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PubMed2005

Identification of zebrafish ARNT1 homologs: 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in the developing zebrafish requires ARNT1.

To use the zebrafish (Danio rerio) as a model to study 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) developmental toxicity, it is essential to know which proteins are involved in mediating toxicity. Previous work has identified zfAHR2 as the receptor that binds TCDD mediating downstream responses. Although zfARNT2b can form a functional heterodimer with zfAHR2 in vitro, zfarnt2 null mutants show no protection against endpoints of TCDD developmental toxicity, demonstrating that zfARNT2b cannot be the physiological dimerization partner for zfAHR2 mediating responses to TCDD in zebrafish embryos. The purpose of the current study was to identify an alternate dimerization partner(s) for zfAHR2 that may function to mediate TCDD developmental toxicity. By searching zebrafish genomic sequence and using the polymerase chain reaction-based rapid amplification of cDNA ends technique, three forms of cDNA that seem to be alternate mRNA splice variants of a zebrafish homolog of ARNT1 were detected. Analysis of the zfARNT1 proteins in vitro demonstrates that the two longest forms of zfARNT1, zfARNT1b and zfARNT1c, can form functional heterodimers with zfAHR2. However, the shortest form, zfARNT1a, seems to be nonfunctional with zfAHR2 in vitro. To determine whether a zfARNT1 protein functions with zfAHR2 in vivo, a morpholino targeted against the 5' end of zfARNT1 (zfarnt1-MO) was used. Injection of the zfarnt1-MO before TCDD treatment significantly decreases the induction of zfCYP1A mRNA and protein. In addition, zfarnt1 morphants show complete protection against TCDD-induced pericardial edema and show partial protection against reduced blood flow and craniofacial malformations caused by TCDD, demonstrating the role of zfARNT1 proteins in mediating these responses.

Amino Acid Sequence↗

PubMed2005

Analysis of early epidermal development in zebrafish.

The zebrafish (Danio rerio) is a member of the teleost (bony fish) lineage that diverged from the mammalian lineage 420 million years ago. Despite this ancient divergence, there are important similarities between the epidermis of fish and mammals. In addition, recent work suggests that the genes required for epidermal development in the zebrafish also are essential for this process in higher vertebrates, indicating that analysis of the zebrafish system will contribute to understanding mouse and human development. The zebrafish is a very useful genetic system, and many mutants with epidermal defects have been obtained in large-scale genetic screens. In addition, zebrafish embryos are transparent, develop externally, and can be collected in abundance. The aim of this chapter is to provide the researcher with basic protocols to examine gene expression, cell proliferation, and cell death in the developing zebrafish. Although only a few specific epidermal markers have been identified in the zebrafish thus far, we also provide a list of useful genes for epidermal analysis.

Animals↗

PubMed1999

Characterization of Ca2+-dependent phospholipase A2 activity during zebrafish embryogenesis.

We have developed a simple fluorescent assay for detection of phospholipase A2 (PLA2) activity in zebrafish embryos that utilizes a fluorescent phosphatidylcholine substrate. By using this assay in conjunction with selective PLA2 inhibitors and Western blot analysis, we identified the principal activity in zebrafish embryogenesis as characteristic of the Ca2+-dependent cytosolic PLA2 (cPLA2) subtype. Embryonic cPLA2 activity remained constant from the 1-cell stage until the onset of somitogenesis, at which time it increased sharply. This increase was preceded by the expression of a previously identified zebrafish cPLA2 homologue (Nalefski, E., Sultzman, L., Martin, D., Kriz, R., Towler, P., Knopf, J., and Clark, J. (1994) J. Biol. Chem. 269, 18239-18249). By using a quenched BODIPY-labeled phosphatidylcholine that fluoresces only upon cleavage by PLA2, lipase activity was visualized in the cells of living embryos where it localized to perinuclear membranes.

Animals↗

PubMed2004

Technology for high-throughput screens: the present and future using zebrafish.

The zebrafish is a popular vertebrate model organism with similar organ systems and gene sequences to humans. Zebrafish embryos are optically transparent enabling organ visualisation, which can be complemented with gene expression analysis at the transcript and protein levels. Furthermore, zebrafish can be treated with small molecules and drugs in a microtitre plate format for high-throughput analysis and for the identification and validation of drugs. High-throughput methodologies for use in zebrafish include phenotype-based visualisation, transcript studies using low-density DNA microarrays and proteomic analysis. These technologies offer significant whole-organism biological value in the drug discovery and drug development pipeline.

