical tank geometries using time lapse imaging. Our results suggest that for both tank geometries, the tanks fitted with self-cleaning unit provided superior sediment cleaning than the tanks fitted with overflow-drain unit. Although the self-cleaning unit could clean the sediment completely from both geometries over prolonged period, the cleaning of sediments was faster in the cylindrical tank than the rectangular tank. In conclusion, cost and efforts of zebrafish maintenance could be significantly reduced through the installation of our self-cleaning unit in any custom-made zebrafish tank.

  1. The Control of Calcium Metabolism in Zebrafish (Danio rerio)

    PubMed Central

    Lin, Chia-Hao; Hwang, Pung-Pung

    2016-01-01

    Zebrafish is an emerging model for the research of body fluid ionic homeostasis. In this review, we focus on current progress on the regulation of Ca2+ uptake in the context of Ca2+ sensing and hormonal regulation in zebrafish. Na+-K+-ATPase-rich cells (NaRCs), the specialized ionocytes in the embryonic skin and adult gills, play a dominant role in Ca2+ uptake in zebrafish. Transepithelial Ca2+ transport in NaRC, through apical epithelial Ca2+ channels (ECaC), basolateral plasma membrane Ca2+-ATPase (PMCA), and Na+/Ca2+ exchanger (NCX), is analogous to mammalian renal and intestinal Ca2+-absorption cells. Several hormones were demonstrated to differentially regulate Ca2+ uptake through modulating the expression of Ca2+ transporters and/or the proliferation/differentiation of NaRC in zebrafish. In addition, the counterbalance among these hormones is associated with the maintenance of body fluid Ca2+ homeostasis. Calcium-sensing receptor (CaSR) is expressed in several hormone-secreting tissues in zebrafish, and activated CaSR differentially controls calciotropic hormones. The major principles of Ca2+ transport and the hormonal control appear to be conserved from zebrafish to other vertebrates including mammals. The new knowledge gained from zebrafish studies provides new insights into the related issues in vertebrates. PMID:27792163

  2. Developmental biology. Rocks that roll zebrafish.

    PubMed

    Fekete, Donna M

    2003-10-10

    The vestibular organs of the inner ear of higher vertebrates control balance, and their counterparts in fish control both balance and hearing. Essential to the operation of these sensory organs are the biomineralized structures--otoconia in higher vertebrates or otoliths in fish--that deflect the sensory hair bundles situated beneath them. In her Perspective, Fekete explores the fascinating world of otolith biomineralization in zebrafish; revealing the importance of a protein called Starmaker for coordinating the shape and type of crystal in fish otoliths ( Söllner et al.).

  3. Zebrafish Are Able to Detect Ethanol in Their Environment.

    PubMed

    Tran, Steven; Chow, Hayden; Tsang, Benjamin; Facciol, Amanda; Gandhi, Prabhlene; Desai, Priyanka; Gerlai, Robert

    2017-04-01

    Zebrafish have become a popular animal model for studying the development of alcohol addiction. Several behavioral paradigms for studying alcohol addiction have been developed for zebrafish, including conditioned place preference, alcohol-induced tolerance, and withdrawal. However, alcohol choice preference tasks have not been established in zebrafish as of yet. The ability of zebrafish to detect alcohol in their environment is required in alcohol choice or preference tasks. To our knowledge, it is currently unknown whether zebrafish are able to detect alcohol in their environment immediately following bath immersion. In the current study, we analyzed the time course of alcohol-induced behavioral changes of zebrafish while being immersed in alcohol solution in a 1.5 L tank. We recorded each trial in high-definition and quantified behavioral responses using automated video tracking-based and manual observation-based methods to quantify temporal changes in alcohol-induced behaviors. As alcohol is known to require several minutes of bath immersion to reach the brain in zebrafish, we argued that behavioral responses before this time point would prove zebrafish's ability to detect this substance in the water. Our results show that a 60-min exposure to 1% alcohol alters behavioral responses in a time-dependent manner. Notably, alcohol exposure significantly increased absolute turn angle, decreased distance to bottom, and variance of distance to bottom within the first 3 min immediately following exposure, a response that occurred before alcohol could reach the brain of the subjects in measurable amounts. These results imply that zebrafish are able to detect alcohol in their environment immediately following immersion into the drug solution.

  4. Using engineered endonucleases to create knockout and knockin zebrafish models.

    PubMed

    Bedell, Victoria M; Ekker, Stephen C

    2015-01-01

    Over the last few years, the technology to create targeted knockout and knockin zebrafish animals has exploded. We have gained the ability to create targeted knockouts through the use of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR associated system (CRISPR/Cas). Furthermore, using the high-efficiency TALEN system, we were able to create knockin zebrafish using a single-stranded DNA (ssDNA) protocol described here. Through the use of these technologies, the zebrafish has become a valuable vertebrate model and an excellent bridge between the invertebrate and mammalian model systems for the study of human disease.

  5. Heme Reversibly Damps PERIOD2 Rhythms in Mouse Suprachiasmatic Nucleus Explants

    PubMed Central

    Guenthner, Casey J.; Bickar, David; Harrington, Mary E.

    2009-01-01

    The hypothalamic suprachiasmatic nucleus (SCN), which in mammals serves as the master circadian pacemaker by synchronizing autonomous clocks in peripheral tissues, is composed of coupled single-cell oscillators that are driven by interlocking positive/negative transcriptional/translational feedback loops. Several studies have suggested that heme, a common prosthetic group that is synthesized and degraded in a circadian manner in the SCN, may modulate the function of several feedback loop components, including the REV-ERB nuclear receptors and PERIOD2 (PER2). We found that ferric heme (hemin, 3-100 μM) dose-dependently and reversibly damped luminescence rhythms in SCN explants from mice expressing a PER2∷LUCIFERASE (PER2∷LUC) fusion protein. Inhibitors of heme oxygenases (HOs, which degrade heme to biliverdin, carbon monoxide, and iron) mimicked heme’s effects on PER2 rhythms. In contrast, heme and HO inhibition did not damp luminescence rhythms in thymus and esophagus explants and had only a small effect on PER2∷LUC damping in spleen explants, suggesting that heme’s effects are tissue-specific. Analysis of the effects of heme’s degradation products on SCN PER2∷LUC rhythms indicated that they probably were not responsible for heme’s effects on rhythms. The heme synthesis inhibitor N-methylprotoporphyrin IX (NMP) lengthened the circadian period of SCN PER2∷LUC rhythms by about an hour. These data are consistent with an important role for heme in the circadian system. PMID:19698763

  6. Chevron formation of the zebrafish muscle segments.

    PubMed

    Rost, Fabian; Eugster, Christina; Schröter, Christian; Oates, Andrew C; Brusch, Lutz

    2014-11-01

    The muscle segments of fish have a folded shape, termed a chevron, which is thought to be optimal for the undulating body movements of swimming. However, the mechanism shaping the chevron during embryogenesis is not understood. Here, we used time-lapse microscopy of developing zebrafish embryos spanning the entire somitogenesis period to quantify the dynamics of chevron shape development. By comparing such time courses with the start of movements in wildtype zebrafish and analysing immobile mutants, we show that the previously implicated body movements do not play a role in chevron formation. Further, the monotonic increase of chevron angle along the anteroposterior axis revealed by our data constrains or rules out possible contributions by previously proposed mechanisms. In particular, we found that muscle pioneers are not required for chevron formation. We put forward a tension-and-resistance mechanism involving interactions between intra-segmental tension and segment boundaries. To evaluate this mechanism, we derived and analysed a mechanical model of a chain of contractile and resisting elements. The predictions of this model were verified by comparison with experimental data. Altogether, our results support the notion that a simple physical mechanism suffices to self-organize the observed spatiotemporal pattern in chevron formation.

  7. Parametric analyses of anxiety in zebrafish scototaxis.

    PubMed

    Maximino, Caio; de Brito, Thiago Marques; Colmanetti, Rafael; Pontes, Alvaro Antonio Assis; de Castro, Henrique Meira; de Lacerda, Renata Inah Tavares; Morato, Silvio; Gouveia, Amauri

    2010-06-26

    Scototaxis, the preference for dark environments in detriment of bright ones, is an index of anxiety in zebrafish. In this work, we analyzed avoidance of the white compartment by analysis of the spatiotemporal pattern of exploratory behavior (time spent in the white compartment of the apparatus and shuttle frequency between compartments) and swimming ethogram (thigmotaxis, freezing and burst swimming in the white compartment) in four experiments. In Experiment 1, we demonstrate that spatiotemporal measures of white avoidance and locomotion do not habituate during a single 15-min session. In Experiments 2 and 3, we demonstrate that locomotor activity habituates to repeated exposures to the apparatus, regardless of whether inter-trial interval is 15-min or 24-h; however, no habituation of white avoidance was observed in either experiment. In Experiment 4, we confined animals for three 15-min sessions in the white compartment prior to recording spatiotemporal and ethogram measures in a standard preference test. After these forced exposures, white avoidance and locomotor activity showed no differences in relation to non-confined animals, but burst swimming, thigmotaxis and freezing in the white compartment were all decreased. These results suggest that neither avoidance of the white compartment nor approach to the black compartment account for the behavior of zebrafish in the scototaxis test.

  8. Identification of polarized macrophage subsets in zebrafish.

    PubMed

    Nguyen-Chi, Mai; Laplace-Builhe, Béryl; Travnickova, Jana; Luz-Crawford, Patricia; Tejedor, Gautier; Phan, Quang Tien; Duroux-Richard, Isabelle; Levraud, Jean-Pierre; Kissa, Karima; Lutfalla, Georges; Jorgensen, Christian; Djouad, Farida

    2015-07-08

    While the mammalian macrophage phenotypes have been intensively studied in vitro, the dynamic of their phenotypic polarization has never been investigated in live vertebrates. We used the zebrafish as a live model to identify and trail macrophage subtypes. We generated a transgenic line whose macrophages expressing tumour necrosis factor alpha (tnfa), a key feature of classically activated (M1) macrophages, express fluorescent proteins Tg(mpeg1:mCherryF/tnfa:eGFP-F). Using 4D-confocal microscopy, we showed that both aseptic wounding and Escherichia coli inoculation triggered macrophage recruitment, some of which started to express tnfa. RT-qPCR on Fluorescence Activated Cell Sorting (FACS)-sorted tnfa(+) and tnfa(-) macrophages showed that they, respectively, expressed M1 and alternatively activated (M2) mammalian markers. Fate tracing of tnfa(+) macrophages during the time-course of inflammation demonstrated that pro-inflammatory macrophages converted into M2-like phenotype during the resolution step. Our results reveal the diversity and plasticity of zebrafish macrophage subsets and underline the similarities with mammalian macrophages proposing a new system to study macrophage functional dynamic.

  9. Multidimensional In Vivo Hazard Assessment Using Zebrafish

    PubMed Central

    Tanguay, Robert L.

    2014-01-01

    There are tens of thousands of man-made chemicals in the environment; the inherent safety of most of these chemicals is not known. Relevant biological platforms and new computational tools are needed to prioritize testing of chemicals with limited human health hazard information. We describe an experimental design for high-throughput characterization of multidimensional in vivo effects with the power to evaluate trends relating to commonly cited chemical predictors. We evaluated all 1060 unique U.S. EPA ToxCast phase 1 and 2 compounds using the embryonic zebrafish and found that 487 induced significant adverse biological responses. The utilization of 18 simultaneously measured endpoints means that the entire system serves as a robust biological sensor for chemical hazard. The experimental design enabled us to describe global patterns of variation across tested compounds, evaluate the concordance of the available in vitro and in vivo phase 1 data with this study, highlight specific mechanisms/value-added/novel biology related to notochord development, and demonstrate that the developmental zebrafish detects adverse responses that would be missed by less comprehensive testing strategies. PMID:24136191

  10. Identification of polarized macrophage subsets in zebrafish

    PubMed Central

    Nguyen-Chi, Mai; Laplace-Builhe, Béryl; Travnickova, Jana; Luz-Crawford, Patricia; Tejedor, Gautier; Phan, Quang Tien; Duroux-Richard, Isabelle; Levraud, Jean-Pierre; Kissa, Karima; Lutfalla, Georges

    2015-01-01

    While the mammalian macrophage phenotypes have been intensively studied in vitro, the dynamic of their phenotypic polarization has never been investigated in live vertebrates. We used the zebrafish as a live model to identify and trail macrophage subtypes. We generated a transgenic line whose macrophages expressing tumour necrosis factor alpha (tnfa), a key feature of classically activated (M1) macrophages, express fluorescent proteins Tg(mpeg1:mCherryF/tnfa:eGFP-F). Using 4D-confocal microscopy, we showed that both aseptic wounding and Escherichia coli inoculation triggered macrophage recruitment, some of which started to express tnfa. RT-qPCR on Fluorescence Activated Cell Sorting (FACS)-sorted tnfa+ and tnfa− macrophages showed that they, respectively, expressed M1 and alternatively activated (M2) mammalian markers. Fate tracing of tnfa+ macrophages during the time-course of inflammation demonstrated that pro-inflammatory macrophages converted into M2-like phenotype during the resolution step. Our results reveal the diversity and plasticity of zebrafish macrophage subsets and underline the similarities with mammalian macrophages proposing a new system to study macrophage functional dynamic. DOI: http://dx.doi.org/10.7554/eLife.07288.001 PMID:26154973

  11. Afferent Connectivity of the Zebrafish Habenulae

    PubMed Central

    Turner, Katherine J.; Hawkins, Thomas A.; Yáñez, Julián; Anadón, Ramón; Wilson, Stephen W.; Folgueira, Mónica

    2016-01-01

    The habenulae are bilateral nuclei located in the dorsal diencephalon that are conserved across vertebrates. Here we describe the main afferents to the habenulae in larval and adult zebrafish. We observe afferents from the subpallium, nucleus rostrolateralis, posterior tuberculum, posterior hypothalamic lobe, median raphe; we also see asymmetric afferents from olfactory bulb to the right habenula, and from the parapineal to the left habenula. In addition, we find afferents from a ventrolateral telencephalic nucleus that neurochemical and hodological data identify as the ventral entopeduncular nucleus (vENT), confirming and extending observations of Amo et al. (2014). Fate map and marker studies suggest that vENT originates from the diencephalic prethalamic eminence and extends into the lateral telencephalon from 48 to 120 hour post-fertilization (hpf). No afferents to the habenula were observed from the dorsal entopeduncular nucleus (dENT). Consequently, we confirm that the vENT (and not the dENT) should be considered as the entopeduncular nucleus “proper” in zebrafish. Furthermore, comparison with data in other vertebrates suggests that the vENT is a conserved basal ganglia nucleus, being homologous to the entopeduncular nucleus of mammals (internal segment of the globus pallidus of primates) by both embryonic origin and projections, as previously suggested by Amo et al. (2014). PMID:27199671

  12. Variability in mitochondria of zebrafish photoreceptor ellipsoids.

    PubMed

    Tarboush, R; Novales Flamarique, I; Chapman, G B; Connaughton, V P

    2014-01-01

    Ultrastructural examination of photoreceptor inner segment ellipsoids in larval (4, 8, and 15 days postfertilization; dpf) and adult zebrafish identified morphologically different types of mitochondria. All photoreceptors had mitochondria of different sizes (large and small). At 4 dpf, rods had small, moderately stained electron-dense mitochondria (E-DM), and two cone types could be distinguished: (1) those with electron-lucent mitochondria (E-LM) and (2) those with mitochondria of moderate electron density. These distinctions were also apparent at later ages (8 and 15 dpf). Rods from adult fish had fewer mitochondria than their corresponding cones. The ellipsoids of some fully differentiated single and double cones contained large E-DM with few cristae; these were surrounded by small E-LM with typical internal morphology. The mitochondria within the ellipsoids of other single cones showed similar electron density. Microspectrophotometry of cone ellipsoids from adult fish indicated that the large E-DM had a small absorbance peak (∼0.03 OD units) and did not contain cytochrome-c, but crocetin, a carotenoid found in old world monkeys. Crocetin functions to prevent oxidative damage to photoreceptors, suggesting that the ellipsoid mitochondria in adult zebrafish cones protect against apoptosis and function metabolically, rather than as a light filter.

  13. Toxicity of chlorine to zebrafish embryos.

    PubMed

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

    2014-01-16

    Surface disinfection of fertilized fish eggs is widely used in aquaculture to reduce extraovum pathogens that may be released from brood fish during spawning, and this is routinely used in zebrafish Danio rerio research laboratories. Most laboratories use approximately 25 to 50 ppm unbuffered chlorine solution for 5 to 10 min. Treatment of embryos with chlorine has significant germicidal effects for many Gram-negative bacteria, viruses, and trophozoite stages of protozoa, but is less effective against cyst or spore stages of protozoa and certain Mycobacterium spp. Therefore, we evaluated the toxicity of unbuffered and buffered chlorine solutions to embryos exposed at 6 or 24 h post-fertilization (hpf) to determine whether higher concentrations can be used for treating zebrafish embryos. Most of our experiments entailed using an outbred line (5D), with both mortality and malformations as endpoints. We found that 6 hpf embryos consistently were more resistant than 24 hpf embryos to the toxic effects of chlorine. Chlorine is more toxic and germicidal at lower pH, and chlorine causes elevated pH. Consistent with this, we found that unbuffered chlorine solutions (pH ca. 8-9) were less toxic at corresponding concentrations than solutions buffered to pH 7. Based on our findings here, we recommend treating 6 hpf embryos for 10 min and 24 hpf embryos for 5 min with unbuffered chlorine solution at 100 ppm.

  14. Elucidating Cannabinoid Biology in Zebrafish (Danio rerio)

    PubMed Central

    Krug, Randall G.; Clark, Karl J.

    2015-01-01

    The number of annual cannabinoid users exceeds 100,000,000 globally and an estimated 9 % of these individuals will suffer from dependency. Although exogenous cannabinoids, like those contained in marijuana, are known to exert their effects by disrupting the endocannabinoid system, a dearth of knowledge exists about the potential toxicological consequences on public health. Conversely, the endocannabinoid system represents a promising therapeutic target for a plethora of disorders because it functions to endogenously regulate a vast repertoire of physiological functions. Accordingly, the rapidly expanding field of cannabinoid biology has sought to leverage model organisms in order to provide both toxicological and therapeutic insights about altered endocannabinoid signaling. The primary goal of this manuscript is to review the existing field of cannabinoid research in the genetically tractable zebrafish model—focusing on the cannabinoid receptor genes, cnr1 and cnr2, and the genes that produce enzymes for synthesis and degradation of the cognate ligands anandamide and 2-arachidonylglycerol. Consideration is also given to research that has studied the effects of exposure to exogenous phytocannabinoids and synthetic cannabinoids that are known to interact with cannabinoid receptors. These results are considered in the context of either endocannabinoid gene expression or endocannabinoid gene function, and are integrated with findings from rodent studies. This provides the framework for a discussion of how zebrafish may be leveraged in the future to provide novel toxicological and therapeutic insights in the field of cannabinoid biology, which has become increasingly significant given recent trends in cannabis legislation. PMID:26192460

  15. Radiographic analysis of zebrafish skeletal defects.

    PubMed

    Fisher, Shannon; Jagadeeswaran, Pudur; Halpern, Marnie E

    2003-12-01

    Systematic identification of skeletal dysplasias in model vertebrates provides insight into the pathogenesis of human skeletal disorders and can aid in the identification of orthologous human genes. We are undertaking a mutagenesis screen for skeletal dysplasias in adult zebrafish, using radiography to detect abnormalities in skeletal anatomy and bone morphology. We have isolated chihuahua, a dominant mutation causing a general defect in bone growth. Heterozygous chihuahua fish have phenotypic similarities to human osteogenesis imperfecta, a skeletal dysplasia caused by mutations in the type I collagen genes. Mapping and molecular characterization of the chihuahua mutation indicates that the defect resides in the gene encoding the collagen I(alpha1) chain. Thus, chihuahua accurately models osteogenesis imperfecta at the biologic and molecular levels, and will prove an important resource for studies on the disease pathophysiology. Radiography is a practical screening tool to detect subtle skeletal abnormalities in the adult zebrafish. The identification of chihuahua demonstrates that mutant phenotypes analogous to human skeletal dysplasias will be discovered.

  16. Zebrafish biosensor for toxicant induced muscle hyperactivity

    PubMed Central

    Shahid, Maryam; Takamiya, Masanari; Stegmaier, Johannes; Middel, Volker; Gradl, Marion; Klüver, Nils; Mikut, Ralf; Dickmeis, Thomas; Scholz, Stefan; Rastegar, Sepand; Yang, Lixin; Strähle, Uwe

    2016-01-01

    Robust and sensitive detection systems are a crucial asset for risk management of chemicals, which are produced in increasing number and diversity. To establish an in vivo biosensor system with quantitative readout for potential toxicant effects on motor function, we generated a transgenic zebrafish line TgBAC(hspb11:GFP) which expresses a GFP reporter under the control of regulatory elements of the small heat shock protein hspb11. Spatiotemporal hspb11 transgene expression in the musculature and the notochord matched closely that of endogenous hspb11 expression. Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression. Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency. The hspb11 transgene up-regulation induced by either chemicals or heat shock was eliminated after co-application of the anaesthetic MS-222. TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function. PMID:27029555

  17. Zebrafish biosensor for toxicant induced muscle hyperactivity.

    PubMed

    Shahid, Maryam; Takamiya, Masanari; Stegmaier, Johannes; Middel, Volker; Gradl, Marion; Klüver, Nils; Mikut, Ralf; Dickmeis, Thomas; Scholz, Stefan; Rastegar, Sepand; Yang, Lixin; Strähle, Uwe

    2016-03-31

    Robust and sensitive detection systems are a crucial asset for risk management of chemicals, which are produced in increasing number and diversity. To establish an in vivo biosensor system with quantitative readout for potential toxicant effects on motor function, we generated a transgenic zebrafish line TgBAC(hspb11:GFP) which expresses a GFP reporter under the control of regulatory elements of the small heat shock protein hspb11. Spatiotemporal hspb11 transgene expression in the musculature and the notochord matched closely that of endogenous hspb11 expression. Exposure to substances that interfere with motor function induced a dose-dependent increase of GFP intensity beginning at sub-micromolar concentrations, while washout of the chemicals reduced the level of hspb11 transgene expression. Simultaneously, these toxicants induced muscle hyperactivity with increased calcium spike height and frequency. The hspb11 transgene up-regulation induced by either chemicals or heat shock was eliminated after co-application of the anaesthetic MS-222. TgBAC(hspb11:GFP) zebrafish embryos provide a quantitative measure of muscle hyperactivity and represent a robust whole organism system for detecting chemicals that affect motor function.

  18. Cadherin-6 Function in Zebrafish Retinal Development

    PubMed Central

    Liu, Qin; Londraville, Richard; Marrs, James A.; Wilson, Amy L.; Mbimba, Thomas; Murakami, Tohru; Kubota, Fumitaka; Zheng, Weiping; Fatkins, David G.

    2008-01-01

    Cadherin cell adhesion molecules play crucial roles in vertebrate development including the development of the visual system. Most studies have focused on examining functions of classical type I cadherins (e.g. cadherin-2) in visual system development. There is little information on the function of classical type II cadherins (e.g. cadherin-6) in the development of the vertebrate visual system. To gain insight into cadherin-6 role in the formation of the retina, we analyzed differentiation of retinal ganglion cells, amacrine cells and photoreceptors in zebrafish embryos injected with cadherin-6 specific antisense morpholino oligonucleotides. Differentiation of the retinal neurons in cadherin-6 knockdown embryos (cdh6 morphants) was analyzed using multiple markers. We found that expression of transcription factors important for retinal development was greatly reduced, and expression of Notch-Delta genes and proneural gene ath5 was altered in the cdh6 morphant retina. The retinal lamination was present in the morphants, although the morphant eyes were significantly smaller than control embryos due mainly to decreased cell proliferation. Differentiation of the retinal ganglion cells, amacrine cells and photoreceptors was severely disrupted in the cdh6 morphants due to a significant delay in neuronal differentiation. Our results suggest that cadherin-6 plays an important role in the normal formation of the zebrafish retina. PMID:18506771

  19. Chevron formation of the zebrafish muscle segments

    PubMed Central

    Rost, Fabian; Eugster, Christina; Schröter, Christian; Oates, Andrew C.; Brusch, Lutz

    2014-01-01

    The muscle segments of fish have a folded shape, termed a chevron, which is thought to be optimal for the undulating body movements of swimming. However, the mechanism shaping the chevron during embryogenesis is not understood. Here, we used time-lapse microscopy of developing zebrafish embryos spanning the entire somitogenesis period to quantify the dynamics of chevron shape development. By comparing such time courses with the start of movements in wildtype zebrafish and analysing immobile mutants, we show that the previously implicated body movements do not play a role in chevron formation. Further, the monotonic increase of chevron angle along the anteroposterior axis revealed by our data constrains or rules out possible contributions by previously proposed mechanisms. In particular, we found that muscle pioneers are not required for chevron formation. We put forward a tension-and-resistance mechanism involving interactions between intra-segmental tension and segment boundaries. To evaluate this mechanism, we derived and analysed a mechanical model of a chain of contractile and resisting elements. The predictions of this model were verified by comparison with experimental data. Altogether, our results support the notion that a simple physical mechanism suffices to self-organize the observed spatiotemporal pattern in chevron formation. PMID:25267843

  20. Evidence for a core gut microbiota in the zebrafish

    PubMed Central

    Roeselers, Guus; Mittge, Erika K; Stephens, W Zac; Parichy, David M; Cavanaugh, Colleen M; Guillemin, Karen; Rawls, John F

    2011-01-01

    Experimental analysis of gut microbial communities and their interactions with vertebrate hosts is conducted predominantly in domesticated animals that have been maintained in laboratory facilities for many generations. These animal models are useful for studying coevolved relationships between host and microbiota only if the microbial communities that occur in animals in lab facilities are representative of those that occur in nature. We performed 16S rRNA gene sequence-based comparisons of gut bacterial communities in zebrafish collected recently from their natural habitat and those reared for generations in lab facilities in different geographic locations. Patterns of gut microbiota structure in domesticated zebrafish varied across different lab facilities in correlation with historical connections between those facilities. However, gut microbiota membership in domesticated and recently caught zebrafish was strikingly similar, with a shared core gut microbiota. The zebrafish intestinal habitat therefore selects for specific bacterial taxa despite radical differences in host provenance and domestication status. PMID:21472014

  1. Persistent impaired glucose metabolism in a zebrafish hyperglycemia model.

    PubMed

    Capiotti, Katiucia Marques; Antonioli, Régis; Kist, Luiza Wilges; Bogo, Maurício Reis; Bonan, Carla Denise; Da Silva, Rosane Souza

    2014-05-01

    Diabetes mellitus (DM) affects over 10% of the world's population. Hyperglycemia is the main feature for the diagnosis of this disease. The zebrafish (Danio rerio) is an established model organism for the study of various metabolic diseases. In this paper, hyperglycemic zebrafish, when immersed in a 111 mM glucose solution for 14 days, developed increased glycation of proteins from the eyes, decreased mRNA levels of insulin receptors in the muscle, and a reversion of high blood glucose level after treatment with anti-diabetic drugs (glimepiride and metformin) even after 7 days of glucose withdrawal. Additionally, hyperglycemic zebrafish developed an impaired response to exogenous insulin, which was recovered after 7 days of glucose withdrawal. These data suggest that the exposure of adult zebrafish to high glucose concentration is able to induce persistent metabolic changes probably underlined by a hyperinsulinemic state and impaired peripheral glucose metabolism.

  2. Teratogenic potential of antiepileptic drugs in the zebrafish model.

    PubMed

    Lee, Sung Hak; Kang, Jung Won; Lin, Tao; Lee, Jae Eun; Jin, Dong Il

    2013-01-01

    The zebrafish model is an attractive candidate for screening of developmental toxicity during early drug development. Antiepileptic drugs (AEDs) arouse concern for the risk of teratogenicity, but the data are limited. In this study, we evaluated the teratogenic potential of seven AEDs (carbamazepine (CBZ), ethosuximide (ETX), valproic acid (VPN), lamotrigine (LMT), lacosamide (LCM), levetiracetam (LVT), and topiramate (TPM)) in the zebrafish model. Zebrafish embryos were exposed to AEDs from initiation of gastrula (5.25 hours post-fertilization (hpf)) to termination of hatching (72 hpf) which mimic the mammalian teratogenic experimental design. The lethality and teratogenic index (TI) of AEDs were determined and the TI values of each drug were compared with the US FDA human pregnancy categories. Zebrafish model was useful screening model for teratogenic potential of antiepilepsy drugs and was in concordance with in vivo mammalian data and human clinical data.

  3. Developmental Toxicity of Louisiana Crude Oiled Sediment to Zebrafish

    EPA Science Inventory

    Embryonic exposures to polycyclic aromatic hydrocarbons (PAHs) and petroleum products cause a characteristic suite of developmental defects in a variety of fish species. We exposed zebrafish embryos to sediment mixed with laboratory weathered South Louisiana crude oil. Oiled sedi...

