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Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells.

Identification and manipulation of the germ line are important to the study of model organisms. Although zebrafish has recently emerged as a model for vertebrate development, the primordial germ cells (PGCs) in this organism have not been previously described. To identify a molecular marker for the zebrafish PGCs, we cloned the zebrafish homologue of the Drosophila vasa gene, which, in the fly, encodes a germ-cell-specific protein. Northern blotting revealed that zebrafish vasa homologue (vas) transcript is present in embryos just after fertilization, and hence it is probably maternally supplied. Using whole-mount in situ hybridization, we investigated the expression pattern of vas RNA in zebrafish embryos from the 1-cell stage to 10 days of development. Here we present evidence that vas RNA is a germ-cell-specific marker, allowing a description of the zebrafish PGCs for the first time. Furthermore, vas transcript was detected in a novel pattern, localized to the cleavage planes in 2- and 4-cell-stage embryos. During subsequent cleavages, the RNA is segregated as subcellular clumps to a small number of cells that may be the future germ cells. These results suggest new ways in which one might develop techniques for the genetic manipulation of zebrafish. Furthermore, they provide the basis for further studies on this novel RNA localization pattern and on germ-line development in general.

Amino Acid Sequence↗

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↗

Isolation of zebrafish gdf7 and comparative genetic mapping of genes belonging to the growth/differentiation factor 5, 6, 7 subgroup of the TGF-beta superfamily.

The Growth/differentiation factor (Gdf) 5, 6, 7 genes form a closely related subgroup belonging to the TGF-beta superfamily. In zebrafish, there are three genes that belong to the Gdf5, 6, 7 subgroup that have been named radar, dynamo, and contact. The genes radar and dynamo both encode proteins most similar to mouse GDF6. The orthologous identity of these genes on the basis of amino acid similarities has not been clear. We have identified gdf7, a fourth zebrafish gene belonging to the Gdf5, 6, 7 subgroup. To assign correct orthologies and to investigate the evolutionary relationships of the human, mouse, and zebrafish Gdf5, 6, 7 subgroup, we have compared genetic map positions of the zebrafish and mammalian genes. We have mapped zebrafish gdf7 to linkage group (LG) 17, contact to LG9, GDF6 to human chromosome (Hsa) 8 and GDF7 to Hsa2p. The radar and dynamo genes have been localized previously to LG16 and LG19, respectively. A comparison of syntenies shared among human, mouse, and zebrafish genomes indicates that gdf7 is the ortholog of mammalian GDF7/Gdf7. LG16 shares syntenic relationships with mouse chromosome (Mmu) 4, including Gdf6. Portions of LG16 and LG19 appear to be duplicate chromosomes, thus suggesting that radar and dynamo are both orthologs of Gdf6. Finally, the mapping data is consistent with contact being the zebrafish ortholog of mammalian GDF5/Gdf5.

Amino Acid Sequence↗

Expression pattern of two otx genes suggests a role in specifying anterior body structures in zebrafish.

We isolated two zebrafish sequences containing a homeobox related to orthodenticle (otd), a gene expressed in the developing head of Drosophila. One of these is clearly homologous to Otx1, a homeobox gene previously reported to be expressed in the developing rostral brain of the mouse. We termed this zebrafish gene otx1. The second gene is not as closely related to Otx1 and is equally divergent from Otx2, a second homeobox gene expressed in the developing rostral brain of the mouse. We termed it otx3, even if a corresponding murine Otx3 gene has not been reported yet. Both genes are expressed in early-gastrula zebrafish embryos in the involuting presumptive anterior mesendoderm. With the extension of the body axis, the expression domain of both genes extends to neuroectodermal regions fated to become fore- and mid-brain. From this stage the expression domains of the two genes differ slightly from each other but both cover the rostral brain with a sharp posterior boundary coinciding with that between midbrain and hind-brain. This late expression closely corresponds to that of the murine Otx1 gene, whereas the earliest expression of both zebrafish otx genes is different from that of Otx1 and reminiscent of that of Otx2 in the mouse. In this light, the zebrafish otx1 and otx3 genes appear to share some expression features of both murine Otx1 and Otx2. It will be of considerable interest to study the specific role of the various genes of the otx family in the development of the zebrafish brain regions. The peculiar spatio-temporal pattern of these genes during early zebrafish gastrulation suggests a role of this gene family in interactions between anterior mesendoderm and neuroectoderm.