Animals↗

PubMed2006

Methanol alters ecto-nucleotidases and acetylcholinesterase in zebrafish brain.

Methanol is a neurotoxic compound that is responsible for serious damage on CNS. Besides being found as an environmental contaminant, this alcohol is also employed as a component of cryoprotector solutions for zebrafish embryos. Here we tested the acute effect of methanol on ecto-nucleotidase (NTPDase, ecto-5'-nucleotidase) and acetylcholinesterase (AChE) activities in zebrafish brain. After acute treatment, there were significant decreases on ATP (26% and 45%) and ADP hydrolysis (26% and 30%) at 0.5% and 1.0%, respectively. However, no significant alteration on ecto-5'-nucleotidase activity was verified in zebrafish brain. A significant inhibition on AChE activity (39%, 33% and 30%) was observed at the range of 0.25% to 1.0% methanol exposure. Four NTPDase sequences were identified from phylogenetic analyses, which one is similar to NTPDase1 and the others to NTPDase2. Methanol was able to inhibit NTPDase1, two isoforms of NTPDase2 and AChE transcripts. To evaluate if methanol affects directly these enzymes activities, we have performed in vitro assays. ATP hydrolysis presented a significant inhibition (19% and 34%) at 1.5% and 3.0%, respectively, and ADP hydrolysis decreased only at 3.0% (29.2%). Nevertheless, AMP hydrolysis and AChE were not altered after in vitro exposure. The inhibitory effect observed on these enzymes could contribute to the neurodegenerative events promoted by methanol in zebrafish brain.

5'-Nucleotidase↗

PubMed2002

Cell migration in the postembryonic development of the fish lateral line.

We examine at the cellular level the postembryonic development of the posterior lateral line in the zebrafish. We show that the first wave of secondary neuromasts is laid down by a migrating primordium, primII. This primordium originates from a cephalic region much like the primordium that formed the primary line during embryogenesis. PrimII contributes to both the lateral and the dorsal branches of the posterior lateral line. Once they are deposited by the primordium, the differentiating neuromasts induce the specialisation of overlying epidermal cells into a pore-forming annulus, and the entire structure begins to migrate ventrally across the epithelium. Thus the final two-dimensional pattern depends on the combination of two orthogonal processes: anteroposterior waves of neuromast formation and dorsoventral migration of individual neuromasts. Finally, we examine how general these migratory processes can be by describing two fish species with very different adult patterns, Astyanax fasciatus (Mexican blind cavefish) and Oryzias latipes (medaka). We show that their primary patterns are nearly identical to that observed in zebrafish embryos, and that their postembryonic growth relies on the same combination of migratory processes that we documented in the case of the zebrafish.

Animals↗

PubMed2002

Analysis of xanthophore and pterinosome biogenesis in zebrafish using methylene blue and pteridine autofluorescence.

We have identified two simple methods to analyse xanthophore and pterinosome biogenesis in zebrafish. The first uses methylene blue (methylthionium chloride), a redox dye which specifically labels xanthophores and pterinosomes, while the second uses autofluorescence to detect pteridine levels; these methods may be used to detect the number, location and shape of xanthophores and pterinosomes. These assays were applied to two zebrafish mutants--brie and yobo--and revealed that both mutants have pterinosome biogenesis and pteridine synthesis defects. Additionally, using capillary electrophoresis, we provide evidence that sepiapterin is responsible for the yellow colour and blue-light induced fluorescence in zebrafish embryos.

Animals↗

PubMed2006

Enhancer detection in the zebrafish using pseudotyped murine retroviruses.

Vectors based on murine retroviruses are among the most efficient means to insert reporter constructs into the context of a vertebrate chromosome with the aim to visualize cis-regulatory information available to a basal promoter at the site of insertion. In combination with using the zebrafish embryo as a readout for the activity of regulatory elements, enhancer detection becomes a powerful technique for gene discovery and for the mapping of the extent of regulatory domains in a vertebrate genome. Our laboratory has performed the only large-scale enhancer detection screen to date in any vertebrate and we describe in this paper the methods we developed to generate viral particles, to insert reporter constructs into the zebrafish germ line, the screening of detection events in heterozygous F1 embryos, and the isolation of genomic sequence flanking the inserted vector for the purpose of genomic mapping. Given sufficient scale, the technology described here can be used to obtain cis-regulatory information across the entire zebrafish genome for any given basal promoter.

Animals↗

PubMed2006

Embryonic zebrafish neuronal growth is not affected by an applied electric field in vitro.