  4. Study of Host–Microbe Interactions in Zebrafish

    PubMed Central

    Milligan-Myhre, Kathryn; Charette, Jeremy R.; Phennicie, Ryan T.; Stephens, W. Zac; Rawls, John F.; Guillemin, Karen; Kim, Carol H.

    2015-01-01

    All animals are ecosystems, home to diverse microbial populations. Animal-associated microbes play important roles in the normal development and physiology of their hosts, but can also be agents of infectious disease. Traditionally, mice have been used to study pathogenic and beneficial associations between microbes and vertebrate animals. The zebrafish is emerging as a valuable new model system for host-microbe interaction studies, affording researchers with the opportunity to survey large populations of hosts and to visualize microbe-host associations at a cellular level in living animals. This chapter provides detailed protocols for the analysis of zebrafish-associated microbial communities, the derivation and husbandry of germ-free zebrafish, and the modeling of infectious disease in different stages of zebrafish development via different routes of inoculation. These protocols offer a starting point for researchers to address a multitude of questions about animals’ coexistence with microorganisms. PMID:21951527

  5. Myomaker mediates fusion of fast myocytes in zebrafish embryos.

    PubMed

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

    2014-09-05

    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.

  6. The zebrafish as a model for complex tissue regeneration.

    PubMed

    Gemberling, Matthew; Bailey, Travis J; Hyde, David R; Poss, Kenneth D

    2013-11-01

    For centuries, philosophers and scientists have been fascinated by the principles and implications of regeneration in lower vertebrate species. Two features have made zebrafish an informative model system for determining mechanisms of regenerative events. First, they are highly regenerative, able to regrow amputated fins, as well as a lesioned brain, retina, spinal cord, heart, and other tissues. Second, they are amenable to both forward and reverse genetic approaches, with a research toolset regularly updated by an expanding community of zebrafish researchers. Zebrafish studies have helped identify new mechanistic underpinnings of regeneration in multiple tissues and, in some cases, have served as a guide for contemplating regenerative strategies in mammals. Here, we review the recent history of zebrafish as a genetic model system for understanding how and why tissue regeneration occurs.

  7. REVIEW: Zebrafish: A Renewed Model System For Functional Genomics

    NASA Astrophysics Data System (ADS)

    Wen, Xiao-Yan

    2008-01-01

    In the post genome era, a major goal in molecular biology is to determine the function of the many thousands of genes present in the vertebrate genome. The zebrafish (Danio rerio) provides an almost ideal genetic model to identify the biological roles of these novel genes, in part because their embryos are transparent and develop rapidly. The zebrafish has many advantages over mouse for genome-wide mutagenesis studies, allowing for easier, cheaper and faster functional characterization of novel genes in the vertebrate genome. Many molecular research tools such as chemical mutagenesis, transgenesis, gene trapping, gene knockdown, TILLING, gene targeting, RNAi and chemical genetic screen are now available in zebrafish. Combining all the forward, reverse, and chemical genetic tools, it is expected that zebrafish will make invaluable contribution to vertebrate functional genomics in functional annotation of the genes, modeling human diseases and drug discoveries.

  8. The genetics of ocular disorders: insights from the zebrafish.

    PubMed

    Morris, Ann C

    2011-09-01

    Proper formation of the vertebrate eye requires a precisely coordinated sequence of morphogenetic events that integrate the developmental contributions of the skin ectoderm, neuroectoderm, and head mesenchyme. Disruptions in this process result in ocular malformations or retinal degeneration and can cause significant visual impairment. The zebrafish is an excellent vertebrate model for the study of eye development and disease due to the transparency of the embryo, its ex utero development, and its amenability to forward genetic screens. This review will present an overview of the genetic methodologies utilized in the zebrafish, a description of several zebrafish models of congenital ocular diseases, and a discussion of the utility of the zebrafish for assessing the pathogenicity of candidate disease alleles.

  9. Evolution of complexity in the zebrafish synapse proteome.

    PubMed

    Bayés, Àlex; Collins, Mark O; Reig-Viader, Rita; Gou, Gemma; Goulding, David; Izquierdo, Abril; Choudhary, Jyoti S; Emes, Richard D; Grant, Seth G N

    2017-03-02

    The proteome of human brain synapses is highly complex and is mutated in over 130 diseases. This complexity arose from two whole-genome duplications early in the vertebrate lineage. Zebrafish are used in modelling human diseases; however, its synapse proteome is uncharacterized, and whether the teleost-specific genome duplication (TSGD) influenced complexity is unknown. We report the characterization of the proteomes and ultrastructure of central synapses in zebrafish and analyse the importance of the TSGD. While the TSGD increases overall synapse proteome complexity, the postsynaptic density (PSD) proteome of zebrafish has lower complexity than mammals. A highly conserved set of ∼1,000 proteins is shared across vertebrates. PSD ultrastructural features are also conserved. Lineage-specific proteome differences indicate that vertebrate species evolved distinct synapse types and functions. The data sets are a resource for a wide range of studies and have important implications for the use of zebrafish in modelling human synaptic diseases.

  10. Social learning of an associative foraging task in zebrafish

    NASA Astrophysics Data System (ADS)

    Zala, Sarah M.; Määttänen, Ilmari

    2013-05-01

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

  11. Functional inhibition of UQCRB suppresses angiogenesis in zebrafish

    SciTech Connect

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

    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.

  12. Social learning of an associative foraging task in zebrafish.

    PubMed

    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.

  13. Evolution of complexity in the zebrafish synapse proteome

    PubMed Central

    Bayés, Àlex; Collins, Mark O.; Reig-Viader, Rita; Gou, Gemma; Goulding, David; Izquierdo, Abril; Choudhary, Jyoti S.; Emes, Richard D.; Grant, Seth G. N.

    2017-01-01

    The proteome of human brain synapses is highly complex and is mutated in over 130 diseases. This complexity arose from two whole-genome duplications early in the vertebrate lineage. Zebrafish are used in modelling human diseases; however, its synapse proteome is uncharacterized, and whether the teleost-specific genome duplication (TSGD) influenced complexity is unknown. We report the characterization of the proteomes and ultrastructure of central synapses in zebrafish and analyse the importance of the TSGD. While the TSGD increases overall synapse proteome complexity, the postsynaptic density (PSD) proteome of zebrafish has lower complexity than mammals. A highly conserved set of ∼1,000 proteins is shared across vertebrates. PSD ultrastructural features are also conserved. Lineage-specific proteome differences indicate that vertebrate species evolved distinct synapse types and functions. The data sets are a resource for a wide range of studies and have important implications for the use of zebrafish in modelling human synaptic diseases. PMID:28252024

  14. The zebrafish as a model for complex tissue regeneration

    PubMed Central

    Gemberling, Matthew; Bailey, Travis J.; Hyde, David R.; Poss, Kenneth D.

    2013-01-01

    For centuries, philosophers and scientists have been fascinated by the principles and implications of regeneration in lower vertebrate species. Two features have made zebrafish an informative model system for determining mechanisms of regenerative events. First, they are highly regenerative, able to regrow amputated fins, as well as a lesioned brain, retina, spinal cord, heart, and other tissues. Second, they are amenable to both forward and reverse genetic approaches, with a research toolset regularly updated by an expanding community of zebrafish researchers. Zebrafish studies have helped identify new mechanistic underpinnings of regeneration in multiple tissues, and in some cases have served as a guide for contemplating regenerative strategies in mammals. Here, we review the recent history of zebrafish as a genetic model system for understanding how and why tissue regeneration occurs. PMID:23927865

  15. The Dorsal Pallium in Zebrafish, Danio rerio (Cyprinidae, Teleostei)

    PubMed Central

    Mueller, Thomas; Dong, Zhiqiang; Berberoglu, Michael A.; Guo, Su

    2011-01-01

    Zebrafish as a neurogenetic model system depends on the correct neuroanatomical understanding of its brain organization. Here, we address the unresolved question regarding a possible zebrafish homologue of the dorsal pallial division, the region that in mammals gives rise to the isocortex. Analyzing the distributions of nicotine adenine dinucleotide phosphate diphorase (NADPHd) activity and parvalbumin in the anterior zebrafish telencephalon, we show that against previous assumptions the central (Dc) zone possesses its own germinative region in the dorsal proliferative zone. We define the central (Dc) zone as topologically corresponding to the dorsal pallial division of other vertebrates (mammalian isocortex). In addition, we confirm through BrdU-labeling experiments that the posterior (Dp) zone is formed by radial migration and homologous to the mammalian piriform cortex. Based on our results, we propose a new developmental and organizational model of the zebrafish pallium—one which is the result of a complex outward-inward folding. PMID:21219890

  16. Think Small: Zebrafish as a Model System of Human Pathology

    PubMed Central

    Goldsmith, J. R.; Jobin, Christian

    2012-01-01

    Although human pathologies have mostly been modeled using higher mammal systems such as mice, the lower vertebrate zebrafish has gained tremendous attention as a model system. The advantages of zebrafish over classical vertebrate models are multifactorial and include high genetic and organ system homology to humans, high fecundity, external fertilization, ease of genetic manipulation, and transparency through early adulthood that enables powerful imaging modalities. This paper focuses on four areas of human pathology that were developed and/or advanced significantly in zebrafish in the last decade. These areas are (1) wound healing/restitution, (2) gastrointestinal diseases, (3) microbe-host interactions, and (4) genetic diseases and drug screens. Important biological processes and pathologies explored include wound-healing responses, pancreatic cancer, inflammatory bowel diseases, nonalcoholic fatty liver disease, and mycobacterium infection. The utility of zebrafish in screening for novel genes important in various pathologies such as polycystic kidney disease is also discussed. PMID:22701308

  17. Phenotype classification of zebrafish embryos by supervised learning.

    PubMed

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

    2015-01-01

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

  18. Flotillins control zebrafish epiboly through their role in cadherin-mediated cell-cell adhesion.

    PubMed

    Morris, Eduardo A Rios; Bodin, Stéphane; Delaval, Bénédicte; Comunale, Franck; Georget, Virginie; Costa, Manoel L; Lutfalla, Georges; Gauthier-Rouvière, Cécile

    2017-02-22

    Zebrafish gastrulation and particularly epiboly that involves coordinated movements of several cell layers is a dynamic process for which regulators remain to be identified. We show here that Flotillin 1 and 2, ubiquitous and highly conserved proteins, are required for epiboly. Flotillins knockdown compromised embryo survival, strongly delayed epiboly and impaired deep cell radial intercalation and directed collective migration without affecting enveloping layer cell movement. At the molecular level, we identified that Flotillins are required for the formation of E-cadherin-mediated cell-cell junctions. These results provide the first in vivo evidence that Flotillins regulate E-cadherin-mediated cell-cell junctions to allow epiboly progression.

  19. Targeted mutagenesis of zebrafish: use of zinc finger nucleases.

    PubMed

    Leong, Ivone Un San; Lai, Daniel; Lan, Chuan-Ching; Johnson, Ross; Love, Donald R; Johnson, Ross; Love, Donald R

    2011-09-01

    The modeling of human disease in the zebrafish (Danio rerio) is moving away from chemical mutagensis and transient downregulation using morpholino oligomers to more targeted and stable transgenic methods. In this respect, zinc finger nucleases offer a means of introducing mutations at targeted sites at high efficiency. We describe here the development of zinc finger nucleases and their general use in model systems with a focus on the zebrafish.

  20. Unique and conserved aspects of gut development in zebrafish.

    PubMed

    Wallace, Kenneth N; Pack, Michael

    2003-03-01

    Although the development of the digestive system of humans and vertebrate model organisms has been well characterized, relatively little is known about how the zebrafish digestive system forms. We define developmental milestones during organogenesis of the zebrafish digestive tract, liver, and pancreas and identify important differences in the way the digestive endoderm of zebrafish and amniotes is organized. Such differences account for the finding that the zebrafish digestive system is assembled from individual organ anlagen, whereas the digestive anlagen of amniotes arise from a primitive gut tube. Despite differences of organ morphogenesis, conserved molecular programs regulate pharynx, esophagus, liver, and pancreas development in teleosts and mammals. Specifically, we show that zebrafish faust/gata-5 is a functional ortholog of gata-4, a gene that is essential for the formation of the mammalian and avian foregut. Further, extraembryonic gata activity is required for this function in zebrafish as has been shown in other vertebrates. We also show that a loss-of-function mutation that perturbs sonic hedgehog causes defects in the development of the esophagus that parallel those associated with targeted disruption of this gene in mammals. Perturbation of sonic hedgehog also affects zebrafish liver and pancreas development, and these effects occur in a reciprocal fashion, as has been described during mammalian liver and ventral pancreas development. Together, these data define aspects of digestive system development necessary for the characterization of zebrafish mutants. Given the similarities of teleost and mammalian digestive physiology and anatomy, these findings have implications for developmental and evolutionary studies as well as research of human diseases, such as diabetes, liver cirrhosis, and cancer.

  1. Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

    PubMed Central

    Chrispell, Jared D.; Rebrik, Tatiana I.; Weiss, Ellen R.

    2015-01-01

    The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals. PMID

  2. Electroretinogram analysis of the visual response in zebrafish larvae.

    PubMed

    Chrispell, Jared D; Rebrik, Tatiana I; Weiss, Ellen R

    2015-03-16

    The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

  3. Conserved gene regulation during acute inflammation between zebrafish and mammals

    PubMed Central

    Forn-Cuní, G.; Varela, M.; Pereiro, P.; Novoa, B.; Figueras, A.

    2017-01-01

    Zebrafish (Danio rerio), largely used as a model for studying developmental processes, has also emerged as a valuable system for modelling human inflammatory diseases. However, in a context where even mice have been questioned as a valid model for these analysis, a systematic study evaluating the reproducibility of human and mammalian inflammatory diseases in zebrafish is still lacking. In this report, we characterize the transcriptomic regulation to lipopolysaccharide in adult zebrafish kidney, liver, and muscle tissues using microarrays and demonstrate how the zebrafish genomic responses can effectively reproduce the mammalian inflammatory process induced by acute endotoxin stress. We provide evidence that immune signaling pathways and single gene expression is well conserved throughout evolution and that the zebrafish and mammal acute genomic responses after lipopolysaccharide stimulation are highly correlated despite the differential susceptibility between species to that compound. Therefore, we formally confirm that zebrafish inflammatory models are suited to study the basic mechanisms of inflammation in human inflammatory diseases, with great translational impact potential. PMID:28157230

  4. Toxic effects of polychlorinated biphenyls on cardiac development in zebrafish.

    PubMed

    Li, Mengmeng; Wang, Xuejie; Zhu, Jingai; Zhu, Shasha; Hu, Xiaoshan; Zhu, Chun; Guo, Xirong; Yu, Zhangbin; Han, Shuping

    2014-12-01

    Polychlorinated biphenyls (PCBs) are ubiquitous environmental pollutants that may pose significant health-risks to various organisms including humans. Although the mixed PCB Aroclor 1254 is widespread in the environment, its potential toxic effect on heart development and the mechanism underlying its developmental toxicity have not been previously studied. Here, we used the zebrafish as a toxicogenomic model to examine the effects of Aroclor 1254 on heart development. We found that PCB exposure during zebrafish development induced heart abnormalities including pericardial edema and cardiac looping defects. Further malformations of the zebrafish embryo were observed and death of the larvae occurred in a time- and dose-dependent manner. Our mechanistic studies revealed that abnormalities in the arylhydrocarbon receptor, Wnt and retinoic acid signaling pathways may underlie the effects of PCBs on zebrafish heart development. Interestingly, co-administration of Aroclor 1254 and diethylaminobenzaldehyde, an inhibitor of retinaldehyde dehydrogenase, partially rescued the toxic effects of PCBs on zebrafish heart development. In conclusion, PCBs can induce developmental defects in the zebrafish heart, which may be mediated by abnormal RA signaling.

  5. Enhanced hyperplasia in muscles of transgenic zebrafish expressing Follistatin1.

    PubMed

    Li, Xi; Nie, Fen; Yin, Zhan; He, JiangYan

    2011-02-01

    Myostatin is a member of the transforming growth factor-β (TGF-β) super-family and functions as a negative regulator of muscle growth. Binding of the specific receptor, Activin receptor IIB (Act RIIB), with myostatin or other related TGF-β members, could be inhibited by the activin-binding protein follistatin (Fst) in mammals. Overexpressing Fst in mouse skeletal muscle leads to muscle hypertrophy and hyperplasia. To determine if Fst has similar roles in fish, we generated transgenic zebrafish expressing high levels of zebrafish Fst1 using the promoter of the zebrafish skeletal muscle-specific gene, myosin, light polypeptide 2, skeletal muscle (Mylz2). Independent transgenic zebrafish lines exhibited elevated expression levels of myogenic regulatory genes MyoD and Pax7 in muscle cells. Adult Fst1 overexpressing transgenic zebrafish exhibited a slight body weight increase. The high level of Fst1 expression dramatically increased myofiber numbers in skeletal muscle, without significantly changing the fiber size. Our findings suggest that Fst1 overexpression can promote zebrafish muscle growth by enhancing myofiber hyperplasia.

  6. Microarray Noninvasive Neuronal Seizure Recordings from Intact Larval Zebrafish

    PubMed Central

    Meyer, Michaela; Dhamne, Sameer C.; LaCoursiere, Christopher M.; Tambunan, Dimira; Poduri, Annapurna; Rotenberg, Alexander

    2016-01-01

    Zebrafish epilepsy models are emerging tools in experimental epilepsy. Zebrafish larvae, in particular, are advantageous because they can be easily genetically altered and used for developmental and drug studies since agents applied to the bath penetrate the organism easily. Methods for electrophysiological recordings in zebrafish are new and evolving. We present a novel multi-electrode array method to non-invasively record electrical activity from up to 61 locations of an intact larval zebrafish head. This method enables transcranial noninvasive recording of extracellular field potentials (which include multi-unit activity and EEG) to identify epileptic seizures. To record from the brains of zebrafish larvae, the dorsum of the head of an intact larva was secured onto a multi-electrode array. We recorded from individual electrodes for at least three hours and quantified neuronal firing frequency, spike patterns (continuous or bursting), and synchrony of neuronal firing. Following 15 mM potassium chloride- or pentylenetetrazole-infusion into the bath, spike and burst rate increased significantly. Additionally, synchrony of neuronal firing across channels, a hallmark of epileptic seizures, also increased. Notably, the fish survived the experiment. This non-invasive method complements present invasive zebrafish neurophysiological techniques: it affords the advantages of high spatial and temporal resolution, a capacity to measure multiregional activity and neuronal synchrony in seizures, and fish survival for future experiments, such as studies of epileptogenesis and development. PMID:27281339

  7. Normal anatomy and histology of the adult zebrafish.

    PubMed

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

    2011-08-01

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

  8. Whole-body and multispectral photoacoustic imaging of adult zebrafish

    NASA Astrophysics Data System (ADS)

    Huang, Na; Xi, Lei

    2016-10-01

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

  9. Turning Rate Dynamics of Zebrafish Exposed to Ethanol

    NASA Astrophysics Data System (ADS)

    Mwaffo, Violet; Porfiri, Maurizio

    2015-06-01

    Zebrafish is emerging as a species of choice in alcohol-related pharmacological studies. In these studies, zebrafish are often exposed to acute ethanol treatments and their activity scored during behavioral assays. Computational modeling of zebrafish behavior is expected to positively impact these efforts by offering a predictive toolbox to plan hypothesis-driven studies, reduce the number of subjects, perform pilot trials, and refine behavioral screening. In this work, we demonstrate the use of the recently proposed jump persistent turning walker to model the turning rate dynamics of zebrafish exposed to acute ethanol administration. This modeling framework is based on a stochastic mean reverting jump process to capture the sudden and large changes in orientation of swimming zebrafish. The model is calibrated on an available experimental dataset of 40 subjects, tested at different ethanol concentrations. We demonstrate that model parameters are modulated by ethanol administration, whereby both the relaxation rate and jump frequency of the turning rate dynamics are influenced by ethanol concentration. This effort offers a first evidence for the possibility of complementing zebrafish pharmacological research with computational modeling of animal behavior.

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

    PubMed Central

    Harrison, Nicholas R.; Laroche, Fabrice J.F.; Gutierrez, Alejandro

    2016-01-01

    Insights concerning leukemic pathophysiology have been acquired in various animal models and further efforts to understand the mechanisms underlying leukemic treatment resistance and disease relapse promise to improve therapeutic strategies. The zebrafish (Danio rerio) is a vertebrate organism with a conserved hematopoietic program and unique experimental strengths suiting it for the investigation of human leukemia. Recent technological advances in zebrafish research including efficient transgenesis, precise genome editing, and straightforward transplantation techniques have led to the generation of a number of leukemia models. The transparency of the zebrafish when coupled with improved lineage-tracing and imaging techniques has revealed exquisite details of leukemic initiation, progression, and regression. With these advantages, the zebrafish represents a unique experimental system for leukemic research and additionally, advances in zebrafish-based high-throughput drug screening promise to hasten the discovery of novel leukemia therapeutics. To date, investigators have accumulated knowledge of the genetic underpinnings critical to leukemic transformation and treatment resistance and without doubt, zebrafish are rapidly expanding our understanding of disease mechanisms and helping to shape therapeutic strategies for improved outcomes in leukemic patients. PMID:27165361

  11. Fundamental Approaches to the Study of Zebrafish Nutrition

    PubMed Central

    Watts, Stephen A.; Powell, Mickie; D’Abramo, Louis R.

    2014-01-01

    The value of the zebrafish model has been well established. However, culture variability within and among laboratories remains a concern, particularly as it relates to nutrition. Investigators using rodent models addressed this concern several decades ago and have developed strict nutritional regimes to which their models adhere. These investigators decreased the variability associated with nutrition in most studies by developing standardized reference and open formulation diets. Zebrafish investigators have not embraced this approach. In this article, we address the problems associated with the lack of nutritional information and standardization in the zebrafish research community. Based on the knowledge gained from studies of other animals, including traditional research models, other fish species, domesticated and companion animals, and humans, we have proposed an approach that seeks to standardize nutrition research in zebrafish. We have identified a number of factors for consideration in zebrafish nutrition studies and have suggested a number of proposed outcomes. The long term-goal of nutrition research will be to identify the daily nutritional requirements of the zebrafish and to develop appropriate standardized reference and open formulation diets. PMID:23382346

  12. Synchronized oscillations in a mathematical model of segmentation in zebrafish

    NASA Astrophysics Data System (ADS)

    Liao, Kang-Ling; Shih, Chih-Wen; Tseng, Jui-Pin

    2012-04-01

    Somitogenesis is a process for the development of somites which are transient, segmental structures that lie along the anterior-posterior axis of vertebrate embryos. The pattern of somites is governed by the segmentation clock and its timing is controlled by the clock genes which undergo synchronous oscillation over adjacent cells in the posterior presomitic mesoderm (PSM). In this paper, we analyze a mathematical model which depicts the kinetics of the zebrafish segmentation clock genes subject to direct autorepression by their own products under time delay, and cell-to-cell interaction through Delta-Notch signalling. Our goal is to elucidate how synchronous oscillations are generated for the cells in the posterior PSM, and how oscillations are arrested for the cells in the anterior PSM. For this system of delayed equations, an iteration technique is employed to derive the global convergence to the synchronous equilibrium, which corresponds to the oscillation-arrested. By applying the delay Hopf bifurcation theory and the center manifold theorem, we derive the criteria for the existence of stable synchronous oscillations for the cells at the tail bud of the PSM. Our analysis provides the basic parameter ranges and delay magnitudes for stable synchronous, asynchronous oscillation and oscillation-arrested. We exhibit how synchronous oscillations are affected by the degradation rates and delays. Extended from the analytic theory, further numerical findings linked to the segmentation process are presented.

  13. EP300 Protects from Light-Induced Retinopathy in Zebrafish

    PubMed Central

    Kawase, Reiko; Nishimura, Yuhei; Ashikawa, Yoshifumi; Sasagawa, Shota; Murakami, Soichiro; Yuge, Mizuki; Okabe, Shiko; Kawaguchi, Koki; Yamamoto, Hiroshi; Moriyuki, Kazumi; Yamane, Shinsaku; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Hara, Hideaki; Tanaka, Toshio

    2016-01-01

    Exposure of rhodopsin to bright white light can induce photoreceptor cell damage and degeneration. However, a comprehensive understanding of the mechanisms underlying light-induced retinopathy remains elusive. In this study, we performed comparative transcriptome analysis of three rodent models of light-induced retinopathy, and we identified 37 genes that are dysregulated in all three models. Gene ontology analysis revealed that this gene set is significantly associated with a cytokine signaling axis composed of signal transducer and activator of transcription 1 and 3 (STAT1/3), interleukin 6 signal transducer (IL6ST), and oncostatin M receptor (OSMR). Furthermore, the analysis suggested that the histone acetyltransferase EP300 may be a key upstream regulator of the STAT1/3–IL6ST/OSMR axis. To examine the role of EP300 directly, we developed a larval zebrafish model of light-induced retinopathy. Using this model, we demonstrated that pharmacological inhibition of EP300 significantly increased retinal cell apoptosis, decreased photoreceptor cell outer segments, and increased proliferation of putative Müller cells upon exposure to intense light. These results suggest that EP300 may protect photoreceptor cells from light-induced damage and that activation of EP300 may be a novel therapeutic approach for the treatment of retinal degenerative diseases. PMID:27242532

  14. A dedicated visual pathway for prey detection in larval zebrafish

    PubMed Central

    Semmelhack, Julia L; Donovan, Joseph C; Thiele, Tod R; Kuehn, Enrico; Laurell, Eva; Baier, Herwig

    2014-01-01

    Zebrafish larvae show characteristic prey capture behavior in response to small moving objects. The neural mechanism used to recognize objects as prey remains largely unknown. We devised a machine learning behavior classification system to quantify hunting kinematics in semi-restrained animals exposed to a range of virtual stimuli. Two-photon calcium imaging revealed a small visual area, AF7, that was activated specifically by the optimal prey stimulus. This pretectal region is innervated by two types of retinal ganglion cells, which also send collaterals to the optic tectum. Laser ablation of AF7 markedly reduced prey capture behavior. We identified neurons with arbors in AF7 and found that they projected to multiple sensory and premotor areas: the optic tectum, the nucleus of the medial longitudinal fasciculus (nMLF) and the hindbrain. These findings indicate that computations in the retina give rise to a visual stream which transforms sensory information into a directed prey capture response. DOI: http://dx.doi.org/10.7554/eLife.04878.001 PMID:25490154

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

    PubMed

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

    2016-09-01

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

  16. Position- and quantity-dependent responses in zebrafish turning behavior

    PubMed Central

    Umeda, Keiko; Ishizuka, Toru; Yawo, Hiromu; Shoji, Wataru

    2016-01-01

    Neural reflexes are stereotypical automatic responses often modulated by both intrinsic and environmental factors. We report herein that zebrafish larval C-shaped turning is modulated by the stimulated position of Rohon-Beard (RB) neurons. Targeted stimulation of more anterior RB neurons produces larger trunk flexion, which anticipates adult escape behavior by coordinated turning toward the appropriate direction. We also demonstrated that turning laterality varies with the numbers of stimulated neurons. Multi-cell stimulation of RB neurons elicits contralateral turning, as seen in the touch response to physical contact, while minimum input from single-cell stimulation induces ipsilateral turning, a phenomenon not previously reported. This ipsilateral response, but not the contralateral one, is impaired by transecting the ascending neural tract known as the dorsolateral fascicule (DLF), indicating that two, distinct neural circuits trigger these two responses. Our results suggest that RB neurons transmit the position and quantity of sensory information, which are then processed separately to modulate behavioral strength and to select turning laterality. PMID:27292818

  17. A proteome map of the zebrafish (Danio rerio) lens reveals similarities between zebrafish and mammalian crystallin expression

    PubMed Central

    Hawke, Molly; LaCava, Carrie; Prince, Courtney J.; Bellanco, Nicholas R.; Corbin, Rebecca W.

    2008-01-01

    Purpose To characterize the crystallin content of the zebrafish lens using two-dimensional gel electrophoresis (2-DE). These data will facilitate future investigations of vertebrate lens development, function, and disease. Methods Adult zebrafish lens proteins were separated by 2-DE, and the resulting spots were identified by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The relative proportion of each crystallin was quantified by image analysis, and phosphospecific staining was used to identify phosphorylated α-crystallins. The proportion of each crystallin in the soluble and insoluble fraction of the lens was also determined by resolving these lens fractions separately by 2-DE. Results α-, β-, and γ-crystallins comprised 7.8, 36.0, and 47.2% of the zebrafish lens, respectively. While the α-crystallin content of the zebrafish lens is less than the amounts found in the human lens, the ratio of αA:αB crystallin is very similar. The phosphorylation pattern of zebrafish αA-crystallins was also similar to that of humans. The most abundant γ-crystallins were the diverse γMs, comprising 30.5% of the lens. Intact zebrafish crystallins were generally more common in the soluble fraction with truncated versions more common in the insoluble fraction. Conclusions While the total α- and γ-crystallin content of the zebrafish lens differs from that of humans, similarities in α-crystallin ratios and modifications and a link between crystallin truncation and insolubility suggest that the zebrafish is a suitable model for the vertebrate lens. The proteome map provided here will be of value to future studies of lens development, function, and disease. PMID:18449354

  18. Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy.