Amino Acid Sequence↗

R-cadherin expression in the developing and adult zebrafish visual system.

Cell adhesion molecules in the cadherin family have been implicated in histogenesis and maintenance of cellular structure and function in several organs. Zebrafish have emerged as an important new developmental model, but only three zebrafish cadherin molecules have been identified to date (N-cadherin, paraxial protocadherin, and VN-cadherin). We began a systematic study to identify other zebrafish cadherins by screening zebrafish cDNA libraries using an antibody raised to the cytoplasmic domain of mouse E-cadherin. Here, we report a partial cDNA with extensive sequence homology to R-cadherin. Spatial and temporal expression of this putative zebrafish R-cadherin was examined in embryos and adults by Northern analysis, RNase protection, and in situ hybridization. R-cadherin message increased during embryogenesis up to 80 hours postfertilization (hpf) and persisted in adults. In the embryonic brain, R-cadherin was first expressed in groups of cells in the diencephalon and pretectum. In adult zebrafish brain, R-cadherin continued to be expressed in several specific regions including primary visual targets. In the retina, R-cadherin was first detected at about 33 hours postfertilization in the retinal ganglion cell layer and the inner part of the inner nuclear layer. Expression levels were highest during periods of axon outgrowth and synaptogenesis. Retrograde labeling of the optic nerve with 1,1'-dioctadecyl-3,3,3',3', tetramethylindocarbocyanine perchlorate (DiI) followed by in situ hybridization confirmed that a subset of retinal ganglion cells in the embryo expressed R-cadherin message. In the adult, R-cadherin expression continued in a subpopulation of retinal ganglion cells. These results suggest that R-cadherin-mediated adhesion plays a role in development and maintenance of neuronal connections in zebrafish visual system.

Age Factors↗

Oligodendrocyte development and myelination in GFP-transgenic zebrafish.

Green fluorescent protein (GFP) transgenic zebrafish technology has been employed to directly visualize and analyze dynamic developmental processes, such as cell migration and morphogenesis. Stable transgenic zebrafish that express GFP in oligodendrocytes can be a valuable tool to visualize complex myelination processes in vivo, as well as to conduct rapid mutagenesis screens for defective myelination mutants. We investigated whether two myelin gene promoters, the zebrafish P0 promoter and the mouse proteolipid protein (PLP) promoter, drive GFP expression in zebrafish oligodendrocytes. Transiently, both promoters drive enhanced GFP (EGFP) expression in morphologically identifiable oligodendrocytes, premyelinating oligodendrocytes, and possible oligodendrocyte precursors. We have established a stable transgenic zebrafish line, tg(plp:EGFP) zebrafish, at the F1 generation, which expresses enhanced GFP (EGFP) driven by the mouse PLP promoter. In this transgenic line, EGFP-expressing cells are visually detectable around 24-hr postfertilization (hpf), and later at 54 hpf, these cells start exhibiting the clear morphologic characteristics of oligodendrocytes. Shortly afterward, EGFP-expressing oligodendrocytes establish a ventral dominant distribution pattern throughout the central nervous system. This transgenic zebrafish line is likely to serve as a useful tool, in which normal myelination as well as abnormal myelination can be recorded under time-lapse confocal microscopy. Furthermore, it has the potential to greatly facilitate mutagenesis screening for novel dysmyelinating mutants.

Animals↗

Follistatin and noggin are excluded from the zebrafish organizer.

