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PubMed2005

Novel use of zebrafish as a vertebrate model to screen radiation protectors and sensitizers.

PURPOSE: Zebrafish (Danio rerio) embryos provide a unique vertebrate model to screen therapeutic agents easily and rapidly because of their relatively close genetic relationship to humans, ready abundance and accessibility, short embryonal development, and optical clarity. To validate zebrafish embryos as a screen for radiation modifiers, we evaluated the effects of ionizing radiation in combination with a known radioprotector (free radical scavenger Amifostine) or radiosensitizing agent (tyrosine kinase inhibitor AG1478). METHODS AND MATERIALS: Viable zebrafish embryos were exposed to 0-10 Gy single-fraction 250 kVp X-rays with or without either Amifostine (0-4 mM) or AG1478 (0-10 microM) at defined developmental stages from 1-24 h postfertilization (hpf). Embryos were examined for morphologic abnormalities and viability until 144 hpf. RESULTS: Radiation alone produced a time- and dose-dependent perturbation of normal embryonic development and survival with maximal sensitivity at doses > or =4 Gy delivered before 4 hpf. Amifostine markedly attenuated this effect, whereas AG1478 enhanced teratogenicity and lethality, particularly at therapeutically relevant (2-6 Gy) radiation doses. CONCLUSIONS: Collectively, these data validate the use of zebrafish as a vertebrate model to assess the effect of radiation alone or with radiation response modulators. Zebrafish embryos may thus provide a rapid, facile system to screen novel agents ultimately intended for human use in the context of therapeutic or accidental radiation exposure.

Amifostine↗

PubMed2000

Direct action of the nodal-related signal cyclops in induction of sonic hedgehog in the ventral midline of the CNS.

The secreted molecule Sonic hedgehog (Shh) is crucial for floor plate and ventral brain development in amniote embryos. In zebrafish, mutations in cyclops (cyc), a gene that encodes a distinct signal related to the TGF(beta) family member Nodal, result in neural tube defects similar to those of shh null mice. cyc mutant embryos display cyclopia and lack floor plate and ventral brain regions, suggesting a role for Cyc in specification of these structures. cyc mutants express shh in the notochord but lack expression of shh in the ventral brain. Here we show that Cyc signalling can act directly on shh expression in neural tissue. Modulation of the Cyc signalling pathway by constitutive activation or inhibition of Smad2 leads to altered shh expression in zebrafish embryos. Ectopic activation of the shh promoter occurs in response to expression of Cyc signal transducers in the chick neural tube. Furthermore an enhancer of the shh gene, which controls ventral neural tube expression, is responsive to Cyc signal transducers. Our data imply that the Nodal related signal Cyc induces shh expression in the ventral neural tube. Based on the differential responsiveness of shh and other neural tube specific genes to Hedgehog and Cyc signalling, a two-step model for the establishment of the ventral midline of the CNS is proposed.

Animals↗

PubMed1998

The nieuwkoid gene characterizes and mediates a Nieuwkoop-center-like activity in the zebrafish.

BACKGROUND: In amphibians, the Nieuwkoop center--a primary inducing region--has a central role in the induction of dorsal mesodermal cells to form the Spemann organizer. In teleosts, such as the zebrafish, Danio rerio, the functional equivalent of the amphibian Spemann organizer is the dorsal shield. Historically, a small region of the teleost yolk syncytial layer (YSL), an extraembryonic tissue that underlies the entire blastoderm, has been implicated in dorsal shield specification. Difficulties in transplanting discrete regions of the YSL and the previous lack of localized expression patterns unique to the YSL have, however, hindered efforts to prove definitively that the YSL possesses Nieuwkoop-center-like activities. RESULTS: Here, we describe the isolation and analysis of a new homeobox gene, called nieuwkoid, which is first expressed immediately following the mid-blastula transition on the dorsal side of the zebrafish pregastrula embryo. We found that, by the onset of gastrulation, nieuwkoid expression becomes localized to a restricted region of the YSL, directly underlying the future dorsal shield. Mis-expression of nieuwkoid in early zebrafish embryos was found to be sufficient for the induction of ectopic organizer regions and secondary axes. Mis-expression of nieuwkoid by cell transplantation or by direct injection into the YSL led to the non-autonomous induction of ectopic organizer gene expression. CONCLUSIONS: The dynamic and restricted expression of the nieuwkoid gene, combined with its potent dorsalizing activity, suggests that nieuwkoid is an important component in the regionalization of the gastrula organizer, possibly characterizing and mediating an organizer-inducing/Nieuwkoop-center-like activity.

