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PubMed2001

Zebrafish genes rx1 and rx2 help define the region of forebrain that gives rise to retina.

Zebrafish retinal homeobox genes rx1 and rx2 are expressed exclusively in the optic primordia and then in cone photoreceptors of the differentiated neural retina. In this study, we show that the rx expression domain is coextensive with the region identified as the retinal field in published fate maps of the neural plate in zebrafish embryos. Analysis of the spatiotemporal relationships between retinal and forebrain precursors suggests that lateral movement of retinal precursors is responsible for evagination of the optic primordia. Overexpression of either rx1 or rx2 results in the loss of forebrain tissue and the ectopic formation of retinal tissue. We asked whether the deletion of forebrain and expansion of retinal tissue could be explained by the death of telencephalic precursors and enhanced proliferation of retinal precursors, and we found that it could not. Instead, our data are consistent with a change in cell fate of forebrain precursors associated with reduced expression of telencephalic markers (emx1 and BF-1) and ectopic expression of retinal markers (rx1/2/3, pax6, six6, and vsx2) at the neural keel stage. The rx homeodomain alone is sufficient to induce ectopic retinal tissue, although weakly so, and this observation, together with results from deletion constructs, suggests that interactions with unidentified transcriptional regulators are important for rx1 and rx2 function during early eye development. We conclude that regulated expression of zebrafish rx1 and rx2 helps to define the region of the forebrain fated to give rise to retinal tissue and may be involved in the cellular migrations that lead to splitting of the retinal field and formation of the optic primordia.

Animals↗

PubMed2000

Roles for Fgf signaling during zebrafish fin regeneration.

Following amputation of a urodele limb or teleost fin, the formation of a blastema is a crucial step in facilitating subsequent regeneration. Using the zebrafish caudal fin regeneration model, we have examined the hypothesis that fibroblast growth factors (Fgfs) initiate blastema formation from fin mesenchyme. We find that fibroblast growth factor receptor 1 (fgfr1) is expressed in mesenchymal cells underlying the wound epidermis during blastema formation and in distal blastemal tissue during regenerative outgrowth. fgfr1 transcripts colocalize with those of msxb and msxc, putative markers for undifferentiated, proliferating cells. A zebrafish Fgf member, designated wfgf, is expressed in the regeneration epidermis during outgrowth. Furthermore, we show that a specific inhibitor of Fgfr1 applied immediately following fin amputation blocks blastema formation, without obvious effects on wound healing. This inhibitor blocks the proliferation of blastemal cells and the onset of msx gene transcription. Inhibition of Fgf signaling during ongoing fin regeneration prevents further outgrowth while downregulating the established expression of blastemal msx genes and epidermal sonic hedgehog. Our findings indicate that zebrafish fin blastema formation and regenerative outgrowth require Fgf signaling.

Amputation, Surgical↗

PubMed2001

Zebrafish mdk2, a novel secreted midkine, participates in posterior neurogenesis.

Patterning the neural plate in vertebrates depends on complex interactions between a variety of secreted growth factors. Here we describe a novel secreted factor in zebrafish, named mdk2, related to the midkine family of heparin-binding growth factors that is involved in posterior neural development. mdk2 is expressed shortly after the onset of gastrulation in the presumptive neural plate cells of the epiblast, and this expression is enhanced by exogenous retinoic acid. Ectopic expression of mdk2 enhances neural crest cell fates at the lateral edges of the caudal neural plate, concomitant with a repression of anterior structures and mesendodermal and ectodermal markers. Reciprocally, ectopic expression of a dominant negative mdk2 results in severe deficiencies of structures posterior to the midbrain-hindbrain boundary, with negligible effects on anterior structures. In these embryos, the expression of hindbrain and neural crest markers is strongly reduced, and the formation of posterior primary moto- and sensory neurons is blocked. Analyses in mutant zebrafish embryos shows that expression of mdk2 is independent of FGF8 and nodal-related-1 signaling, but is under negative control of BMP signaling. These data support the hypothesis that mdk2 participates in posterior neural development in zebrafish.