Naturally occurring electric fields (EFs) have been implicated in cell guidance during embryonic development and adult wound healing. Embryonic Xenopus laevis neurons sprout preferentially towards the cathode, turn towards the cathode, and migrate faster towards the cathode in the presence of an external EF in vitro. A recent Phase 1 clinical trial has investigated the effects of oscillating EFs on human spinal cord regeneration. The purpose of this study was to investigate whether embryonic zebrafish neurons respond to an applied EF, and thus extend this research into another vertebrate system. Neural tubes of zebrafish embryos (16-17 somites) were dissected and dissociated neuroblasts were plated onto laminin-coated glass. A 100 mV/mm EF was applied to cell cultures for 4 or 20 h and the responses of neurons to the applied EFs were investigated. After 4h in an EF neurites were significantly shorter than control neurites. No other statistically significant effects were observed. After 20 h, control and EF-exposed neurites were no different in length. No length difference was seen between cathodally- and anodally-sprouted neurites. Application of an EF did not affect the average number of neurons in a chamber. Growth cones did not migrate preferentially towards either pole of the EF and no asymmetry was seen in neurite sprout sites. We conclude that zebrafish neurons do not respond to a 100 mV/mm applied EF in vitro. This suggests that neurons of other vertebrate species may not respond to applied EFs in the same ways as Xenopus laevis neurons.

Animals↗

PubMed2007

Transcriptional regulation of arylalkylamine-N-acetyltransferase-2 gene in the pineal gland of the gilthead seabream.

Pineal serotonin-N-acetyltransferase (arylalkylamine-N-acetyltransferase; AANAT) is considered the key enzyme in the generation of circulating melatonin rhythms; the rate of melatonin production is determined by AANAT activity. In all the examined species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2. DNA constructs were prepared in which green fluorescent protein was driven by the aanat2 promoters of seabream and Northern pike. In vivo transient expression analyses in zebrafish embryos indicated that these promoters contain the necessary elements to drive enhanced expression in the pineal gland. In the light-entrainable clock-containing PAC-2 zebrafish cell line, a stably transfected seabream aanat2 promoter-luciferase DNA construct exhibited a clock-controlled circadian rhythm of luciferase activity, characteristic for an E-box-driven expression. In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms.

Animals↗

PubMed2006

Analysis and visualization of cell movement in the developing zebrafish brain.

Detailed reconstruction of the spatiotemporal history of embryonic cells is key to understanding tissue formation processes but is often complicated by the large number of cells involved, particularly so in vertebrates. Through a combination of high-resolution time-lapse lineage tracing and antibody staining, we have analyzed the movement of mesencephalic and metencephalic cell populations in the early zebrafish embryo. To facilitate the analysis of our cell tracking data, we have created TracePilot, a software tool that allows interactive manipulation and visualization of tracking data. We demonstrate its utility by showing novel visualizations of cell movement in the developing zebrafish brain. TracePilot (http://www.mpi-cbg.de/tracepilot) is Java-based, available free of charge, and has a program structure that allows the incorporation of additional analysis tools.

Animals↗

PubMedDate not supplied

Ethanol- and acetaldehyde-mediated developmental toxicity in zebrafish.

Ethanol is a well-established developmental toxicant; however, the mechanism(s) of this toxicity remains unclear. Zebrafish are becoming an important model system for the evaluation of chemical and drug toxicity. In this study, zebrafish embryos were utilized to compare the developmental toxicity resulting from either ethanol or acetaldehyde exposure. Embryos were exposed to waterborne ethanol concentrations for various lengths of time but encompassed the earliest stages of embryogenesis. The waterborne ethanol concentration that causes 50% mortality (LC(50)) following a 45-h ethanol exposure was approximately 340 mM (1.98% v/v). A number of reproducible endpoints resulted from ethanol exposure and included pericardial edema, yolk sac edema, axial malformations, otolith defects, delayed development, and axial blistering. When the exposure period was reduced, similar signs of toxicity were produced at nearly identical ethanol concentrations. To estimate the embryonic dose following a given waterborne ethanol concentration, a kinetic alcohol dehydrogenase (ADH) assay was adapted. The average embryonic ethanol dose was calculated to be a fraction of the waterborne concentration. Embryos exposed to waterborne acetaldehyde resulted in similar, but not identical, endpoints as those induced by ethanol. Embryos were however, almost three orders of magnitude more sensitive to acetaldehyde than to ethanol. Ethanol and acetaldehyde both negatively impact embryonic development; however, ethanol is more teratogenic based on teratogenic indices (TIs). These results demonstrate that the zebrafish model will provide an opportunity to further evaluate the mechanism of action of ethanol on vertebrate development.