    PubMed

    Panier, Thomas; Romano, Sebastián A; Olive, Raphaël; Pietri, Thomas; Sumbre, Germán; Candelier, Raphaël; Debrégeas, Georges

    2013-01-01

    The optical transparency and the small dimensions of zebrafish at the larval stage make it a vertebrate model of choice for brain-wide in-vivo functional imaging. However, current point-scanning imaging techniques, such as two-photon or confocal microscopy, impose a strong limit on acquisition speed which in turn sets the number of neurons that can be simultaneously recorded. At 5 Hz, this number is of the order of one thousand, i.e., approximately 1-2% of the brain. Here we demonstrate that this limitation can be greatly overcome by using Selective-plane Illumination Microscopy (SPIM). Zebrafish larvae expressing the genetically encoded calcium indicator GCaMP3 were illuminated with a scanned laser sheet and imaged with a camera whose optical axis was oriented orthogonally to the illumination plane. This optical sectioning approach was shown to permit functional imaging of a very large fraction of the brain volume of 5-9-day-old larvae with single- or near single-cell resolution. The spontaneous activity of up to 5,000 neurons was recorded at 20 Hz for 20-60 min. By rapidly scanning the specimen in the axial direction, the activity of 25,000 individual neurons from 5 different z-planes (approximately 30% of the entire brain) could be simultaneously monitored at 4 Hz. Compared to point-scanning techniques, this imaging strategy thus yields a ≃20-fold increase in data throughput (number of recorded neurons times acquisition rate) without compromising the signal-to-noise ratio (SNR). The extended field of view offered by the SPIM method allowed us to directly identify large scale ensembles of neurons, spanning several brain regions, that displayed correlated activity and were thus likely to participate in common neural processes. The benefits and limitations of SPIM for functional imaging in zebrafish as well as future developments are briefly discussed.

  19. Zebrafish Development: High-throughput Test Systems to Assess Developmental Toxicity

    EPA Science Inventory

    Abstract Because of its developmental concordance, ease of handling and rapid development, the small teleost, zebrafish (Danio rerio), is frequently promoted as a vertebrate model for medium-throughput developmental screens. This present chapter discusses zebrafish as an altern...

  20. Modular organization of axial microcircuits in zebrafish

    PubMed Central

    Bagnall, Martha W.; McLean, David L.

    2014-01-01

    Locomotion requires precise control of spinal networks. In tetrapods and bipeds, dynamic regulation of locomotion is simplified by the modular organization of spinal limb circuits, but it is not known whether their predecessors, fish axial circuits, are similarly organized. Here, we demonstrate that the larval zebrafish spinal cord contains distinct, parallel microcircuits for independent control of dorsal and ventral musculature on each side of the body. During normal swimming, dorsal and ventral microcircuits are equally active; but during postural correction, fish differentially engage these microcircuits to generate torque for self-righting. These findings reveal greater complexity in the axial spinal networks responsible for swimming than previously recognized and suggest an early template of modular organization for more complex locomotor circuits in later vertebrates. PMID:24408436

  1. Small zebrafish in a big chemical pond.

    PubMed

    Helenius, I Taneli; Yeh, J-R Joanna

    2012-07-01

    The number of possible small organic molecules of different structure is virtually limitless. One of the main goals of chemical biologists is to identify, from this "chemical space", entities that affect biological processes or systems in a specific manner. This can lead to a better understanding of the regulation and components of various biological machineries, as well as provide insights into efficacious therapeutic targets and drug candidates. However, the challenges confronting chemical biologists are multiple. How do we efficiently identify compounds that possess desirable activities without unwanted off-target effects? Once a candidate compound has been found, how do we determine its mode of action? In this Prospects piece, we call attention to recent studies using embryonic and larval zebrafish to illustrate the breadth and depth of questions in chemical biology that may be addressed using this model, and hope that they can serve as catalysts for future investigational ideas.

  2. Zebrafish learn to forage in the dark.

    PubMed

    Carrillo, Andres; McHenry, Matthew J

    2016-02-01

    A large diversity of fishes struggle early in life to forage on zooplankton while under the threat of predation. Some species, such as zebrafish (Danio rerio), acquire an ability to forage in the dark during growth as larvae, but it is unclear how this is achieved. We investigated the functional basis of this foraging by video-recording larval and juvenile zebrafish as they preyed on zooplankton (Artemia sp.) under infrared illumination. We found that foraging improved with age, to the extent that 1-month-old juveniles exhibited a capture rate that was an order of magnitude greater than that of hatchlings. At all ages, the ability to forage in the dark was diminished when we used a chemical treatment to compromise the cranial superficial neuromasts, which facilitate flow sensing. However, a morphological analysis showed no developmental changes in these receptors that could enhance sensitivity. We tested whether the improvement in foraging with age could instead be a consequence of learning by raising fish that were naïve to the flow of prey. After 1 month of growth, both groups foraged with a capture rate that was significantly less than that of fish that had the opportunity to learn and indistinguishable from that of fish with no ability to sense flow. This suggests that larval fish learn to use water flow to forage in the dark. This ability could enhance resource acquisition under reduced competition and predation. Furthermore, our findings offer an example of learning in a model system that offers promise for understanding its neurophysiological basis.

  3. Developmental toxicity of cartap on zebrafish embryos.

    PubMed

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

    2009-12-13

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

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

    PubMed

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

    2015-10-08

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

  5. Single Low-Dose Ionizing Radiation Induces Genotoxicity in Adult Zebrafish and its Non-Irradiated Progeny.

    PubMed

    Lemos, J; Neuparth, T; Trigo, M; Costa, P; Vieira, D; Cunha, L; Ponte, F; Costa, P S; Metello, L F; Carvalho, A P

    2017-02-01

    This study investigated to what extent a single exposure to low doses of ionizing radiation can induce genotoxic damage in irradiated adult zebrafish (Danio rerio) and its non-irradiated F1 progeny. Four groups of adult zebrafish were irradiated with a single dose of X-rays at 0 (control), 100, 500 and 1000 mGy, respectively, and couples of each group were allowed to reproduce following irradiation. Blood of parental fish and whole-body offspring were analysed by the comet assay for detection of DNA damage. The level of DNA damage in irradiated parental fish increased in a radiation dose-dependent manner at day 1 post-irradiation, but returned to the control level thereafter. The level of DNA damage in the progeny was directly correlated with the parental irradiation dose. Results highlight the genotoxic risk of a single exposure to low-dose ionizing radiation in irradiated individuals and also in its non-irradiated progeny.

  6. Phase variance optical coherence microscopy for label-free imaging of the developing vasculature in zebrafish embryos

    NASA Astrophysics Data System (ADS)

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

    2016-12-01

    A phase variance optical coherence microscope (pvOCM) has been created to image blood flow in the microvasculature of zebrafish embryos, without the use of exogenous labels. The pvOCM imaging system has axial and lateral resolutions of 2.8 μm in tissue and imaging depth of more than 100 μm. Images of 2 to 5 days postfertilization zebrafish embryos identified the detailed anatomical structure based on OCM intensity contrast. Phase variance contrast offered visualization of blood flow in the arteries, veins, and capillaries. The pvOCM images of the vasculature were confirmed by direct comparisons with fluorescence microscopy images of transgenic embryos in which the vascular endothelium is labeled with green fluorescent protein. The ability of pvOCM to capture activities of regional blood flow permits it to reveal functional information that is of great utility for the study of vascular development.

  7. The social zebrafish: Behavioral responses to conspecific, heterospecific, and computer animated fish

    PubMed Central

    Saverino, Cristina; Gerlai, Robert

    2008-01-01

    Zebrafish has been in the forefront of developmental biology and genetics, but only recently has interest in their behavior increased. Zebrafish are small and prolific, which lends this species to high throughput screening applications. A typical feature of zebrafish is its propensity to aggregate in groups, a behavior known as shoaling. Thus zebrafish has been proposed as a possible model organism appropriate for the analysis of the genetics of vertebrate social behavior. However, shoaling behavior is not well characterized in zebrafish. Here, using a recently developed software application, we first investigate how zebrafish respond to conspecific and heterospecific fish species that differ in coloration and/or shoaling tendencies. We found that zebrafish shoaled with their own species but not with two heterospecific species, one of which was a shoaling the other a non-shoaling species. In addition, we have started the analysis of visual stimuli that zebrafish may utilize to determine whether to shoal with a fish or not. We systematically modified the color, the location, the pattern, and the body shape of computer animated zebrafish images and presented them to experimental zebrafish. The subjects responded differentially to some of these stimuli showing preference for yellow and avoidance of elongated zebrafish images. Our results suggest that computerized stimulus presentation and automated behavioral quantification of zebrafish responses are feasible, which in turn implies that high throughput forward genetic mutation or drug screening will be possible in the analysis of social behavior with this model organism. PMID:18423643

  8. Absence of rapid eye movements during sleep in adult zebrafish.

    PubMed

    Árnason, B B; Þorsteinsson, H; Karlsson, K Æ

    2015-09-15

    Sleep is not a uniform phenomenon, but is organized in alternating, fundamentally different states, rapid eye movement sleep and non-rapid eye movement sleep. Zebrafish (Danio rerio) have recently emerged as an excellent model for sleep research. Zebrafish are well characterized in terms of development, neurobiology and genetics. Moreover, there are many experimental tools not easily applied in mammalian models that can be readily applied to zebrafish, making them a valuable additional animal model for sleep research. Sleep in zebrafish is defined behaviorally and exhibits the hallmarks of mammalian sleep (e.g. sleep homeostasis and pressure). To our knowledge no attempts have been made to discern if sleep in zebrafish entails alternations of REM-NREM sleep cycles which are critical for further development of the model. In the current experiment we quantify two key REM sleep components, rapid eye movements and respiratory rates, across sleep-wake cycles. We find no sleep-related rapid eye movements. During sleep respiratory rates, however, are reduced and become less regular, further establishing that the behavioral definition used truly captures a change in the fish's physiology. We thus fail to find evidence for REM-NREM sleep cycles in zebrafish but demonstrate a physiological change that occurs concomitantly with the previously defined behavioral state of sleep. We do not rule out that other phasic REM components (e.g. atonia, cardiac arrhythmias, myoclonic twitches or desynchronized EEG) are coherently expressed during sleep but we conclude that adult zebrafish do not have REM-sleep-related rapid eye movements.

  9. Prolactin regulates transcription of the ion uptake Na+/Cl- cotransporter (ncc) gene in zebrafish gill

    USGS Publications Warehouse

    Breves, Jason P.; Serizier, Sandy B.; Goffin, Vincent; McCormick, Stephen D.; Karlstrom, Rolf O.

    2013-01-01

    Prolactin (PRL) is a well-known regulator of ion and water transport within osmoregulatory tissues across vertebrate species, yet how PRL acts on some of its target tissues remains poorly understood. Using zebrafish as a model, we show that ionocytes in the gill directly respond to systemic PRL to regulate mechanisms of ion uptake. Ion-poor conditions led to increases in the expression of PRL receptor (prlra), Na+/Cl− cotransporter (ncc; slc12a10.2), Na+/H+ exchanger (nhe3b; slc9a3.2), and epithelial Ca2+ channel (ecac; trpv6) transcripts within the gill. Intraperitoneal injection of ovine PRL (oPRL) increased ncc and prlra transcripts, but did not affect nhe3b or ecac. Consistent with direct PRL action in the gill, addition of oPRL to cultured gill filaments stimulated ncc in a concentration-dependent manner, an effect blocked by a pure human PRL receptor antagonist (Δ1-9-G129R-hPRL). These results suggest that PRL signaling through PRL receptors in the gill regulates the expression of ncc, thereby linking this pituitary hormone with an effector of Cl− uptake in zebrafish for the first time.

  10. Acute exposure to 2,4-dinitrophenol alters zebrafish swimming performance and whole body triglyceride levels.

    PubMed

    Marit, Jordan S; Weber, Lynn P

    2011-06-01

    While swimming endurance (critical swimming speed or U(crit)) and lipid stores have both been reported to acutely decrease after exposure to a variety of toxicants, the relationship between these endpoints has not been clearly established. In order to examine these relationships, adult zebrafish (Danio rerio) were aqueously exposed to solvent control (ethanol) or two nominal concentrations of 2,4-dinitrophenol (DNP), a mitochondrial electron transport chain uncoupler, for a 24-h period. Following exposure, fish were placed in a swim tunnel in clean water for swimming testing or euthanized immediately without testing, followed by analysis of whole body triglyceride levels. U(crit) decreased in both the 6 mg/L and 12 mg/L DNP groups, with 12 mg/L approaching the LC₅₀. A decrease in tail beat frequency was observed without a significant change in tail beat amplitude. In contrast, triglyceride levels were elevated in a concentration-dependent manner in the DNP exposure groups, but only in fish subjected to swimming tests. This increase in triglyceride stores may be due to a direct interference of DNP on lipid catabolism as well as increased triglyceride production when zebrafish were subjected to the co-stressors of swimming and toxicant exposure. Future studies should be directed at determining how acute DNP exposure combines with swimming to cause alterations in triglyceride accumulation.

  11. Visualisation and Quantification of Morphogen Gradient Formation in the Zebrafish

    PubMed Central

    Harvey, Steven A; Smith, James C

    2009-01-01

    During embryonic development, signalling molecules known as morphogens act in a concentration-dependent manner to provide positional information to responding tissues. In the early zebrafish embryo, graded signalling by members of the nodal family induces the formation of mesoderm and endoderm, thereby patterning the embryo into three germ layers. Nodal signalling has also been implicated in the establishment of the dorso-ventral axis of the embryo. Although one can infer the existence of nodal gradients by comparing gene expression patterns in wild-type embryos and embryos in which nodal signalling is diminished or augmented, real understanding can only come from directly observing the gradients. One approach is to determine local ligand concentrations in the embryo, but this is technically challenging, and the presence of inhibitors might cause the effective concentration of a ligand to differ from its actual concentration. We have therefore taken two approaches to visualise a direct response to nodal signalling. In the first, we have used transgenic embryos to study the nuclear accumulation of a Smad2-Venus fusion protein, and in the second we have used bimolecular fluorescence complementation to visualise the formation of a complex between Smad2 and Smad4. This has allowed us to visualise, in living embryos, the formation of a graded distribution of nodal signalling activity. We have quantified the formation of the gradient in time and space, and our results not only confirm that nodal signalling patterns the embryo into three germ layers, but also shed light on its role in patterning the dorso-ventral axis and highlight unexpected complexities of mesodermal patterning. PMID:19419239

  12. Paired patch clamp recordings from motor-neuron and target skeletal muscle in zebrafish.

    PubMed

    Wen, Hua; Brehm, Paul

    2010-11-20

    Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

  13. Inducible Sterilization of Zebrafish by Disruption of Primordial Germ Cell Migration

    PubMed Central

    Wong, Ten-Tsao; Collodi, Paul

    2013-01-01

    During zebrafish development, a gradient of stromal-derived factor 1a (Sdf1a) provides the directional cue that guides the migration of the primordial germ cells (PGCs) to the gonadal tissue. Here we describe a method to produce large numbers of infertile fish by inducing ubiquitous expression of Sdf1a in zebrafish embryos resulting in disruption of the normal PGC migration pattern. A transgenic line of zebrafish, Tg(hsp70:sdf1a-nanos3, EGFP), was generated that expresses Sdf1a under the control of the heat-shock protein 70 (hsp70) promoter and nanos3 3?UTR. To better visualize the PGCs, the Tg(hsp70:sdf1a-nanos3, EGFP) fish were crossed with another transgenic line, Tg(kop:DsRed-nanos3), that expresses DsRed driven by the PGC-specific kop promoter. Heat treatment of the transgenic embryos caused an induction of Sdf1a expression throughout the embryo resulting in the disruption of their normal migration. Optimal embryo survival and disruption of PGC migration was achieved when transgenic embryos at the 4- to 8-cell stage were incubated at 34.5°C for 18 hours. Under these conditions, disruption of PGC migration was observed in 100% of the embryos. Sixty-four adult fish were developed from three separate batches of heat-treated embryos and all were found to be infertile males. When each male was paired with a wild-type female, only unfertilized eggs were produced and histological examination revealed that each of the adult male fish possessed severely under-developed gonads that lacked gametes. The results demonstrate that inducible Sdf1a expression is an efficient and reliable strategy to produce infertile fish. This approach makes it convenient to generate large numbers of infertile adult fish while also providing the capability to maintain a fertile brood stock. PMID:23826390

  14. Quantitative investigation of the mechanisms of microplastics and nanoplastics toward zebrafish larvae locomotor activity.

    PubMed

    Chen, Qiqing; Gundlach, Michael; Yang, Shouye; Jiang, Jing; Velki, Mirna; Yin, Daqiang; Hollert, Henner

    2017-04-15

    This study investigated the direct and indirect toxic effects of microplastics and nanoplastics toward zebrafish (Danio rerio) larvae locomotor activity. Results showed that microplastics alone exhibited no significant effects except for the upregulated zfrho visual gene expression; whereas nanoplastics inhibited the larval locomotion by 22% during the last darkness period, and significantly reduced larvae body length by 6%, inhibited the acetylcholinesterase activity by 40%, and upregulated gfap, α1-tubulin, zfrho and zfblue gene expression significantly. When co-exposed with 2μg/L 17 α-ethynylestradiol (EE2), microplastics led to alleviation on EE2's inhibition effect on locomotion, which was probably due to the decreased freely dissolved EE2 concentration. However, though nanoplastics showed stronger adsorption ability for EE2, the hypoactivity phenomenon still existed in the nanoplastics co-exposure group. Moreover, when co-exposed with a higher concentration of EE2 (20μg/L), both plastics showed an enhanced effect on the hypoactivity. Principal component analysis was performed to reduce data dimensions and four principal components were reconstituted in terms of oxidative stress, body length, nervous and visual system related genes explaining 84% of total variance. Furthermore, oxidative damage and body length reduction were evaluated to be main reasons for the hypoactivity. Therefore, nanoplastics alone suppressed zebrafish larvae locomotor activity and both plastic particles can change the larvae swimming behavior when co-exposed with EE2. This study provides new insights into plastic particles' effects on zebrafish larvae, improving the understanding of their environmental risks to the aquatic environment.

  15. Stimulus dependence of the development of the zebrafish (Danio rerio) vestibular system.

    PubMed

    Moorman, S J; Burress, C; Cordova, R; Slater, J

    1999-02-05

    It has been suggested that stimulus dependence is a general feature of all developing sensory systems. We tested this idea for the developing zebrafish vestibular system using a bioreactor the National Aeronautic and Space Agency designed to simulate microgravity for cells in culture on earth. We replaced the culture medium with aquarium water and maintained zebrafish eggs/hatchlings in the bioreactor for either 72 or 96 h postfertilization. These experimental animals displayed a swimming behavior that was indistinguishable from the control animals when illuminated from above. However, when illuminated from below, experimental animals swam not only dorsal surface up, but also lying on their side; they corkscrewed, swam vertical loops, and occasionally even swam upside down. When incubated in the bioreactor for 96 h, the saccular otolith was significantly smaller than normal, suggesting that otolith development was either delayed or slower than normal. When incubated in the bioreactor for 72 h, some animals were missing one or more otoliths. In contrast, control animals all had two otoliths on each side. This supports the idea that otolith development was delayed. Immediately upon removal from the bioreactor at 96 h, experimental animals showed some signs of compensatory eye rotation, but with a much less clear relationship between the orientation of the eye and the direction of gravity than the age-matched control animals. This difference was still obvious 1 day later. These results support the idea that development of the vestibular system in zebrafish is dependent on the presence of the normal stimulus the system is designed to detect.

  16. Lactobacillus rhamnosus GG Effect on Behavior of Zebrafish During Chronic Ethanol Exposure

    PubMed Central

    Schneider, Ana Claudia Reis; Rico, Eduardo Pacheco; de Oliveira, Diogo Losch; Rosemberg, Denis Broock; Guizzo, Ranieli; Meurer, Fábio; da Silveira, Themis Reverbel

    2016-01-01

    Abstract Ethanol is a widely consumed drug, which acts on the central nervous system to induce behavioral alterations ranging from disinhibition to sedation. Recent studies have produced accumulating evidence for the therapeutic role of probiotic bacteria in behavior. We aimed to investigate the effect of Lactobacillus rhamnosus GG (LGG) on the behavior of adult zebrafish chronically exposed to ethanol. Adult wild-type zebrafish were randomly divided into four groups, each containing 15 fish. The following groups were formed: Control (C), received unsupplemented feed during the trial period; Probiotic (P), fed with feed supplemented with LGG; Ethanol (E), received unsupplemented feed and 0.5% of ethanol directly added to the tank water; and Probiotic+Ethanol (P+E), group under ethanol exposure (0.5%) and fed with LGG supplemented feed. After 2 weeks of exposure, the novel tank test was used to evaluate fish behavior, which was analyzed using computer-aided video tracking. LGG alone did not alter swimming behavior of the fish. Ethanol exposure led to robust behavioral effects in the form of reduced anxiety levels, as indicated by increased vertical exploration and more time spent in the upper region of the novel tank. The group exposed to ethanol and treated with LGG behaved similarly to animals exposed to ethanol alone. Taken together, these results show that zebrafish behavior was not altered by LGG per se, as seen in murine models. This was the first study to investigate the effects of a probiotic diet on behavior after a chronic ethanol exposure. PMID:26862467

  17. Developmental and reproductive toxicity of PVP/PEI-coated silver nanoparticles to zebrafish.

    PubMed

    Orbea, Amaia; González-Soto, Nagore; Lacave, José María; Barrio, Irantzu; Cajaraville, Miren P

    2017-03-05

    Cellular and molecular mechanisms of toxicity of silver nanoparticles (NPs) and their toxicity to fish embryos after waterborne exposure have been widely investigated, but much less information is available regarding the effect of Ag NPs on physiological functions such as growth or reproduction. In this work, the effects of waterborne exposure of adult zebrafish (Danio rerio) to PVP/PEI coated Ag NPs (~5nm) on reproduction (fecundity) were investigated. Moreover, the development of the embryos after parental exposure was compared with the development of embryos after direct waterborne exposure to the NPs. For this, two experiments were run: 1) embryos from unexposed parents were treated for 5days with Ag NPs (10μg Ag L(-1)-10mg Ag L(-1)) and development was monitored, and 2) selected breeding zebrafish were exposed for 3weeks to 100ng Ag L(-1) (environmentally relevant concentration) or to 10μg Ag L(-1) of Ag NPs, fecundity was scored and development of resulting embryos was monitored up to 5 d. Waterborne exposure of embryos to Ag NPs resulted highly toxic (LC50 at 120h=50μg Ag L(-1)), causing 100% mortality during the first 24h of exposure at 0.1mg Ag L(-1). Exposure of adults, even at the environmentally relevant silver concentration, caused a significant reduction of fecundity by the second week of treatment and resulting embryos showed a higher prevalence of malformations than control embryos. Exposed adult females presented higher prevalence of vacuolization in the liver. These results show that Ag NPs at an environmentally relevant concentration are able to affect population level parameters in zebrafish.

  18. Latent TGFβ binding protein 3 identifies a second heart field in zebrafish

    PubMed Central

    Zhou, Yong; Cashman, Timothy J.; Nevis, Kathleen R.; Obregon, Pablo; Carney, Sara A.; Liu, Yan; Gu, Aihua; Mosimann, Christian; Sondalle, Samuel; Peterson, Richard E.; Heideman, Warren; Burns, Caroline E.; Burns, C. Geoffrey

    2012-01-01

    The four-chambered mammalian heart develops from two fields of cardiac progenitor cells (CPCs) distinguished by their spatiotemporal patterns of differentiation and contributions to the definitive heart [1–3]. The first heart field differentiates earlier in lateral plate mesoderm, generates the linear heart tube and ultimately gives rise to the left ventricle. The second heart field (SHF) differentiates later in pharyngeal mesoderm, elongates the heart tube, and gives rise to the outflow tract (OFT) and much of the right ventricle. Because hearts in lower vertebrates contain a rudimentary OFT but not a right ventricle [4], the existence and function of SHF-like cells in these species has remained a topic of speculation [4–10]. Here we provide direct evidence from Cre/Lox-mediated lineage tracing and loss of function studies in zebrafish, a lower vertebrate with a single ventricle, that latent-TGFβ binding protein 3 (ltbp3) transcripts mark a field of CPCs with defining characteristics of the anterior SHF in mammals. Specifically, ltbp3+ cells differentiate in pharyngeal mesoderm after formation of the heart tube, elongate the heart tube at the outflow pole, and give rise to three cardiovascular lineages in the OFT and myocardium in the distal ventricle. In addition to expressing Ltbp3, a protein that regulates the bioavailability of TGFβ ligands [11], zebrafish SHF cells co-express nkx2.5, an evolutionarily conserved marker of CPCs in both fields [4]. Embryos devoid of ltbp3 lack the same cardiac structures derived from ltbp3+ cells due to compromised progenitor proliferation. Additionally, small-molecule inhibition of TGFβ signaling phenocopies the ltbp3-morphant phenotype whereas expression of a constitutively active TGFβ type I receptor rescues it. Taken together, our findings uncover a requirement for ltbp3-TGFβ signaling during zebrafish SHF development, a process that serves to enlarge the single ventricular chamber in this species. PMID:21623370

  19. Venous-derived angioblasts generate organ-specific vessels during zebrafish embryonic development

    PubMed Central

    Hen, Gideon; Nicenboim, Julian; Mayseless, Oded; Asaf, Lihee; Shin, Masahiro; Busolin, Giorgia; Hofi, Roy; Almog, Gabriella; Tiso, Natascia; Lawson, Nathan D.; Yaniv, Karina

    2015-01-01

    Formation and remodeling of vascular beds are complex processes orchestrated by multiple signaling pathways. Although it is well accepted that vessels of a particular organ display specific features that enable them to fulfill distinct functions, the embryonic origins of tissue-specific vessels and the molecular mechanisms regulating their formation are poorly understood. The subintestinal plexus of the zebrafish embryo comprises vessels that vascularize the gut, liver and pancreas and, as such, represents an ideal model in which to investigate the early steps of organ-specific vessel formation. Here, we show that both arterial and venous components of the subintestinal plexus originate from a pool of specialized angioblasts residing in the floor of the posterior cardinal vein (PCV). Using live imaging of zebrafish embryos, in combination with photoconvertable transgenic reporters, we demonstrate that these angioblasts undergo two phases of migration and differentiation. Initially, a subintestinal vein forms and expands ventrally through a Bone Morphogenetic Protein-dependent step of collective migration. Concomitantly, a Vascular Endothelial Growth Factor-dependent shift in the directionality of migration, coupled to the upregulation of arterial markers, is observed, which culminates with the generation of the supraintestinal artery. Together, our results establish the zebrafish subintestinal plexus as an advantageous model for the study of organ-specific vessel development and provide new insights into the molecular mechanisms controlling its formation. More broadly, our findings suggest that PCV-specialized angioblasts contribute not only to the formation of the early trunk vasculature, but also to the establishment of late-forming, tissue-specific vascular beds. PMID:26525671

  20. Correct anteroposterior patterning of the zebrafish neurectoderm in the absence of the early dorsal organizer

    PubMed Central

    2011-01-01

    Background The embryonic organizer (i.e., Spemann organizer) has a pivotal role in the establishment of the dorsoventral (DV) axis through the coordination of BMP signaling. However, as impaired organizer function also results in anterior and posterior truncations, it is of interest to determine if proper anteroposterior (AP) pattern can be obtained even in the absence of early organizer signaling. Results Using the ventralized, maternal effect ichabod (ich) mutant, and by inhibiting BMP signaling in ich embryos, we provide conclusive evidence that AP patterning is independent of the organizer in zebrafish, and is governed by TGFβ, FGF, and Wnt signals emanating from the germ-ring. The expression patterns of neurectodermal markers in embryos with impaired BMP signaling show that the directionality of such signals is oriented along the animal-vegetal axis, which is essentially concordant with the AP axis. In addition, we find that in embryos inhibited in both Wnt and BMP signaling, the AP pattern of such markers is unchanged from that of the normal untreated embryo. These embryos develop radially organized trunk and head tissues, with an outer neurectodermal layer containing diffusely positioned neuronal precursors. Such organization is reflective of the presumed eumetazoan ancestor and might provide clues for the evolution of centralization in the nervous system. Conclusions Using a zebrafish mutant deficient in the induction of the embryonic organizer, we demonstrate that the AP patterning of the neuroectoderm during gastrulation is independent of DV patterning. Our results provide further support for Nieuwkoop's "two step model" of embryonic induction. We also show that the zebrafish embryo can form a radial diffuse neural sheath in the absence of both BMP signaling and the early organizer. PMID:21575247

  1. Silver nanoparticles induce endoplasmatic reticulum stress response in zebrafish

    SciTech Connect

    Christen, Verena; Capelle, Martinus; Fent, Karl

    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.