Amino Acid Sequence↗

PubMed2001

Dispersion of cyclin B mRNA aggregation is coupled with translational activation of the mRNA during zebrafish oocyte maturation.

Cyclin B mRNA stored in immature zebrafish oocytes is translationally activated upon the stimulation of 17alpha,20beta-dihydroxy-4-pregnen-3-one (17alpha,20beta-DP), an event prerequisite for initiating oocyte maturation in this species. We investigated localization of cyclin B mRNA in zebrafish oocytes. Cyclin B mRNA was found to be exclusively localized as an aggregation along the cytoplasm at the animal pole of full-grown immature oocytes. When oocytes were treated with 17alpha,20beta-DP, a meshwork of microfilaments in the oocyte cortex disappeared and the aggregation of cyclin B mRNA dispersed just prior to the initiation of cyclin B synthesis and germinal vesicle breakdown (GVBD). Cytochalasin B, but not nocodazole or taxol, deformed the aggregation of cyclin B mRNA, indicating the involvement of microfilaments in organizing this form. Like 17alpha,20beta-DP, cytochalasin B (10 microg/ml) induced both complete dispersion of the aggregation and translational activation of cyclin B mRNA, forcing the oocytes to undergo GVBD without 17alpha,20beta-DP. Conversely, disturbance of the aggregation of cyclin B mRNA with a low concentration (1 microg/ml) of cytochalasin B inhibited 17alpha,20beta-DP-induced GVBD. These results suggest that the direct change in cyclin B mRNA from the aggregated form to the dispersed form is responsible for translational activation of the mRNA during zebrafish oocyte maturation.

Animals↗

PubMed2001

Hedgehog signalling is required for maintenance of myf5 and myoD expression and timely terminal differentiation in zebrafish adaxial myogenesis.

Hedgehog proteins have been implicated in the control of myogenesis in the medial vertebrate somite. In the mouse, normal epaxial expression of the myogenic transcription factor gene myf5 is dependent on Sonic hedgehog. Here we examine in zebrafish the interaction between Hedgehog signals, the expression of myoD family genes, including the newly cloned zebrafish myf5, and slow myogenesis. We show that Sonic hedgehog is necessary for normal expression of both myf5 and myoD in adaxial slow muscle precursors, but not in lateral paraxial mesoderm. Expression of both genes is initiated normally in rostral presomitic mesoderm in sonic you mutants, which lack all Sonic hedgehog. Similar initiation continues during tailbud outgrowth when the cells forming caudal somites are generated. However, adaxial cells in sonic you embryos are delayed in terminal differentiation and caudal adaxial cells fail to maintain myogenic regulatory factor expression. Despite these defects, other signals are able to maintain, or reinitiate, some slow muscle development in sonic you mutants. In the cyclops mutant, the absence of floorplate-derived Tiggywinkle hedgehog and Sonic hedgehog has no discernible effect on slow adaxial myogenesis. Similarly, the absence of notochord-derived Sonic hedgehog and Echidna hedgehog in mutants lacking notochord delays, but does not prevent, adaxial slow muscle development. In contrast, removal of both Sonic hedgehog and a floorplate signal, probably Tiggywinkle hedgehog, from the embryonic midline in cyclops;sonic you double mutants essentially abolishes slow myogenesis. We conclude that several midline signals, likely to be various Hedgehogs, collaborate to maintain adaxial slow myogenesis in the zebrafish embryo. Moreover, the data demonstrate that, in the absence of this required Hedgehog signalling, expression of myf5 and myoD is insufficient to commit cells to adaxial myogenesis.

Amino Acid Sequence↗

PubMed2001

Tracing transgene expression in living zebrafish embryos.