Amino Acid Sequence↗

PubMed2006

Mycobacterium marinum infection of adult zebrafish causes caseating granulomatous tuberculosis and is moderated by adaptive immunity.

The zebrafish, a genetically tractable model vertebrate, is naturally susceptible to tuberculosis caused by Mycobacterium marinum, a close genetic relative of the causative agent of human tuberculosis, Mycobacterium tuberculosis. We previously developed a zebrafish embryo-M. marinum infection model to study host-pathogen interactions in the context of innate immunity. Here, we have constructed a flowthrough fish facility for the large-scale longitudinal study of M. marinum-induced tuberculosis in adult zebrafish where both innate and adaptive immunity are operant. We find that zebrafish are exquisitely susceptible to M. marinum strain M. Intraperitoneal injection of five organisms produces persistent granulomatous tuberculosis, while the injection of approximately 9,000 organisms leads to acute, fulminant disease. Bacterial burden, extent of disease, pathology, and host mortality progress in a time- and dose-dependent fashion. Zebrafish tuberculous granulomas undergo caseous necrosis, similar to human tuberculous granulomas. In contrast to mammalian tuberculous granulomas, zebrafish lesions contain few lymphocytes, calling into question the role of adaptive immunity in fish tuberculosis. However, like rag1 mutant mice infected with M. tuberculosis, we find that rag1 mutant zebrafish are hypersusceptible to M. marinum infection, demonstrating that the control of fish tuberculosis is dependent on adaptive immunity. We confirm the previous finding that M. marinum DeltaRD1 mutants are attenuated in adult zebrafish and extend this finding to show that DeltaRD1 predominantly produces nonnecrotizing, loose macrophage aggregates. This observation suggests that the macrophage aggregation defect associated with DeltaRD1 attenuation in zebrafish embryos is ongoing during adult infection.

Animals↗

PubMed2006

Development of a new rapid measurement technique for fish embryo membrane permeability studies using impedance spectroscopy.

Information on fish embryo membrane permeability is vital in their cryopreservation. Whilst conventional volumetric measurement based assessment methods have been widely used in fish embryo membrane permeability studies, they are lengthy and reduce the capacity for multi-embryo measurement during an experimental run. A new rapid 'real-time' measurement technique is required to determine membrane permeability during cryoprotectant treatment. In this study, zebrafish (Danio rerio) embryo membrane permeability to cryoprotectants was investigated using impedance spectroscopy. An embryo holding cell, capable of holding up to 10 zebrafish embryos was built incorporating the original system electrods for measuring the impedance spectra. The holding cell was tested with deionised water and a series of KCl solutions with known conductance values to confirm the performance of the modified system. Untreated intact embryos were then tested to optimise the loading capacity and sensitivity of the system. To study the impedance changes of zebrafish embryos during cryoprotectant exposure, three, six or nine embryos at 50% epiboly stage were loaded into the holding cell in egg water, which was then removed and replaced by 0.5, 1.0, 2.0 or 3M methanol or dimethyl sulfoxide (DMSO). The impedance changes of the loaded embryos in different cryoprotectant solutions were monitored over 30 min at 22 degrees C, immediately following embryo exposure to cryoprotectants, at the frequency range of 10-10(6)Hz. The impedance changes of the embryos in egg water were used as controls. Results from this study showed that the optimum embryo loading level was six embryos per cell for each experimental run. The optimum frequency was identified at 10(3.14) or 1,380 Hz which provided good sensitivity and reproducibility. Significant impedance changes were detected after embryos were exposed to different concentrations of cryoprotectants. The results agreed well with those obtained from conventional volumetric based studies.

Animals↗

PubMed1991

Toxicity of mercury, copper, nickel, lead, and cobalt to embryos and larvae of zebrafish, Brachydanio rerio.