  2. Comparative studies of Toll-like receptor signalling using zebrafish.

    PubMed

    Kanwal, Zakia; Wiegertjes, Geert F; Veneman, Wouter J; Meijer, Annemarie H; Spaink, Herman P

    2014-09-01

    Zebrafish model systems for infectious disease are increasingly used for the functional analysis of molecular pattern recognition processes. These studies benefit from the high conservation level of all innate immune factors in vertebrates. Zebrafish studies are strategically well positioned for this because of the ease of comparisons with studies in other fish species of which the immune system also has been intensively studied, but that are currently still less amendable to detailed genetic or microscopic studies. In this paper we focus on Toll-like receptor (TLR) signalling factors, which currently are the best characterized in mammalian systems. We review the knowledge on TLR signalling in the context of recent advances in zebrafish studies and discuss possibilities for future approaches that can complement studies in cell cultures and rodent models. A focus in these comparisons is the role of negative control mechanisms in immune responses that appear very important in a whole organism to keep adverse systemic responses in check. We also pay much attention to comparisons with studies in common carp that is highly related to zebrafish and that because of its large body mass can complement immune studies in zebrafish.

  3. Zebrafish as a Model to Investigate Dynamin 2-Related Diseases

    PubMed Central

    Bragato, Cinzia; Gaudenzi, Germano; Blasevich, Flavia; Pavesi, Giulio; Maggi, Lorenzo; Giunta, Michele; Cotelli, Franco; Mora, Marina

    2016-01-01

    Mutations in the dynamin-2 gene (DNM2) cause autosomal dominant centronuclear myopathy (CNM) and dominant intermediate Charcot-Marie-Tooth (CMT) neuropathy type B (CMTDIB). As the relation between these DNM2-related diseases is poorly understood, we used zebrafish to investigate the effects of two different DNM2 mutations. First we identified a new alternatively spliced zebrafish dynamin-2a mRNA (dnm2a-v2) with greater similarity to human DNM2 than the deposited sequence. Then we knocked-down the zebrafish dnm2a, producing defects in muscle morphology. Finally, we expressed two mutated DNM2 mRNA by injecting zebrafish embryos with human mRNAs carrying the R522H mutation, causing CNM, or the G537C mutation, causing CMT. Defects arose especially in secondary motor neuron formation, with incorrect branching in embryos injected with CNM-mutated mRNA, and total absence of branching in those injected with CMT-mutated mRNA. Muscle morphology in embryos injected with CMT-mutated mRNA appeared less regularly organized than in those injected with CNM-mutated mRNA. Our results showing, a continuum between CNM and CMTDIB phenotypes in zebrafish, similarly to the human conditions, confirm this animal model to be a powerful tool to investigate mutations of DNM2 in vivo. PMID:26842864

  4. Genetic evidence for shared mechanisms of epimorphic regeneration in zebrafish.

    PubMed

    Qin, Zhao; Barthel, Linda K; Raymond, Pamela A

    2009-06-09

    In a microarray-based gene profiling analysis of Müller glia-derived retinal stem cells in light-damaged retinas from adult zebrafish, we found that 2 genes required for regeneration of fin and heart tissues in zebrafish, hspd1 (heat shock 60-kDa protein 1) and mps1 (monopolar spindle 1), were up-regulated. Expression of both genes in the neurogenic Müller glia and progenitors was independently verified by quantitative reverse transcriptase PCR and in situ hybridization. Functional analysis of temperature-sensitive mutants of hspd1 and mps1 revealed that both are necessary for Müller glia-based cone photoreceptor regeneration in adult zebrafish retina. In the amputated fin, hspd1 is required for the induction of mesenchymal stem cells and blastema formation, whereas mps1 is required at a later step for rapid cell proliferation and outgrowth. This temporal sequence of hspd1 and mps1 function is conserved in the regenerating retina. Comparison of gene expression profiles from regenerating zebrafish retina, caudal fin, and heart muscle revealed additional candidate genes potentially implicated in injury-induced epimorphic regeneration in diverse zebrafish tissues.

  5. Isolation and Characterization of Single Cells from Zebrafish Embryos

    PubMed Central

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

    2017-01-01

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

  6. Death-associated odors induce stress in zebrafish.

    PubMed

    Oliveira, Thiago Acosta; Koakoski, Gessi; da Motta, Adriana Costa; Piato, Angelo Luis; Barreto, Rodrigo Egydio; Volpato, Gilson Luiz; Barcellos, Leonardo José Gil

    2014-04-01

    Living animals exploit information released from dead animals to conduct adaptive biological responses. For instance, a recently published study has shown that avoidance behavior is triggered by death-associated odors in zebrafish. Stress can clearly act as an adaptive response that allows an organism to deal with an imminent threat. However, it has not been demonstrated whether these chemical cues are stressful for fish. Here, we confirmed that dead zebrafish scents induce defensive behavior in live conspecifics. Additionally, we show for the first time in fish that these scents increase cortisol in conspecifics. To reach this conclusion, firstly, we exposed zebrafish to multi-sensorial cues (e.g., visual, tactile, chemical cues) from dead conspecifics that displayed defensive behaviors and increased cortisol. Also, when we limited zebrafish to chemical cues from dead conspecifics, similar responses arose. These responses coincide with the decaying destruction of epidermal cells, indicating that defensive and stress responses could take place as an effect of substances emanating from decaying flesh, as well as alarm substance released due to rupture of epidermal cells. Taken together, these results illustrate that living zebrafish utilize cues from dead conspecific to avoid or to cope with danger and ensure survival.

  7. Modeling Pancreatic Endocrine Cell Adaptation and Diabetes in the Zebrafish

    PubMed Central

    Maddison, Lisette A.; Chen, Wenbiao

    2017-01-01

    Glucose homeostasis is an important element of energy balance and is conserved in organisms from fruit fly to mammals. Central to the control of circulating glucose levels in vertebrates are the endocrine cells of the pancreas, particularly the insulin-producing β-cells and the glucagon producing α-cells. A feature of α- and β-cells is their plasticity, an ability to adapt, in function and number as a response to physiological and pathophysiological conditions of increased hormone demand. The molecular mechanisms underlying these adaptive responses that maintain glucose homeostasis are incompletely defined. The zebrafish is an attractive model due to the low cost, high fecundity, and amenability to genetic and compound screens, and mechanisms governing the development of the pancreatic endocrine cells are conserved between zebrafish and mammals. Post development, both β- and α-cells of zebrafish display plasticity as in mammals. Here, we summarize the studies of pancreatic endocrine cell adaptation in zebrafish. We further explore the utility of the zebrafish as a model for diabetes, a relevant topic considering the increase in diabetes in the human population. PMID:28184214

  8. Atlas of Cellular Dynamics during Zebrafish Adult Kidney Regeneration

    PubMed Central

    McCampbell, Kristen K.; Springer, Kristin N.; Wingert, Rebecca A.

    2015-01-01

    The zebrafish is a useful animal model to study the signaling pathways that orchestrate kidney regeneration, as its renal nephrons are simple, yet they maintain the biological complexity inherent to that of higher vertebrate organisms including mammals. Recent studies have suggested that administration of the aminoglycoside antibiotic gentamicin in zebrafish mimics human acute kidney injury (AKI) through the induction of nephron damage, but the timing and details of critical phenotypic events associated with the regeneration process, particularly in existing nephrons, have not been characterized. Here, we mapped the temporal progression of cellular and molecular changes that occur during renal epithelial regeneration of the proximal tubule in the adult zebrafish using a platform of histological and expression analysis techniques. This work establishes the timing of renal cell death after gentamicin injury, identifies proliferative compartments within the kidney, and documents gene expression changes associated with the regenerative response of proliferating cells. These data provide an important descriptive atlas that documents the series of events that ensue after damage in the zebrafish kidney, thus availing a valuable resource for the scientific community that can facilitate the implementation of zebrafish research to delineate the mechanisms that control renal regeneration. PMID:26089919

  9. A jump persistent turning walker to model zebrafish locomotion

    PubMed Central

    Mwaffo, Violet; Anderson, Ross P.; Butail, Sachit; Porfiri, Maurizio

    2015-01-01

    Zebrafish are gaining momentum as a laboratory animal species for the investigation of several functional and dysfunctional biological processes. Mathematical models of zebrafish behaviour are expected to considerably aid in the design of hypothesis-driven studies by enabling preliminary in silico tests that can be used to infer possible experimental outcomes without the use of zebrafish. This study is motivated by observations of sudden, drastic changes in zebrafish locomotion in the form of large deviations in turn rate. We demonstrate that such deviations can be captured through a stochastic mean reverting jump diffusion model, a process that is commonly used in financial engineering to describe large changes in the price of an asset. The jump process-based model is validated on trajectory data of adult subjects swimming in a shallow circular tank obtained from an overhead camera. Through statistical comparison of the empirical distribution of the turn rate against theoretical predictions, we demonstrate the feasibility of describing zebrafish as a jump persistent turning walker. The critical role of the jump term is assessed through comparison with a simplified mean reversion diffusion model, which does not allow for describing the heavy-tailed distributions observed in the fish turn rate. PMID:25392396

  10. Finding clues to the riddle of sex determination in zebrafish.

    PubMed

    Nagabhushana, A; Mishra, Rakesh K

    2016-03-01

    How sex is determined has been one of the most intriguing puzzles in biology since antiquity. Although a fundamental process in most metazoans, there seems to be myriad of ways in which sex can be determined - from genetic to environmental sex determination. This variation is limited mainly to upstream triggers with the core of sex determination pathway being conserved. Zebrafish has gained prominence as a vertebrate model system to study development and disease. However, very little is known about its primary sex determination mechanism. Here we review our current understanding of the sex determination in zebrafish. Zebrafish lack identifiable heteromorphic sex chromosomes and sex is determined by multiple genes, with some influence from the environment. Recently, chromosome 4 has been identified as sex chromosome along with few sex-linked loci on chromosomes 5 and 16. The identities of candidate sex-linked genes, however, have remained elusive. Sex in zebrafish is also influenced by the number of meiotic oocytes in the juvenile ovary, which appear to instruct retention of the ovarian fate. The mechanism and identity of this instructive signal remain unknown. We hypothesize that sex in zebrafish is a culmination of combinatorial effects of the genome, germ cells and the environment with inputs from epigenetic factors translating the biological meaning of this interaction.

  11. Cyp1a reporter zebrafish reveals target tissues for dioxin.

    PubMed

    Kim, Kun-Hee; Park, Hye-Jeong; Kim, Jin Hee; Kim, Suhyun; Williams, Darren R; Kim, Myeong-Kyu; Jung, Young Do; Teraoka, Hiroki; Park, Hae-Chul; Choy, Hyon E; Shin, Boo Ahn; Choi, Seok-Yong

    2013-06-15

    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.

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

    PubMed

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

    2015-12-01

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

  13. Myomaker mediates fusion of fast myocytes in zebrafish embryos

    SciTech Connect

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

    2014-09-05

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

  14. Pharmacological Analyses of Learning and Memory in Zebrafish (Danio rerio)

    PubMed Central

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

    2015-01-01

    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 has 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 spans 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. PMID:25792292

  15. Zebrafish: An Important Tool for Liver Disease Research

    PubMed Central

    Goessling, Wolfram; Sadler, Kirsten C.

    2016-01-01

    As the incidence of hepatobiliary diseases increases, we must improve our understanding of the molecular, cellular, and physiological factors that contribute to the pathogenesis of liver disease. Animal models help us identify disease mechanisms that might be targeted therapeutically. Zebrafish (Danio rerio) have traditionally been used to study embryonic development but are also important to the study of liver disease. Zebrafish embryos develop rapidly; all of their digestive organs are mature in larvae by 5 days of age. At this stage, they can develop hepatobiliary diseases caused by developmental defects or toxin- or ethanol-induced injury and manifest premalignant changes within weeks. Zebrafish are similar to humans in hepatic cellular composition, function, signaling, and response to injury as well as the cellular processes that mediate liver diseases. Genes are highly conserved between humans and zebrafish, making them a useful system to study the basic mechanisms of liver disease. We can perform genetic screens to identify novel genes involved in specific disease processes and chemical screens to identify pathways and compounds that act on specific processes. We review how studies of zebrafish have advanced our understanding of inherited and acquired liver diseases as well as liver cancer and regeneration. PMID:26319012

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

    NASA Astrophysics Data System (ADS)

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

    2009-05-01

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

  17. A jump persistent turning walker to model zebrafish locomotion.

    PubMed

    Mwaffo, Violet; Anderson, Ross P; Butail, Sachit; Porfiri, Maurizio

    2015-01-06

    Zebrafish are gaining momentum as a laboratory animal species for the investigation of several functional and dysfunctional biological processes. Mathematical models of zebrafish behaviour are expected to considerably aid in the design of hypothesis-driven studies by enabling preliminary in silico tests that can be used to infer possible experimental outcomes without the use of zebrafish. This study is motivated by observations of sudden, drastic changes in zebrafish locomotion in the form of large deviations in turn rate. We demonstrate that such deviations can be captured through a stochastic mean reverting jump diffusion model, a process that is commonly used in financial engineering to describe large changes in the price of an asset. The jump process-based model is validated on trajectory data of adult subjects swimming in a shallow circular tank obtained from an overhead camera. Through statistical comparison of the empirical distribution of the turn rate against theoretical predictions, we demonstrate the feasibility of describing zebrafish as a jump persistent turning walker. The critical role of the jump term is assessed through comparison with a simplified mean reversion diffusion model, which does not allow for describing the heavy-tailed distributions observed in the fish turn rate.

  18. Regulation of hypocretin (orexin) expression in embryonic zebrafish.

    PubMed

    Faraco, Juliette H; Appelbaum, Lior; Marin, Wilfredo; Gaus, Stephanie E; Mourrain, Philippe; Mignot, Emmanuel

    2006-10-06

    Hypocretins/orexins are neuropeptides involved in the regulation of sleep and energy balance in mammals. Conservation of gene sequence, hypothalamic localization of cell bodies, and projection patterns in adult zebrafish suggest that the architecture and function of the hypocretin system are conserved in fish. We report on the complete genomic structure of the zebrafish and Tetraodon hypocretin genes and the complete predicted hypocretin protein sequences from five teleosts. Using whole mount in situ hybridization, we have traced the development of hypocretin cells in zebrafish from onset of expression at 22 h post-fertilization through the first week of development. Promoter elements of similar size from zebrafish and Tetraodon were capable of driving efficient and specific expression of enhanced green fluorescent protein in developing zebrafish embryos, thus defining a minimal promoter region able to accurately mimic the native hypocretin pattern. This enhanced green fluorescent protein expression also revealed a complex pattern of projections within the hypothalamus, to the midbrain, and to the spinal cord. To further analyze the promoter, a series of deletion and substitution constructs were injected into embryos, and resulting promoter activity was monitored in the first week of development. A critical region of 250 base pairs was identified containing a core 13-base pair element essential for hypocretin expression.

  19. Toxicity Evaluation of New Engineered Nanomaterials in Zebrafish.

    PubMed

    Brundo, Maria V; Pecoraro, Roberta; Marino, Fabio; Salvaggio, Antonio; Tibullo, Daniele; Saccone, Salvatore; Bramanti, Vincenzo; Buccheri, Maria A; Impellizzeri, Giuliana; Scuderi, Viviana; Zimbone, Massimo; Privitera, Vittorio

    2016-01-01

    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/TiO2 nano-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 an excellent model for the environmental biomonitoring of aquatic environments and the Zebrafish Embryo Toxicity Test (ZFET) 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.

  20. Toxicity Evaluation of New Engineered Nanomaterials in Zebrafish

    PubMed Central

    Brundo, Maria V.; Pecoraro, Roberta; Marino, Fabio; Salvaggio, Antonio; Tibullo, Daniele; Saccone, Salvatore; Bramanti, Vincenzo; Buccheri, Maria A.; Impellizzeri, Giuliana; Scuderi, Viviana; Zimbone, Massimo; Privitera, Vittorio

    2016-01-01

    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/TiO2 nano-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 an excellent model for the environmental biomonitoring of aquatic environments and the Zebrafish Embryo Toxicity Test (ZFET) 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. PMID:27148069

  1. Structured illumination fluorescence correlation spectroscopy for velocimetry in Zebrafish embryos

    NASA Astrophysics Data System (ADS)

    Pozzi, Paolo; Rossetti, Leone; Sironi, Laura; Freddi, Stefano; D'Alfonso, Laura; Caccia, Michele; Bouzin, Margaux; Collini, Maddalena; Chirico, Giuseppe

    2013-02-01

    The vascular system of Zebrafish embryos is studied by means of Fluorescence Correlation and Image Correlation Spectroscopy. The long term project addresses biologically relevant issues concerning vasculogenesis and cardiogenesis and in particular mechanical interaction between blood flow and endothelial cells. To this purpose we use Zebrafish as a model system since the transparency of its embryos facilitates morphological observation of internal organs in-vivo. The correlation analysis provides quantitative characterization of fluxes in blood vessels in vivo. We have pursued and compared two complementary routes. In a first one we developed a two-spots two-photon setup in which the spots are spaced at adjustable micron-size distances (1-40 μm) along a vessel and the endogenous (autofluorescence) or exogenous (dsRed transgenic erythrocytes) signal is captured with an EM-CCD and cross-correlated. In this way we are able to follow the morphology of the Zebrafish embryo, simultaneously measure the heart pulsation, the velocity of red cells and of small plasma proteins. These data are compared to those obtained by image correlations on Zebrafish vessels. The two methods allows to characterize the motion of plasma fluids and erythrocytes in healthy Zebrafish embryos to be compared in the future to pathogenic ones.

  2. Zebrafish: an important tool for liver disease research.

    PubMed

    Goessling, Wolfram; Sadler, Kirsten C

    2015-11-01

    As the incidence of hepatobiliary diseases increases, we must improve our understanding of the molecular, cellular, and physiological factors that contribute to the pathogenesis of liver disease. Animal models help us identify disease mechanisms that might be targeted therapeutically. Zebrafish (Danio rerio) have traditionally been used to study embryonic development but are also important to the study of liver disease. Zebrafish embryos develop rapidly; all of their digestive organs are mature in larvae by 5 days of age. At this stage, they can develop hepatobiliary diseases caused by developmental defects or toxin- or ethanol-induced injury and manifest premalignant changes within weeks. Zebrafish are similar to humans in hepatic cellular composition, function, signaling, and response to injury as well as the cellular processes that mediate liver diseases. Genes are highly conserved between humans and zebrafish, making them a useful system to study the basic mechanisms of liver disease. We can perform genetic screens to identify novel genes involved in specific disease processes and chemical screens to identify pathways and compounds that act on specific processes. We review how studies of zebrafish have advanced our understanding of inherited and acquired liver diseases as well as liver cancer and regeneration.

  3. Retinoid regulation of the zebrafish cyp26a1 promoter.

    PubMed

    Hu, Ping; Tian, Miao; Bao, Jie; Xing, Guangdong; Gu, Xingxing; Gao, Xiang; Linney, Elwood; Zhao, Qingshun

    2008-12-01

    Cyp26A1 is a major enzyme that controls retinoic acid (RA) homeostasis by metabolizing RA into bio-inactive metabolites. Previous research revealed that the mouse Cyp26A1 promoter has two canonical RA response elements (RAREs) that underlie the regulation of the gene by RA. Analyzing the 2,533-base pairs (2.5 k) genomic sequence upstream of zebrafish cyp26a1 start codon, we report that the two RAREs are conserved in zebrafish cyp26a1 promoter. Mutagenesis demonstrated that the two RAREs work synergistically in RA inducibility of cyp26a1. Fusing the 2.5 k (kilobase pairs) fragment to the enhanced yellow fluorescent protein (eYFP) reporter gene, we have generated two transgenic lines of zebrafish [Tg(cyp26a1:eYFP)]. The transgenic zebrafish display expression patterns similar to that of cyp26a1 gene in vivo. Consistent with the in vitro results, the reporter activity is RA inducible in embryos. Taken together, our results demonstrate that the 2.5 k fragment underlies the regulation of the zebrafish cyp26a1 gene by RA.

  4. LPS response and tolerance in the zebrafish (Danio rerio).

    PubMed

    Novoa, B; Bowman, T V; Zon, L; Figueras, A

    2009-02-01

    Zebrafish (Danio rerio) has been used in the present work to study the fish response to bacterial lipopolysaccharide (LPS) exposure and LPS tolerance. These mechanisms are not completely understood in mammals and, presently, are totally unknown in fish. Zebrafish larval survival was assessed following treatment with various types of LPS at a variety of concentrations to determine the sensitivity of zebrafish to LPS-induced immune activation. In addition, fish pretreated with a sublethal concentration of LPS did not die after exposure to a lethal concentration of LPS demonstrating, for the first time that LPS tolerance also happens in fish. The time interval between pretreatment and secondary exposure as well as the type of pretreatment dictated the strength of protection. Since zebrafish are in intimate contact with microorganisms, the high resistance of fish to LPS suggests that there must be a tight control of the LPS receptor cluster in order to avoid an excess of inflammation. One of these components is CXCR4, which has previously been shown to regulate the signal transduced by TLR4. Treating fish with AMD3100, a specific inhibitor of CXCR4, increased LPS treatment associated mortality. Blocking CXCR4 via chemical or genetic inhibition resulted in a reversion of LPS tolerance, thus further supporting the negative regulatory role of CXCR4 in this inflammatory response. In support of an inhibitory role for CXCR4 in the inflammatory cascade, IL-1 transcript levels were elevated in both unstimulated and LPS stimulated zebrafish Odysseus (CXCR4 deficient mutant) larvae.

  5. The Zebrafish Breathes New Life into the Study of Tuberculosis

    PubMed Central

    Myllymäki, Henna; Bäuerlein, Carina A.; Rämet, Mika

    2016-01-01

    Tuberculosis (TB) is a global health emergency. Up to one-third of the world’s population is infected with Mycobacterium tuberculosis, and the pathogen continues to kill 1.5 million people annually. Currently, the means for preventing, diagnosing, and treating TB are unsatisfactory. One of the main reasons for the poor progress in TB research has been a lack of good animal models to study the latency, dormancy, and reactivation of the disease. Although sophisticated in vitro and in silico methods suitable for TB research are constantly being developed, they cannot reproduce the complete vertebrate immune system and its interplay with pathogens and vaccines. However, the zebrafish has recently emerged as a useful alternative to more traditional models, such as mice, rabbits, guinea pigs, and non-human primates, for studying the complex pathophysiology of a mycobacterial infection. The model is based on the similarity between Mycobacterium marinum – a natural fish pathogen – and M. tuberculosis. In both zebrafish larvae and adult fish, an infection with M. marinum leads to the formation of macrophage aggregates and granulomas, which resemble the M. tuberculosis infections in humans. In this review, we will summarize the current status of the zebrafish model in TB research and highlight the advantages of using zebrafish to dissect mycobacterial virulence strategies as well as the host immune responses elicited against them. In addition, we will discuss the possibilities of using the adult zebrafish model for studying latency, dormancy, and reactivation in a mycobacterial infection. PMID:27242801

  6. Zebrafish Craniofacial Development: A Window into Early Patterning

    PubMed Central

    Mork, Lindsey; Crump, Gage

    2016-01-01

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

  7. Chiral bioaccumulation behavior of tebuconazole in the zebrafish (Danio rerio).

    PubMed

    Liu, Na; Dong, Fengshou; Xu, Jun; Liu, Xingang; Zheng, Yongquan

    2016-04-01

    Tebuconazole is an effective chiral fungicide, and previous studies have demonstrated that tebuconazole enantiomers exhibit enantioselective toxicity to non-target aquatic organisms. Thus, the aim of the present study was to investigate the chiral bioaccumulation behavior of tebuconazole in zebrafish (Danio rerio). Two exposure concentrations (0.107 and 1.07 mg/L) of tebuconazole were used. The uptake experiments lasted for 8 days, and subsequently, the zebrafish were transferred to another clean tank containing water without tebuconazole for depuration experiments (up to 14 days). A significant trend in enantioselective bioaccumulation was observed in these zebrafish with the preferential accumulation of (-)-R-tebuconazole at two dose levels. The results of the depuration experiments indicated that the degradation of (-)-R-tebuconazole in zebrafish was slower than that of (+)-S-tebuconazole. The BCFk values for (+)-S-tebuconazole and (-)-R-tebuconazole in a low dose of this chemical were 11.22 and 16.25, respectively, while at a high dose, these values were 9.79 and 10.31, respectively. The enantiomer fraction of tebuconazole in zebrafish and water ranged from 0.31-0.49. Hence, future research should focus on the fate of tebuconazole in the aquatic environment at the enantiomer levels.

  8. Strategies to Mitigate a Mycobacterium marinum Outbreak in a Zebrafish Research Facility.

    PubMed

    Mason, Timothy; Snell, Kathy; Mittge, Erika; Melancon, Ellie; Montgomery, Rebecca; McFadden, Marcie; Camoriano, Javier; Kent, Michael L; Whipps, Christopher M; Peirce, Judy

    2016-07-01

    In 2011, the zebrafish research facility at the University of Oregon experienced an outbreak of Mycobacterium marinum that affected both research fish and facility staff. A thorough review of risks to personnel, the zebrafish veterinary care program, and zebrafish husbandry procedures at the research facility followed. In the years since 2011, changes have been implemented throughout the research facility to protect the personnel, the fish colony, and ultimately the continued success of the zebrafish model research program. In this study, we present the history of the outbreak, the changes we implemented, and recommendations to mitigate pathogen outbreaks in zebrafish research facilities.

  9. Strategies to Mitigate a Mycobacterium marinum Outbreak in a Zebrafish Research Facility

    PubMed Central

    Snell, Kathy; Mittge, Erika; Melancon, Ellie; Montgomery, Rebecca; McFadden, Marcie; Camoriano, Javier; Kent, Michael L.; Whipps, Christopher M.; Peirce, Judy

    2016-01-01

    Abstract In 2011, the zebrafish research facility at the University of Oregon experienced an outbreak of Mycobacterium marinum that affected both research fish and facility staff. A thorough review of risks to personnel, the zebrafish veterinary care program, and zebrafish husbandry procedures at the research facility followed. In the years since 2011, changes have been implemented throughout the research facility to protect the personnel, the fish colony, and ultimately the continued success of the zebrafish model research program. In this study, we present the history of the outbreak, the changes we implemented, and recommendations to mitigate pathogen outbreaks in zebrafish research facilities. PMID:27351618

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

    PubMed

    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.