Ectopic expression by injection of plasmid DNA is rarely used in zebrafish embryos due to a low frequency of cells expressing a transgene of interest at detectable levels. Furthermore, the mosaic nature of ectopic expression by plasmid injection requires the direct detection of transgene-expressing cells. We have used the transcriptional activator Gal4-VP16 to amplify transgene expression in living zebrafish embryos. In comparison to conventional expression vectors, Gal4-VP16-amplified expression results in a significant higher number of cells which express a transgene at detectable levels. The Gal4-VP16-activator and the Gal4-VP16-dependent transgene can be placed on a single expression vector. Using tissue-specific regulatory elements, we show that expression of a Gal4-VP16-dependent transgene can be reliably restricted to muscle, notochordal, or neuronal tissues. Furthermore, Gal4-VP16 can drive the expression of two or more transgenes from the same construct resulting in simultaneous coexpression of both genes in virtually all expressing cells. The reported expression system works effectively not only in zebrafish embryos but also in Xenopus embryos, chicken, mouse, and human cultured cells and is thus applicable to a broad variety of vertebrates. The high frequency of transgene expression together with the linked coexpression of more than one transgene opens the possibility of easily monitoring the behavior of individual transgene-expressing cells in real time by labeling them with the fluorescent reporter GFP. The combinatorial nature of the expression system greatly facilitates changing the tissue-specificity, the transgene expressed, or the cell compartment-specific GFP reporter, making it simpler to address a gene's function in different tissues as well as its cell biological consequences.

Animals↗

PubMed2002

Gene transfer into zebrafish by sperm nuclear transplantation.

A technique for fertilizing zebrafish eggs by injection of sperm nuclei is described. Eggs that cleave normally can develop into swimming larvae and give rise to fertile adults. If sperm nuclei are preincubated for 20 min with DNA encoding the green fluorescent protein, transgene expression can be detected in all cells of the embryo. The use of condensed sperm nuclei allows injection with a small bore pipette, which is critical for successful injection of the relatively small zebrafish egg. This technique enables the generation of ubiquitously expressing transgenic zebrafish directly by microinjection. Hence, experiments involving transgenic fish can be completed in days, without the need for growing and breeding founders. This technique may also be used to generate transgenic lines, as transgene expression was visible in the offspring of transgenic founders. The method described here is likely to be applicable to other teleosts, such as medaka and salmon.

Animals↗

PubMed1994

Identification of RAPD primers that reveal extensive polymorphisms between laboratory strains of zebrafish.

The zebrafish has recently emerged as a useful system for understanding vertebrate developmental genetics, despite the lack of a linkage map. To identify DNA-based genetic polymorphisms for constructing a genetic map, we have screened a collection of RAPD primers for their utility in identifying genetic polymorphisms between two laboratory strains of zebrafish. Here, we report 116 primers that identify 721 strain-specific genetic markers and show how they can be used in haploid genetics of zebrafish.

Animals↗

PubMed1995

Microsatellite-centromere mapping in the zebrafish (Danio rerio).

Ten (CA)n microsatellite simple sequence repeat (SSR) markers, 1, 2, 12, 14, 16, 18, 20, 22, 26, and 29, were used to show high chiasma interference and to determine centromere-marker map distances in the zebrafish (Danio rerio). Of these, SSR 12 exhibited no recombinant tetratypes among 175 half-tetrad embryos, placing this marker within 1 cM of the centromere of Linkage Group XVII. Fractions of heterozygous half-tetrads for the remaining nine markers ranged from 0.64 to 0.89. Of these, six recombinant fractions were more than 0.67 (P < 0.05), indicating strong chiasma interference during female meiosis in the zebrafish. Consistent with previous mapping data, SSRs 2 and 20 of Linkage Group VI were tightly linked. Half-tetrad analysis will allow the mapping of the remaining centromeres and may be useful in the mapping of new genes and mutations in the zebrafish.

Animals↗

PubMed1998

Zebrafish genomic library in yeast artificial chromosomes.

We have constructed a zebrafish yeast artificial chromosome (YAC) library using genomic DNA isolated from the inbred AB zebrafish strain. The average insert size is 470 kb, estimated from analysis of 155 random selected YACs. The library consists of 17,000 clones, providing about a 4.7-fold coverage of zebrafish genome. The YAC clones have been arrayed in individual wells of 96-well microplates and also pooled to permit rapid polymerase chain reaction screening of the entire library. We have also found that the YAC ends can be easily rescued and sequenced from pRML1/pRML2-based mini-YAC clones.

Animals↗

PubMed1997

CYP1A induction by beta-naphthoflavone, Aroclor 1254, and 2,3,7,8-tetrachlorodibenzo-p-dioxin and its influence on aflatoxin B1 metabolism and DNA adduction in zebrafish.