The toxicity of mercury (HgCl2), copper (CuCl2: 5 H20), nickel (NiSO4: 6 H2O), lead (Pb(CH3COO)2: 3 H2O) and cobalt (CoCl2: 6 H2O) was studied under standardized conditions in embryos and larvae of the zebrafish, Brachydanio rerio. Exposures were started at the blastula stage (2-4 h after spawning) and the effects on hatching and survival were monitored daily for 16 days. Copper and nickel were more specific inhibitors of hatching than cobalt, lead, and mercury. Nominal "no effect" concentrations determined from the dose-response relationships (ZEPs, Zero Equivalent Points) for effect on hatching time were 0.05 microgram Cu/L, 10 micrograms Hg/L, 20 micrograms Pb/L, 40 micrograms Ni/L and 3,840 micrograms Co/L, and those for effect on survival time were 0.25 microgram Cu/L, 1.2 micrograms Hg/L, 30 micrograms Pb/L, 80 micrograms Ni/L, and 60 micrograms Co/L. The "no effect" concentrations for Ni, Hg and Pb are consistent with previously reported MATC values for sensitive species of fish. The "no effect" concentrations for copper are 1-2 orders of magnitude lower than previously reported values. The major reason for the latter discrepancy was considered to be the absence of organics that can complex copper ions in the reconstituted water that we used, which had a hardness of 100 mg/L (as CaCO3) and a pH of 7.5-7.7. Unexposed controls were started with embryos from different parental zebrafishes and the parental-caused variability in early embryo mortality, median hatching time and median survival time were estimated.

Animals↗

PubMed1997

Sclerotome development and peripheral nervous system segmentation in embryonic zebrafish.

Vertebrate embryos display segmental patterns in many trunk structures, including somites and peripheral nervous system elements. Previous work in avian embryos suggests a role for somite-derived sclerotome in segmental patterning of the peripheral nervous system. We investigated sclerotome development and tested its role in patterning motor axons and dorsal root ganglia in embryonic zebrafish. Individual somite cells labeled with vital fluorescent dye revealed that some cells of a ventromedial cell cluster within each somite produced mesenchymal cells that migrated to positions expected for sclerotome. Individual somites showed anterior/posterior distinctions in several aspects of development: (1) anterior ventromedial cluster cells produced only sclerotome, (2) individual posterior ventromedial cluster cells produced both sclerotome and muscle, and (3) anterior sclerotome migrated earlier and along a more restricted path than posterior sclerotome. Vital labeling showed that anterior sclerotome colocalized with extending identified motor axons and migrating neural crest cells. To investigate sclerotome involvement in peripheral nervous system patterning, we ablated the ventromedial cell cluster and observed subsequent development of peripheral nervous system elements. Primary motor axons were essentially unaffected by sclerotome ablation, although in some cases outgrowth was delayed. Removal of sclerotome did not disrupt segmental pattern or development of dorsal root ganglia or peripheral nerves to axial muscle. We propose that peripheral nervous system segmentation is established through interactions with adjacent paraxial mesoderm which develops as sclerotome in some vertebrate species and myotome in others.

Animals↗

PubMed2002

Altering fish embryos with aquaporin-3: an essential step toward successful cryopreservation.

Fish populations are globally threatened by overharvesting and habitat degradation. The ability to bank fish embryos by cryopreservation could be crucial for preserving species diversity, for aquaculture (allowing circannual fish farming), and for managing fish models used in human biomedical research. However, no nonmammalian embryo has ever been successfully cryopreserved. For fish, low membrane permeability prevents cryoprotectants from entering the yolk to prevent cryodamage. Here, we present evidence of a membrane mechanism hindering cryopreservation of fish and propose a novel solution to this obstacle. Zebrafish (Danio rerio) embryos have rectifying membranes that allow water to leave but not to reenter readily. This feature may be an evolutionary trait that allows freshwater embryos to grow in hypoosmotic environments without osmoregulatory organs. However, this trait may also prevent successful fish embryo cryopreservation because both water and cryoprotectants must move into and out of cells. As a solution, we injected zebrafish embryos with mRNA for the aquaporin-3 water channel protein and demonstrated increased membrane permeability to water and to a cryoprotectant. Modeling indicates that sufficient cryoprotectant enters aquaporin-3-expressing zebrafish embryos to allow cryopreservation.