Abnormalities, Multiple↗

PubMed2005

Wortmannin induces zebrafish cardia bifida through a mechanism independent of phosphoinositide 3-kinase and myosin light chain kinase.

Cardia bifida is an anomaly of the embryonic heart in which the bilateral myocardial rudiments fail to travel to the midline, resulting in the formation of two separate hearts in lateral positions. In zebrafish, eight loci responsible for the cardia bifida phenotype were identified in the large-scale genetic screen. Wortmannin has been reported to be a highly selective inhibitor of phosphoinositide 3-kinase and myosin light chain kinase activity. We provide the first evidence that wortmannin treatment of zebrafish embryos can induce cardia bifida in a dose-dependent manner and that wortmannin alters cardiac development between 6 and 16 h post-fertilization. In addition, we demonstrate that wortmannin induces zebrafish cardia bifida through a mechanism independent of phosphoinositide 3-kinase and myosin light chain kinase. Our findings may provide new insights into the cardiomyocyte function and disfunction.

Androstadienes↗

PubMed2006

Antisense targeting of CXXC finger protein 1 inhibits genomic cytosine methylation and primitive hematopoiesis in zebrafish.

CXXC finger protein 1 (CFP1) binds to unmethylated CpG dinucleotides and is a component of the Set1 histone methyltransferase complex. Mice lacking CFP1 suffer a peri-implantation lethal phenotype, and CFP1-deficient embryonic stem cells are viable but unable to differentiate and exhibit a 60-80% decrease in genomic cytosine methylation. A zebrafish homolog of CFP1 has been identified, is approximately 70% similar to murine CFP1, and is widely expressed during development. Zebrafish embryos treated with a zCFP1 antisense morpholino oligonucleotide had little or no circulating red blood cells and exhibited abnormal yolk sac morphology at 48 h post-fertilization. Many of the antisense-treated zebrafish also exhibited cardiac edema, and 14% were dead at 24 h post-fertilization. Morphant zebrafish also exhibited elevated levels of apoptosis, particularly in the intermediate cell mass, the site of primitive erythropoiesis, as well as aberrations in vascular development. Genomic DNA isolated from morphant embryos exhibited a 60% reduction of global genomic cytosine methylation. A similar phenotype was observed with an independent zCFP1 antisense morpholino oligonucleotide, but not following injection of an unrelated control oligonucleotide. The morphant phenotype was rescued when mRNA encoding murine CFP1 was co-injected with the antisense oligonucleotide. Genomic data base analysis reveals the presence of a second version of zebrafish CFP1 (zCFP1b). However, the morphant phenotype observed following specific depletion of zCFP1 indicates that these related genes have nonredundant functions controlling normal zebrafish hematopoiesis and epigenetic regulation. These findings establish the importance of CFP1 during postgastrulation development.

Amino Acid Motifs↗

PubMed2002

Characterization of muscle-regulatory genes, Myf5 and myogenin, from striped bass and promoter analysis of muscle-specific expression.

Myf5 and Myogenin are basic helix-loop-helix transcription factors that belong to the muscle regulatory factor (MRF) gene family, which plays important roles in regulating skeletal muscle development and growth. Members of the MRF family, including Myf5, MyoD, Myogenin, and MRF4 are specifically expressed in skeletal muscle cells. They have the remarkable property of converting a variety of cells into myoblasts and myotubes when ectopically expressed in other cell types. To better understand their role and regulation of expression in fish muscle cells, Myf5 and myogenin genomic genes were isolated from striped bass (Morone saxatilis). Sequence analysis revealed that these 2 genes shared similar structures. They both contained 3 exons and 2 introns, and a highly conserved basic helix-loop-helix domain. Promoter analysis identified several putative E box sites in both Myf5 and myogenin promoters that might confer muscle-specific expression. To determine if the striped bass Myf5 and myogenin promoters could control muscle-specific expression, the Myf5 or myogenin promoter was linked with the green fluorescent protein (GFP) reporter gene, and their promoter activity was analyzed in zebrafish embryos by transient expression assay. Our data showed that both striped bass Myf5 and myogenin promoters could drive muscle-specific GFP expression in zebrafish. These data demonstrated that a muscle-specific regulatory element or elements were located within the striped bass Myf5 and myogenin promoters, and were conserved between striped bass and zebrafish. Moreover, these data suggested the muscle-specific regulatory element could function across fish species in regulating gene expression.