The patterning activity of the Spemann organizer in early amphibian embryos has been characterized by a number of organizer-specific secreted proteins including Chordin, Noggin, and Follistatin, which all share the same inductive properties. They can neuralize ectoderm and dorsalize ventral mesoderm by blocking the ventralizing signals Bmp2 and Bmp4. In the zebrafish, null mutations in the chordin gene, named chordino, lead to a severe reduction of organizer activity, indicating that Chordino is an essential, but not the only, inductive signal generated by the zebrafish organizer. A second gene required for zebrafish organizer function is mercedes, but the molecular nature of its product is not known as yet. To investigate whether and how Follistatin and Noggin are involved in dorsoventral (D-V) patterning of the zebrafish embryo, we have now isolated and characterized their zebrafish homologues. Overexpression studies demonstrate that both proteins have the same dorsalizing properties as their Xenopus homologues. However, unlike the Xenopus genes, zebrafish follistatin and noggin are not expressed in the organizer region, nor are they linked to the mercedes mutation. Expression of both genes starts at midgastrula stages. While no patterned noggin expression was detectable by in situ hybridization during gastrulation stages, later expression is confined to presumptive cartilage cells in the branchial arches and the neurocranium and to proximal regions of the pectoral fin buds. follistatin transcripts in gastrulating embryos are confined to anterior paraxial regions, which give rise to head mesoderm and the first five somites. The dorsolateral extent of this expression domain is regulated by Bmp2b, Chordino, and Follistatin itself. In addition, transient expression was observed in a subset of cells in the posterior notochord anlage. Later, follistatin is expressed in brain, eyes, and somites. Comparison of the spatiotemporal expression pattern of follistatin and noggin with those of bmp2b and bmp4 and overexpression studies suggest that Noggin and Follistatin may function as Bmp antagonists in later processes of zebrafish development, including late phases of D-V patterning, to refine the early pattern set up by the interaction of Chordino and Bmp2/4. It thus appears that many, but not all, aspects of early dorsoventral patterning are shared among different vertebrate species.

Amino Acid Sequence↗

Gene mapping in zebrafish using single-strand conformation polymorphism analysis.

To exploit fully the power of the zebrafish system as a model for vertebrate development, it will be necessary to develop efficient tools for genomic analysis. In this report we have tested whether single-strand conformation polymorphism analysis (SSCP) can be utilized for gene mapping in zebrafish. Over 100 primer pairs derived from noncoding regions of known genes and partially characterized cDNAs were analyzed, and a polymorphism frequency of approximately 50% was detected in zebrafish strains used for genetic mapping studies. A subset of these polymorphic cDNAs was localized on the zebrafish map. SSCP thus represents an efficient strategy for mapping transcribed sequences with a high resolution in the zebrafish genome, which will facilitate the integration of existing zebrafish framework maps, the generation of a zebrafish EST map, and the application of alternative gene localization strategies such as comparative mapping.

3' Untranslated Regions↗

In vivo aflatoxin B1 metabolism and hepatic DNA adduction in zebrafish (Danio rerio).

The zebrafish (Danio rerio) is assuming prominence in developmental genetics research. By comparison, little is known of tumorigenesis and nothing is known of carcinogen metabolism in this species. This study evaluated the ability of zebrafish to metabolize a well-characterized human carcinogen, aflatoxin B1 (AFB1), to phase I and phase II metabolites and assessed hepatic AFB1-DNA adduction in vivo. Fish i.p. injected with 50-400 micrograms [3H]AFB1/kg body wt displayed a linear dose response for hepatic DNA binding at 24 hr. AFB1-DNA adduct levels among treatments showed no statistical difference over the period from 1 to 21 days after injection, suggesting poor adduct repair in this species. DNA binding in female fish was 1-7-fold higher than that in males (p < 0.01). An in vitro AFB1 metabolism assay verified that zebrafish liver extracts oxidize AFB1 to the 8,9-epoxide proximate electrophile (Km = 79.0 +/- 16.4 microM, Vmax = 11.7 +/- 1.4 pmol/min/mg protein at 28 degrees C). The excretion of AFB1 and its metabolites was also examined by HPLC. As is typical of other fish studied, major metabolites excreted were aflatoxicol (AFL) and aflatoxicol-glucuronide (AFL-g), followed by unreacted AFB1. AFL appeared as early as 5 min after injection, whereas AFL-g was a significant metabolite after 18 hr. This study shows that in vivo administration of AFB1 to zebrafish results in moderate adduction of the carcinogen to liver DNA and that zebrafish have the capacity for both phase I and phase II metabolism of AFB1. The approximate fourfold difference between rainbow trout and zebrafish AFB1-DNA covalent binding index appears insufficient to explain the relative resistance of zebrafish to dietary AFB1 hepatocarcinogenicity.