Animals↗

PubMed2003

The maternal-effect gene futile cycle is essential for pronuclear congression and mitotic spindle assembly in the zebrafish zygote.

Embryos have been successfully used for the general study of the cell cycle. Although there are significant differences between the early embryonic and the somatic cell cycle in vertebrates, the existence of specialised factors that play a role during the early cell cycles has remained elusive. We analysed a lethal recessive maternal-effect mutant, futile cycle (fue), isolated in a maternal-effect screen for nuclear division defects in the zebrafish (Danio rerio). The pronuclei fail to congress in zygotes derived from homozygous fue mothers. In addition, a defect in the formation of chromosomal microtubules prevents mitotic spindle assembly and thus chromosome segregation in fue zygotes. However, centrosomal functions do not appear to be affected in fue embryos, suggesting this mutant blocks a subset of microtubule functions. Cleavage occurs normally for several divisions resulting in many anucleate cells, thus showing that nuclear- and cell division can be uncoupled genetically. Therefore, we propose that in mitotic spindle assembly chromosome-dependent microtubule nucleation is essential for the coupling of nuclear and cell division.

Animals↗

PubMed1993

[Ventral and posterior expression of the homeo box gene eve1 in zebrafish (Brachydanio rerio) is repressed in dorsalized embryos].

We have identified and characterized the zebrafish eve1 homeo box gene, a member of the Drosophila even-skipped (eve) gene family. eve1 is expressed in the most posterior part of the tail bud during somitogenesis. During gastrulation, transcripts are confined to ventral and lateral cells of the marginal zone of the embryo. We show that LiCl, known to dorsalize Xenopus embryos, has the same effect in zebrafish embryos. In LiCl-treated embryos, eve1 transcripts are completely absent, suggesting that eve1 marks the ventral specification of mesoderm in zebrafish gastrulae.

Animals↗

PubMed2001

Pre-pattern in the pronephric kidney field of zebrafish.

Vertebrate embryos use a series of transient kidneys to regulate fluid balance, osmolarity and metabolic waste during development. The first kidney to form in the embryo is the pronephros. This kidney is composed of several cell types with very different functions and is organized into discrete segments: glomerulus, tubules and nephric duct. The site of origin of these cells is poorly understood, as are their lineage relationships. We have defined regions of the intermediate mesoderm as candidates for the pronephric field by expression patterns of the Wilms' Tumor suppressor gene (wt1), single-minded 1 (sim1) and pax2.1. All of these potential kidney markers are expressed in a stripe of intermediate mesoderm, with distinct, overlapping antero-posterior borders. We labeled small groups of cells in this area by laser uncaging of a fluorescent dextran, and then tracked their fates. We found that there was a bounded contiguous region of the intermediate mesoderm that provides pronephric progenitors. As is true for other organ fields, the pronephric field regulates after focal destruction, such that a normal pronephros forms after laser-mediated removal of the wt1 domain. The progenitors for podocytes, tubular cells and duct are restricted to subdomains within the pronephric field. The most anterior cells in the pronephric field give rise to podocytes. This corresponds to the wt1-expressing region. The next more posterior cells contribute to the tubule, and express both wt1 and pax2.1. The most posterior cells contribute to the nephric duct, and these express pax2.1 and sim1, but not wt1. Thus, there is a field for the pronephric kidney with classical attributes of defined border, pre-pattern and regulation. The pattern of the fate map reflects particular combinations of transcription factors.

Animals↗

PubMed1996

Inhibition of protein kinase A phenocopies ectopic expression of hedgehog in the CNS of wild-type and cyclops mutant embryos.

The zebrafish hedgehog (hh) family members tiggy-winkle hedgehog (twhh) and sonic hedgehog (shh) are involved in patterning the ventral CNS and proximal eye. Using a dominant negative protein kinase A regulatory subunit mutant, we show that these hh activities are mediated by protein kinase A. The effects of dominant negative protein kinase A on pax2 expression appear to be cell nonautonomous, suggesting that cells can respond to regulation of hh signaling by modulating an additional cell-cell signaling pathway. We also investigate the potential involvement of cyclops in the hh signaling pathway and conclude that although cyclops mutant cells can respond to hh signaling, neither hh nor dominant negative protein kinase A rescues the phenotypes associated with cyclops.

