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PubMed2004

Cloning of somatolactin alpha and beta cDNAs in zebrafish and phylogenetic analysis of two distinct somatolactin subtypes in fish.

Somatolactin (SL) is a pituitary hormone belonging to the growth hormone/prolactin superfamily, with recognizable homologues in all fish taxa examined to date. Although sequences from most fish share reasonably high sequence identity, several more highly divergent SLs have been reported. Goldfish SL and a second SL protein found in rainbow trout (rtSLP) are remarkably different from each other and also dissimilar to other SLs. It has been unclear whether rtSLP is a recent paralogue restricted to rainbow trout, or reflects a more ancient duplication of the SL gene, and whether it is related to the goldfish sequence. Here we report the cloning of two different zebrafish SL cDNAs, which share only 57.5% nucleotide and 47.7% deduced amino acid identities. One copy, designated zebrafish SLalpha (zfSLalpha), displays a typical range of sequence similarity to most other SLs. The other copy, zebrafish SLbeta (zfSLbeta), shows low identity to most other SLs; surprisingly, it is most similar to the divergent SL sequence from goldfish. The mRNAs of zfSLalpha and zfSLbeta were expressed specifically in two distinct regions of the pars intermedia in zebrafish. Cells expressing zfSLalpha are located at the posterior pars intermedia, bordering the neurohypophysis, whereas zfSLbeta is expressed in the anterior part of the pars intermedia, bordering the pars distalis. Phylogenetic analyses indicate that zfSLbeta, goldfish SL and rtSLP all belong to the SL hormone family; however, along with the genes from eel and catfish, these divergent sequences form a group that is clearly distinct from all other SLs. These results suggest the presence of two distinct SL families, SLalpha and SLbeta, which may trace back to a teleost genome duplication prior to divergence of the cyprinids and salmonids.

Amino Acid Sequence↗

PubMed2006

A novel contact assay for testing genotoxicity of chemicals and whole sediments in zebrafish embryos.

Broad consensus exists that whole-sediment exposure protocols represent the most realistic scenario to simulate in situ exposure conditions. So far, however, several endpoints including genotoxicity in vertebrate-based systems could be tested only after transfer of particle-bound substances into the aqueous phase. The present study was carried out to develop a protocol for generating a suspension of single cells from sediment-exposed zebrafish embryos that is suitable for detecting particle-bound genotoxicity in the alkaline single cell gel electrophoresis (comet assay). In this solid-phase genotoxicity assay, a whole-body cell suspension derived from zebrafish embryos exposed to native (whole) sediments is assayed in the comet assay. Several chemical and mechanical isolation procedures were compared to optimize cell yield and minimize DNA damage by the method itself. If compared to collagenase isolation, mechanical cell dissociation gave less DNA damage; trypsinization resulted in similarly low DNA damage but significantly lower cell yield. In order to test the optimized protocol, effects of well-known genotoxicants (4-nitroquinoline-N-oxide, nitrofurantoin, hydrogen peroxide, benzo[a]pyrene) and of two sediments from the upper Rhine River (Germany) on zebrafish embryos were investigated. Results documented clear-cut genotoxicity for all four substances and for one of the two whole-sediment samples. An ultraviolet (UV) light exposure of whole embryos and primary cultures from embryos elucidated only minor effects for the whole embryos compared to the primary cells. Consequently, UV irradiation cannot be suggested as a positive control in intact zebrafish embryos. In conclusion, the newly developed sediment contact assay can be recommended for the detection of both single substances but also the bioavailable fraction of the total hazard potential of sediments.

Animals↗

PubMed2002

The zebrafish as a model system for human disease.

The zebrafish (Danio rerio) has been widely utilised for the study of developmental biology, which has lead to the evolution of sophisticated cellular and molecular approaches. More recently, the rapid progress of various zebrafish genomic infrastructure initiatives is facilitating the development of zebrafish models of human disease. This review aims to describe several representative examples of how the zebrafish can be successfully used to identify novel genes and assign gene function, providing invaluable clues to human pathophysiology.

Animals↗

PubMed1994

Induction of zebrafish (Brachydanio rerio) P450 in vivo and in cell culture.

