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PubMed1993

Immunohistochemical localization of the cytochrome P450 isozymes LMC2 and LM4B (P4501A1) in 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated zebrafish (Brachydanio rerio).

Dibenzo-p-dioxins are persistent pollutants in our environment that exert a variety of biological and toxic effects in various species, in particular developmental toxicity in fish. Using zebrafish (Brachydanio rerio) as a model system, we analyzed the expression and inducibility of a constitutive isozyme (LMC2) and a dioxin-inducible isozyme (LM4B or P4501A1) of cytochrome (cyt.) P450 in different organs by immunohistochemical techniques. In untreated zebrafish, LMC2 was constitutively expressed at high levels in the liver, kidney, skin, and oral mucosa, while moderate expression was detected in gills, pseudobranch, intestine, and ovaries. LM4B was either not expressed in these organs or was found at comparatively low levels. After treatment of zebrafish with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; total dose of 15 ng per fish), LM4B expression was strongly induced in livers and kidneys and less pronounced in gills and pseudobranches, whereas no or only marginal induction was seen in skin, intestine, oral mucosa, and ovaries. In addition, cyt. P450 catalyzed 7-ethoxyresorufin-O-deethylation and phenol UDP-glucuronosyltransferase activity were also found to be inducible in liver and kidney. Our results demonstrate that zebrafish respond to inducing stimuli of TCDD in a fashion similar to that observed with related compounds in other fish species, suggesting that zebrafish may be a useful experimental model for studying biological and toxic effects of TCDD and other environmental pollutants in fish.

Animals↗
PubMedDate not supplied

Culture of cells from zebrafish (Brachydanio rerio) embryo and adult tissues.

The zebrafish is a popular model for studies of vertebrate development and toxicology. However, in vitro approaches with this organism have not been fully exploited because cell culture systems have been unavailable. We developed methods for the culture of cells from blastula-stage diploid and haploid zebrafish embryos, as well as cells from the caudal and pelvic fin, gill, liver, and viscera of adult fish. The haploid embryo-derived cells differentiated in culture to a pigmented phenotype and expressed, upon exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin, a protein that was immunologically and functionally similar to rainbow trout cytochrome P450IA1. Zebrafish cultures were grown in a complex basal nutrient medium supplemented with insulin, trout embryo extract, and low concentrations of trout and fetal bovine serum; they could not be maintained in conventional culture medium containing a high concentration of mammalian serum. Using calcium phosphate-mediated transfection, a plasmid constructed for use in mammalian cells was introduced into zebrafish embryo cell cultures and expressed in a stable manner. These results indicated that the transfection procedures utilized in mammalian systems can also be applied to zebrafish cell cultures, providing a means for in vitro alteration of the genotype and phenotype of the cells.

Animals↗
PubMed1999

Identification and characterization of zebrafish thrombocytes.

To analyse primary haemostasis in the zebrafish we have identified and characterized the zebrafish thrombocyte by morphologic, immunologic and functional approaches. Novel methods were developed for harvesting zebrafish blood with preservation of thrombocytes, and assaying whole blood adhesion/aggregation responses in microtitre plates. Light and electron microscopy of the thrombocyte illustrated morphological characteristics including the formation of aggregates, pseudopodia, and surface-connected vesicles analagous to the platelet canalicular system. Immunostaining with polyclonal antisera versus human platelet glycoproteins demonstrated the presence of glycoprotein Ib and IIb/IIIa-like complexes on the thrombocyte surface. Whole blood assays for adhesion/aggregation and ATP release showed ristocetin-induced adhesion without ATP release, and platelet agonist (collagen, arachidonic acid) induced aggregation with ATP release. Blood harvested from zebrafish treated with aspirin demonstrated inhibition of arachidonic acid induced aggregation and agonist induced ATP release, consistent with at least partial dependence on an intact cyclo oxygenase pathway. The combined morphologic immunologic and functional evidence suggest that the zebrafish thrombocyte is the haemostatic homologue of the mammalian platelet. Conservation of major haemostatic pathways involved in platelet function and coagulation suggests that the zebrafish is a relevant model for mammalian haemostasis and thrombosis.

Animals↗
PubMed2001

Myelopoiesis in the zebrafish, Danio rerio.