This study investigated the inductive response of cytochrome P4501A (CYP1A) in the zebrafish (Danio rerio) following exposure to Aroclor 1254, beta-naphthoflavone (betaNF), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and then investigated TCDD modulation of aflatoxin B1 (AFB1) metabolism and hepatic AFB1-DNA adduction. Aroclor 1254 fed at 500 ppm for 1 to 9 days or intraperitoneal (ip) injection of 75-200 mg Aroclor 1254/kg body weight failed to induce CYP1A protein or associated 7-ethoxyresorufin-O-deethylase (EROD) activity. By contrast, dietary betaNF at 500 ppm for 3 or 7 days induced CYP1A protein and EROD activity approximately threefold above controls. A single ip injection of 150 mg/kg betaNF showed maximal induction of CYP1A protein and EROD activity near 24 hr, both of which decreased to control levels during the next 6 days. Single ip administration of 25, 50, 100, or 150 mg betaNF/kg body weight provided dose-responsive increases in CYP1A and EROD activity. Dietary exposure to 0.75 ppm TCDD for 3 days also significantly induced CYP1A and EROD. The effect of TCDD on the metabolism of [3H]AFB1 in zebrafish was then investigated. The major [3H]AFB1 metabolites excreted in water over 24 hr in the control group were aflatoxicol, aflatoxicol-glucuronide, and parent AFB1. By contrast, the predominant metabolites in the TCDD-pretreated group were aflatoxicol-M1-glucuronide, aflatoxicol, aflatoxin M1 plus aflatoxicol-M1 (unresolved), aflatoxicol-glucuronide, and parent AFB1. Surprisingly, hepatic AFB1-DNA adduction was approximately fourfold higher in the TCDD treated group than in controls. This significant difference could not be explained by increased capacity for bioactivation of AFB1 as measured by an in vitro AFB1-exo-8, 9-epoxide trapping assay. However, it was demonstrated that both control and induced zebrafish have high capacity to bioactivate aflatoxin M1 to a reactive intermediate, such that secondary bioactivation of this genotoxic intermediate may be responsible for the increased DNA binding.

Aflatoxin B1↗

PubMed1999

Transactivation activity of human, zebrafish, and rainbow trout aryl hydrocarbon receptors expressed in COS-7 cells: greater insight into species differences in toxic potency of polychlorinated dibenzo-p-dioxin, dibenzofuran, and biphenyl congeners.

Transactivation assays were used to compare the potency and efficacy of polychlorinated dibenzo-p-dioxin (PCDD), dibenzofuran (PCDF), and biphenyl (PCB) congeners in activating aryl hydrocarbon receptors (AhRs) from rainbow trout (rtAhR2alpha and rtAhR2beta), zebrafish (zfAhR2), and human (huAhR), respectively. All AhRs were expressed with their species-specific AhR nuclear translocator (ARNT) in COS-7 cells. Transactivation activity was determined for two PCDD, two PCDF, and seven PCB congeners with each of the four AhR/ARNT pairs using prt1Aluc, a luciferase reporter driven by two dioxin-responsive enhancer elements (DREs) from the rainbow trout cyp1A gene. Maximal-fold induction, EC50, and relative potency values were calculated for congeners that exhibited dose-related activity in the assay. Of the four AhR/ARNT pairs tested with PCDD, PCDF, and non-ortho PCB congeners, three exhibited high activity (rainbow trout AhR2alpha, zebrafish AhR2, and human AhR), while rainbow trout AhR2beta had very weak or no activity. Comparisons between these AhRs showed that while mono-ortho PCBs were able to activate the human AhR, they were generally ineffective in activating rainbow trout and zebrafish AhR2s. This supports the hypothesis that structural differences between mammalian and fish AhRs may account for differences in relative potencies of the mono-ortho PCBs between mammals and fish. Another important finding was a significant difference in transactivation activity between the two rainbow trout AhR2 isoforms despite the fact that they are 95% identical at the amino acid level. For all PCDD, PCDF, and PCB agonists tested, rainbow trout AhR2alpha was significantly more active than AhR2beta. However, rainbow trout AhR2beta is active as a 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-activated transcription factor, with enhancer elements from the mouse cyp1A gene. This suggests that AhR2beta may have evolved to serve a different physiological function than AhR2alpha in salmonid fish species.