1. Induction of zebrafish P450 by 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) was studied in liver tissue, primary liver cell culture and multipassage cell culture derived from zebrafish haploid and diploid embryos and liver. 2. TCDD induced two hepatic proteins (54 and 50 kDa) in vivo which were recognized by anti-trout P4501A1 IgG. The 54-kDa protein was induced by TCDD in primary and multipassage hepatocyte cultures and in haploid and diploid embryo-derived cells. The proteins in liver homogenates were not induced by aqueous exposure of zebrafish to beta-naphthoflavone (BNF). 3. Homogenates of zebrafish liver, cultured hepatocytes and embryo-derived cells also exhibited increased ethoxyresorufin O-deethylase (EROD) and 7-12-dimethyl-benz[a]anthracene (DMBA) hydroxylase activity following TCDD exposure.

Animals↗

PubMed1999

The zebrafish swimbladder: A simple model for lung elastin injury and repair.

In this communication we offer data to suggest that the zebrafish swimbladder may provide a simple model of elastin injury and repair which is amenable to genetic analysis and pertinent to lung physiology. In situ hybridization of zebrafish embryos illustrated that elastin gene expression is evident in the developing gut tract prior to swimbladder morphogenesis. Northern blot analysis demonstrated that the major zebrafish elastin mRNA is 2.0 kb which is significantly smaller than its higher vertebrate counterpart. Amino acid analysis of alkali-resistant protein from the anterior chamber of the adult zebrafish swimbladder showed a composition similar to higher vertebrate elastins including significant amounts of desmosine crosslinks. Electron microscopic investigations of the swimbladder wall indicate a simple structure with an inner layer of elastin fibers. Elastase delivery to the swimbladder in vitro resulted in significant fragmentation of elastin in the anterior chamber providing an environment for studying elastin repair within the tissue.

Air Sacs↗

PubMedDate not supplied

Cloning of zebrafish cDNA for 3beta-hydroxysteroid dehydrogenase and P450scc.

P450scc and 3beta-HSD cDNA were isolated from a zebrafish lambda gt10 cDNA library using trout SCC and 3beta-HSD cDNA as the probes. The zebrafish SCC cDNA encodes a protein of 509 amino acids, which shares a 78% similarities with the trout SCC and 58% with the human SCC. As for 3beta-HSD, two forms of cDNA were isolated, termed HSD 5 and HSD 17, which may have resulted from alternative splicing. HSD 5 and HSD 17 encode proteins of 374 and 341 amino acids respectively. Both share 77% amino acid similarities with trout 3beta-HSD and 53% similarities with the mouse 3beta-HSD. Zebrafish has been increasingly used as a genetic model system to study organ development and to investigate human diseases. The cloning and the characterization of zebrafish P450scc and 3beta-HSD should facilitate study of steroidogenesis and human disease associated with steroid imbalance.

3-Hydroxysteroid Dehydrogenases↗

PubMed1998

Assembly of a complete zebrafish mitochondrial 16S rRNA gene from overlapping expressed sequence tags.

A complete zebrafish mitochondrial 16S ribosomal RNA gene was assembled from our existing zebrafish EST clones by aligning them with the carp mitochondria 16S rRNA sequence. The overall homology between our assembled zebrafish mt 16S rRNA and the carp mt 16S rRNA is 83.5%. As the number of zebrafish ESTs grows, the assembly of more full-length cDNA sequences from overlapping EST data could be expected.

Animals↗

PubMed2000

Conservation of Mhc class III region synteny between zebrafish and human as determined by radiation hybrid mapping.

In the HLA, H2, and other mammalian MHC:, the class I and II loci are separated by the so-called class III region comprised of approximately 60 genes that are functionally and evolutionarily unrelated to the class I/II genes. To explore the origin of this island of unrelated loci in the middle of the MHC: 19 homologues of HLA class III genes, we identified 19 homologues of HLA class III genes as well as 21 additional non-class I/II HLA homologues in the zebrafish and mapped them by testing a panel of 94 zebrafish-hamster radiation hybrid cell lines. Six of the HLA class III and eight of the flanking homologues were found to be linked to the zebrafish class I (but not class II) loci in linkage group 19. The remaining homologous loci were found to be scattered over 14 zebrafish linkage groups. The linkage group 19 contains at least 25 genes (not counting the class I loci) that are also syntenic on human chromosome 6. This gene assembly presumably represents the pre-MHC: that existed before the class I/II genes arose. The pre-MHC: may not have contained the complement and other class III genes involved in immune response.

Animals↗

PubMed1999

Gene duplication of zebrafish JAK2 homologs is accompanied by divergent embryonic expression patterns: only jak2a is expressed during erythropoiesis.