  11. Zebrafish P54 RNA helicases are cytoplasmic granule residents that are required for development and stress resilience

    PubMed Central

    Zampedri, Cecilia; Tinoco-Cuellar, Maryana; Carrillo-Rosas, Samantha; Diaz-Tellez, Abigail; Ramos-Balderas, Jose Luis; Pelegri, Francisco

    2016-01-01

    ABSTRACT Stress granules are cytoplasmic foci that directly respond to the protein synthesis status of the cell. Various environmental insults, such as oxidative stress or extreme heat, block protein synthesis; consequently, mRNA will stall in translation, and stress granules will immediately form and become enriched with mRNAs. P54 DEAD box RNA helicases are components of RNA granules such as P-bodies and stress granules. We studied the expression, in cytoplasmic foci, of both zebrafish P54 RNA helicases (P54a and P54b) during development and found that they are expressed in cytoplasmic granules under both normal conditions and stress conditions. In zebrafish embryos exposed to heat shock, some proportion of P54a and P54b helicases move to larger granules that exhibit the properties of genuine stress granules. Knockdown of P54a and/or P54b in zebrafish embryos produces developmental abnormalities restricted to the posterior trunk; further, these embryos do not form stress granules, and their survival upon exposure to heat-shock conditions is compromised. Our observations fit the model that cells lacking stress granules have no resilience or ability to recover once the stress has ended, indicating that stress granules play an essential role in the way organisms adapt to a changing environment. PMID:27489304

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

    PubMed Central

    Choi, Jin Soo; Kim, Ryeo-Ok; Yoon, Seokjoo

    2016-01-01

    Zinc oxide nanoparticles (ZnO NPs) are being utilized in an increasing number of fields and commercial applications. While their general toxicity and associated oxidative stress have been extensively studied, the toxicological pathways that they induce in developmental stages are still largely unknown. In this study, the developmental toxicity of ZnO NPs to embryonic/larval zebrafish was investigated. The transcriptional expression profiles induced by ZnO NPs were also investigated to ascertain novel genomic responses related to their specific toxicity pathway. Zebrafish embryos were exposed to 0.01, 0.1, 1, and 10 mg/L ZnO NPs for 96 h post-fertilization. The toxicity of ZnO NPs, based on their Zn concentration, was quite similar to that in embryonic/larval zebrafish exposed to corresponding ZnSO4 concentrations. Pericardial edema and yolk-sac edema were the principal malformations induced by ZnO NPs. Gene-expression profiling using microarrays demonstrated 689 genes that were differentially regulated (fold change >1.5) following exposure to ZnO NPs (498 upregulated, 191 downregulated). Several genes that were differentially regulated following ZnO NP exposure shared similar biological pathways with those observed with ZnSO4 exposure, but six genes (aicda, cyb5d1, edar, intl2, ogfrl2 and tnfsf13b) associated with inflammation and the immune system responded specifically to ZnO NPs (either in the opposite direction or were unchanged in ZnSO4 exposure). Real-time reverse-transcription quantitative polymerase chain reaction confirmed that the responses of these genes to ZnO NPs were significantly different from their response to ZnSO4 exposure. ZnO NPs may affect genes related to inflammation and the immune system, resulting in yolk-sac edema and pericardia edema in embryonic/larval developmental stages. These results will assist in elucidating the mechanisms of toxicity of ZnO NPs during development of zebrafish. PMID:27504894

  13. Definition of the zebrafish genome using flow cytometry and cytogenetic mapping

    PubMed Central

    Freeman, Jennifer L; Adeniyi, Adeola; Banerjee, Ruby; Dallaire, Stephanie; Maguire, Sean F; Chi, Jianxiang; Ng, Bee Ling; Zepeda, Cinthya; Scott, Carol E; Humphray, Sean; Rogers, Jane; Zhou, Yi; Zon, Leonard I; Carter, Nigel P; Yang, Fengtang; Lee, Charles

    2007-01-01

    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. PMID:17597531

  14. Wnt/β-catenin signaling promotes regeneration after adult zebrafish spinal cord injury.

    PubMed

    Strand, Nicholas S; Hoi, Kimberly K; Phan, Tien M T; Ray, Catherine A; Berndt, Jason D; Moon, Randall T

    2016-09-02

    Unlike mammals, zebrafish can regenerate their injured spinal cord and regain control of caudal tissues. It was recently shown that Wnt/β-catenin signaling is necessary for spinal cord regeneration in the larval zebrafish. However, the molecular mechanisms of regeneration may or may not be conserved between larval and adult zebrafish. To test this, we assessed the role of Wnt/β-catenin signaling after spinal cord injury in the adult zebrafish. We show that Wnt/β-catenin signaling is increased after spinal cord injury in the adult zebrafish. Moreover, overexpression of Dkk1b inhibited Wnt/β-catenin signaling in the regenerating spinal cord of adult zebrafish. Dkk1b overexpression also inhibited locomotor recovery, axon regeneration, and glial bridge formation in the injured spinal cord. Thus, our data illustrate a conserved role for Wnt/β-catenin signaling in adult and larval zebrafish spinal cord regeneration.

  15. Saccadic and Postsaccadic Disconjugacy in Zebrafish Larvae Suggests Independent Eye Movement Control

    PubMed Central

    Chen, Chien-Cheng; Bockisch, Christopher J.; Straumann, Dominik; Huang, Melody Ying-Yu

    2016-01-01

    Spontaneous eye movements of zebrafish larvae in the dark consist of centrifugal saccades that move the eyes from a central to an eccentric position and postsaccadic centripetal drifts. In a previous study, we showed that the fitted single-exponential time constants of the postsaccadic drifts are longer in the temporal-to-nasal (T->N) direction than in the nasal-to-temporal (N->T) direction. In the present study, we further report that saccadic peak velocities are higher and saccadic amplitudes are larger in the N->T direction than in the T->N direction. We investigated the underlying mechanism of this ocular disconjugacy in the dark with a top-down approach. A mathematic ocular motor model, including an eye plant, a set of burst neurons and a velocity-to-position neural integrator (VPNI), was built to simulate the typical larval eye movements in the dark. The modeling parameters, such as VPNI time constants, neural impulse signals generated by the burst neurons and time constants of the eye plant, were iteratively adjusted to fit the average saccadic eye movement. These simulations suggest that four pools of burst neurons and four pools of VPNIs are needed to explain the disconjugate eye movements in our results. A premotor mechanism controls the synchronous timing of binocular saccades, but the pools of burst and integrator neurons in zebrafish larvae seem to be different (and maybe separate) for both eyes and horizontal directions, which leads to the observed ocular disconjugacies during saccades and postsaccadic drifts in the dark. PMID:27761109

  16. Hyaluronic acid synthesis is required for zebrafish tail fin regeneration

    PubMed Central

    Ouyang, Xiaohu; Panetta, Nicholas J.; Talbott, Maya D.; Payumo, Alexander Y.; Halluin, Caroline; Longaker, Michael T.

    2017-01-01

    Using genome-wide transcriptional profiling and whole-mount expression analyses of zebrafish larvae, we have identified hyaluronan synthase 3 (has3) as an upregulated gene during caudal fin regeneration. has3 expression is induced in the wound epithelium within hours after tail amputation, and its onset and maintenance requires fibroblast growth factor, phosphoinositide 3-kinase, and transforming growth factor-ß signaling. Inhibition of hyaluronic acid (HA) synthesis by the small molecule 4-methylumbelliferone (4-MU) impairs tail regeneration in zebrafish larvae by preventing injury-induced cell proliferation. In addition, 4-MU reduces the expression of genes associated with wound epithelium and blastema function. Treatment with glycogen synthase kinase 3 inhibitors rescues 4-MU-induced defects in cell proliferation and tail regeneration, while restoring a subset of wound epithelium and blastema markers. Our findings demonstrate a role for HA biosynthesis in zebrafish tail regeneration and delineate its epistatic relationships with other regenerative processes. PMID:28207787

  17. Alcohol impairs predation risk response and communication in zebrafish.

    PubMed

    Oliveira, Thiago Acosta; Koakoski, Gessi; Kreutz, Luiz Carlos; Ferreira, Daiane; da Rosa, João Gabriel Santos; de Abreu, Murilo Sander; Giacomini, Ana Cristina Vendrametto; Oliveira, Ricardo Pimentel; Fagundes, Michele; Piato, Angelo Luis; Barreto, Rodrigo Egydio; Barcellos, Leonardo José Gil

    2013-01-01

    The effects of ethanol exposure on Danio rerio have been studied from the perspectives of developmental biology and behavior. However, little is known about the effects of ethanol on the prey-predator relationship and chemical communication of predation risk. Here, we showed that visual contact with a predator triggers stress axis activation in zebrafish. We also observed a typical stress response in zebrafish receiving water from these conspecifics, indicating that these fish chemically communicate predation risk. Our work is the first to demonstrate how alcohol effects this prey-predator interaction. We showed for the first time that alcohol exposure completely blocks stress axis activation in both fish seeing the predator and in fish that come in indirect contact with a predator by receiving water from these conspecifics. Together with other research results and with the translational relevance of this fish species, our data points to zebrafish as a promising animal model to study human alcoholism.

  18. Cell adhesion in zebrafish embryos is modulated by March 8.

    PubMed

    Kim, Mi Ha; Rebbert, Martha L; Ro, Hyunju; Won, Minho; Dawid, Igor B

    2014-01-01

    March 8 is a member of a family of transmembrane E3 ubiquitin ligases that have been studied mostly for their role in the immune system. We find that March 8 is expressed in the zebrafish egg and early embryo, suggesting a role in development. Both knock-down and overexpression of March 8 leads to abnormal development. The phenotype of zebrafish embryos and Xenopus animal explants overexpressing March 8 implicates impairment of cell adhesion as a cause of the effect. In zebrafish embryos and in cultured cells, overexpression of March 8 leads to a reduction in the surface levels of E-cadherin, a major cell-cell adhesion molecule. Experiments in cell culture further show that E-cadherin can be ubiquitinated by March 8. On the basis of these observations we suggest that March 8 functions in the embryo to modulate the strength of cell adhesion by regulating the localization of E-cadherin.

  19. A modular, low-cost robot for zebrafish handling.

    PubMed

    Pfriem, Alexander; Pylatiuk, Christian; Alshut, Rüdiger; Ziegener, Bertram; Schulz, Stefan; Bretthauer, Georg

    2012-01-01

    The zebrafish (danio rerio) is one of the most important model organisms in modern drug discovery and disease modeling. Handling and analyzing large numbers of zebrafish larvae require an immense manpower and involve time-consuming manual processes. A novel modular, robotic platform for high-throughput screening is being developed at BioRobotLab (KIT). In this article the fish sorter, which is a robotic device for the automation of a manual process in bio analysis, is presented. The fish sorter detects randomly spread zebrafish eggs and larvae up to an age of 120 hours post fertilization (hpf) in Petri dishes and transfers them to standard 96- or 384- well plates. The robot is controlled by an advanced algorithm with sensor-based process control. Fast and precise hardware components lead to a high working speed and success rate >= 95%.

  20. Exploring Hallucinogen Pharmacology and Psychedelic Medicine with Zebrafish Models.

    PubMed

    Kyzar, Evan J; Kalueff, Allan V

    2016-10-01

    After decades of sociopolitical obstacles, the field of psychiatry is experiencing a revived interest in the use of hallucinogenic agents to treat brain disorders. Along with the use of ketamine for depression, recent pilot studies have highlighted the efficacy of classic serotonergic hallucinogens, such as lysergic acid diethylamide and psilocybin, in treating addiction, post-traumatic stress disorder, and anxiety. However, many basic pharmacological and toxicological questions remain unanswered with regard to these compounds. In this study, we discuss psychedelic medicine as well as the behavioral and toxicological effects of hallucinogenic drugs in zebrafish. We emphasize this aquatic organism as a model ideally suited to assess both the potential toxic and therapeutic effects of major known classes of hallucinogenic compounds. In addition, novel drugs with hallucinogenic properties can be efficiently screened using zebrafish models. Well-designed preclinical studies utilizing zebrafish can contribute to the reemerging treatment paradigm of psychedelic medicine, leading to new avenues of clinical exploration for psychiatric disorders.

  1. Aquatic blues: modeling depression and antidepressant action in zebrafish.

    PubMed

    Nguyen, Michael; Stewart, Adam Michael; Kalueff, Allan V

    2014-12-03

    Depression is a serious psychiatric condition affecting millions of patients worldwide. Unipolar depression is characterized by low mood, anhedonia, social withdrawal and other severely debilitating psychiatric symptoms. Bipolar disorder manifests in alternating depressed mood and 'hyperactive' manic/hypomanic states. Animal experimental models are an invaluable tool for research into the pathogenesis of bipolar/unipolar depression, and for the development of potential treatments. Due to their high throughput value, genetic tractability, low cost and quick reproductive cycle, zebrafish (Danio rerio) have emerged as a promising new model species for studying brain disorders. Here, we discuss the developing utility of zebrafish for studying depression disorders, and outline future areas of research in this field. We argue that zebrafish represent a useful model organism for studying depression and its behavioral, genetic and physiological mechanisms, as well as for anti-depressant drug discovery.

  2. The Hypocretin/Orexin Neuronal Networks in Zebrafish.

    PubMed

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

    2016-12-24

    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.

  3. Using engineered endonucleases to create knockout and knockin zebrafish models

    PubMed Central

    Bedell, Victoria M.; Ekker, Stephen C.

    2015-01-01

    Summary Over the last few years, the technology to create targeted knockout and knockin zebrafish animals has exploded. We have gained the ability to create targeted knockouts through the use of zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats/CRISPR associated system (CRISPR/Cas). Furthermore, using the high-efficiency TALEN system, we were able to create knockin zebrafish using a single-stranded DNA (ssDNA) protocol described here. Through the use of these technologies, the zebrafish has become a valuable vertebrate model and an excellent bridge between the invertebrate and mammalian model systems for the study of human disease. PMID:25408414

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

    PubMed Central

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

    2017-01-01

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

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

    PubMed Central

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

    2012-01-01

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

  6. Zebrafish embryos as an alternative to animal experiments--a commentary on the definition of the onset of protected life stages in animal welfare regulations.

    PubMed

    Strähle, Uwe; Scholz, Stefan; Geisler, Robert; Greiner, Petra; Hollert, Henner; Rastegar, Sepand; Schumacher, Axel; Selderslaghs, Ingrid; Weiss, Carsten; Witters, Hilda; Braunbeck, Thomas

    2012-04-01

    Worldwide, the zebrafish has become a popular model for biomedical research and (eco)toxicology. Particularly the use of embryos is receiving increasing attention, since they are considered as replacement method for animal experiments. Zebrafish embryos allow the analysis of multiple endpoints ranging from acute and developmental toxicity determination to complex functional genetic and physiological analysis. Particularly the more complex endpoints require the use of post-hatched eleutheroembryo stages. According to the new EU Directive 2010/63/EU on the protection of animals used for scientific purposes, the earliest life-stages of animals are not defined as protected and, therefore, do not fall into the regulatory frameworks dealing with animal experimentation. Independent feeding is considered as the stage from which free-living larvae are subject to regulations for animal experimentation. However, despite this seemingly clear definition, large variations exist in the interpretation of this criterion by national and regional authorities. Since some assays require the use of post-hatched stages up to 120 h post fertilization, the literature and available data are reviewed in order to evaluate if this stage could still be considered as non-protected according to the regulatory criterion of independent feeding. Based on our analysis and by including criteria such as yolk consumption, feeding and swimming behavior, we conclude that zebrafish larvae can indeed be regarded as independently feeding from 120 h after fertilization. Experiments with zebrafish should thus be subject to regulations for animal experiments from 120 h after fertilization onwards.

  7. The Effect of Zeaxanthin on the Visual Acuity of Zebrafish.

    PubMed

    Saidi, Eric A; Davey, Pinakin Gunvant; Cameron, D Joshua

    2015-01-01

    Oral supplementation of carotenoids such as zeaxanthin or lutein which naturally occur in human retina have been shown to improve vision and prevent progression of damage to advanced AMD in some studies. The zebrafish eye shares many physiological similarities with the human eye and is increasingly being used as model for vision research. We hypothesized that injection of zeaxanthin into the zebrafish eye would improve the visual acuity of the zebrafish over time. Visual acuity, calculated in cycles per degree, was measured in adult zebrafish to establish a consistent baseline using the optokinetic response. Zeaxanthin dissolved into phosphate buffered saline (PBS) or PBS only was injected into the anterior chamber of the right and left eyes of the Zebrafish. Visual acuities were measured at 1 week and 3, 8 and 12 weeks post-injection to compare to baseline values. Repeated measures ANOVA was used to compare visual acuities between fish injected with PBS and zeaxanthin. A significant improvement in visual acuity, 14% better than before the injection (baseline levels), was observed one week after injection with zeaxanthin (p = 0.04). This improvement peaked at more than 30% for some fish a few weeks after the injection and improvement in vision persisted at 3 weeks after injection (p = 0.006). The enhanced visual function was not significantly better than baseline at 8 weeks (p = 0.19) and returned to baseline levels 12 weeks after the initial injection (p = 0.50). Zeaxanthin can improve visual acuity in zebrafish eyes. Further studies are required to develop a better understanding of the role zeaxanthin and other carotenoids play during normal visual function.

  8. Zebrafish Model for Functional Screening of Flow-Responsive Genes

    PubMed Central

    Serbanovic-Canic, Jovana; de Luca, Amalia; Warboys, Christina; Ferreira, Pedro F.; Luong, Le A.; Hsiao, Sarah; Gauci, Ismael; Mahmoud, Marwa; Feng, Shuang; Souilhol, Celine; Bowden, Neil; Ashton, John-Paul; Walczak, Henning; Firmin, David; Krams, Rob; Mason, Justin C.; Haskard, Dorian O.; Sherwin, Spencer; Ridger, Victoria; Chico, Timothy J.A.

    2017-01-01

    Objective— Atherosclerosis is initiated at branches and bends of arteries exposed to disturbed blood flow that generates low shear stress. This mechanical environment promotes lesions by inducing endothelial cell (EC) apoptosis and dysfunction via mechanisms that are incompletely understood. Although transcriptome-based studies have identified multiple shear-responsive genes, most of them have an unknown function. To address this, we investigated whether zebrafish embryos can be used for functional screening of mechanosensitive genes that regulate EC apoptosis in mammalian arteries. Approach and Results— First, we demonstrated that flow regulates EC apoptosis in developing zebrafish vasculature. Specifically, suppression of blood flow in zebrafish embryos (by targeting cardiac troponin) enhanced that rate of EC apoptosis (≈10%) compared with controls exposed to flow (≈1%). A panel of candidate regulators of apoptosis were identified by transcriptome profiling of ECs from high and low shear stress regions of the porcine aorta. Genes that displayed the greatest differential expression and possessed 1 to 2 zebrafish orthologues were screened for the regulation of apoptosis in zebrafish vasculature exposed to flow or no-flow conditions using a knockdown approach. A phenotypic change was observed in 4 genes; p53-related protein (PERP) and programmed cell death 2–like protein functioned as positive regulators of apoptosis, whereas angiopoietin-like 4 and cadherin 13 were negative regulators. The regulation of perp, cdh13, angptl4, and pdcd2l by shear stress and the effects of perp and cdh13 on EC apoptosis were confirmed by studies of cultured EC exposed to flow. Conclusions— We conclude that a zebrafish model of flow manipulation coupled to gene knockdown can be used for functional screening of mechanosensitive genes in vascular ECs, thus providing potential therapeutic targets to prevent or treat endothelial injury at atheroprone sites. PMID:27834691

  9. Graph Theoretical Model of a Sensorimotor Connectome in Zebrafish

    PubMed Central

    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. PMID:22624008

  10. Defective Glycinergic Synaptic Transmission in Zebrafish Motility Mutants

    PubMed Central

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

    2009-01-01

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

  11. Genetic Analysis of Histamine Signaling in Larval Zebrafish Sleep

    PubMed Central

    Oikonomou, Grigorios

    2017-01-01

    Abstract Pharmacological studies in mammals and zebrafish suggest that histamine plays an important role in promoting arousal. However, genetic studies using rodents with disrupted histamine synthesis or signaling have revealed only subtle or no sleep/wake phenotypes. Studies of histamine function in mammalian arousal are complicated by its production in cells of the immune system and its roles in humoral and cellular immunity, which can have profound effects on sleep/wake states. To avoid this potential confound, we used genetics to explore the role of histamine in regulating sleep in zebrafish, a diurnal vertebrate in which histamine production is restricted to neurons in the brain. Similar to rodent genetic studies, we found that zebrafish that lack histamine due to mutation of histidine decarboxylase (hdc) exhibit largely normal sleep/wake behaviors. Zebrafish containing predicted null mutations in several histamine receptors also lack robust sleep/wake phenotypes, although we are unable to verify that these mutants are completely nonfunctional. Consistent with some rodent studies, we found that arousal induced by overexpression of the neuropeptide hypocretin (Hcrt) or by stimulation of hcrt-expressing neurons is not blocked in hdc or hrh1 mutants. We also found that the number of hcrt-expressing or histaminergic neurons is unaffected in animals that lack histamine or Hcrt signaling, respectively. Thus, while acute pharmacological manipulation of histamine signaling has been shown to have profound effects on zebrafish and mammalian sleep, our results suggest that chronic loss of histamine signaling due to genetic mutations has only subtle effects on sleep in zebrafish, similar to rodents. PMID:28275716

  12. Persistent behavioral impairment caused by embryonic methylphenidate exposure in zebrafish.

    PubMed

    Levin, Edward D; Sledge, Damiyon; Roach, Stephanie; Petro, Ann; Donerly, Susan; Linney, Elwood

    2011-01-01

    As more adults take the stimulant medication methylphenidate to treat attention deficit hyperactivity disorder (ADHD) residual type, the risk arises with regard to exposure during early development if people taking the medication become pregnant. We studied the neurobehavioral effects of methylphenidate in zebrafish. Zebrafish offer cellular reporter systems, continuous visual access and molecular interventions such as morpholinos to help determine critical mechanisms underlying neurobehavioral teratogenicity. Previously, we had seen that persisting neurobehavioral impairment in zebrafish with developmental chlorpyrifos exposure was associated with disturbed dopamine systems. Because methylphenidate is an indirect dopamine agonist, it was thought that it might also cause persistent behavioral impairment after developmental exposure. Zebrafish embryos were exposed to the ADHD stimulant medication methylphenidate 0-5 days post fertilization (12.5-50mg/l). They were tested for long-term behavioral effects as adults. Methylphenidate exposure (50mg/l) caused significant increases in dopamine, norepinepherine and serotonin on day 6 but not day 30 after fertilization. In the novel tank diving test of predatory avoidance developmental methylphenidate (50mg/l) caused a significant reduction in the normal diving response. In the three-chamber spatial learning task early developmental methylphenidate (50mg/l) caused a significant impairment in choice accuracy. These data show that early developmental exposure of zebrafish to methylphenidate causes a long-term impairment in neurobehavioral plasticity. The identification of these functional deficits in zebrafish enables further studies with this model to determine how molecular and cellular mechanisms are disturbed to arrive at this compromised state.

  13. Immunocytochemical characterisation of olfactory ensheathing cells of zebrafish

    PubMed Central

    Lazzari, Maurizio; Bettini, Simone; Franceschini, Valeria

    2014-01-01

    Continuous lifelong neurogenesis is typical of the vertebrate olfactory system. The regenerative ability of olfactory receptor neurons is dependent on the glial cell type specific to the olfactory pathway, designated ‘olfactory ensheathing cells'. Several studies to date have focused on mammalian olfactory ensheathing cells, owing to their potential roles in cell-based therapy for spinal cord injury repair. However, limited information is available regarding this glial cell type in non-mammalian vertebrates, particularly anamniotes. In the current immunocytochemical study, we analysed the features of olfactory ensheathing cells in the zebrafish, Danio rerio. Fish provide a good model for studying glial cells associated with the olfactory pathway of non-mammalian vertebrates. In particular, zebrafish has numerous valuable features that enable its use as a prime model organism for genetic, neurobiological and developmental studies, as well as toxicology and genomics research. Paraffin sections from decalcified heads of zebrafish were processed immunocytochemically to detect proteins used in the research on mammalian olfactory ensheathing cells, including glial fibrillary acid protein (GFAP), S100, neural cell adhesion molecule (NCAM), polysialylated NCAM (PSA-NCAM), vimentin (VIM), p75NTR and galactin (Gal)-1. Notably, GFAP, S100, NCAM and Gal-1 were clearly observed, whereas no vimentin staining was detected. Weak immunostaining for PSA-NCAM and p75NTR was evident. Moreover the degree of marker expression was not uniform in various tracts of the zebrafish olfactory pathway. The immunostaining patterns of the zebrafish olfactory system are distinct from those of other fish to some extent, suggesting interspecific differences. We also showed that the olfactory pathway of zebrafish expresses markers of mammalian olfactory ensheathing cells. The olfactory systems of vertebrates have similarities but there are also marked variations between them. The issue of whether

  14. Graph theoretical model of a sensorimotor connectome in zebrafish.

    PubMed

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

    2012-01-01

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

  15. The development of zebrafish tendon and ligament progenitors.

    PubMed

    Chen, Jessica W; Galloway, Jenna L

    2014-05-01

    Despite the importance of tendons and ligaments for transmitting movement and providing stability to the musculoskeletal system, their development is considerably less well understood than that of the tissues they serve to connect. Zebrafish have been widely used to address questions in muscle and skeletal development, yet few studies describe their tendon and ligament tissues. We have analyzed in zebrafish the expression of several genes known to be enriched in mammalian tendons and ligaments, including scleraxis (scx), collagen 1a2 (col1a2) and tenomodulin (tnmd), or in the tendon-like myosepta of the zebrafish (xirp2a). Co-expression studies with muscle and cartilage markers demonstrate the presence of scxa, col1a2 and tnmd at sites between the developing muscle and cartilage, and xirp2a at the myotendinous junctions. We determined that the zebrafish craniofacial tendon and ligament progenitors are neural crest derived, as in mammals. Cranial and fin tendon progenitors can be induced in the absence of differentiated muscle or cartilage, although neighboring muscle and cartilage are required for tendon cell maintenance and organization, respectively. By contrast, myoseptal scxa expression requires muscle for its initiation. Together, these data suggest a conserved role for muscle in tendon development. Based on the similarities in gene expression, morphology, collagen ultrastructural arrangement and developmental regulation with that of mammalian tendons, we conclude that the zebrafish tendon populations are homologous to their force-transmitting counterparts in higher vertebrates. Within this context, the zebrafish model can be used to provide new avenues for studying tendon biology in a vertebrate genetic system.

  16. The art of fin regeneration in zebrafish.

    PubMed

    Pfefferli, Catherine; Jaźwińska, Anna

    2015-04-01

    The zebrafish fin provides a valuable model to study the epimorphic type of regeneration, whereby the amputated part of the appendage is nearly perfectly replaced. To accomplish fin regeneration, two reciprocally interacting domains need to be established at the injury site, namely a wound epithelium and a blastema. The wound epithelium provides a supporting niche for the blastema, which contains mesenchyme-derived progenitor cells for the regenerate. The fate of blastemal daughter cells depends on their relative position with respect to the fin margin. The apical compartment of the outgrowth maintains its undifferentiated character, whereas the proximal descendants of the blastema progressively switch from the proliferation program to the morphogenesis program. A delicate balance between self-renewal and differentiation has to be continuously adjusted during the course of regeneration. This review summarizes the current knowledge about the cellular and molecular mechanisms of blastema formation, and discusses several studies related to the regulation of growth and morphogenesis during fin regeneration. A wide range of canonical signaling pathways has been implicated during the establishment and maintenance of the blastema. Epigenetic mechanisms play a crucial role in the regulation of cellular plasticity during the transition between differentiation states. Ion fluxes, gap-junctional communication and protein phosphatase activity have been shown to coordinate proliferation and tissue patterning in the caudal fin. The identification of the downstream targets of the fin regeneration signals and the discovery of mechanisms integrating the variety of input pathways represent exciting future aims in this fascinating field of research.

  17. The art of fin regeneration in zebrafish

    PubMed Central

    Pfefferli, Catherine

    2015-01-01

    Abstract The zebrafish fin provides a valuable model to study the epimorphic type of regeneration, whereby the amputated part of the appendage is nearly perfectly replaced. To accomplish fin regeneration, two reciprocally interacting domains need to be established at the injury site, namely a wound epithelium and a blastema. The wound epithelium provides a supporting niche for the blastema, which contains mesenchyme‐derived progenitor cells for the regenerate. The fate of blastemal daughter cells depends on their relative position with respect to the fin margin. The apical compartment of the outgrowth maintains its undifferentiated character, whereas the proximal descendants of the blastema progressively switch from the proliferation program to the morphogenesis program. A delicate balance between self‐renewal and differentiation has to be continuously adjusted during the course of regeneration. This review summarizes the current knowledge about the cellular and molecular mechanisms of blastema formation, and discusses several studies related to the regulation of growth and morphogenesis during fin regeneration. A wide range of canonical signaling pathways has been implicated during the establishment and maintenance of the blastema. Epigenetic mechanisms play a crucial role in the regulation of cellular plasticity during the transition between differentiation states. Ion fluxes, gap‐junctional communication and protein phosphatase activity have been shown to coordinate proliferation and tissue patterning in the caudal fin. The identification of the downstream targets of the fin regeneration signals and the discovery of mechanisms integrating the variety of input pathways represent exciting future aims in this fascinating field of research. PMID:27499869

  18. Cadmium affects retinogenesis during zebrafish embryonic development

    SciTech Connect

    Hen Chow, Elly Suk; Yu Hui, Michelle Nga; Cheng, Chi Wa; Cheng, Shuk Han

    2009-02-15

    Ocular malformations are commonly observed in embryos of aquatic species after exposure to toxicants. Using zebrafish embryos as the model organism, we showed that cadmium exposure from sphere stage (4 hpf) to end of segmentation stage (24 hpf) induced microphthalmia in cadmium-treated embryos. Embryos with eye defects were then assessed for visual abilities. Cadmium-exposed embryos were behaviorally blind, showing hyperpigmentation and loss of camouflage response to light. We investigated the cellular basis of the formation of the small eyes phenotype and the induction of blindness by studying retina development and retinotectal projections. Retinal progenitors were found in cadmium-treated embryos albeit in smaller numbers. The number of retinal ganglion cells (RGC), the first class of retinal cells to differentiate during retinogenesis, was reduced, while photoreceptor cells, the last batch of retinal neurons to differentiate, were absent. Cadmium also affected the propagation of neurons in neurogenic waves. The neurons remained in the ventronasal area and failed to spread across the retina. Drastically reduced RGC axons and disrupted optic stalk showed that the optic nerves did not extend from the retina beyond the chiasm into the tectum. Our data suggested that impairment in neuronal differentiation of the retina, disruption in RGC axon formation and absence of cone photoreceptors were the causes of microphthalmia and visual impairment in cadmium-treated embryos.