Genome-wide chemical mutagenesis screens in the zebrafish (Danio rerio) have led to the identification of novel genes affecting vertebrate erythropoiesis. In determining if this approach could also be used to clarify the molecular genetics of myelopoiesis, it was found that the developmental hierarchy of myeloid precursors in the zebrafish kidney is similar to that in human bone marrow. Zebrafish neutrophils resembled human neutrophils, possessing segmented nuclei and myeloperoxidase-positive cytoplasmic granules. The zebrafish homologue of the human myeloperoxidase (MPO) gene, which is specific to cells of the neutrophil lineage, was cloned and used to synthesize antisense RNA probes for in situ hybridization analyses of zebrafish embryos. Granulocytic cells expressing zebrafish mpo were first evident at 18 hours after fertilization (hpf) in the posterior intermediate cell mass (ICM) and on the anterior yolk sac by 20 hpf. By 24 hpf, mpo-expressing cells were observed along the ICM and within the developing vascular system. Thus, the mpo gene should provide a useful molecular probe for identifying zebrafish mutants with defects in granulopoiesis. The expression of zebrafish homologues was also examined in 2 other mammalian hematopoietic genes, Pu.1, which appears to initiate a commitment step in normal mammalian myeloid development, and L-Plastin, a gene expressed by human monocytes and macrophages. The results demonstrate a high level of conservation of the spatio-temporal expression patterns of these genes between zebrafish and mammals. The morphologic and molecular genetic evidence presented here supports the zebrafish as an informative model system for the study of normal and aberrant human myelopoiesis. (Blood. 2001;98:643-651)

Amino Acid Sequence↗
PubMed2005

Matrix metalloproteinase expression and function during fin regeneration in zebrafish: analysis of MT1-MMP, MMP2 and TIMP2.

Matrix metalloproteinases (MMPs) play key roles in the turnover of extracellular matrix (ECM) and, thereby, function as key regulators of cell-ECM interactions during development. In spite of their importance during developmental processes, relatively little has been reported about the role of these metalloproteinases during limb development and regeneration. To approach the problem of cell-ECM interactions during limb (fin) regeneration, we have utilized zebrafish as an experimental model. Based on previous MMP cloning studies from our laboratory, the current study has focused on the expression of membrane-type 1 metalloproteinase (MT1-MMP), gelatinase A (MMP-2) and endogenous tissue inhibitor 2 of metalloproteinases (TIMP-2) during fin regeneration in adult zebrafish. In situ analysis indicated co-expression of zmt1-mmp, zmmp-2, and ztimp-2 mRNA transcripts in regenerating caudal fins. In situ gelatin-zymography confirmed the presence of active metalloproteinases in regenerating fins. zmt1-mmp, zmmp-2, and ztimp-2 mRNA transcripts were expressed in the blastema and basal epithelium during caudal fin regeneration while expression of type IV collagen [zcol-IV(a5)] transcripts (a basal lamina component) was restricted to the basal epithelium. Fin outgrowth was greatly reduced in the presence of GM6001 (an inhibitor of MMP activity) indicating the importance of these enzymes during fin regeneration. Previous studies by Itoh (EMBO, 2001) indicated that expression of a vertebrate MT1-MMP construct containing only the hemopexin-transmembrane-cytoplasmic domains (MT1HPX) resulted in blockage of MT1-MMP homophilic complex formation and subsequent inhibition of pro-MMP-2 activation. Interference with homophilic complex formation was attributed to expression of the hemopexin domain at the cell surface. Building upon these earlier findings, the current study found that ectopic expression of MT1HPX in fin regenerates inhibited the regeneration process and resulted in a reduction in cell proliferation in the blastema. Taken together, these results indicate that MMPs have an important role during fin regeneration in zebrafish.

Animals↗
PubMed2003

The crypt neurons in the olfactory epithelium of the adult zebrafish express TrkA-like immunoreactivity.