Members of the JAK family of protein tyrosine kinase (PTK) proteins are required for the transmission of signals from a variety of cell surface receptors, particularly those of the cytokine receptor family. JAK function has been implicated in hematopoiesis and regulation of the immune system, and recent data suggest that the vertebrate JAK2 gene may play a role in leukemia. We have isolated and characterized jak cDNAs from the zebrafish Danio rerio. The zebrafish genome possesses 2 jak2 genes that occupy paralogous chromosome segments in the zebrafish genome, and these segments conserve syntenic relationships with orthologous genes in mammalian genomes, suggesting an ancient duplication in the zebrafish lineage. The jak2a gene is expressed at high levels in erythroid precursors of primitive and definitive waves and at a lower level in early central nervous system and developing fin buds. jak2b is expressed in the developing lens and nephritic ducts, but not in hematopoietic tissue. The expression of jak2a was examined in hematopoietic mutants and found to be disrupted in cloche and spadetail, suggesting an early role in hematopoiesis. Taken together with recent gene knockout data in the mouse, we suggest that jak2a may be functionally equivalent to mammalian Jak2, with a role in early erythropoiesis.

Alleles↗

PubMed2000

Involvement of L1.1 in memory consolidation after active avoidance conditioning in zebrafish.

To investigate the involvement of the cell adhesion molecules L1.1, L1.2, NCAM, and tenascin-C in memory formation, zebrafish (Brachydanio rerio) were trained in an active avoidance paradigm to cross a hurdle to avoid mild electric shocks after a light signal. Application of [(14)C]deoxyglucose prior to the training session revealed an increased energy demand in the optic tectum during acquisition of the active avoidance response compared with untrained fish and with fish not learning the task (nonlearners). In situ hybridization with digoxigenin-labeled cRNA probes directed against zebrafish L1.1, L1.2, NCAM, and tenascin-C revealed an enhanced expression of L1.1 and NCAM mRNA in the optic tectum of learners 3 h after acquisition of the task compared with untrained fish, nonlearners, overtrained fish, and learners decapitated 1 or 6 h after acquisition. Levels of L1.2 mRNA were not significantly increased in the tectum 3 h after learning. Tenascin-C was neither expressed in the optic tectum of untrained fish nor in the tectum of learners. To test for a possible involvement of L1.1 in memory consolidation, antibodies were injected intracerebroventricularly 1 h after the last training trial. Two days later, injected zebrafish were tested for recall and evaluated by a retention score (RS), ranging from 1.0 for immediate recall to 0.0 indicating no savings. The average retention score of L1.1 antibody-injected fish (RS = 0. 29) was different from that of tenascin-C antibody-injected (RS = 0. 71) or uninjected fish (RS = 0.78), indicating a pivotal function of L1.1 in long-term memory formation in zebrafish.

Animals↗

PubMed2000

Diagnosis and management of atypical Mycobacterium spp. infections in established laboratory zebrafish (Brachydanio rerio) facilities.

Two established zebrafish colonies experienced increased mortality and decreased reproductive performance. Initial examination of several fish from one facility revealed hyperemic gills, petechia around the opercula, abdominal distention, and emaciation. Affected fish had congested liver with inflammation and multifocal hepatic necrosis. Large numbers of acid-fast-positive, rod-shaped bacteria were evident in multiple tissues and the blood. Mycobacterium fortuitum was subsequently isolated from several fish. Zebrafish from the second facility had skin erosions and ulceration along the flank just caudal to the pectoral fins. Large numbers of acid-fast-positive, rod-shaped bacteria were observed within the necrotic centers of well-demarcated, multifocal granulomas in gonads, liver, and peritoneum from affected fish. Mycobacterium abscessus and M. chelonae were isolated and identified biochemically. Definitive diagnosis in these outbreaks was obtained by culture on selective media. Because Mycobacterium spp. grow extremely slowly and positive confirmation may require 45 to 60 days, Mycobacterium species-specific polymerase chain reaction analysis was used to provide a rapid screening assay for Mycobacterium spp. as well as for verification of culture results. To our knowledge, this is the first documentation of mycobacterial infection in laboratory-maintained zebrafish and provides guidelines for diagnosis, management, and prevention of atypical mycobacteriosis in laboratory zebrafish colonies.