  19. Hydroxylated PBDEs induce developmental arrest in zebrafish.

    PubMed

    Usenko, Crystal Y; Hopkins, David C; Trumble, Stephen J; Bruce, Erica D

    2012-07-01

    The ubiquitous spread of polybrominated diphenyl ethers (PBDEs) has led to concerns regarding the metabolites of these congeners, in particular hydroxylated PBDEs. There are limited studies regarding the biological interactions of these chemicals, yet there is some concern they may be more toxic than their parent compounds. In this study three hydroxylated PBDEs were assessed for toxicity in embryonic zebrafish: 3-OH-BDE 47, 5-OH-BDE 47, and 6-OH-BDE 47. All three congeners induced developmental arrest in a concentration-dependent manner; however, 6-OH-BDE 47 induced adverse effects at lower concentrations than the other congeners. Furthermore, all three induced cell death; however apoptosis was not observed. In short-term exposures (24-28 hours post fertilization), all hydroxylated PBDEs generated oxidative stress in the region corresponding to the cell death at 5 and 10 ppm. To further investigate the short-term effects that may be responsible for the developmental arrest observed in this study, gene regulation was assessed for embryos exposed to 0.625 ppm 6-OH-BDE 47 from 24 to 28 hpf. Genes involved in stress response, thyroid hormone regulation, and neurodevelopment were significantly upregulated compared to controls; however, genes related to oxidative stress were either unaffected or downregulated. This study suggests that hydroxylated PBDEs disrupt development, and may induce oxidative stress and potentially disrupt the cholinergic system and thyroid hormone homeostasis.

  20. Zebrafish Genomic Instability Mutants and Cancer Susceptibility

    PubMed Central

    Moore, Jessica L.; Rush, Lindsay M.; Breneman, Carol; Mohideen, Manzoor-Ali P. K.; Cheng, Keith C.

    2006-01-01

    Somatic loss of tumor suppressor gene function comprising the second hit of Knudson's two-hit hypothesis is important in human cancer. A genetic screen was performed in zebrafish (Danio rerio) to find mutations that cause genomic instability (gin), as scored by Streisinger's mosaic-eye assay that models this second hit. The assay, based on a visible test for loss of wild-type gene function at a single locus, golden, is representative of genomewide events. Twelve ENU-induced genomic instability (gin) mutations were isolated. Most mutations showed weak dominance in heterozygotes and all showed a stronger phenotype in homozygotes. Trans-heterozygosity for 7 of these mutations showed greatly enhanced instability. A variety of spontaneous tumors were found in heterozygous adults from all gin lines, consistent with the expectation that genomic instability (mutator) mutations can accelerate carcinogenesis. The incidence of spontaneous cancer at 30–34 months was increased 9.6-fold in heterozygotes for the mutant with the strongest phenotype, gin-10. Tumors were seen in skin, colon, kidney, liver, pancreas, ovary, testis, and neuronal tissues, with multiple tumors in some fish. The study of these mutants will add to our understanding of the mechanisms of somatic loss of gene function and how those mechanisms contribute to cancer susceptibility. PMID:16888336

  1. Sensitivity to Dioxin Decreases as Zebrafish Mature

    PubMed Central

    Lanham, Kevin A.; Peterson, Richard E.; Heideman, Warren

    2012-01-01

    The embryos of teleost fish are exquisitely sensitive to the toxic effects of exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, several lines of evidence suggest that adults are less sensitive to TCDD. To better understand and characterize this difference between early life stage and adults, we exposed zebrafish (Danio rerio) to graded TCDD concentrations at different ages. The LD50 for embryos exposed at 1 day post-fertilization (dpf) was more than an order of magnitude lower than it was for juveniles exposed at 30 dpf. The latency between exposure and response also increased with age. Embryo toxicity was characterized by marked cardiovascular collapse and heart malformation, whereas juveniles exposed at 30 dpf had no detectable cardiovascular toxicity. In juveniles, the effects of TCDD exposure included stunted growth, altered pigmentation, and skeletal malformations. Furthermore, the transcriptional profile produced in hearts exposed to TCDD as embryos had very little overlap with the transcriptional changes induced by TCDD at 30 dpf. The early cardiotoxic response was associated with fish exposed prior to metamorphosis from the larval to the adult body plan at approximately 14 dpf. Our results show conclusively that the developmental stage at the time of exposure controls the toxic response to TCDD. PMID:22403156

  2. Neural alterations from lead exposure in zebrafish.

    PubMed

    Roy, Nicole M; DeWolf, Sarah; Schutt, Alexius; Wright, Ashia; Steele, Latina

    2014-01-01

    Lead was used extensively as a gas additive and pesticide, in paints, batteries, lead shot, pipes, canning and toy manufacturing. Although uses of lead have been restricted, lead persists in our environment especially in older homes, and generally in soil and water. Although extensive studies have determined that fetal and childhood exposures to lead have been associated with childhood and adolescent memory impairments and learning disabilities, there are limited studies investigating early neural and morphological effects that may lead to these behavioral and learning abnormalities. Here we utilize the zebrafish vertebrate model system to study early effects of lead exposure on the brain. We treat embryos with 0.2mM lead for 24, 48 and 72 h and analyze neural structures through live imagery and transgenic approaches. We find structural abnormalities in the hindbrain region as well as changes in branchiomotor neuron development and altered neural vasculature. Additionally, we find areas of increased apoptosis. We conclude that lead is developmentally neurotoxic to a specific region of the brain, the hindbrain and is toxic to branchiomotor neurons residing in rhombomeres 2 through 7 of the hindbrain and hindbrain central artery vasculature.

  3. Ca2+ dynamics in zebrafish morphogenesis

    PubMed Central

    Tsutsui, Kenta; Ogawa, Tomohisa

    2017-01-01

    Intracellular calcium ion (Ca2+) signaling is heavily involved in development, as illustrated by the use of a number of Ca2+ indicators. However, continuous Ca2+ patterns during morphogenesis have not yet been studied using fluorescence resonance energy transfer to track the Ca2+ sensor. In the present study, we monitored Ca2+ levels during zebrafish morphogenesis and differentiation with yellow cameleon, YC2.12. Our results show not only clear changes in Ca2+ levels but also continuous Ca2+ patterns at 24 hpf and later periods for the first time. Serial Ca2+dynamics during early pharyngula period (Prim-5-20; 24–33 hpf) was successfully observed with cameleon, which have not reported anywhere yet. In fact, high Ca2+ level occurred concurrently with hindbrain development in segmentation and pharyngula periods. Ca2+ patterns in the late gastrula through segmentation periods which were obtained with cameleon, were similar to those obtained previously with other Ca2+sensor. Our results suggested that the use of various Ca2+ sensors may lead to novel findings in studies of Ca2+ dynamics. We hope that these results will prove valuable for further research in Ca2+ signaling. PMID:28133572

  4. Toxicity of silver nanoparticles in zebrafish models.

    PubMed

    Asharani, P V; Lian Wu, Yi; Gong, Zhiyuan; Valiyaveettil, Suresh

    2008-06-25

    This study was initiated to enhance our insight on the health and environmental impact of silver nanoparticles (Ag-np). Using starch and bovine serum albumin (BSA) as capping agents, silver nanoparticles were synthesized to study their deleterious effects and distribution pattern in zebrafish embryos (Danio rerio). Toxicological endpoints like mortality, hatching, pericardial edema and heart rate were recorded. A concentration-dependent increase in mortality and hatching delay was observed in Ag-np treated embryos. Additionally, nanoparticle treatments resulted in concentration-dependent toxicity, typified by phenotypes that had abnormal body axes, twisted notochord, slow blood flow, pericardial edema and cardiac arrhythmia. Ag(+) ions and stabilizing agents showed no significant defects in developing embryos. Transmission electron microscopy (TEM) of the embryos demonstrated that nanoparticles were distributed in the brain, heart, yolk and blood of embryos as evident from the electron-dispersive x-ray analysis (EDS). Furthermore, the acridine orange staining showed an increased apoptosis in Ag-np treated embryos. These results suggest that silver nanoparticles induce a dose-dependent toxicity in embryos, which hinders normal development.

  5. The neural basis of visual behaviors in the larval zebrafish.

    PubMed

    Portugues, Ruben; Engert, Florian

    2009-12-01

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

  6. Systemic Fluorescence Imaging of Zebrafish Glycans with Bioorthogonal Chemistry.

    PubMed

    Agarwal, Paresh; Beahm, Brendan J; Shieh, Peyton; Bertozzi, Carolyn R

    2015-09-21

    Vertebrate glycans constitute a large, important, and dynamic set of post-translational modifications that are notoriously difficult to manipulate and image. Although the chemical reporter strategy has been used in conjunction with bioorthogonal chemistry to image the external glycosylation state of live zebrafish and detect tumor-associated glycans in mice, the ability to image glycans systemically within a live organism has remained elusive. Here, we report a method that combines the metabolic incorporation of a cyclooctyne-functionalized sialic acid derivative with a ligation reaction of a fluorogenic tetrazine, allowing for the imaging of sialylated glycoconjugates within live zebrafish embryos.

  7. The neural basis of visual behaviors in the larval zebrafish

    PubMed Central

    Portugues, Ruben; Engert, Florian

    2015-01-01

    We review visually guided behaviors in larval zebrafish and summarise what is known about the neural processing that results in these behaviors, paying particular attention to the progress made in the last 2 years. Using the examples of the optokinetic reflex, the optomotor response, prey tracking and the visual startle response, we illustrate how the larval zebrafish presents us with a very promising model vertebrate system that allows neurocientists to integrate functional and behavioral studies and from which we can expect illuminating insights in the near future. PMID:19896836

  8. Phenylthiourea alters toxicity of mercury compounds in zebrafish larvae.

    PubMed

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

    2015-10-01

    In recent years larval stage zebrafish have been emerging as a standard vertebrate model in a number of fields, ranging from developmental biology to pharmacology and toxicology. The tyrosinase inhibitor 1-phenyl-2-thiourea (PTU) is used very widely with larval zebrafish to generate essentially transparent organisms through inhibition of melanogenesis, which has enabled many elegant studies in areas ranging from neurological development to cancer research. Here we show that PTU can have dramatic synergistic and antagonistic effects on the chemical toxicology of different mercury compounds. Our results indicate that extreme caution should be used when employing PTU in toxicological studies, particularly when studying toxic metal ions.

  9. Next generation mothers: Maternal control of germline development in zebrafish.

    PubMed

    Dosch, Roland

    2015-01-01

    In many animals, factors deposited by the mother into the egg control the earliest events in development of the zygote. These maternal RNAs and proteins play critical roles in oocyte development and the earliest steps of embryogenesis such as fertilization, cell division and embryonic patterning. Here, this article summarizes recent discoveries made on the maternal control of germline specification in zebrafish. Moreover, this review will discuss the major gaps remaining in our understanding of this process and highlight recent technical innovations in zebrafish, which allow tackling some of these questions in the near future.

  10. astray, a zebrafish roundabout homolog required for retinal axon guidance.

    PubMed

    Fricke, C; Lee, J S; Geiger-Rudolph, S; Bonhoeffer, F; Chien, C B

    2001-04-20

    As growing retinotectal axons navigate from the eye to the tectum, they sense guidance molecules distributed along the optic pathway. Mutations in the zebrafish astray gene severely disrupt retinal axon guidance, causing anterior-posterior pathfinding defects, excessive midline crossing, and defasciculation of the retinal projection. Eye transplantation experiments show that astray function is required in the eye. We identify astray as zebrafish robo2, a member of the Roundabout family of axon guidance receptors. Retinal ganglion cells express robo2 as they extend axons. Thus, robo2 is required for multiple axon guidance decisions during establishment of the vertebrate visual projection.

  11. Insights into kidney stem cell development and regeneration using zebrafish

    PubMed Central

    Drummond, Bridgette E; Wingert, Rebecca A

    2016-01-01

    Kidney disease is an escalating global health problem, for which the formulation of therapeutic approaches using stem cells has received increasing research attention. The complexity of kidney anatomy and function, which includes the diversity of renal cell types, poses formidable challenges in the identification of methods to generate replacement structures. Recent work using the zebrafish has revealed their high capacity to regenerate the integral working units of the kidney, known as nephrons, following acute injury. Here, we discuss these findings and explore the ways that zebrafish can be further utilized to gain a deeper molecular appreciation of renal stem cell biology, which may uncover important clues for regenerative medicine. PMID:26981168

  12. Uranium-induced sensory alterations in the zebrafish Danio rerio.

    PubMed

    Faucher, K; Floriani, M; Gilbin, R; Adam-Guillermin, C

    2012-11-15

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

  13. Distinct Functions of Different scl Isoforms in Zebrafish Definitive Hematopoietic Stem Cell Initiation and Maintenance

    NASA Astrophysics Data System (ADS)

    Lan, Yahui

    2011-07-01

    The establishment of entire blood system relies on the multi-potent hematopoietic stem cells (HSCs), thus identifying the molecular mechanism in HSC generation is of importance for not only complementing the fundamental knowledge in stem cell biology, but also providing insights to the regenerative therapies. Recent researches have documented the formation of nascent HSCs through a direct transition from ventral aortic endothelium, named as endothelial hematopoietic transition (EHT) process. However, the precise genetic program engaged in this process remains largely elusive. The transcription factor scl plays pivotal and conserved roles in embryonic and adult hematopoiesis from teleosts to mammals. Our lab have previously identified a new truncated scl isoform, scl-beta, which is indispensible for the specification of HSCs in the ventral wall of dorsal aorta (VDA), the zebrafish equivalent of mammalian fetal hematopoietic organ. Here we observe that, by combining time-lapse confocal imaging of transgenic zebrafish and genetic epistasis analysis, scl-beta is expressed in a subset of ventral aortic endothelial cells and critical for their forthcoming transformation to hemogenic endothelium; in contrast, runx1 is required downstream to govern the successful egress of the hemogenic endothelial cells to become naive HSCs. In addition, the traditional known full-length scl-alpha isoform is firstly evidenced to be required for the maintenance or survival of newly formed HSCs in VDA. Collectively our data has established the genetic hierarchy controlling discrete steps in the consecutive process of HSC formation from endothelial cells and further development in VDA.

  14. Spinocerebellar ataxia type 13 mutant potassium channel alters neuronal excitability and causes locomotor deficits in zebrafish.

    PubMed

    Issa, Fadi A; Mazzochi, Christopher; Mock, Allan F; Papazian, Diane M

    2011-05-04

    Whether changes in neuronal excitability can cause neurodegenerative disease in the absence of other factors such as protein aggregation is unknown. Mutations in the Kv3.3 voltage-gated K(+) channel cause spinocerebellar ataxia type 13 (SCA13), a human autosomal-dominant disease characterized by locomotor impairment and the death of cerebellar neurons. Kv3.3 channels facilitate repetitive, high-frequency firing of action potentials, suggesting that pathogenesis in SCA13 is triggered by changes in electrical activity in neurons. To investigate whether SCA13 mutations alter excitability in vivo, we expressed the human dominant-negative R420H mutant subunit in zebrafish. The disease-causing mutation specifically suppressed the excitability of Kv3.3-expressing, fast-spiking motor neurons during evoked firing and fictive swimming and, in parallel, decreased the precision and amplitude of the startle response. The dominant-negative effect of the mutant subunit on K(+) current amplitude was directly responsible for the reduced excitability and locomotor phenotype. Our data provide strong evidence that changes in excitability initiate pathogenesis in SCA13 and establish zebrafish as an excellent model system for investigating how changes in neuronal activity impair locomotor control and cause cell death.

  15. In-vivo cell tracking to quantify endothelial cell migration during zebrafish angiogenesis

    NASA Astrophysics Data System (ADS)

    Menon, Prahlad G.; Rochon, Elizabeth R.; Roman, Beth L.

    2016-03-01

    The mechanism of endothelial cell migration as individual cells or collectively while remaining an integral component of a functional blood vessel has not been well characterized. In this study, our overarching goal is to define an image processing workflow to facilitate quantification of how endothelial cells within the first aortic arch and are proximal to the zebrafish heart behave in response to the onset of flow (i.e. onset of heart beating). Endothelial cell imaging was conducted at this developmental time-point i.e. ~24-28 hours post fertilization (hpf) when flow first begins, using 3D+time two-photon confocal microscopy of a live, wild-type, transgenic, zebrafish expressing green fluorescent protein (GFP) in endothelial cell nuclei. An image processing pipeline comprised of image signal enhancement, median filtering for speckle noise reduction, automated identification of the nuclei positions, extraction of the relative movement of nuclei between consecutive time instances, and finally tracking of nuclei, was designed for achieving the tracking of endothelial cell nuclei and the identification of their movement towards or away from the heart. Pilot results lead to a hypothesis that upon the onset of heart beat and blood flow, endothelial cells migrate collectively towards the heart (by 21.51+/-10.35 μm) in opposition to blood flow (i.e. subtending 142.170+/-21.170 with the flow direction).

  16. IQGAP3 Is Essential for Cell Proliferation and Motility During Zebrafish Embryonic Development

    PubMed Central

    Fang, Xiaolan; Zhang, Bianhong; Thisse, Bernard; Bloom, George S.; Thisse, Christine

    2015-01-01

    IQGAPs are scaffolding proteins that regulate actin assembly, exocyst function, cell motility, morphogenesis, adhesion and division. Vertebrates express 3 family members: IQGAP1, IQGAP2 and IQGAP3. IQGAP1 is known to stimulate nucleation of branched actin filaments through N-WASP and the Arp2/3 complex following direct binding to cytoplasmic tails of ligand-activated growth factor receptors, including EGFR, VEGFR2 and FGFR1. By contrast, little is known about functions of IQGAP2 or IQGAP3. Using in situ hybridization on whole mount zebrafish (Danio rerio) embryos, we show that IQGAP1 and IQGAP2 are associated with discrete tissues and organs, while IQGAP3 is mainly expressed in proliferative cells throughout embryonic and larval development. Morpholino knockdowns of IQGAP1 and IQGAP2 have little effect on embryo morphology while loss of function of IQGAP3 affects both cell proliferation and cell motility. IQGAP3 morphant phenotypes are similar to those resulting from overexpression of dominant negative forms of Ras or of Fibroblast Growth Factor Receptor 1 (FGFR1), suggesting that IQGAP3 plays a role in FGFR1-Ras-ERK signaling. In support of this hypothesis, dominant negative forms of FGFR1 or Ras could be rescued by co-injection of zebrafish IQGAP3 mRNA, strongly suggesting that IQGAP3 acts as a downstream regulator of the FGFR1-Ras signaling pathway. PMID:26286209

  17. Benzo[a]pyrene decreases global and gene specific DNA methylation during zebrafish development

    PubMed Central

    Fang, Xiefan; Thornton, Cammi; Scheffler, Brian E.; Willett, Kristine L.

    2013-01-01

    DNA methylation is important for gene regulation and is vulnerable to early-life exposure to environmental contaminants. We found that direct waterborne benzo[a]pyrene (BaP) exposure at 24 μg/L from 2.5 to 96 hours post fertilization (hpf) to zebrafish embryos significantly decreased global cytosine methylation by 44.8% and promoter methylation in vasa by 17%. Consequently, vasa expression was significantly increased by 33%. In contrast, BaP exposure at environmentally relevant concentrations did not change CpG island methylation or gene expression in cancer genes such as ras-association domain family member 1 (rassf1), telomerase reverse transcriptase (tert), c-jun, and c-myca. Similarly, BaP did not change gene expression of DNA methyltransferase 1 (dnmt1) and glycine N-methyltransferase (gnmt). While total DNMT activity was not affected, GNMT enzyme activity was moderately increased. In summary, BaP is an epigenetic modifier for global and gene specific DNA methylation status in zebrafish larvae. PMID:23542452

  18. Sox10-dependent neural crest origin of olfactory microvillous neurons in zebrafish

    PubMed Central

    Saxena, Ankur; Peng, Brian N; Bronner, Marianne E

    2013-01-01

    The sense of smell in vertebrates is detected by specialized sensory neurons derived from the peripheral nervous system. Classically, it has been presumed that the olfactory placode forms all olfactory sensory neurons. In contrast, we show that the cranial neural crest is the primary source of microvillous sensory neurons within the olfactory epithelium of zebrafish embryos. Using photoconversion-based fate mapping and live cell tracking coupled with laser ablation, we followed neural crest precursors as they migrated from the neural tube to the nasal cavity. A subset that coexpressed Sox10 protein and a neurogenin1 reporter ingressed into the olfactory epithelium and differentiated into microvillous sensory neurons. Timed loss-of-function analysis revealed a critical role for Sox10 in microvillous neurogenesis. Taken together, these findings directly demonstrate a heretofore unknown contribution of the cranial neural crest to olfactory sensory neurons in zebrafish and provide important insights into the assembly of the nascent olfactory system. DOI: http://dx.doi.org/10.7554/eLife.00336.001 PMID:23539289

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

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

    2017-02-01

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

  1. Fibronectin is deposited by injury-activated epicardial cells and is necessary for zebrafish heart regeneration

    PubMed Central

    Wang, Jinhu; Karra, Ravi; Dickson, Amy L.; Poss, Kenneth D.

    2013-01-01

    Unlike adult mammals, adult zebrafish vigorously regenerate lost heart muscle in response to injury. The epicardium, a mesothelial cell layer enveloping the myocardium, is activated to proliferate after cardiac injury and can contribute vascular support cells or provide mitogens to regenerating muscle. Here, we applied proteomics to identify secreted proteins that are associated with heart regeneration. We found that Fibronectin, a main component of the extracellular matrix, is induced and deposited after cardiac damage. In situ hybridization and transgenic reporter analyses indicated that expression of two fibronectin paralogues, fn1 and fn1b, are induced by injury in epicardial cells, while the itgb3 receptor is induced in cardiomyocytes near the injury site. fn1, the more dynamic of these paralogs, is induced chamber-wide within one day of injury before localizing epicardial Fn1 synthesis to the injury site. fn1 loss-of-function mutations disrupted zebrafish heart regeneration, as did induced expression of a dominant-negative Fibronectin cassette, defects that were not attributable to direct inhibition of cardiomyocyte proliferation. These findings reveal a new role for the epicardium in establishing an extracellular environment that supports heart regeneration. PMID:23988577

  2. Structure and function relationship of Zebrafish embryonic heart from confocal microscopy images

    NASA Astrophysics Data System (ADS)

    Moghaddam, Abbas N.; Forouhar, Arian; Liebling, Michael; Tsai, Huai-Jen; Gharib, Morteza

    2006-03-01

    Confocal microscopy enables us to track myocytes in the embryonic zebrafish heart. The Zeiss LSM 5 Live high speed confocal microscope has been used to take optical sections (at 3 μm intervals and 151 frames per second) through a fluorescently labeled zebrafish heart at two developmental stages (26 and 34 hours post fertilization (hpf)). This data provides unique information allowing us to conjecture on the morphology and biomechanics of the developing vertebrate heart. Nevertheless, the myocytes, whose positions could be determined in a reliable manner, were located sparsely and mostly in one side of the heart tube. This difficulty was overcome using computational methods, that give longitudinal, radial and circumferential displacements of the myocytes as well as their contractile behavior. Applied strain analysis has shown that in the early embryonic heart tube, only the caudal region (near the in-flow) and another point in the middle of the tube can be active; the rest appears to be mostly passive. This statement is based on the delay between major strain and displacement which a material point experiences. Wave-like propagation of all three components of the displacement, especially in the circumferential direction, as well as the almost-periodic changes of the maximum strain support the hypothesis of helical muscle structure embedded in the tube. Changes of geometry in the embryonic heart after several hours are used to verify speculations about the structure based on the earlier images and aforementioned methods.

  3. Developmental toxicity of the common UV filter, benophenone-2, in zebrafish embryos.

    PubMed

    Fong, Henry C H; Ho, Jeff C H; Cheung, Angela H Y; Lai, K P; Tse, William K F

    2016-12-01

    Benozophenone (BP) type UV filters are extensively used in the personal care products to provide protection against the harmful effects of UV radiation. BPs are one of the primary components in the UV filter family, in which benophenone-2 (BP2) is widely used as a UV filter reagent in the sunscreen. Humans used these personal care products directly on skin and the chemicals will be washed away to the water system. BP2 has been identified as one of the endocrine disruptor chemicals, which can inference the synthesis, metabolism, and action of endogenous hormones. Environmentally, it has been found to contaminate water worldwide. In this study, we aimed to unfold the possible developmental toxicology of this chemical. Zebrafish are used as the screening model to perform in situ hybridization staining to investigate the effects of BP2 on segmentation, brain regionalization, and facial formation at four developmental stages (10-12 somite, prim-5, 2 and 5 days post-fertilization). Results showed 40 μM (9.85 mg L(-1)) or above BP2 exposure in zebrafish embryos for 5 days resulted in lipid accumulation in the yolk sac and facial malformation via affecting the lipid processing and the expression of cranial neural crest cells respectively. To conclude, the study alarmed its potential developmental toxicities at high dosage exposure.

  4. Analysis of the dynamic co-expression network of heart regeneration in the zebrafish

    NASA Astrophysics Data System (ADS)

    Rodius, Sophie; Androsova, Ganna; Götz, Lou; Liechti, Robin; Crespo, Isaac; Merz, Susanne; Nazarov, Petr V.; de Klein, Niek; Jeanty, Céline; González-Rosa, Juan M.; Muller, Arnaud; Bernardin, Francois; Niclou, Simone P.; Vallar, Laurent; Mercader, Nadia; Ibberson, Mark; Xenarios, Ioannis; Azuaje, Francisco

    2016-05-01

    The zebrafish has the capacity to regenerate its heart after severe injury. While the function of a few genes during this process has been studied, we are far from fully understanding how genes interact to coordinate heart regeneration. To enable systematic insights into this phenomenon, we generated and integrated a dynamic co-expression network of heart regeneration in the zebrafish and linked systems-level properties to the underlying molecular events. Across multiple post-injury time points, the network displays topological attributes of biological relevance. We show that regeneration steps are mediated by modules of transcriptionally coordinated genes, and by genes acting as network hubs. We also established direct associations between hubs and validated drivers of heart regeneration with murine and human orthologs. The resulting models and interactive analysis tools are available at http://infused.vital-it.ch. Using a worked example, we demonstrate the usefulness of this unique open resource for hypothesis generation and in silico screening for genes involved in heart regeneration.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25758224

  6. Manipulation of BK channel expression is sufficient to alter auditory hair cell thresholds in larval zebrafish

    PubMed Central

    Rohmann, Kevin N.; Tripp, Joel A.; Genova, Rachel M.; Bass, Andrew H.

    2014-01-01

    Non-mammalian vertebrates rely on electrical resonance for frequency tuning in auditory hair cells. A key component of the resonance exhibited by these cells is an outward calcium-activated potassium current that flows through large-conductance calcium-activated potassium (BK) channels. Previous work in midshipman fish (Porichthys notatus) has shown that BK expression correlates with seasonal changes in hearing sensitivity and that pharmacologically blocking these channels replicates the natural decreases in sensitivity during the winter non-reproductive season. To test the hypothesis that reducing BK channel function is sufficient to change auditory thresholds in fish, morpholino oligonucleotides (MOs) were used in larval zebrafish (Danio rerio) to alter expression of slo1a and slo1b, duplicate genes coding for the pore-forming α-subunits of BK channels. Following MO injection, microphonic potentials were recorded from the inner ear of larvae. Quantitative real-time PCR was then used to determine the MO effect on slo1a and slo1b expression in these same fish. Knockdown of either slo1a or slo1b resulted in disrupted gene expression and increased auditory thresholds across the same range of frequencies of natural auditory plasticity observed in midshipman. We conclude that interference with the normal expression of individual slo1 genes is sufficient to increase auditory thresholds in zebrafish larvae and that changes in BK channel expression are a direct mechanism for regulation of peripheral hearing sensitivity among fishes. PMID:24803460

  7. Localizing Organomercury Uptake And Accumulation in Zebrafish Larvae at the Tissue And Cellular Level

    SciTech Connect

    Korbas, M.; Blechinger, S.R.; Krone, P.H.; Pickering, I.J.; George, G.N.

    2009-05-20

    Using synchrotron x-ray fluorescence mapping, we have examined the uptake and localization of organic mercury in zebrafish larvae. Strikingly, the greatest accumulation of methyl and ethyl mercury compounds was highly localized in the rapidly dividing lens epithelium, with lower levels going to brain, optic nerve, and various other organs. The data suggest that the reported impairment of visual processes by mercury may arise not only from previously reported neurological effects, but also from direct effects on the ocular tissue. This novel approach is a powerful tool for directly investigating the molecular toxicology of heavy metals, and should be equally applicable to the study of a wide range of elements in developing embryos.