Aflatoxin B1↗

The zebrafish brain: a neuroanatomical comparison with the goldfish.

The zebrafish Danio rerio is an important model system for genetic and developmental studies of the vertebrate central nervous system. Considerable knowledge concerning the embryonic development of the central nervous system of the zebrafish has accumulated in recent years. However, there is an apparent lack of information on the organization of the adult zebrafish brain. We have therefore recently studied in detail the neuroanatomy of the adult zebrafish. Here we compare the brains of the zebrafish and of the closely related and neurobiologically well-investigated goldfish, Carassius auratus. Two sensory systems, the visual and the gustatory systems, were identified as differing on the gross morphological and histological levels in the two species. The goldfish shows the simple (evolutionarily reduced) pattern of pretectal organization, and its gustatory system is massively enlarged. The pretectum of the zebrafish conforms to this simplified visual pretectal pattern, although the retention of some ancestral pretectal characters indicates a lesser degree of reduction of the visual system compared to the goldfish. The gustatory system shows many similarities with the evolutionarily derived and functionally specialized gustatory system of the goldfish. However, some peripheral and central gustatory characters are missing in the zebrafish, indicating a less specialized gustatory system.

Animals↗

Biochemical identification and tissue-specific expression patterns of keratins in the zebrafish Danio rerio.

We have identified a number of type I and type II keratins in the zebrafish Danio rerio by two-dimensional polyacrylamide gel electrophoresis, complementary keratin blot-binding assay and immunoblotting. These keratins range from 56 kDa to 46 kDa in molecular mass and from pH 6.6 to pH 5.2 in isoelectric point. Type II zebrafish keratins exhibit significantly higher molecular masses (56-52 kDa) compared with the type I keratins (50-48 kDa), but the isoelectric points show no significant difference between the two keratin subclasses (type II: pH 6.0-5.5; type I: pH 6.1-5.2). According to their occurrence in various zebrafish tissues, the identified keratins can be classified into "E" (epidermal) and "S" (simple epithelial) proteins. A panel of monoclonal anti-keratin antibodies has been used for immunoblotting of zebrafish cytoskeletal preparations and immunofluorescence microscopy of frozen tissue sections. These antibodies have revealed differential cytoplasmic expression of keratins; this not only includes epithelia, but also a variety of mesenchymally derived cells and tissues. Thus, previously detected fundamental differences in keratin expression patterns between higher vertebrates and a salmonid, the rainbow trout Oncorhynchus mykiss, also apply between vertebrates and the zebrafish, a cyprinid. However, in spite of notable similarities, trout and zebrafish keratins differ from each other in many details. The present data provide a firm basis from which the application of keratins as cell differentiation markers in the well-established genetic model organism, the zebrafish, can be developed.

Animals↗

Zebrafish as a versatile model in biomedical research, from disease modeling to regenerative medicine: a review.