The olfactory neurons of vertebrates, including teleosts, regenerate through adult life. This ability seems to depend partially on neurotrophic factors, which include some neurotrophins, mainly nerve growth factor (NGF), and their cognate Trk receptors. Zebrafish is an ideal model for studying neurogenesis in the olfactory system, but what kinds of olfactory sensory neurons (ciliated, microvillous or crypt neurons) are under the control of neurotrophins during adult life is unknown. Here we examined the expression of Trk proteins in the olfactory epithelium of adult zebrafish using immunohistochemistry. TrkA-like immunoreactivity was the only Trk detected, and it was restricted to the somata of crypt sensory neurons, their central processes being apparently unreactive. These results provide indirect evidence for a possible role of NGF-like molecules (i.e. neurotrophin-6 and neurotrophin-7) in the peripheral olfactory system of adult zebrafish, acting on TrkA-like receptor expressing crypt neurons. It remains to be established whether or not TrkA-like receptors and their ligands play a function in the neurogenesis or differentiation of these cells.

Animals↗
PubMed2003

Apoptosis following peripheral sensory deafferentation in the olfactory bulb of adult zebrafish.

Removal of the olfactory organ in adult zebrafish results in a significant decrease in volume of the ipsilateral olfactory bulb. The current study investigated the potential role of apoptosis in this phenomenon. It was hypothesized that cells in the adult olfactory bulb normally undergo minimal apoptosis and that apoptosis increases upon removal of sensory stimulation. By using both the terminal transferase-mediated deoxyuridine nick-end labeling method and bis-benzimide labeling, the current study showed that, in the normal adult olfactory bulb, cells exhibiting apoptotic profiles were scarce and were localized to the outer layers of the bulb. However, in deafferented animals, there was a significant increase in the number of apoptotic cells. The apoptotic response occurred in two phases and was confined to the rostral half of the bulb. The first phase of cell death peaked at 1 hour postsurgery. These apoptotic profiles appeared to be primarily nonneuronal in nature, in that they exhibited no immunohistochemical labeling to the neuron-specific protein Hu. The second phase of cell death peaked at 24 hours and declined to normal levels by 1 week. At the 24 hour time point, only a fraction of the apoptotic cells was neuronal in nature. Thus, apoptosis of nonneuronal and neuronal elements accounts for at least part of the deafferentation-induced volume decrease in the zebrafish olfactory bulb. This model of anterograde transneuronal degeneration will be useful in elucidating the afferent signals involved in survival and maintenance of mature brain neurons.

Animals↗
PubMed2001

Morphology and cell type heterogeneities of the inner ear epithelia in adult and juvenile zebrafish (Danio rerio).

Although the zebrafish has become an important model for genetic analysis of the vertebrate auditory system, a comprehensive description of the zebrafish ear has been provided for embryonic and larval development only (Haddon and Lewis [1996] J. Comp. Neurol. 365:113). Here we describe the development of sensory maculae in juvenile fish and the morphology of the adult zebrafish ear. This description was obtained via three-dimensional reconstruction of serial sections and confocal microscopy of immunolabeled preparations and includes the Weberian ossicles and fluid spaces. Phalloidin staining, which labels actin filaments of stereocilia, was used to delineate the sensory epithelia, to visualize the distribution of hair cells, to estimate their density in different areas of the maculae, and to perform hair cell counts. Morphology of ciliary bundles in different regions of the lagena, saccule, utricle, macula neglecta, and cristae was characterized with an anti-acetylated tubulin antibody and by phalloidin staining. We have identified two antibodies characterized by region-specific staining patterns in the inner ear epithelia. Zn-1 antibody staining largely correlates with the presence of short-bundle hair cells in the peripheral regions of sensory epithelia. Zn-4 antibody, on the other hand, labels a zone of epithelial cells surrounding the sensory maculae. These analyses extend previous observations of cell-type heterogeneity in both sensory and nonsensory epithelia of the fish ear.

Age Factors↗
PubMed2005

Zebrafish IkappaB kinase 1 negatively regulates NF-kappaB activity.