Animals↗

PubMed2002

DarT: The embryo test with the Zebrafish Danio rerio--a general model in ecotoxicology and toxicology.

The acute fish test is an animal test whose ecotoxicological relevance is worthy of discussion. The primary aim of protection in ecotoxicology is the population and not the individual. Furthermore the concentration of pollutants in the environment is normally not in the lethal range. Therefore the acute fish test covers solely the situation after chemical spills. Nevertheless, acute fish toxicity data still belong to the base set used for the assessment of chemicals. The embryo test with the zebrafish Danio rerio (DarT) is recommended as a substitute for the acute fish test. For validation an international laboratory comparison test was carried out. A summary of the results is presented in this paper. Based on the promising results of testing chemicals and waste water the test design was validated by the DIN-working group "7.6 Fischei-Test". A normed test guideline for testing waste water with fish is available. The test duration is short (48 h) and within the test different toxicological endpoints can be examined. Endpoints from the embryo test are suitable for QSAR-studies. Besides the use in ecotoxicology the introduction as a toxicological model was investigated. Disturbance of pigmentation and effects on the frequency of heart-beat were examined. A further important application is testing of teratogenic chemicals. Based on the results DarT could be a screening test within preclinical studies.

Animal Rights↗

PubMed2004

Higher harmonic generation microscopy for developmental biology.

Optical higher harmonic generation, including second harmonic generation and third harmonic generation, leaves no energy deposition to its interacted matters due to an energy-conservation characteristic, providing the "noninvasiveness" nature desirable for biological studies. Combined with its nonlinearity, higher harmonic generation microscopy provides excellent three-dimensional (3D) sectioning capability, offering new insights into the studies of embryonic morphological changes and complex developmental processes. By choosing a laser working in the biological penetration window, here we present a noninvasive in vivo light microscopy with sub-micron 3D resolution and millimeter penetration, utilizing endogenous higher harmonic generation signals in live specimens. Noninvasive imaging was performed in live zebrafish (Danio rerio) embryos. The complex developmental processes within > 1-mm-thick zebrafish embryos can be observed in vivo without any treatment. No optical damage was found even with high illumination after long-term observations and the examined embryos all developed normally at least to the larval stage. The excellent 3D resolution of the demonstrated technology allows us to capture the subtle developmental information on the cellular or sub-cellular levels occurring deep inside the live embryos and larvae. This technique can not only provide in vivo observation of the cytoarchitecture dynamics during embryogenesis with submicron resolution and millimeter penetration depth, but would also make strong impact in developmental and structural biology studies.

Animals↗

PubMed1994

Assessment of pMTL construct for detection in vivo of luciferase expression and fate of the transgene in the zebrafish, Brachydanio rerio.

Early embryos of the zebrafish Brachydanio rerio were cytoplasmically microinjected with pMTL plasmid containing firefly luciferase gene, in both linearized- and supercoiled-plasmid forms, to evaluate in vivo expression, pattern of integration and germ-line transmission of the transgene in the host fish. It was possible to detect luciferase expression in vivo, and the pattern of time-course expression was similar in both linearized- and supercoiled-plasmid injected groups. Strong luciferase activity was detected 15-20 hours after injection, coinciding with early somitogenesis. Expression was detectable in a few 1 week-old individuals but was not detectable in all adults and in F1 progeny. In vivo screening for expression of the transgene in the developing embryo using luciferase assay as a method for detecting the presence of the transgenic fish compares favourably, with PCR and Southern blot analysis (SBA). No integration of the introduced DNA into the genome of treated fish and their progeny, was detected, instead it remained in extrachromosomal form. Most of the first generation founders were mosaic. Germline transmission was observed in one individual only. A probable reason for the absence of integration in this study when compared to the varying frequencies reported earlier in the same fish is discussed.

Animals↗

PubMed2004

MNNG-induced mutations in the adult gill and hepatopancreas and in embryos of rpsL transgenic zebrafish.