Zebrafish are an effective animal model widely utilized in biomedical research. They are known for their rapid reproduction and substantial genetic similarity to humans. Their transparent embryos directly enable the visualization of developmental processes and disease progression. This makes zebrafish invaluable for studying a broad range of human diseases, including cancer, cardiovascular disorders, and neurodegenerative conditions. Compared with other vertebrate models, zebrafish offer several advantages, including ease of genome editing, cost-effective maintenance, and suitability for high-throughput drug screening. Recent advancements have expanded the use of zebrafish in disease modeling and regenerative medicine, providing deeper insights into the genetic and cellular mechanisms underlying human pathologies. Zebrafish provide a robust platform for evaluating the safety, efficacy, and regenerative potential of both natural and synthetic biomaterials, including hydroxyapatite, bioactive glass nanoparticles, and bioceramics. This capability facilitates the creation of artificial tissues that closely resemble native structures. Additionally, integrating artificial intelligence technologies has improved automated data analysis and phenotyping in zebrafish studies, enhancing both accuracy and throughput. This review highlights current applications of zebrafish in disease modeling, drug discovery, regenerative medicine, and biomaterial assessment, emphasizing their evolving role as a versatile preclinical platform supported by advanced genetic and computational tools.

Animals↗

Impacts of 17beta-estradiol, including environmentally relevant concentrations, on reproduction after exposure during embryo-larval-, juvenile- and adult-life stages in zebrafish (Danio rerio).

Zebrafish (Danio rerio) were exposed for 3 weeks to low concentrations of estradiol including environmentally relevant concentrations (5, 25 and 100 ng/l), encompassing either their embryo-larvae (from fertilization to 21 day post-fertilization (dpf)), juvenile (from 21 to 42 dpf) or adult life stages (>200 dpf) with a view to investigating the most sensitive life stage of the zebrafish to 17beta-estradiol (E2). At all sampling points, whole-body vitellogenin concentrations and gonadal development were analyzed in order to investigate the effects of estrogen exposure on these endpoint in the zebrafish. In the adult stage, additional endpoints were measured including secondary sexual characteristics (manifestation of the uro-genital papillae (UGP) in males), gonadal growth (the gonado-somatic index (GSI)) and sex ratio. For all the different life stage exposures, reproductive performance of the F0 generation was assessed (egg production) and survival and development of the F1 embryo-larvae. Exposure to low concentrations of E2 resulted in vitellogenin induction whatever the life stage exposed but these effects were reversible after depuration. The effective concentration for vitellogenin induction in zebrafish early life stages was 100 ng E2/l, and in adult male zebrafish the effective concentration for vitellogenin induction (between 5 and 25 ng/l) was lower than for the early life stage fish. Exposure to E2 prior to (from fertilization to 21 dpf) and during the time of sex differentiation (from 21 to 42 dpf) also caused disruptions in the process of sexual differentiation (resulting in formation of a retrogonadal cavity in presumptive male, germ cell development and leading to a significant change of the sex ratio towards the female sex at the dose of 100 ng E2/l for the fish exposure as embryo-larvae) and altered patterns of egg production in the subsequent adults. Exposure of adult fish to E2 resulted in a modification of the secondary sexual characteristic in males at 25 and 100 ng E2/l as well as a dose-dependent inhibition of egg production. The findings from this study show that the nature and intensity of the reproductive effects of E2 are dependent of the time and concentration of exposures of zebrafish to E2, some of these effects being permanent (effect on the sexual differentiation) while others being reversible (effect on the Vtg induction). This study demonstrated that early life stages of zebrafish are sensitive to low concentrations of E2 and provides relevant data that could be used for the adaptation of existing fish early life stage test for the in vivo testing of estrogenic compounds. The data presented raise further concerns about the effects of steroid estrogens in the environment on fish reproductive health.

Age Factors↗

cDNA-directed expression of a functional zebrafish CYP1A in yeast.