The IkappaB kinase (IKK) activity is critical for processing IkappaB inhibitory proteins and activating the NF-kappaB signaling, which is involved in a series of physiological and developmental steps in vertebrates. The IKK activity resides in two catalytic subunits, IKK1 and IKK2, and two regulatory subunits, NEMO and ELKS. IKK2 is the major cytokine-responsive IkappaB kinase because depletion of IKK1 does not interfere with the IKK activity. In fact, IKK1-/- mice display morphological abnormalities that are independent of its kinase activity and NF-kappaB activation. Hence, using zebrafish (Danio rerio) as a model, we examined the evolutionary role of IKK1 in modulating NF-kappaB. Ikk1-/- zebrafish embryos present head and tail malformations and, surprisingly, show upregulation of NF-kappaB-responsive genes and increased NF-kappaB-dependent apoptosis. Overexpression of ikk1 leads to midline structure defects that resemble NF-kappaB blockage in vivo. Zebrafish Ikk1 forms complexes with NEMO that represses NF-kappaB in vertebrate cells. Indeed, truncation of its NEMO binding domain (NBD) restores NF-kappaB-dependent transcriptional activity and, consequently, the ikk1-overexpressing phenotype. Here, we report that Ikk1 negatively regulates NF-kappaB by sequestering NEMO from active IKK complexes, indicating that IKK1 can function as a repressor of NF-kappaB.

Animals↗
PubMed2006

Methods for reverse genetic screening in zebrafish by resequencing and TILLING.

Animal models provide an in vivo system to study gene function by transgenic and knockout approaches. Targeted knockout approaches have been very successful in mice, but are currently not feasible in zebrafish due to the inability to grow embryonic stem cells. As an alternative, a reverse genetic approach that utilizes screening by resequencing and/or TILLING (Targeting Induced Local Lesions INGenomes) of mutagenized genomes has recently gained popularity in the zebrafish field. Spermatogonia of healthy males are mutagenized using ENU (N-ethyl-N-nitrosourea) and F1 progeny is collected by breeding treated males with healthy wild type females. Sperm and DNA banks are generated from F1 males. DNA is screened for ENU-induced mutations by sequencing or TILLING. These mutations can then be studied by in vitro fertilization (IVF) from the cryopreserved sperm of the corresponding F1 male followed by breeding to homozygosity. A high-throughput method of screening for rare heterozygotes and efficient recovery of mutant lines are important in identification of a large number of mutations using this approach. This article provides optimized protocols for resequencing and TILLING based on our experiences. We performed a pilot screen on 1235 F1 males by resequencing 54 exons from 17 genes and analyzed the sequencing data using multiple programs to maximize the mutation detection with minimal false positive detection. As an alternative to sequencing, we developed the protocols for TILLING by capillary electrophoresis using an ABI Genetic analyzer 3100 platform followed by fragment analysis using GeneScan and Genotyper softwares. PCR products generated by fluorescently labeled universal primers and tailed exon-specific primers were pooled 4-fold prior to heteroduplex formation. Overall, our pilot screen shows that a combination of TILLING and sequencing is optimal for achieving cost-effective, high-throughput screening of a large number of samples. Amplicons with fewer common SNPs are ideal for TILLING whereas amplicons with multiple SNPs and in/del polymorphisms are best suited for sequencing followed by analysis with SNPdetector.

Animals↗
PubMed2000

The syntenic relationship of the zebrafish and human genomes.

The zebrafish is an important vertebrate model for the mutational analysis of genes effecting developmental processes. Understanding the relationship between zebrafish genes and mutations with those of humans will require understanding the syntenic correspondence between the zebrafish and human genomes. High throughput gene and EST mapping projects in zebrafish are now facilitating this goal. Map positions for 523 zebrafish genes and ESTs with predicted human orthologs reveal extensive contiguous blocks of synteny between the zebrafish and human genomes. Eighty percent of genes and ESTs analyzed belong to conserved synteny groups (two or more genes linked in both zebrafish and human) and 56% of all genes analyzed fall in 118 homology segments (uninterrupted segments containing two or more contiguous genes or ESTs with conserved map order between the zebrafish and human genomes). This work now provides a syntenic relationship to the human genome for the majority of the zebrafish genome.

Animals↗
PubMed2006

The developmental neurotoxicity of fipronil: notochord degeneration and locomotor defects in zebrafish embryos and larvae.