Animals↗

PubMed2001

Zebrafish keratin 8 is expressed at high levels in the epidermis of regenerating caudal fin.

Cytokeratins are structural proteins of the intermediate filament family and are mainly expressed in epithelial cells. In several vertebrates it has been shown that keratin 8 is expressed in simple epithelial tissues, some non-epithelial tissue and in hyper-proliferative tissues during development and tumor transformation. We previously cloned and characterised the zebrafish (Danio rerio) homologous cytokeratin 8 cDNA (zfk8) which was described as an epidermal marker during zebrafish development. It has been found that the zfk8 gene is normally expressed in simple epithelia in embryonic and mature zebrafish. Using whole-mount in situ hybridisation, we show in this report that expression of zfk8 is tightly linked to the regeneration of caudal fin and exclusively observed in epidermal cells. It is strongly expressed in the epidermis overlaying the inter-rays zone of regenerating caudal fin. Our results indicate that in zebrafish, cytokeratin 8 is a suitable epidermal marker during regeneration.

Animals↗

PubMedDate not supplied

Differential expression of cadherin-2 and cadherin-4 in the developing and adult zebrafish visual system.

Cadherins are homophilic cell adhesion molecules that control development of a variety of tissues and maintenance of adult structures. Although cadherins have been implicated in the development of the brain, including the visual system, in several vertebrate species, little is known of their role in zebrafish. In this study, we examined distribution of cadherin-2 (Cdh2, N-cadherin) in the visual system of developing and adult zebrafish using both immunocytochemical and in situ hybridization methods, and we compared Cdh2 distribution to that of the previously reported and closely related cadherin-4 (Cdh4, R-cadherin). As in other vertebrates, in zebrafish embryos Cdh2 was widely expressed in the early nervous system, but its expression became more restricted as development proceeded. Cdh4 was not detectable until later in development, at about the time when the first ganglion cells are generated. Cdh2 and Cdh4 were expressed in distinct regions of developing visual structures, including the lens. We hypothesize that the differential expression of these two cadherins in developing zebrafish visual structures reflects functionally different roles in the development of the vertebrate visual system.

Animals↗

PubMed2002

Developmental biology of zebrafish myeloid cells.

The zebrafish (Danio rerio) has emerged as an informative vertebrate model for developmental studies, particularly those employing genetic approaches such as mutagenesis and screening. Zebrafish myelopoiesis has recently been characterized, paving the way for the experimental strengths of this model organism to contribute to an improved understanding of the genetic regulation of myeloid development. Zebrafish have a multi-lineage myeloid compartment with two types of granulocyte (heterophil/neutrophil and eosinophil granulocytes), and monocyte/macrophages, each with characteristic morphological features and histochemical staining properties. Molecular markers have been characterised for various myeloid cell types and their precursor cells, for example: stem cells (scl, hhex, lmo2), myeloid lineage precursors (spi1/pu.1, c/ebp1), heterophil granulocytes (mpx/mpo), macrophages (L-plastin, fms). In zebrafish, the sites of early myeloid and erythroid commitment are anatomically separated, being located in the rostral and caudal lateral plate mesoderm respectively. Functional macrophages appear before cells displaying granulocytic markers. By the second day of life, cells expressing granulocyte- and macrophage-specific genes are scattered throughout the embryo, but tend to aggregate in the ventral venous plexus, which may be a site of their production or a preferred site for their residence. Even in early embryos, macrophages are phagocytically active, and granulocytes participate in acute inflammation. Equipped with an understanding of the developmental biology of these various myeloid cells and a set of tools for their identification and functional study, we will now be able to exploit the experimental strengths of this model organism to better understand the genetic regulation of myelopoiesis.

Animals↗

PubMed2002

Regulation by glycogen synthase kinase-3beta of the arborization field and maturation of retinotectal projection in zebrafish.