  8. Neurodevelopment. Live imaging of adult neural stem cell behavior in the intact and injured zebrafish brain.

    PubMed

    Barbosa, Joana S; Sanchez-Gonzalez, Rosario; Di Giaimo, Rossella; Baumgart, Emily Violette; Theis, Fabian J; Götz, Magdalena; Ninkovic, Jovica

    2015-05-15

    Adult neural stem cells are the source for restoring injured brain tissue. We used repetitive imaging to follow single stem cells in the intact and injured adult zebrafish telencephalon in vivo and found that neurons are generated by both direct conversions of stem cells into postmitotic neurons and via intermediate progenitors amplifying the neuronal output. We observed an imbalance of direct conversion consuming the stem cells and asymmetric and symmetric self-renewing divisions, leading to depletion of stem cells over time. After brain injury, neuronal progenitors are recruited to the injury site. These progenitors are generated by symmetric divisions that deplete the pool of stem cells, a mode of neurogenesis absent in the intact telencephalon. Our analysis revealed changes in the behavior of stem cells underlying generation of additional neurons during regeneration.

  9. Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond.

    PubMed

    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.

  10. Towards a Comprehensive Catalog of Zebrafish Behavior 1.0 and Beyond

    PubMed Central

    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

    2013-01-01

    Abstract 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. PMID:23590400

  11. Phenylthiourea specifically reduces zebrafish eye size.

    PubMed

    Li, Zeran; Ptak, Devon; Zhang, Liyun; Walls, Elwood K; Zhong, Wenxuan; Leung, Yuk Fai

    2012-01-01

    Phenylthiourea (PTU) is commonly used for inhibiting melanization of zebrafish embryos. In this study, the standard treatment with 0.2 mM PTU was demonstrated to specifically reduce eye size in larval fish starting at three days post-fertilization. This effect is likely the result of a reduction in retinal and lens size of PTU-treated eyes and is not related to melanization inhibition. This is because the eye size of tyr, a genetic mutant of tyrosinase whose activity is inhibited in PTU treatment, was not reduced. As PTU contains a thiocarbamide group which is presented in many goitrogens, suppressing thyroid hormone production is a possible mechanism by which PTU treatment may reduce eye size. Despite the fact that thyroxine level was found to be reduced in PTU-treated larvae, thyroid hormone supplements did not rescue the eye size reduction. Instead, treating embryos with six goitrogens, including inhibitors of thyroid peroxidase (TPO) and sodium-iodide symporter (NIS), suggested an alternative possibility. Specifically, three TPO inhibitors, including those that do not possess thiocarbamide, specifically reduced eye size; whereas none of the NIS inhibitors could elicit this effect. These observations indicate that TPO inhibition rather than a general suppression of thyroid hormone synthesis is likely the underlying cause of PTU-induced eye size reduction. Furthermore, the tissue-specific effect of PTU treatment might be mediated by an eye-specific TPO expression. Compared with treatment with other tyrosinase inhibitors or bleaching to remove melanization, PTU treatment remains the most effective approach. Thus, one should use caution when interpreting results that are obtained from PTU-treated embryos.

  12. Hydroxylated PBDEs induce developmental arrest in zebrafish

    SciTech Connect

    Usenko, Crystal Y. Hopkins, David C.; Trumble, Stephen J. Bruce, Erica D.

    2012-07-01

    The ubiquitous spread of polybrominated diphenyl ethers (PBDEs) has led to concerns regarding the metabolites of these congeners, in particular hydroxylated PBDEs. There are limited studies regarding the biological interactions of these chemicals, yet there is some concern they may be more toxic than their parent compounds. In this study three hydroxylated PBDEs were assessed for toxicity in embryonic zebrafish: 3-OH-BDE 47, 5-OH-BDE 47, and 6-OH-BDE 47. All three congeners induced developmental arrest in a concentration-dependent manner; however, 6-OH-BDE 47 induced adverse effects at lower concentrations than the other congeners. Furthermore, all three induced cell death; however apoptosis was not observed. In short-term exposures (24–28 hours post fertilization), all hydroxylated PBDEs generated oxidative stress in the region corresponding to the cell death at 5 and 10 ppm. To further investigate the short-term effects that may be responsible for the developmental arrest observed in this study, gene regulation was assessed for embryos exposed to 0.625 ppm 6-OH-BDE 47 from 24 to 28 hpf. Genes involved in stress response, thyroid hormone regulation, and neurodevelopment were significantly upregulated compared to controls; however, genes related to oxidative stress were either unaffected or downregulated. This study suggests that hydroxylated PBDEs disrupt development, and may induce oxidative stress and potentially disrupt the cholinergic system and thyroid hormone homeostasis. -- Highlights: ► OH-PBDEs induce developmental arrest in a concentration-dependent manner. ► Hydroxyl group location influences biological interaction. ► OH-PBDEs induce oxidative stress. ► Thyroid hormone gene regulation was disrupted following exposure. ► To our knowledge, this is the first whole organism study of OH-PBDE toxicity.

  13. Biliary atresia: From Australia to the zebrafish.

    PubMed

    Davenport, Mark

    2016-02-01

    This review is based upon an invited lecture for the 52nd Annual Meeting of the British Association of Paediatric Surgeons, July 2015. The aetiology of biliary atresia (BA) is at best obscure, but it is probable that a number of causes or pathophysiological mechanisms may be involved leading to the final common phenotype we recognise clinically. By way of illustration, similar conditions to human BA are described, including biliary agenesis, which is the normal state and peculiar final pattern of bile duct development in the jawless fish, the lamprey. Furthermore, there have been remarkable outbreaks in the Australian outback of BA in newborn lambs whose mothers were exposed to and grazed upon a particular plant species (Dysphania glomulifera) during gestation. More recent work using a zebrafish model has isolated a toxic isoflavonoid, now named Biliatresone, thought to be responsible for these outbreaks. Normal development of the bile ducts is reviewed and parallels drawn with two clinical variants thought to definitively have their origins in intrauterine life: Biliary Atresia Splenic Malformation syndrome (BASM) and Cystic Biliary Atresia (CBA). For both variants there is sufficient clinical evidence, including associated anomalies and antenatal detection, respectively, to warrant their aetiological attribution as developmental BA. CMV IgM +ve associated BA is a further variant that appears separate with distinct clinical, histological, and immunohistochemical features. In these it seems possible that this involves perinatal obliteration of a normally formed duct system. Although still circumstantial, this evidence appears convincing enough to perhaps warrant a different treatment strategy. This then still leaves the most common (more than 60% in Western series) variant, now termed Isolated BA, whereby origins can only be alluded to.

  14. A phenotypic screen in zebrafish identifies a novel small-molecule inducer of ectopic tail formation suggestive of alterations in non-canonical Wnt/PCP signaling.

    PubMed

    Gebruers, Evelien; Cordero-Maldonado, María Lorena; Gray, Alexander I; Clements, Carol; Harvey, Alan L; Edrada-Ebel, Ruangelie; de Witte, Peter A M; Crawford, Alexander D; Esguerra, Camila V

    2013-01-01

    Zebrafish have recently emerged as an attractive model for the in vivo bioassay-guided isolation and characterization of pharmacologically active small molecules of natural origin. We carried out a zebrafish-based phenotypic screen of over 3000 plant-derived secondary metabolite extracts with the goal of identifying novel small-molecule modulators of the BMP and Wnt signaling pathways. One of the bioactive plant extracts identified in this screen - Jasminum gilgianum, an Oleaceae species native to Papua New Guinea - induced ectopic tails during zebrafish embryonic development. As ectopic tail formation occurs when BMP or non-canonical Wnt signaling is inhibited during the tail protrusion process, we suspected a constituent of this extract to act as a modulator of these pathways. A bioassay-guided isolation was carried out on the basis of this zebrafish phenotype, identifying para-coumaric acid methyl ester (pCAME) as the active compound. We then performed an in-depth phenotypic analysis of pCAME-treated zebrafish embryos, including a tissue-specific marker analysis of the secondary tails. We found pCAME to synergize with the BMP-inhibitors dorsomorphin and LDN-193189 in inducing ectopic tails, and causing convergence-extension defects in compound-treated embryos. These results indicate that pCAME may interfere with non-canonical Wnt signaling. Inhibition of Jnk, a downstream target of Wnt/PCP signaling (via morpholino antisense knockdown and pharmacological inhibition with the kinase inhibitor SP600125) phenocopied pCAME-treated embryos. However, immunoblotting experiments revealed pCAME to not directly inhibit Jnk-mediated phosphorylation of c-Jun, suggesting additional targets of SP600125, and/or other pathways, as possibly being involved in the ectopic tail formation activity of pCAME. Further investigation of pCAME's mechanism of action will help determine this compound's pharmacological utility.

  15. Digestive enzymatic activity during ontogenetic development in zebrafish (Danio rerio).

    PubMed

    Guerrera, Maria Cristina; De Pasquale, Francesca; Muglia, Ugo; Caruso, Gabriella

    2015-12-01

    Despite the growing importance of zebrafish (Danio rerio) as an experimental model in biomedical research, some aspect of physiological and related morphological age dependent changes in digestive system during larval development are still unknown. In this paper, a biochemical and morphological study of the digestive tract of zebrafish was undertaken to record the functional changes occurring in this species during its ontogenetic development, particularly from 24 hr to 47 days post fertilization (dpf). Endo- and exo-proteases, as well as α-amylase enzymes, were quantified in zebrafish larvae before first feeding (7 dpf). The most morphologically significant events during the ontogenesis of the gut occurred between 3 dpf (mouth opening) and 7 dpf (end of exocrine pancreas differentiation). The presence of a wide range of digestive enzymes, already active at earlier zebrafish larval stages, closely related with the omnivorous diet of this species. Increasing enzyme activities were found with increasing age, probably in relation with intestinal mucosa folding and consequent absorption surface increase. J. Exp. Zool. (Mol. Dev. Evol.) 324B: 699-706, 2015. © 2015 Wiley Periodicals, Inc.

  16. Imaging Subcellular Structures in the Living Zebrafish Embryo.

    PubMed

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

    2016-04-02

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

  17. Ontogeny of Classical and Operant Learning Behaviors in Zebrafish

    ERIC Educational Resources Information Center

    Valente, Andre; Huang, Kuo-Hua; Portugues, Ruben; Engert, Florian

    2012-01-01

    The performance of developing zebrafish in both classical and operant conditioning assays was tested with a particular focus on the emergence of these learning behaviors during development. Strategically positioned visual cues paired with electroshocks were used in two fully automated assays to investigate both learning paradigms. These allow the…

  18. Automated image-based phenotypic analysis in zebrafish embryos

    PubMed Central

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

    2009-01-01

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

  19. Effects of Habitat Complexity on Pair-Housed Zebrafish.

    PubMed

    Keck, Victoria A; Edgerton, Dale S; Hajizadeh, Susan; Swift, Larry L; Dupont, William D; Lawrence, Christian; Boyd, Kelli L

    2015-07-01

    Sexually mature zebrafish were housed as single male-female pairs with or without plastic vegetation for 1, 5, or 10 d for comparison of whole-body cortisol measured by radioimmunoassay. Individually housed male zebrafish were used as controls. In the fish that were pair-housed without vegetation (NVeg), one animal died in 5 of 24 pairs, and one animal was alive but wounded in an additional pair. No deaths or wounds occurred in the fish that were pair-housed with vegetation (Veg). Cortisol levels did not differ between the treatment groups on day 1. On day 5, cortisol values were higher in the Veg group than in the individually housed fish (P < 0.0005) and the NVeg fish (P = 0.004). On day 10, the relationships were inversed: cortisol levels had risen in the individually housed and NVeg groups and had fallen to baseline levels in the Veg group. Cortisol values on day 10 were lower in the Veg group than in the individually housed (P = 0.004) and NVeg (P = 0.05) groups. Cortisol levels in individually housed male zebrafish increased over time. Although this study did not demonstrate a reduction in cortisol levels associated with providing vegetation, this enrichment prevented injury and death from fighting. These findings show how commonly used housing situations may affect the wellbeing of laboratory zebrafish.

  20. Microfluidics expands the zebrafish potentials in pharmaceutically relevant screening.

    PubMed

    Chen, Chia-Yuan; Cheng, Chao-Min

    2014-06-01

    The objective of this study is to enlarge the impact of microfluidics on the pharmaceutical industry by highlighting the reported scientific work on the synergistic relationship between zebrafish and microfluidics, and furthering that effort to shed light on how microfluidics can facilitate the use of zebrafish as a gene screening tool. Zebrafish is ranked the third most important animal model after rats and mice, according to a National Institutes of Health (NIH) announcement in 2003. It has become a staple for scientists to examine and subsequently begin to unravel the mystery of human diseases, and is increasingly used in toxicological studies for new drug development. The unique characteristics that this tiny fish possesses, including rapid growth rate, prodigious numbers of offspring, and eggs that develop outside the body, make it an invaluable genetic tool. Evidently, these advantages can be broadened with the addition of a properly designed microfluidic circuit. By means of the presented illustrations and demonstrated applications, the goal is to spark interest in the development of more novel microfluidic platform designs that can leverage the attributes of zebrafish and quickly come to commercial fruition.

  1. Strategies for Analyzing Cardiac Phenotypes in the Zebrafish Embryo

    PubMed Central

    Houk, Andrew R.; Yelon, Deborah

    2017-01-01

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

  2. Toxicity of Vascular Disrupting Chemicals to Developing Zebrafish

    EPA Science Inventory

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

  3. Trim69 regulates zebrafish brain development by ap-1 pathway

    PubMed Central

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

    2016-01-01

    Proteins belonging to the TRIM family have been implicated in a variety of cellular processes such as apoptosis, differentiation, neurogenesis, muscular physiology and innate immune responses. Trim69, previously identified as a novel gene cloned from a human testis cDNA library, has a homologous gene in zebrafish and this study focused on investigating the function of trim69 in zebrafish neurogenesis. Trim69 was found to be expressed in zebrafish embryo brain at the early stages. Knockdown of trim69 led to deformed brain development, obvious signs of apoptosis present in the head, and decreased expression of neuronal differentiation and stem cell markers. This phenotype was rescued upon co-injection of human mRNA together along with the trim69 knockdown. Results of this study also showed an interaction between TRIM69 and c-Jun in human cells, and upon TRIM69 knock down c-Jun expression subsequently increased, whereas the over-expression of TRIM69 led to the down-regulation of c-Jun. Additionally, knockdown both c-Jun and trim69 can rescue the deformed brain, evident cellular apoptosis in the head and decreased expression of neuronal differentiation and stem cell markers. Overall, our results support a role for trim69 in the development of the zebrafish brain through ap-1 pathway. PMID:27050765

  4. Behavorial Screens for Detecting Developmental Neurotoxicity in Larval Zebrafish

    EPA Science Inventory

    As part of the EPA's effort to develop an in vivo, vertebrate screen for toxic chemicals, we have characterized basic behaviors of 6-day post-fertilization (dpf) zebrafish (Danio rerio) larvae in a microtiter plate format. Our main goal is to develop a convenient, reproducible me...

  5. Preference for ethanol in zebrafish following a single exposure.

    PubMed

    Mathur, Priya; Berberoglu, Michael A; Guo, Su

    2011-02-02

    Ethanol is one of the most widely abused drugs in the world. Its addictive property is believed to primarily stem from its ability to influence the brain reinforcement pathway evolved for mediating natural rewards. Although dopamine is a known component of the reinforcement pathway, clear molecular and cellular compositions of this pathway and its sensitivity to ethanol remain not well understood. Zebrafish has been increasingly used to model and understand human disease states, due to its genetic tractability and ease of maintenance. In this study, we determine whether adult zebrafish develop ethanol preference after a single exposure using a conditioned place preference (CPP) paradigm. Moreover, we establish a procedure that can be carried out in an automated and relatively high-throughput fashion. We find that zebrafish of the AB strain display significantly increased preference for the compartment where they received ethanol during a single 20-min exposure. The largest increase in preference is in response to a 1.5% ethanol administered in the tank water. The results demonstrate robust ethanol preference in zebrafish. Such a relatively high-throughput assay with automated tracking and response to a single ethanol exposure provides a potential means for a large-scale screening aimed at understanding the brain reinforcement pathway and its sensitivity to ethanol in this genetically tractable vertebrate.

  6. The mitochondrial thioredoxin is required for liver development in zebrafish.

    PubMed

    Zhang, J; Cui, X; Wang, L; Liu, F; Jiang, T; Li, C; Li, D; Huang, M; Liao, S; Wang, J; Chen, J; Jia, H; He, J; Tang, Z; Yin, Z; Liu, M

    2014-01-01

    Thioredoxins (Trxs) are a class of small molecular redox proteins that play an important role in scavenging abnormally accumulated reactive oxygen species (ROS). Thioredoxin 2 (Trx2) is one member of this family located in mitochondria. Trx2 protects cells from increased oxidative stress and has anti-apoptosis function. Knockout of Trx2 in mice led to early embryonic lethality. However, the essential role of Trx2 during embryogenesis remains unclear. To further investigate the role of Trx2 during embryonic development, we performed Trx2 knockdown in zebrafish and investigated the regulation role of Trx2 during embryonic development. Our results indicate that Trx2 had a high expression in early zebrafish embryos and its knockdown in zebrafish led to defective liver development mainly due to increased hepatic cell death. The increased ROS and the imbalance of members of the Bcl-2 family were involved in cell death induced by Trx2 suppression in zebrafish. The dysregulation of Bax, puma and Bcl-xl promoted the reduction of mitochondrial trans-membrane potential and the mitochondria membrane permeabilization (MMP), which initiated the mitochondrial apoptosis pathway. Additionally, we found that the increase of relocated GAPDH in mitochondria may be another factor responsible for the mitochondrial catastrophe.

  7. A Sketch of the Taiwan Zebrafish Core Facility.

    PubMed

    You, May-Su; Jiang, Yun-Jin; Yuh, Chiou-Hwa; Wang, Chien-Ming; Tang, Chih-Hao; Chuang, Yung-Jen; Lin, Bo-Hung; Wu, Jen-Leih; Hwang, Sheng-Ping L

    2016-07-01

    In the past three decades, the number of zebrafish laboratories has significantly increased in Taiwan. The Taiwan Zebrafish Core Facility (TZCF), a government-funded core facility, was launched to serve this growing community. The Core Facility was built on two sites, one located at the National Health Research Institutes (NHRI, called Taiwan Zebrafish Core Facility at NHRI or TZeNH) and the other is located at the Academia Sinica (Taiwan Zebrafish Core Facility at AS a.k.a. TZCAS). The total surface area of the TZCF is about 180 m(2) encompassing 2880 fish tanks. Each site has a separate quarantine room and centralized water recirculating systems, monitoring key water parameters. To prevent diseases, three main strategies have been implemented: (1) imported fish must be quarantined; (2) only bleached embryos are introduced into the main facilities; and (3) working practices were implemented to minimize pathogen transfer between stocks and facilities. Currently, there is no health program in place; however, a fourth measure for the health program, specific regular pathogen tests, is being planned. In March 2015, the TZCF at NHRI has been AAALAC accredited. It is our goal to ensure that we provide "disease-free" fish and embryos to the Taiwanese research community.

  8. Retinoic acid expands the evolutionarily reduced dentition of zebrafish

    PubMed Central

    Seritrakul, Pawat; Samarut, Eric; Lama, Tenzing T. S.; Gibert, Yann; Laudet, Vincent; Jackman, William R.

    2012-01-01

    Zebrafish lost anterior teeth during evolution but retain a posterior pharyngeal dentition that requires retinoic acid (RA) cell-cell signaling for its development. The purposes of this study were to test the sufficiency of RA to induce tooth development and to assess its role in evolution. We found that exposure of embryos to exogenous RA induces a dramatic anterior expansion of the number of pharyngeal teeth that later form and shifts anteriorly the expression patterns of genes normally expressed in the posterior tooth-forming region, such as pitx2 and dlx2b. After RA exposure, we also observed a correlation between cartilage malformations and ectopic tooth induction, as well as abnormal cranial neural crest marker gene expression. Additionally, we observed that the RA-induced zebrafish anterior teeth resemble in pattern and number the dentition of fish species that retain anterior pharyngeal teeth such as medaka but that medaka do not express the aldh1a2 RA-synthesizing enzyme in tooth-forming regions. We conclude that RA is sufficient to induce anterior ectopic tooth development in zebrafish where teeth were lost in evolution, potentially by altering neural crest cell development, and that changes in the location of RA synthesis correlate with evolutionary changes in vertebrate dentitions.—Seritrakul, P., Samarut, E., Lama, T. T. S., Gibert, Y., Laudet, V., Jackman, W. R. Retinoic acid expands the evolutionarily reduced dentition of zebrafish. PMID:22942074

  9. Automated Processing of Zebrafish Imaging Data: A Survey

    PubMed Central

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

    2013-01-01

    Abstract Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines. PMID:23758125

  10. Differential reinforcement of an approach response in zebrafish (Danio rerio).

    PubMed

    Manabe, Kazuchika; Dooling, R J; Takaku, Shinichi

    2013-09-01

    Five zebrafish were trained to approach a target using a fully automated training procedure. During a training session, if the distance between the fish and the target was closer than an arbitrarily set distance, the approach response was reinforced by food. The fish continued to respond under this reinforcement contingency and the distance criterion could be shortened up to eighty times within a 1h session. The initial distance limit was then shortened for the next test training session. Once the initial distance criterion was reduced to a final minimum distance, the distance criterion was fixed at this value for the next nine successive sessions. In a second experiment using different fish, we manipulated approach distances in three conditions. The first condition was identical to the changing criterion training as in Experiment 1. In the second condition, only response distances under a distance criterion were reinforced. And in the last condition, only response distances over the distance criterion were reinforced. Results show that zebrafish can control the distance between themselves and a target. In other words, zebrafish are sensitive to the spatial consequences of their behavior. The present results show that a differential reinforcement paradigm can be successfully applied to zebrafish which therefore enhances their value as a vertebrate model for studies of complex behavior including visuomotor learning.

  11. Simple and Rapid Quantification of Thrombocytes in Zebrafish Larvae

    PubMed Central

    Huarng, Michael C.

    2015-01-01

    Abstract Platelets are a critical component of hemostasis, with disorders of number or function resulting in coagulation disturbances. Insights into these processes have primarily been realized through studies using mammalian models or tissues. Increasingly, zebrafish embryos and larvae have been used to study the protein and cellular components of hemostasis and thrombosis, including the thrombocyte, a nucleated platelet analog. However, investigations of thrombocytes have been somewhat limited due to lack of a robust and simple methodology for quantitation, an important component of platelet studies in mammals. Using video capture, we have devised an assay that produces a rapid, reproducible, and precise measurement of thrombocyte number in zebrafish larvae by counting fluorescently tagged cells. Averaging 1000 frames, we were able to subtract background fluorescence, thus limiting assessment to circulating thrombocytes. This method facilitated rapid assessment of relative thrombocyte counts in a population of 372 zebrafish larvae by a single operator in less than 3 days. This technique requires basic microscopy equipment and rudimentary programming, lends itself to high throughput analysis, and will enhance future studies of thrombopoiesis in the zebrafish. PMID:25790244

  12. Effects of Habitat Complexity on Pair-Housed Zebrafish

    PubMed Central

    Keck, Victoria A; Edgerton, Dale S; Hajizadeh, Susan; Swift, Larry L; Dupont, William D; Lawrence, Christian; Boyd, Kelli L

    2015-01-01

    Sexually mature zebrafish were housed as single male-female pairs with or without plastic vegetation for 1, 5, or 10 d for comparison of whole-body cortisol measured by radioimmunoassay. Individually housed male zebrafish were used as controls. In the fish that were pair-housed without vegetation (NVeg), one animal died in 5 of 24 pairs, and one animal was alive but wounded in an additional pair. No deaths or wounds occurred in the fish that were pair-housed with vegetation (Veg). Cortisol levels did not differ between the treatment groups on day 1. On day 5, cortisol values were higher in the Veg group than in the individually housed fish (P < 0.0005) and the NVeg fish (P = 0.004). On day 10, the relationships were inversed: cortisol levels had risen in the individually housed and NVeg groups and had fallen to baseline levels in the Veg group. Cortisol values on day 10 were lower in the Veg group than in the individually housed (P = 0.004) and NVeg (P = 0.05) groups. Cortisol levels in individually housed male zebrafish increased over time. Although this study did not demonstrate a reduction in cortisol levels associated with providing vegetation, this enrichment prevented injury and death from fighting. These findings show how commonly used housing situations may affect the wellbeing of laboratory zebrafish. PMID:26224437

  13. Discrimination reversal and attentional sets in zebrafish (Danio rerio)

    PubMed Central

    Parker, Matthew O.; Gaviria, Jessica; Haigh, Alastair; Millington, Mollie E.; Brown, Verity J.; Combe, Fraser J.; Brennan, Caroline H.

    2014-01-01

    The potential of zebrafish as a comparative model in behavioural neuroscience is currently hampered only by the lack of reliable and validated behavioural assays available to researchers. In the present experiment, we describe the performance of zebrafish in a test of attentional set formation. The fish were initially trained on a two-choice colour discrimination. Upon reaching acquisition criterion, the reinforced alternative was switched to the previously unreinforced alternative. Again, upon reaching criterion, the cues were replaced with a novel pair of colours (intra-dimensional shift) and reversed again on reaching criteria. We found that zebrafish show a steady decrease in trials-to-criteria over the four phases of the experiment, suggesting that they are forming and maintaining an attentional set, as has previously been demonstrated with mammals. Reversal learning deficits have been implicated in a variety of human psychological disorders (e.g., disorders of impulse control) and as such, we propose that performance of zebrafish in this procedure may represent a useful comparative model to complement existing rodent models. PMID:22561034

  14. Genetic Linkage Mapping of Zebrafish Genes and ESTs

    PubMed Central

    Kelly, Peter D.; Chu, Felicia; Woods, Ian G.; Ngo-Hazelett, Phuong; Cardozo, Timothy; Huang, Hui; Kimm, Frankie; Liao, Lingya; Yan, Yi-Lin; Zhou, Yingyao; Johnson, Steven L.; Abagyan, Ruben; Schier, Alexander F.; Postlethwait, John H.; Talbot, William S.

    2000-01-01

    Genetic screens in zebrafish (Danio rerio) have isolated mutations in hundreds of genes essential for vertebrate development, physiology, and behavior. We have constructed a genetic linkage map that will facilitate the identification of candidate genes for these mutations and allow comparisons among the genomes of zebrafish and other vertebrates. On this map, we have localized 771 zebrafish genes and expressed sequence tags (ESTs) by scoring single-stranded conformational polymorphisms (SSCPs) in a meiotic mapping panel. Of these sequences, 642 represent previously unmapped genes and ESTs. The mapping panel was comprised of 42 homozygous diploid individuals produced by heat shock treatment of haploid embryos at the one-cell stage (HS diploids). This “doubled haploid” strategy combines the advantages of mapping in haploid and standard diploid systems, because heat shock diploid individuals have only one allele at each locus and can survive to adulthood, enabling a relatively large quantity of genomic DNA to be prepared from each individual in the mapping panel. To integrate this map with others, we also scored 593 previously mapped simple-sequence length polymorphisms (SSLPs) in the mapping panel. This map will accelerate the molecular analysis of zebrafish mutations and facilitate comparative analysis of vertebrate genomes. [A table of the mapped genes and ESTs is provided online at http://www.genome.org.] PMID:10779498

  15. Mitragynine attenuates withdrawal syndrome in morphine-withdrawn zebrafish.

    PubMed

    Khor, Beng-Siang; Jamil, Mohd Fadzly Amar; Adenan, Mohamad Ilham; Shu-Chien, Alexander Chong

    2011-01-01

    A major obstacle in treating drug addiction is the severity of opiate withdrawal syndrome, which can lead to unwanted relapse. Mitragynine is the major alkaloid compound found in leaves of Mitragyna speciosa, a plant widely used by opiate addicts to mitigate the harshness of drug withdrawal. A series of experiments was conducted to investigate the effect of mitragynine on anxiety behavior, cortisol level and expression of stress pathway related genes in zebrafish undergoing morphine withdrawal phase. Adult zebrafish were subjected to two weeks chronic morphine exposure at 1.5 mg/L, followed by withdrawal for 24 hours prior to tests. Using the novel tank diving tests, we first showed that morphine-withdrawn zebrafish display anxiety-related swimming behaviors such as decreased exploratory behavior and increased erratic movement. Morphine withdrawal also elevated whole-body cortisol levels, which confirms the phenotypic stress-like behaviors. Exposing morphine-withdrawn fish to mitragynine however attenuates majority of the stress-related swimming behaviors and concomitantly lower whole-body cortisol level. Using real-time PCR gene expression analysis, we also showed that mitragynine reduces the mRNA expression of corticotropin releasing factor receptors and prodynorphin in zebrafish brain during morphine withdrawal phase, revealing for the first time a possible link between mitragynine's ability to attenuate anxiety during opiate withdrawal with the stress-related corticotropin pathway.