A cytochrome P450 1A (CYP1A) cDNA was isolated from an adult zebrafish (Danio rerio) library. The 2580-bp clone (GenBank Accession No. AF210727) contained a 62-bp 5'-unstranslated region (UTR), 1557-bp coding region and 962-bp 3'-UTR. The deduced 519-residue protein (calculated molecular weight 58,556, pI = 7.58) shared 74% identity with rainbow trout CYP1A and 57 and 54% identities with mouse and human CYP1A1s, respectively. The zebrafish CYP1A protein coding region was cloned into the pDONR201 entry vector and then transferred to a yeast expression vector pYES-DEST52. Expression of zebrafish CYP1A in Saccharomyces cerevisiae transformants was induced by galactose to a maximum level of 493 pmol CYP1A per mg microsomal protein or about 8 nmol/l of culture. Recombinant CYP1A protein expressed in yeast was mainly in the denatured P420 form under normal microsomal preparation conditions but when the oxygen concentration was reduced in the buffer by degassing and the yeast cells were maintained at less than 10 degrees C, the integrity of the CYP1A was preserved and it exhibited a characteristic reduced CO-difference spectrum maximum at 448 nm. The recombinant zebrafish CYP1A demonstrated 7-ethoxyresorufin O-deethylase (EROD) activity with an apparent Km (Km(app)) and Vmax values at 30 degrees C of 0.31 +/- 0.04 microM and 0.70 +/- 0.10 nmol/min/nmol CYP, respectively. The recombinant protein also metabolized benzo(a)pyrene with a Km(app) and Vmax values of 5.34 +/- 0.58 microM and 1.16 +/- 0.13 nmol/min/nmol CYP, respectively. These results show the recombinant expression of a functional zebrafish CYP in yeast and validated yeast as a host for heterologous expression of zebrafish CYP1A and potentially for other zebrafish CYPs.

Animals↗

Comparative in vitro metabolism of benzo[a]pyrene by recombinant zebrafish CYP1A and liver microsomes from beta-naphthoflavone-treated rainbow trout.

The zebrafish (Danio rerio) is a sensitive non-mammalian model used for studying polycyclic aromatic hydrocarbon (PAH)-induced chemical carcinogenesis. The susceptibility of zebrafish to PAH-induced carcinogenesis may be related to the ability of the zebrafish P450s to bioactivate these procarcinogens. As a part of our overall effort to identify the various P450 enzymes that are involved in the activation and detoxification of PAHs in zebrafish, therefore, we have examined the ability of recombinant zebrafish CYP1A (zCYP1A) expressed in yeast to metabolize BaP in vitro. Comparison studies also were conducted with liver microsomes from beta-naphthoflavone (BNF)-treated rainbow trout (Oncorhynchus mykiss). Results demonstrated that the trout liver microsomes were almost twice as active as zCYP1A in oxidizing BaP, with Vmax values of 1.7 and 0.94 nmol/min/nmol P450 for trout and zebrafish preparations, respectively. Like trout CYP1A1, cDNA-expressed zCYP1A was found to oxidize BaP to phenols, quinones and diols (BaP-7,8-diol and BaP-9,10-diol) in the presence of exogenous human microsomal epoxide hydrolase (hEH). BaP-7,8-diol is the precursor of the ultimate carcinogen, BaP-7,8-diol-9,10-epoxide (BaPDE). The ability of zCYP1A to bioactivate BaP was confirmed by the formation of DNA adducts when calf thymus DNA was added to the incubation mixture. BaP-DNA binding was enhanced by the addition of hEH to the incubation mixture. HPLC analysis of the [33P]-postlabeled DNA adducts showed the formation of at least four adducts mediated by both zCYP1A and trout liver microsomes, and one of these adducts co-migrated with BaPDE-dG in HPLC analysis. The addition of hEH to the incubation mixture decreased the formation of BaPDE-dG by zCYP1A and by trout liver microsomes while increasing the formation of an unidentified DNA adduct in the case of zCYP1A. zCYP1A also mediated the binding of BaP to protein, providing further evidence that this enzyme is capable of oxidizing BaP to reactive metabolites that bind to macromolecules. It thus appears that zCYP1A may play an important role in BaP-induced carcinogenesis in the zebrafish model by catalyzing the sequential formation of the ultimate diol epoxide carcinogenic metabolite of BaP.