To evaluate the feasibility of a mutagenicity assay using adult rpsL transgenic zebrafish, 4- to 8-month-old females were exposed to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (0, 15 or 30 mg/L in a water bath for 2 h). At 2 weeks after exposure, MNNG showed a concentration-dependent significant increase in mutant frequency (MF) of 8 x 10(-5), 18 x 10(-5), and 51 x 10(-5), respectively, in the gill. DNA sequencing revealed that 60-74% of the induced mutations were G:C to A:T transitions, consistent with the known mutagenic effects of MNNG. A marginal but significant increase in MF was observed in the hepatopancreas only in the group exposed to 30 mg/L, with the induction of some G:C to A:T transitions. A time-course of the appearance of mutations was determined in fish treated with 15 mg/L MNNG. In both, the gill and hepatopancreas, a higher MF was observed at 3 weeks than at 2 weeks, suggesting that an expression time of at least 3 weeks is preferable for the assay. When embryos (29 h post-fertilization) were exposed to MNNG (0, 50, and 150 mg/L) for 1 h, MFs increased significantly with an increase in the concentration of MNNG (5 x 10(-5), 40 x 10(-5), and 144 x 10(-5), respectively) at 3 days after exposure. G:C to A:T transitions were the predominant mutations, and these occurred at the same sites in the rpsL gene as in adult tissues. Thus, MNNG induces typical mutations in the gill and hepatopancreas of adult fish, and in embryos, suggesting that the rpsL zebrafish is a useful tool for monitoring genotoxicity caused by water-borne mutagens.

Animals↗

PubMed1994

lacZ expression in germline transgenic zebrafish can be detected in living embryos.

Use of transgenic technology in zebrafish has been limited by the inability to efficiently express transgenes in early embryos of F1 and subsequent generations and to rapidly detect transgenic fish. We generated transgenic fish by injecting fertilized eggs with the Escherichia coli lacZ gene under the control of the Xenopus elongation factor 1 alpha transcriptional regulatory element. Four of five lines of transgenic fish we obtained express the lacZ gene in early embryos. The pattern of expression was distinct for each line, with two lines showing extensive expression beginning at approximately the midblastula transition, one showing patchy expression and one showing expression almost exclusively in motor neurons. Expression patterns were stable through the F2 generation in the three lines studied to date. The availability of these lines facilitated the development of a reliable and rapid method for live-staining lacZ-expressing embryos using the substrate fluorescein-di-beta-D-galactopyranoside (FDG). Positive embryos of the two most highly lacZ-expressing lines could be identified after 2-3 min of staining in FDG and then picked out and raised. These observations should prove useful for a variety of studies in zebrafish.

Animals↗

PubMed2004

Effect of a vitrification protocol on the lactate dehydrogenase and glucose-6-phosphate dehydrogenase activities and the hatching rates of Zebrafish (Danio rerio) and Turbot (Scophthalmus maximus) embryos.

Vitrification, is the most promising option for the cryopreservation of fish embryos but requires high concentrations of potentially toxic cryoprotectants. In this study, embryos from Turbot and Zebrafish, each in two developmental stages, were submitted to a four stepwise cryoprotectant incorporation protocol. After incubation in the vitrificant solution (5M dimethyl sulfoxide, 2M methanol, 1M ethylen-glycol and 10% sucrose) embryos were loaded in straws and plunged into liquid nitrogen. The activity of two cytoplasmic enzymes, LDH and G6PDH, and the hatching rates were analyzed in control embryos, those subjected to the cryoprotectant solutions and in frozen/thawed embryos. Results showed that the cryoprotectants incorporation protocol did not have important effects on the analyzed enzymatic activities, which remained at similar levels to that in control embryos but significantly reduced the hatching rates. Turbot was less sensitive than Zebrafish to the toxic effect of the cryoprotectants, achieving hatching rates of 74.8% in comparison with fresh control embryos at G stage, whereas in Zebrafish only 17.7% of hatching was reported with five somites-treated embryos. In Turbot, G stage was more resistant to the cryoprotectants and thus more convenient for further vitrification studies. After vitrification no survival was recorded and enzymatic activities dropped significantly, particularly in Zebrafish, indicating cell damage and loss of cytoplasmic enzymes. Nevertheless, total cell lysis was not produced, and once again Turbot was more resistant to the effect of vitrification, particularly at the later stage. In that stage, Turbot embryos showed around 50% of G6PDH activity after vitrification, in comparison with the control, indicating the preservation of some cellular activity after freezing-thawing, despite the loss of developmental ability.

Animals↗