Fipronil is a phenylpyrazole insecticide designed to selectively inhibit insect gamma-aminobutyric acid (GABA) receptors. Although fipronil is often used in or near aquatic environments, few studies have assessed the effects of this neurotoxicant on aquatic vertebrates at sensitive life stages. We explored the toxicological effects of fipronil on embryos and larvae using the zebrafish (Danio rerio) experimental model system. Embryos exposed to fipronil at nominal concentrations at or above 0.7 microM (333 mug/l) displayed notochord degeneration, shortening along the rostral-caudal body axis, and ineffective tail flips and uncoordinated muscle contractions along the body axis in response to touch. This phenotype closely resembles zebrafish locomotor mutants of the accordion class and is consistent with loss of reciprocal inhibitory neurotransmission by glycinergic commissural interneurons in the spinal cord. Consistent with the hypothesis that notochord degeneration may be due to abnormal mechanical stress from muscle tetany, the expression patterns of gene and protein markers specific to notochord development were unaffected by fipronil. Moreover, the degenerative effects of fipronil (1.1 microM) were reversed by coexposure to the sodium channel blocker MS-222 (0.6mM). The notochord effects of fipronil were phenocopied by exposure to 70 microM strychnine, a glycinergic receptor antagonist. In contrast, exposure to gabazine, a potent vertebrate GABA(A) antagonist, resulted in a hyperactive touch response but did not cause notochord degeneration. Although specifically developed to target insect GABA receptors with low vertebrate toxicity, our results suggest that fipronil impairs the development of spinal locomotor pathways in fish by inhibiting a structurally related glycine receptor subtype. This represents an unanticipated and potentially novel mechanism for fipronil toxicity in vertebrates.

Animals↗
PubMed2005

Binding profile of the endogenous novel heptapeptide Met-enkephalin-Gly-tyr in zebrafish and rat brain.

Zebrafish is considered a model organism, not only for the study of the biological functions of vertebrates but also as a tool to analyze the effects of some drugs or toxic agents. Five opioid precursor genes homologous to the mammalian opioid propeptide genes have recently been identified; one of these, the zebrafish proenkephalin, codes a novel heptapeptide, the Met-enkephalin-Gly-Tyr (MEGY). To analyze the pharmacological properties of this novel ligand, we have labeled it with tritium ([(3)H]MEGY). In addition, we have also synthesized two analogs: (d-Ala(2))-MEGY (Y-d-Ala-GFMGY) and (d-Ala(2), Val(5))-MEGY (Y-d-Ala-GFVGY). The binding profile of these three agents has been studied in zebrafish and rat brain membranes. [(3)H]MEGY presents one binding site in zebrafish, as well as in rat brain membranes, although it shows a slight higher affinity in zebrafish brain. The observed saturable binding is displaced by naloxone, thus confirming the opioid nature of the binding sites. Competition binding assays indicate that the methionine residue is essential for high-affinity binding of MEGY and probably of other peptidic agonists in zebrafish, whereas the change of a Gly for a d-Ala does not dramatically affect the ligand affinity. Our results show that the percentage of [(3)H]MEGY displaced by all the ligands studied is higher than 100%, thus inferring that naloxone (used to determine nonspecific binding) does not bind to all the sites labeled by [(3)H]MEGY. Therefore, we can deduct that some of the MEGY binding sites should not be considered classical opioid sites.

Amino Acid Substitution↗
PubMed2003

Sarcoglycans of the zebrafish: orthology and localization to the sarcolemma and myosepta of muscle.

The zebrafish is an established model of vertebrate development and is also receiving increasing attention in terms of human disease modelling. In order to provide experimental support to realize this modelling potential, we report here the identification of apparent orthologues of many critical members of the dystrophin-associated glycoprotein complex (DGC) that have been implicated in a diverse range of neuromuscular disorders. In addition, immunohistochemical studies show the localization of the DGC to the sarcolemma of adult zebrafish muscle and in particular the myosepta. Together, these data suggest that the DGC in adult zebrafish may play a highly conserved functional role in muscle architecture that, when disrupted, could offer insight into human neuromuscular disease processes.

Amino Acid Sequence↗
PubMed2006

Pbx proteins cooperate with Engrailed to pattern the midbrain-hindbrain and diencephalic-mesencephalic boundaries.