  16. Zebrafish antipredatory responses: A future for translational research?

    PubMed Central

    Gerlai, Robert

    2011-01-01

    Human neuropsychiatric conditions associated with abnormally exaggerated or misdirected fear (anxiety disorders and phobias) still represent a large unmet medical need because the biological mechanisms underlying these diseases are not well understood. Animal models have been proposed to facilitate this research. Here I review the literature with a focus on zebrafish, an upcoming laboratory organism in behavioral brain research. I argue that abnormal human fear responses are likely the result of the malfunction of neurobiological mechanisms (brain areas, circuits and/or molecular mechanisms) that originally evolved to support avoidance of predators or other harm in nature. I also argue that the understanding of the normal as well as pathological functioning of such mechanisms may be best achieved if one utilizes naturalistic experimental approaches. In case of laboratory model organisms, this may entail presenting stimuli associated with predators and measuring species-specific antipredatory responses. Although zebrafish is a relatively new subject of such inquiry, I review the recently rapidly increasing number of zebrafish studies in this area, and conclude that zebrafish is a promising research tool for the analysis of the neurobiology and genetics of vertebrate fear responses. PMID:19836422

  17. Mixing and pumping functions of the intestine of zebrafish larvae.

    PubMed

    Yang, Jinyou; Shimogonya, Yuji; Ishikawa, Takuji

    2017-02-07

    Due to its transparency, the intestine of zebrafish larvae has been widely used in studies of gastrointestinal diseases and the microbial flora of the gut. However, transport phenomena in the intestine of zebrafish larvae have not been fully clarified. In this study, therefore, transport caused by peristaltic motion in the intestine of zebrafish larvae was investigated by numerical simulation. An anatomically realistic three-dimensional geometric model of the intestine at various times after feeding was constructed based on the experimental data of Field et al. (2009). The flow of digested chyme was analyzed using the governing equations of fluid mechanics, together with peristaltic motion and long-term contraction of the intestinal wall. The results showed that retrograde peristaltic motion was the main contributor to the mixing function. The dispersion caused by peristalsis over 30min was in the order of 10(-12)m(2)/s, which is greater than the Brownian diffusion of a sphere of 0.4µm diameter. In contrast, anterograde peristaltic motion contributed mainly to the pumping function. The pressure decrease due to peristalsis was in the order of millipascals, which may reduce the activation and maintenance heat of intestinal muscle. These findings enhance our understanding of the mixing and pumping functions of the intestine of zebrafish larvae.

  18. A new model to study visual attention in zebrafish.

    PubMed

    Braida, Daniela; Ponzoni, Luisa; Martucci, Roberta; Sala, Mariaelvina

    2014-12-03

    The major part of cognitive tasks applied to zebrafish has not fully assessed their attentional ability, a process by which the nervous system learns, organizes sensory input and generates coordinated behaviour. In an attempt to maximize the value of zebrafish as an animal model of cognition, we tested the possibility to apply a modified version of novel object recognition test named virtual object recognition test (VORT) using 2D geometrical shapes (square, triangle, circle, cross, etc.) on two iPod 3.5-inch widescreen displays, located on two opposite walls of the water tank. Each fish was subjected to a familiarization trial (T1), and after different time delays (from 5 min to 96 h) to a novel shape recognition trial (T2). A progressive decrease, across time, of memory performance, in terms of mean discrimination index and mean exploration time, was shown. The predictive validity was tested using cholinergic drugs. Nicotine (0.02 mg/kg intraperitoneally, IP) significantly increased, while scopolamine (0.025 mg/kg IP) and mecamylamine decreased, mean discrimination index. Zebrafish discriminated different movements (vertical, horizontal, oblique) and the discrimination index increased significantly when moving poorly discriminated shapes were presented, thus increasing visual attention. Taken together these findings demonstrate that VORT is a viable, fast and useful model to evaluate sustained attention in zebrafish and for predicting the efficacy of pharmacotherapies for cognitive disorders.

  19. Acute Neuroactive Drug Exposures alter Locomotor Activity in Larval Zebrafish

    EPA Science Inventory

    As part of the development of a rapid in vivo screen for prioritization of toxic chemicals, we have begun to characterize the locomotor activity of zebrafish (Danio rerio) larvae by assessing the acute effects of prototypic drugs that act on the central nervous system. Initially,...

  20. Gene and protein expression biomarkers in fungicide exposed zebrafish

    EPA Science Inventory

    In this study, the impact of prochloraz (PCZ) on reproductively mature male and female zebrafish was examined following up to 96 h continuous exposure to a flow-through system to control (water only), low (100 ug/l) and high (500 ug/l) PCZ dose. An imidazole fungicide used to rpo...

  1. Short-term memory in zebrafish (Danio rerio).

    PubMed

    Jia, Jason; Fernandes, Yohaan; Gerlai, Robert

    2014-08-15

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

  2. Vascular wall shear stress in zebrafish model of early atherosclerosis

    NASA Astrophysics Data System (ADS)

    Choi, Woorak; Seo, Eunseok; Yeom, Eunseop; Lee, Sang Joon

    2016-11-01

    Although atherosclerosis is a multifactorial disease, the role of hemodynamic force has strong influence on the outbreak of the disease. Low and oscillating wall shear stress (WSS) is associated with the incidence of atherosclerosis. Many researchers have investigated relationships between WSS and the occurrence of atherosclerosis using in vitro and in vivo models. However, these models possess technological limitations in mimicking real biophysiological conditions and monitoring the temporal progression of atherosclerosis. In this study, a hypercholesterolaemic zebrafish model was established as a novel model to resolve these technical limitations. WSS in blood vessels of 15 days post-fertilisation zebrafish was measured using a micro PIV technique, and the spatial distribution of lipids inside blood vessels was quantitatively visualized using a confocal microscopy. As a result, lipids are mainly deposited in the regions of low WSS. The oscillating WSS is not induced by blood flows in the zebrafish disease model. The present hypercholesterolaemic zebrafish model would be useful for understanding the effect of WSS on the early stage of atherosclerosis. This work was supported by the National Research Foundation of Korea (NRF) under a Grant funded by the Korean government (MSIP) (No. 2008-0061991).

  3. Souffle/Spastizin controls secretory vesicle maturation during zebrafish oogenesis.

    PubMed

    Kanagaraj, Palsamy; Gautier-Stein, Amandine; Riedel, Dietmar; Schomburg, Christoph; Cerdà, Joan; Vollack, Nadine; Dosch, Roland

    2014-06-01

    During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research.

  4. Souffle/Spastizin Controls Secretory Vesicle Maturation during Zebrafish Oogenesis

    PubMed Central

    Riedel, Dietmar; Schomburg, Christoph; Cerdà, Joan; Vollack, Nadine; Dosch, Roland

    2014-01-01

    During oogenesis, the egg prepares for fertilization and early embryogenesis. As a consequence, vesicle transport is very active during vitellogenesis, and oocytes are an outstanding system to study regulators of membrane trafficking. Here, we combine zebrafish genetics and the oocyte model to identify the molecular lesion underlying the zebrafish souffle (suf) mutation. We demonstrate that suf encodes the homolog of the Hereditary Spastic Paraplegia (HSP) gene SPASTIZIN (SPG15). We show that in zebrafish oocytes suf mutants accumulate Rab11b-positive vesicles, but trafficking of recycling endosomes is not affected. Instead, we detect Suf/Spastizin on cortical granules, which undergo regulated secretion. We demonstrate genetically that Suf is essential for granule maturation into secretion competent dense-core vesicles describing a novel role for Suf in vesicle maturation. Interestingly, in suf mutants immature, secretory precursors accumulate, because they fail to pinch-off Clathrin-coated buds. Moreover, pharmacological inhibition of the abscission regulator Dynamin leads to an accumulation of immature secretory granules and mimics the suf phenotype. Our results identify a novel regulator of secretory vesicle formation in the zebrafish oocyte. In addition, we describe an uncharacterized cellular mechanism for Suf/Spastizin activity during secretion, which raises the possibility of novel therapeutic avenues for HSP research. PMID:24967841

  5. Neurochemical anatomy of the zebrafish retina as determined by immunocytochemistry.

    PubMed

    Yazulla, S; Studholme, K M

    2001-07-01

    The zebrafish retina is rapidly becoming a major preparation for the study of molecular genetic mechanisms underlying neural development and visual behavior. Studies utilizing retinal mutants would benefit by the availability of a data base on the distribution of neurotransmitter systems in the wild-type fish. To this end, the neurochemical anatomy of the zebrafish retina was surveyed by light microscopic immunocytochemistry. An extensive series of 60 separate antibodies were used to describe the distribution of major transmitter systems and a variety of neuron-associated membrane channels and proteins. These include markers (i.e., antibodies against enzymes, receptors, transporters) for transmitters: GABA, glycine, glutamate, biogenic amines, acetylcholine, cannabinoids and neuropeptides; as well as a sample of voltage-gated channels and synapse associated membrane proteins. Discussion of the comparative localization of these antibodies is restricted to other teleost fishes, particularly goldfish. Overall, there was great similarity in the distribution of the various markers, as might be expected. However, there were some notable differences, including several antibodies that did not label zebrafish at all, even though goldfish retinas that were processed in parallel, labeled beautifully. This survey is extensive, but not exhaustive, and hopefully will serve as a valuable resource for future studies of the zebrafish retina.

  6. Large-scale zebrafish embryonic heart dissection for transcriptional analysis.

    PubMed

    Lombardo, Verónica A; Otten, Cécile; Abdelilah-Seyfried, Salim

    2015-01-12

    The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

  7. Zebrafish response to a robotic replica in three dimensions

    PubMed Central

    Ruberto, Tommaso; Mwaffo, Violet; Singh, Sukhgewanpreet; Neri, Daniele

    2016-01-01

    As zebrafish emerge as a species of choice for the investigation of biological processes, a number of experimental protocols are being developed to study their social behaviour. While live stimuli may elicit varying response in focal subjects owing to idiosyncrasies, tiredness and circadian rhythms, video stimuli suffer from the absence of physical input and rely only on two-dimensional projections. Robotics has been recently proposed as an alternative approach to generate physical, customizable, effective and consistent stimuli for behavioural phenotyping. Here, we contribute to this field of investigation through a novel four-degree-of-freedom robotics-based platform to manoeuvre a biologically inspired three-dimensionally printed replica. The platform enables three-dimensional motions as well as body oscillations to mimic zebrafish locomotion. In a series of experiments, we demonstrate the differential role of the visual stimuli associated with the biologically inspired replica and its three-dimensional motion. Three-dimensional tracking and information-theoretic tools are complemented to quantify the interaction between zebrafish and the robotic stimulus. Live subjects displayed a robust attraction towards the moving replica, and such attraction was lost when controlling for its visual appearance or motion. This effort is expected to aid zebrafish behavioural phenotyping, by offering a novel approach to generate physical stimuli moving in three dimensions. PMID:27853566

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

    PubMed Central

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

    2017-01-01

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

  9. Zebrafish as a disease model for studying human hepatocellular carcinoma

    PubMed Central

    Lu, Jeng-Wei; Ho, Yi-Jung; Yang, Yi-Ju; Liao, Heng-An; Ciou, Shih-Ci; Lin, Liang-In; Ou, Da-Liang

    2015-01-01

    Liver cancer is one of the world’s most common cancers and the second leading cause of cancer deaths. Hepatocellular carcinoma (HCC), a primary hepatic cancer, accounts for 90%-95% of liver cancer cases. The pathogenesis of HCC consists of a stepwise process of liver damage that extends over decades, due to hepatitis, fatty liver, fibrosis, and cirrhosis before developing fully into HCC. Multiple risk factors are highly correlated with HCC, including infection with the hepatitis B or C viruses, alcohol abuse, aflatoxin exposure, and metabolic diseases. Over the last decade, genetic alterations, which include the regulation of multiple oncogenes or tumor suppressor genes and the activation of tumorigenesis-related pathways, have also been identified as important factors in HCC. Recently, zebrafish have become an important living vertebrate model organism, especially for translational medical research. In studies focusing on the biology of cancer, carcinogen induced tumors in zebrafish were found to have many similarities to human tumors. Several zebrafish models have therefore been developed to provide insight into the pathogenesis of liver cancer and the related drug discovery and toxicology, and to enable the evaluation of novel small-molecule inhibitors. This review will focus on illustrative examples involving the application of zebrafish models to the study of human liver disease and HCC, through transgenesis, genome editing technology, xenografts, drug discovery, and drug-induced toxic liver injury. PMID:26576090

  10. Efficient transgenesis mediated by pigmentation rescue in zebrafish.

    PubMed

    Harrold, Itrat; Carbonneau, Seth; Moore, Bethany M; Nguyen, Gina; Anderson, Nicole M; Saini, Amandeep S; Kanki, John P; Jette, Cicely A; Feng, Hui

    2016-01-01

    The zebrafish represents a revolutionary tool in large-scale genetic and small-molecule screens for gene and drug discovery. Transgenic zebrafish are often utilized in these screens. Many transgenic fish lines are maintained in the heterozygous state due to the lethality associated with homozygosity; thus, their progeny must be sorted to ensure a population expressing the transgene of interest for use in screens. Sorting transgenic embryos under a fluorescence microscope is very labor-intensive and demands fine-tuned motor skills. Here we report an efficient transgenic method of utilizing pigmentation rescue of nacre mutant fish for accurate naked-eye identification of both mosaic founders and stable transgenic zebrafish. This was accomplished by co-injecting two constructs with the I-SceI meganuclease enzyme into pigmentless nacre embryos: I-SceI-mitfa:mitfa-I-SceI to rescue the pigmentation and I-SceI-zpromoter:gene-of-interest-I-SceI to express the gene of interest under a zebrafish promoter (zpromoter). Pigmentation rescue reliably predicted transgene integration. Compared with other transgenic techniques, our approach significantly increases the overall percentage of founders and facilitates accurate naked-eye identification of stable transgenic fish, greatly reducing laborious fluorescence microscope sorting and PCR genotyping. Thus, this approach is ideal for generating transgenic fish for large-scale screens.

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

    EPA Science Inventory

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

  12. Repairing quite swimmingly: advances in regenerative medicine using zebrafish

    PubMed Central

    Goessling, Wolfram; North, Trista E.

    2014-01-01

    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. PMID:24973747

  13. Acute neuroactive drug exposures alter locomotor activity in larval zebrafish

    EPA Science Inventory

    In an effort to develop a rapid in vivo screen for EPA's prioritization of toxic chemicals, we are characterizing the locomotor activity of zebrafish (Danio rerio) larvae after exposure to prototypic drugs that act on the central nervous system. MPTP (1-methyl-4phenyl- 1 ,2,3,6-...

  14. Determinism and stochasticity during maturation of the zebrafish antibody repertoire

    PubMed Central

    Jiang, Ning; Weinstein, Joshua A.; Penland, Lolita; White, Richard A.; Fisher, Daniel S.; Quake, Stephen R.

    2011-01-01

    It is thought that the adaptive immune system of immature organisms follows a more deterministic program of antibody creation than is found in adults. We used high-throughput sequencing to characterize the diversifying antibody repertoire in zebrafish over five developmental time points. We found that the immune system begins in a highly stereotyped state with preferential use of a small number of V (variable) D (diverse) J (joining) gene segment combinations, but that this stereotypy decreases dramatically as the zebrafish mature, with many of the top VDJ combinations observed in 2-wk-old zebrafish virtually disappearing by 1 mo. However, we discovered that, in the primary repertoire, there are strong correlations in VDJ use that increase with zebrafish maturity, suggesting that VDJ recombination involves a level of deterministic programming that is unexpected. This stereotypy is masked by the complex diversification processes of antibody maturation; the variation and lack of correlation in full repertoires between individuals appears to be derived from randomness in clonal expansion during the affinity maturation process. These data provide a window into the mechanisms of VDJ recombination and diversity creation and allow us to better understand how the adaptive immune system achieves diversity. PMID:21393572

  15. Tween-80 and impurity induce anaphylactoid reaction in zebrafish.

    PubMed

    Yang, Rui; Lao, Qiao-Cong; Yu, Hang-Ping; Zhang, Yong; Liu, Hong-Cui; Luan, Lin; Sun, Hui-Min; Li, Chun-Qi

    2015-03-01

    A number of recent reports suspected that Tween-80 in injectable medicines, including traditional Chinese medicine injections could cause life-threatening anaphylactoid reaction, but no sound conclusion was drawn. A drug-induced anaphylactoid reaction is hard to be assayed in vitro and in conventional animal models. In this study, we developed a microplate-based quantitative in vivo zebrafish assay for assessing anaphylactoid reaction and live whole zebrafish mast cell tryptase activity was quantitatively measured at a wavelength of 405 nm using N-benzoyl-dl-arginine p-nitroanilide as a substrate. We assessed 10 batches of Tween-80 solutions from various national and international suppliers and three Tween-80 impurities (ethylene glycol, 2-chloroethanol and hydrogen peroxide) in this model and found that three batches of Tween-80 (nos 2, 20080709 and 20080616) and one Tween-80 impurity, hydrogen peroxide (H2 O2 ), induced anaphylactoid reactions in zebrafish. Furthermore, we found that H2 O2 residue and peroxide value were much higher in Tween-80 samples 2, 20080709 and 20080616. These findings suggest that H2 O2 residue in combination with oxidized fatty acid residues (measured as peroxide value) or more likely the oxidized fatty acid residues in Tween-80 samples, but not Tween-80 itself, may induce anaphylactoid reaction. High-throughput zebrafish tryptase assay developed in this report could be used for assessing safety of Tween-80-containing injectable medicines and potentially for screening novel mast cell-modulating drugs.

  16. Early Retinoic acid deprivation in developing zebrafish results in microphthalmia

    PubMed Central

    Le, Hong-Gam T.; Dowling, John E.; Cameron, D. Joshua

    2013-01-01

    Vitamin A deficiency causes impaired vision and blindness in millions of children around the world. Previous studies in zebrafish have demonstrated that retinoic acid (RA), the acid form of vitamin A, plays a vital role in early eye development. The objective of this study was to describe the effects of early RA deficiency by treating zebrafish with diethylaminobenzaldehyde (DEAB), a potent inhibitor of the enzyme retinaldehyde dehydrogenase (Raldh) that converts retinal to RA. Zebrafish embryos were treated for 2 hours beginning at 9 hours post-fertilization (hpf). Gross morphology and retinal development were examined at regular intervals for 5 days after treatment. The optokinetic reflex (OKR) test, visual background adaptation (VBA) test, and the electroretinogram (ERG) were performed to assess visual function and behavior. Early treatment of zebrafish embryos with 100 μM DEAB (9hr) resulted in reduced eye size and this microphthalmia persisted through larval development. Retinal histology revealed that DEAB eyes, had significant developmental abnormalities but had relatively normal retinal lamination by 5.5 days post-fertilization (dpf). However, the fish showed neither, an OKR or VBA response. Further, the retina did not respond to light as measured by the ERG. We conclude that early deficiency of RA during eye development causes microphthalmia as well as other visual defects, and that timing of the RA deficiency is critical to the developmental outcome. PMID:23013828

  17. Zebrafish as animal model for aquaculture nutrition research

    PubMed Central

    Ulloa, Pilar E.; Medrano, Juan F.; Feijoo, Carmen G.

    2014-01-01

    The aquaculture industry continues to promote the diversification of ingredients used in aquafeed in order to achieve a more sustainable aquaculture production system. The evaluation of large numbers of diets in aquaculture species is costly and requires time-consuming trials in some species. In contrast, zebrafish (Danio rerio) can solve these drawbacks as an experimental model, and represents an ideal organism to carry out preliminary evaluation of diets. In addition, zebrafish has a sequenced genome allowing the efficient utilization of new technologies, such as RNA-sequencing and genotyping platforms to study the molecular mechanisms that underlie the organism’s response to nutrients. Also, biotechnological tools like transgenic lines with fluorescently labeled neutrophils that allow the evaluation of the immune response in vivo, are readily available in this species. Thus, zebrafish provides an attractive platform for testing many ingredients to select those with the highest potential of success in aquaculture. In this perspective article aspects related to diet evaluation in which zebrafish can make important contributions to nutritional genomics and nutritional immunity are discussed. PMID:25309575

  18. Shoaling develops with age in Zebrafish (Danio rerio).

    PubMed

    Buske, Christine; Gerlai, Robert

    2011-08-01

    The biological mechanisms of human social behavior are complex. Animal models may facilitate the understanding of these mechanisms and may help one to develop treatment strategies for abnormal human social behavior, a core symptom in numerous clinical conditions. The zebrafish is perhaps the most social vertebrate among commonly used laboratory species. Given its practical features and the numerous genetic tools developed for it, it should be a promising tool. Zebrafish shoal, i.e. from a tight multimember groups, but the ontogenesis of this behavior has not been described. Analyzing the development of shoaling is a step towards discovering the mechanisms of this behavior. Here we study age-dependent changes of shoaling in zebrafish from day 7 post fertilization to over 5months of age by measuring the distance between all pairs of fish in freely swimming groups of ten subjects. Our longitudinal (repeated measure within subject) and cross sectional (non-repeated measure between subject) analyses both demonstrated a significant increase of shoaling with age (decreased distance between shoal members). Given the sophisticated genetic and developmental biology methods already available for zebrafish, we argue that our behavioral results open a new avenue towards the understanding of the development of vertebrate social behavior and of its mechanisms and abnormalities.

  19. Structural characterization of glycosaminoglycans from zebrafish in different ages

    PubMed Central

    Zhang, Fuming; Zhang, Zhenqing; Thistle, Robert; McKeen, Lindsey; Hosoyama, Saori; Toida, Toshihiko

    2009-01-01

    The zebrafish (Danio rerio) is a popular model organism for the study of developmental biology, disease mechanisms, and drug discovery. Glycosaminoglycans (GAGs), located on animal cell membranes and in the extracellular matrix, are important molecules in cellular communication during development, in normal physiology and pathophysiology. Vertebrates commonly contain a variety of GAGs including chondroitin/dermatan sulfates, heparin/heparan sulfate, hyaluronan and keratan sulfate. Zebrafish might represent an excellent experimental organism to study the biological roles of GAGs. A recent study showing the absence of heparan sulfate in adult zebrafish, suggested a more detailed evaluation of the GAGs present in this important model organism needed to be undertaken. This report aimed at examining the structural alterations of different GAGs at the molecular level at different developmental stages. GAGs were isolated and purified from zebrafish in different stages in development ranging from 0.5 days to adult. The content and disaccharide composition of chondroitin sulfate and heparan sulfate were determined using chemical assays, liquid chromotography and mass spectrometry. The presence of HS in adult fish was also confirmed using 1H-NMR. PMID:18777207

  20. Chamber Specific Gene Expression Landscape of the Zebrafish Heart

    PubMed Central

    Singh, Angom Ramcharan; Sivadas, Ambily; Sabharwal, Ankit; Vellarikal, Shamsudheen Karuthedath; Jayarajan, Rijith; Verma, Ankit; Kapoor, Shruti; Joshi, Adita; Scaria, Vinod; Sivasubbu, Sridhar

    2016-01-01

    The organization of structure and function of cardiac chambers in vertebrates is defined by chamber-specific distinct gene expression. This peculiarity and uniqueness of the genetic signatures demonstrates functional resolution attributed to the different chambers of the heart. Altered expression of the cardiac chamber genes can lead to individual chamber related dysfunctions and disease patho-physiologies. Information on transcriptional repertoire of cardiac compartments is important to understand the spectrum of chamber specific anomalies. We have carried out a genome wide transcriptome profiling study of the three cardiac chambers in the zebrafish heart using RNA sequencing. We have captured the gene expression patterns of 13,396 protein coding genes in the three cardiac chambers—atrium, ventricle and bulbus arteriosus. Of these, 7,260 known protein coding genes are highly expressed (≥10 FPKM) in the zebrafish heart. Thus, this study represents nearly an all-inclusive information on the zebrafish cardiac transcriptome. In this study, a total of 96 differentially expressed genes across the three cardiac chambers in zebrafish were identified. The atrium, ventricle and bulbus arteriosus displayed 20, 32 and 44 uniquely expressing genes respectively. We validated the expression of predicted chamber-restricted genes using independent semi-quantitative and qualitative experimental techniques. In addition, we identified 23 putative novel protein coding genes that are specifically restricted to the ventricle and not in the atrium or bulbus arteriosus. In our knowledge, these 23 novel genes have either not been investigated in detail or are sparsely studied. The transcriptome identified in this study includes 68 differentially expressing zebrafish cardiac chamber genes that have a human ortholog. We also carried out spatiotemporal gene expression profiling of the 96 differentially expressed genes throughout the three cardiac chambers in 11 developmental stages and 6

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

    PubMed

    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.

  2. Verification of intraovum transmission of a microsporidium of vertebrates: Pseudoloma neurophilia infecting the Zebrafish, Danio rerio.

    PubMed

    Sanders, Justin L; Watral, Virginia; Clarkson, Keri; Kent, Michael L

    2013-01-01

    Direct transmission from parents to offspring, referred to as vertical transmission, occurs within essentially all major groups of pathogens. Several microsporidia (Phylum Microsporidia) that infect arthropods employ this mode of transmission, and various lines of evidence have suggested this might occur with certain fish microsporidia. The microsporidium, Pseudoloma neurophilia, is a common pathogen of the laboratory zebrafish, Danio rerio. We previously verified that this parasite is easily transmitted horizontally, but previous studies also indicated that maternal transmission occurs. We report here direct observation of Pseudoloma neurophilia in the progeny of infected zebrafish that were reared in isolation, including microscopic visualization of the parasite in all major stages of development. Histological examination of larval fish reared in isolation from a group spawn showed microsporidian spores in the resorbing yolk sac of a fish. Infections were also observed in three of 36 juvenile fish. Eggs from a second group spawn of 30 infected fish were examined using a stereomicroscope and the infection was observed from 4 to 48 hours post-fertilization in two embryos. Intraovum infections were detected in embryos from 4 of 27 pairs of infected fish that were spawned based on qPCR detection of P. neurophilia DNA. The prevalence of intraovum infections from the four spawns containing infected embryos was low (∼1%) based on calculation of prevalence using a maximum likelihood analysis for pooled samples. Parasite DNA was detected in the water following spawning of 11 of the infected pairs, suggesting there was also potential for extraovum transmission in these spawning events. Our study represents the first direct observation of vertical transmission within a developing embryo of a microsporidian parasite in a vertebrate. The low prevalence of vertical transmission in embryos is consistent with observations of some other fish pathogens that are also readily

  3. Developmental nephrotoxicity of aristolochic acid in a zebrafish model

    SciTech Connect

    Ding, Yu-Ju; Chen, Yau-Hung

    2012-05-15

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

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

    PubMed

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

    2016-02-01

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

  5. Comprehensive and quantitative proteomic analyses of zebrafish plasma reveals conserved protein profiles between genders and between zebrafish and human.

    PubMed

    Li, Caixia; Tan, Xing Fei; Lim, Teck Kwang; Lin, Qingsong; Gong, Zhiyuan

    2016-04-13

    Omic approaches have been increasingly used in the zebrafish model for holistic understanding of molecular events and mechanisms of tissue functions. However, plasma is rarely used for omic profiling because of the technical challenges in collecting sufficient blood. In this study, we employed two mass spectrometric (MS) approaches for a comprehensive characterization of zebrafish plasma proteome, i.e. conventional shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS) for an overview study and quantitative SWATH (Sequential Window Acquisition of all THeoretical fragment-ion spectra) for comparison between genders. 959 proteins were identified in the shotgun profiling with estimated concentrations spanning almost five orders of magnitudes. Other than the presence of a few highly abundant female egg yolk precursor proteins (vitellogenins), the proteomic profiles of male and female plasmas were very similar in both number and abundance and there were basically no other highly gender-biased proteins. The types of plasma proteins based on IPA (Ingenuity Pathway Analysis) classification and tissue sources of production were also very similar. Furthermore, the zebrafish plasma proteome shares significant similarities with human plasma proteome, in particular in top abundant proteins including apolipoproteins and complements. Thus, the current study provided a valuable dataset for future evaluation of plasma proteins in zebrafish.

  6. An Individual-Based Model of Zebrafish Population Dynamics Accounting for Energy Dynamics

    PubMed Central

    Beaudouin, Rémy; Goussen, Benoit; Piccini, Benjamin; Augustine, Starrlight; Devillers, James; Brion, François; Péry, Alexandre R. R.

    2015-01-01

    Developing population dynamics models for zebrafish is crucial in order to extrapolate from toxicity data measured at the organism level to biological levels relevant to support and enhance ecological risk assessment. To achieve this, a dynamic energy budget for individual zebrafish (DEB model) was coupled to an individual based model of zebrafish population dynamics (IBM model). Next, we fitted the DEB model to new experimental data on zebrafish growth and reproduction thus improving existing models. We further analysed the DEB-model and DEB-IBM using a sensitivity analysis. Finally, the predictions of the DEB-IBM were compared to existin