The retinotectal projection is one of the best systems to study the molecular basis of synapse formation in the CNS because of the well characterized topographic connections and activity-dependent refinement. Here, we developed a presynaptic neuron-specific gene manipulation system in the zebrafish retinotectal projection in vivo using the nicotinic acetylcholine receptor beta3 (nAChRbeta3) gene promoter. Enhanced green fluorescent protein (EGFP) expression signals in living transgenic zebrafish lines carrying the nAChRbeta3 gene promoter-directed EGFP expression vector visualized the development of entire retinal ganglion cell (RGC) axon projection to the tectum. Microinjection of the nAChRbeta3 gene promoter-driven double-cassette vectors directing the expression of both dominant-negative glycogen synthase kinase-3beta (dnGSK-3beta) and EGFP enabled us to follow the development of individual RGCs and to examine the effect of the molecule on the axonal arborization and maturation of the same neurons in living zebrafish. We found that the expression of the dominant-negative form of zebrafish GSK-3beta suppressed the arborization field of RGC axon terminals in the tectum as estimated by the reduction of arbor branch length and arbor areas. Furthermore, the suppression of GSK-3beta activity increased the size of vesicle-associated membrane protein 2-EGFP puncta in RGC axon terminals at the early stage of innervation to the tectum. These results suggest that GSK-3beta regulates the arborization field and maturation of RGC axon terminals in vivo.

Animals↗

PubMed2003

Visuomotor behaviors in larval zebrafish after GFP-guided laser ablation of the optic tectum.

The optic tectum is the largest visual center in most vertebrates and the main target for retinal ganglion cells (RGCs) conveying visual information from the eye to the brain. The retinotectal projection has served as an important model in many areas of developmental neuroscience. However, knowledge of the function of the tectum is limited. We began to address this issue using laser ablations and subsequent behavioral testing in zebrafish. We used a transgenic zebrafish line that expresses green-fluorescent protein in RGCs projecting to the tectum. By aiming a laser beam at the labeled retinal fibers demarcating the tectal neuropil, the larval tectum could be selectively destroyed. We tested whether tectum-ablated zebrafish larvae, when presented with large-field movements in their surroundings, displayed optokinetic responses (OKR) or optomotor responses (OMR), two distinct visuomotor behaviors that compensate for self-motion. Neither OKR nor OMR were found to be dependent on intact retinotectal connections. Also, visual acuity remained unaffected. Tectum ablation, however, slowed down the OKR by reducing the frequency of saccades but left tracking velocity, gain, and saccade amplitude unaffected. Removal of the tectum had no effect on the processing of second-order motion, to which zebrafish show both OKR and OMR, suggesting that the tectum is not an integral part of the circuit that extracts higher-order cues in the motion pathway.

Animals↗

PubMed1992

Characterization of AluI repeats of zebrafish (Brachydanio rerio).

Two families of repetitive DNA sequences were isolated from the zebrafish genome and characterized. Eight different sequences were sequenced and classified by two standards, their (G + C) composition and their lengths. For convenience, the sequences were first divided into two types. Type I was (A + T)-rich, was repeated approximately 500,000 times, and constituted approximately 5% of the zebrafish genome. Type II was (G + C)-rich, was reiterated approximately 90,000 times, and comprised approximately 0.5% of the genome. Agarose gel electrophoresis of zebrafish DNA cleaved with AluI revealed three distinguishable bands of repetitive fragments: large (approximately 180 bp, designated RFAL), medium (approximately 140 bp, RFAM), and small (approximately 90 bp, RFAS). The RFAL fragments contained both type I and type II sequences. Limited digestion of genomic DNA indicated that RFAL and RFAM were tandemly arranged in the genome, whereas RFAS showed a mixed pattern of both tandem and interspersed repeated arrangements. Although inclusion of a repetitive sequence in a transgenic construct did not appreciably accelerate homologous integration of transgenes into the zebrafish genome, the AluI sequences could facilitate transgene mapping following chromosomal integration.

Animals↗

PubMed2004

Novel vector systems optimized for injecting in vitro-synthesized mRNA into zebrafish embryos.

Microinjection of nucleic acids or proteins is a useful way of studying embryonic development. In particular, injection of in vitro-transcribed capped RNA is commonly employed to achieve ectopic or increased expression of genes. Two vector systems, pCS2+ and pT7Ts, have been used for this purpose in zebrafish. However, they were initially optimized for Xenopus embryos not for zebrafish. Here we describe a vector, pcGlobin2, optimized for zebrafish, and its derivative, pcGlobin2-GST. This new vector system offers several advantages. First, pcGlobin 2 contains three critical elements 15' and 3' zebrafish beta-globin UTRs, and a poly(A) tail] for generating stable mRNAs and greatly improving translation efficiency. Second, subcloning and preparation of template DNA is easier because of the larger number of restriction sites. Third, protein-binding assays can be performed directly on the injected embryos with pcGlobin2-GST. Lastly, this vector system can be transfected into animal cells without additional subcloning.

Animals↗