Pbx proteins are a family of TALE-class transcription factors that are well characterized as Hox co-factors acting to impart segmental identity to the hindbrain rhombomeres. However, no role for Pbx in establishing more anterior neural compartments has been demonstrated. Studies done in Drosophila show that Engrailed requires Exd (Pbx orthologue) for its biological activity. Here, we present evidence that zebrafish Pbx proteins cooperate with Engrailed to compartmentalize the midbrain by regulating the maintenance of the midbrain-hindbrain boundary (MHB) and the diencephalic-mesencephalic boundary (DMB). Embryos lacking Pbx function correctly initiate midbrain patterning, but fail to maintain eng2a, pax2a, fgf8, gbx2, and wnt1 expression at the MHB. Formation of the DMB is also defective as shown by a caudal expansion of diencephalic epha4a and pax6a expression into midbrain territory. These phenotypes are similar to the phenotype of an Engrailed loss-of-function embryo, supporting the hypothesis that Pbx and Engrailed act together on a common genetic pathway. Consistent with this model, we demonstrate that zebrafish Engrailed and Pbx interact in vitro and that this interaction is required for both the eng2a overexpression phenotype and Engrailed's role in patterning the MHB. Our data support a novel model of midbrain development in which Pbx and Engrailed proteins cooperatively pattern the mesencephalic region of the neural tube.

Animals↗
PubMed2001

Genetic analysis of digestive physiology using fluorescent phospholipid reporters.

Zebrafish are a valuable model for mammalian lipid metabolism; larvae process lipids similarly through the intestine and hepatobiliary system and respond to drugs that block cholesterol synthesis in humans. After ingestion of fluorescently quenched phospholipids, endogenous lipase activity and rapid transport of cleavage products results in intense gall bladder fluorescence. Genetic screening identifies zebrafish mutants, such as fat free, that show normal digestive organ morphology but severely reduced phospholipid and cholesterol processing. Thus, fluorescent lipids provide a sensitive readout of lipid metabolism and are a powerful tool for identifying genes that mediate vertebrate digestive physiology.

Animals↗
PubMed2006

Sesn1 is a novel gene for left-right asymmetry and mediating nodal signaling.

Remarkable progress has been made in understanding the molecular mechanisms underlying left-right asymmetry in vertebrate animal models but little is known on left-right axis formation in humans. Previously, we identified SESN1 (also known as PA26) as a candidate gene for heterotaxia by positional cloning of the breakpoint regions of a de novo translocation in a heterotaxia patient. In this study, we show by means of a zebrafish sesn1-knockdown model that Sesn1 is required for normal embryonic left-right determination. In this model, developmental defects and expression data of genes implicated in vertebrate left-right asymmetry indicate a role for Sesn1 in mediating Nodal signaling. In the lateral plate mesoderm, Nodal signaling plays a central role in left-right axis formation in vertebrates and is mediated by FoxH1 transcriptional induction. In line with this, we show that Sesn1 physically interacts with FoxH1 or a FoxH1-containing complex. Mutation analysis in a panel of 234 patients with isolated heterotaxia did not reveal mutations, indicating that these are only exceptional causes of human heterotaxia. In this study, we identify SESN1 as an indispensable gene for vertebrate left-right asymmetry and a new player in mediating Nodal signaling.

Animals↗
PubMed2004

Targeted knockdown of insulin-like growth factor binding protein-2 disrupts cardiovascular development in zebrafish embryos.

IGF binding protein-2 (IGFBP-2) is an evolutionarily conserved protein that binds IGFs and modulates their biological activities. Although the actions of IGFBP-2 have been well studied in vitro, we have a poor understanding of its in vivo functions, particularly during early development. Using the transparent zebrafish embryo as a model, we show that IGFBP-2 mRNA is expressed in lens epithelium and cranial boundary regions during early embryonic development and becomes localized to the liver by the completion of embryogenesis. Targeted knock-down of IGFBP-2 by antisense morpholino-modified oligonucleotides resulted in delayed development, reduced body growth, reduced IGF-I mRNA levels, and disruptions to cardiovascular development, including reduced blood cell number, reduced blood circulation, cardiac dysfunction, and brain ventricle edema. Detailed examination of vascular tissues, using a stable transgenic line of zebrafish expressing green fluorescent protein in vascular endothelial cells, revealed specific angiogenic (vessel sprouting) defects in IGFBP-2 knockdown embryos, with effects being localized in regions associated with IGFBP-2 mRNA expression. These findings suggest that IGFBP-2 is required for general embryonic development and growth and plays a local role in regulating vascular development in a model vertebrate organism.

Animals↗