Animals↗

PubMed1995

Cloning and characterization of class I Mhc genes of the zebrafish, Brachydanio rerio.

The zebrafish (Brachydanio rerio) offers many advantages for immunological and immunogenetic research and has the potential for becoming one of the most important nonmammalian vertebrate research models. With this in mind, we initiated a systematic study of the zebrafish major histocompatibility complex (Mhc) genes. In this report, we describe the cloning and characteristics of the zebrafish class I A genes coding for the alpha chains of the alpha beta heterodimer and thus complete the identification of all four classes and subclasses of the Mhc in this species. We describe the full class I alpha cDNA sequence as well as the exon-intron organization of the class I A genes, including intron sequences. We identify three families of class I A genes which we designate Brre-UAA, -UBA, and -UCA. The three families originated about the time of the divergence of cyprinid and salmonid fishes. All three families are members of an ancient lineage that diverged from another, older lineage also represented in cyprinid fishes before the radiation of teleost orders. The fish class I A genes therefore evolve differently from mammalian class I A genes, in which the establishment of lineages and families mostly postdates the divergence of orders.

Amino Acid Sequence↗

PubMed1995

Characterization of a Tc1-like transposable element in zebrafish (Danio rerio).

We have characterized Tdr1, a family of Tc1-like transposable elements found in the genome of zebrafish (Danio rerio). The copy number and distribution of the sequence in the zebrafish genome have been determined, and by these criteria Tdr1 can be classified as a moderately repetitive, interspersed element. Examination of the sequences and structures of several copies of Tdr1 revealed that a particular deletion derivative, 1250 bp long, of the transposon has been amplified to become the dominant form of Tdr1. The deletion in these elements encompasses sequences encoding the N-terminal portion of the putative Tdr1 transposase. Sequences corresponding to the deleted region were also detected, and thus allowed prediction of the nucleotide sequence of a hypothetical full-length element. Well conserved segments of Tc1-like transposons were found in the flanking regions of known fish genes, suggesting that these elements have a long evolutionary history in piscine genomes. Tdr1 elements have long, 208 bp inverted repeats, with a short DNA motif repeated four times at the termini of the inverted repeats. Although different from that of the prototype C. elegans transposon Tc1, this inverted repeat structure is shared by transposable elements from salmonid fish species and two Drosophila species. We propose that these transposons form a subgroup within the Tc1-like family. Comparison of Tc1-like transposons supports the hypothesis that the transposase genes and their flanking sequences have been shaped by independent evolutionary constraints. Although Tc1-like sequences are present in the genomes of several strains of zebrafish and in salmonid fishes, these sequences are not conserved in the genus Danio, thus raising the possibility that these elements can be exploited for gene tagging and genome mapping.

Amino Acid Sequence↗

PubMed1994

Culture of cells from zebrafish (Brachydanio rerio) blastula-stage embryos.

The zebrafish has become a popular model for studies of vertebrate development and toxicology. However, in vitro approaches utilizing this organism have not been fully exploited due to the absence of suitable cell culture systems. Previously, we developed methods for the culture of cells derived from zebrafish blastula-stage embryos. One of these cultures, ZEM-2, was derived in a complex medium containing trout embryo extract, trout serum and medium conditioned by buffalo rat liver cells. In this study we describe a zebrafish embryo cell line, ZEM-2A, derived from ZEM-2 following selection for growth in a simplified medium. Optimal growth of ZEM-2A cells is attained in nutrient medium supplemented with 5% fetal bovine serum.

Animals↗

PubMed1998

The response of trout and zebrafish embryos to low and high boron concentrations is U-shaped.