Journal Article↗

PubMed2005

The basic helix-loop-helix olig3 establishes the neural plate boundary of the trunk and is necessary for development of the dorsal spinal cord.

olig genes encode a previously unrecognized group of vertebrate-specific basic helix-loop-helix transcription factors. As shown in mice, chickens, and zebrafish, two members of this group, olig1 and olig2, are involved in the differentiation of motoneurons and oligodendrocytes, but nothing is known about the role of the third member, olig3. Here, we show that olig3 plays an essential role in the establishment of the neural crest-lateral neural plate boundary. In zebrafish embryos, morpholino-induced olig3 inactivation dramatically increases the number of neural crest cells, but lateral neural plate fates (interneurons and astrocytes) are missing. Zebrafish swirl mutants that have impaired bone morphogenetic protein signaling and lack neural crest cells display an expanded olig3 expression domain. Moreover, olig3 is up-regulated in mindbomb mutants lacking the neural crest because of an impaired notch signaling, and olig3 repression in such mutants rescues the neural crest. In addition, olig3 regulates ngn1 and deltaA expression in interneuron precursors. Our results indicate that olig3 has an essential proneural activity in the dorsal spinal cord and cooperates with the Delta/Notch regulatory loop to establish the boundary between the neural crest and the lateral neural plate. Thus, a proper regulation of the olig gene family is essential for the formation of three cell types (oligodendrocytes, astrocytes, and neural crest) that are unique to vertebrates.

Animals↗

PubMed2006

Mutant desmocollin-2 causes arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetically heterogeneous heart-muscle disorder characterized by progressive fibrofatty replacement of right ventricular myocardium and an increased risk of sudden cardiac death. Mutations in desmosomal proteins that cause ARVC have been previously described; therefore, we investigated 88 unrelated patients with the disorder for mutations in human desmosomal cadherin desmocollin-2 (DSC2). We identified a heterozygous splice-acceptor-site mutation in intron 5 (c.631-2A-->G) of the DSC2 gene, which led to the use of a cryptic splice-acceptor site and the creation of a downstream premature termination codon. Quantitative analysis of cardiac DSC2 expression in patient specimens revealed a marked reduction in the abundance of the mutant transcript. Morpholino knockdown in zebrafish embryos revealed a requirement for dsc2 in the establishment of the normal myocardial structure and function, with reduced desmosomal plaque area, loss of the desmosome extracellular electron-dense midlines, and associated myocardial contractility defects. These data identify DSC2 mutations as a cause of ARVC in humans and demonstrate that physiologic levels of DSC2 are crucial for normal cardiac desmosome formation, early cardiac morphogenesis, and cardiac function.

Adult↗

PubMed1996

Characterizing the zebrafish organizer: microsurgical analysis at the early-shield stage.

The appearance of the embryonic shield, a slight thickening at the leading edge of the blastoderm during the formation of the germ ring, is one of the first signs of dorsoventral polarity in the zebrafish embryo. It has been proposed that the shield plays a role in fish embryo patterning similar to that attributed to the amphibian dorsal lip. In a recent study, we fate mapped many of the cells in the region of the forming embryonic shield, and found that neural and mesodermal progenitors are intermingled (Shih, J. and Fraser, S.E. (1995) Development 121, 2755-2765), in contrast to the coherent region of mesodermal progenitors found at the amphibian dorsal lip. Here, we examine the fate and the inductive potential of the embryonic shield to determine if the intermingling reflects a different mode of embryonic patterning than that found in amphibians. Using the microsurgical techniques commonly used in amphibian and avian experimental embryology, we either grafted or deleted the region of the embryonic shield. Homotopic grafting experiments confirmed the fates of cells within the embryonic shield region, showing descendants in the hatching gland, head mesoderm, notochord, somitic mesoderm, endoderm and ventral aspect of the neuraxis. Heterotopic grafting experiments demonstrated that the embryonic shield can organize a second embryonic axis; however, contrary to our expectations based on amphibian research, the graft contributes extensively to the ectopic neuraxis. Microsurgical deletion of the embryonic shield region at the onset of germ ring formation has little effect on neural development: embryos with a well-formed and well-patterned neuraxis develop in the complete absence of notochord cells. While these results show that the embryonic shield is sufficient for ectopic axis formation, they also raise questions concerning the necessity of the shield region for neural induction and embryonic patterning after the formation of the germ ring.

Animals↗