Fish in the embryo-larval stage of development have been shown to be sensitive to boron (B) at both ends of the dose-response curve (1,2). The present study evaluated the health effects of low and high B concentrations on rainbow trout (Oncorhynchus mykiss), a cold water species, and zebrafish (Danio rerio), a warm water species. Rainbow trout embryos were incubated from day 1 until 2 wk posthatch in Type 1 ASTM ultrapure-grade water (12.5 degrees C) supplemented with only B (0-500 microM) as boric acid, or together with CaCO3 (0-2 mM) to increase water hardness. Embryonic growth was stimulated by B in a dose-dependent manner at all Ca concentrations (p < 0.001). Chronic exposures below 9 micromol B/L impaired embryonic growth and above 10 mmol B/L caused death (p < 0.001). Thus, the safe range of exposure for the rainbow trout was between the adverse effect concentrations of 9 micromol B/L and 10 mmol B/L. Zebrafish were maintained for 6 mo in ultrapure water containing <0.2 micromol B/L to determine the effect of low-level exposure. High-level exposure was assessed by exposing zygotes, derived from parents maintained at 46 micromol B/L, to graded concentrations of boric acid up to a concentration of 75 mmol B/L from fertilization until they were free feeding (96 h). Fertilization occurred, but zygotes failed to survive when water contained <0.2 micromol B/L (p < 0.001). Death occurred at and above 9.2 mmol B/L. Thus, the safe range of B exposure for zebrafish was between the adverse effect concentrations of 0.2 micromol B/L and 9.2 mmol B/L. The dose-response for both species was thus U-shaped.

Analysis of Variance↗

PubMed1997

Heat shock protein gene expression during embryonic development of the zebrafish.

Heat shock genes exhibit complex patterns of spatial and temporal regulation during embryonic development of a wide range of organisms. Our laboratory has been involved in an analysis of heat shock gene expression in the zebrafish, a model system which is now utilized extensively for the examination of early embryonic development of vertebrates. Members of the zebrafish hsp47, hsp70 and hsp90 gene families have been cloned and shown to be closely related to their counterparts in higher vertebrates. Expression of these genes has been examined using Northern blot and whole mount in situ hybridization analyses. Both the hsp47 and hsp90 genes are expressed in a highly tissue-restricted manner during normal development. The data raise a number of interesting questions regarding the function and regulation of these heat shock genes during early zebrafish development.

Animals↗

PubMed2007

Hallucinatory and rewarding effect of salvinorin A in zebrafish: kappa-opioid and CB1-cannabinoid receptor involvement.

RATIONALE: The hallucinatory effect and potential abuse of salvinorin A, the major ingredient of Salvia divinorum, has not been documented in animals. OBJECTIVE: The effects of salvinorin A on the zebrafish (Danio rerio) model, through its swimming behavior and conditioned place preference (CPP) task, was studied. MATERIALS AND METHODS: Swimming activity was determined in a squared observational chamber after an i.m. treatment of salvinorin A (0.1-10 microg/kg). For the CPP test, zebrafish were given salvinorin A (0.2 and 1 microg/kg) or vehicle and evaluated in a two-compartment chamber. RESULTS: Salvinorin A (0.1 and 0.2 microg/kg) induced accelerated swimming behavior in comparison with vehicle, whereas a "trance-like" effect, at doses as 5 and 10 microg/kg, was obtained. Pretreatment with the kappa-opioid antagonist, nor-binaltorphimine (nor-BNI; 10 mg/kg) and the cannabinoid type 1 (CB(1)) antagonist, rimonabant (1 mg/kg), blocked salvinorin A-induced both stimulating and depressive effects obtained at a dose of 0.2 and 10 microg/kg, respectively. In the CPP test, salvinorin A (0.2 and 0.5 microg/kg) produced an increase in the time spent in the drug-associated compartment. A dose of 1 microg/kg produced no effect, whereas a dose of 80 microg/kg induced aversion. Pretreatment with nor-BNI or rimonabant fully reversed the reinforcing properties of salvinorin A (0.5 microg/kg). CONCLUSIONS: Taken together, these results indicate that salvinorin A, as is sometimes reported in humans, exhibits rewarding effects, independently from its motor activity, suggesting the usefulness of the zebrafish model to study addictive behavior. These effects appear mediated by activation of both kappa-opioid and cannabinoid CB(1) receptors.

Animals↗