Animals↗

Characterization of the zebrafish vascular endothelial growth factor A gene: comparison with vegf-A genes in mammals and Fugu.

Vascular endothelial growth factor (VEGF-A) is a key angiogenic growth factor which regulates vertebrate embryonic vascularization, adult physiology such as wound healing and reproduction as well as many human diseases. To understand the evolution and regulation of this gene in vertebrates, we have isolated and characterized the zebrafish vegf-A gene and compared it with VEGF-A genes of human, mouse as well as an in silico isolated VEGF-A homologue from pufferfish. Our results indicate that the zebrafish vegf-A gene is organized similarly to mammalian and Fugu VEGF-A genes, with eight exons interrupted by seven introns. However, zebrafish vegf-A introns are generally larger than mammalian introns while Fugu VEGF-A introns are much smaller. Furthermore, zebrafish exon 6 (z6) has a unique sequence while Fugu's exon 6 is highly homologous to the mammalian counterparts. Alternative splicing generates multiple vegf-A mRNA isoforms in zebrafish with Vegf(121) as the dominant isoform in adult and Vegf(165) as the dominant isoform in early embryos. The exon z6 containing isoform Vegf(12345z678) is only detected in heart, muscle, and early embryos while another isoform Vegf-A(1234577)(a)(8) is only detected in heart. Furthermore, no conserved 5' flanking sequences between zebrafish and Fugu were observed while numerous conserved regions exist between human and mouse in this area. These results suggest both conserved and diverged functions of VEGF-A from fish to mammals since the separation of these two groups from their common ancestor about 450 million years ago and a diverged regulation of this gene since the separation of zebrafish from Fugu. These data will be valuable for future studies of VEGF-A gene regulation and function in different vertebrates.

Amino Acid Sequence↗

The use of zebrafish for assessing ototoxic and otoprotective agents.

Zebrafish and other fish exhibit hair cells in the lateral-line neuromasts which are structurally and functionally similar to mammalian inner ear hair cells. To facilitate drug screening for ototoxic or otoprotective agents, we report a straightforward, quantitative in vivo assay to determine potential ototoxicity of drug candidates and to screen otoprotective agents in zebrafish larva. In this study, a fluorescent vital dye, DASPEI (2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide), was used to stain zebrafish hair cells in vivo and morphometric analysis was performed to quantify fluorescence intensity and convert images to numerical endpoints. Various therapeutics, including gentamicin, cisplatin, vinblastine sulfate, quinine, and neomycin, which cause ototoxicity in humans, also resulted in hair cell loss in zebrafish. In addition, protection against cisplatin-induced ototoxicity was observed in zebrafish larva co-treated with cisplatin and different antioxidants including, glutathione (GSH), allopurinol (ALO), N-acetyl l-cysteine (l-NAC), 2-oxothiazolidine-4-carboxylate (OTC) and d-methionine (d-MET). Our data indicate that results of ototoxicity and otoprotection in zebrafish correlated with results in humans, supporting use of zebrafish for preliminary drug screening.

Animals↗

Zebrafish (Danio rerio) whole kidney assays to measure neutrophil extracellular trap release and degranulation of primary granules.

The zebrafish (Danio rerio) is an excellent model system for studies in developmental biology, genetics, and toxicology, and is increasingly gaining importance in the field of immunology. The use of whole zebrafish kidneys as source of neutrophils for degranulation assays and detection of neutrophil extracellular traps is described for the first time. Neutrophils from zebrafish kidneys released neutrophil extracellular traps (NETs) and myeloperoxidase (MPO) upon stimulation with calcium ionophore, phorbol myristate acetate, and beta-glucan. Immunocytochemical study of zebrafish kidney cells revealed that NETs are made of DNA fibers associated with neutrophil granular proteins, but not with cytoskeleton. Rapid, direct MPO and extracellular DNA detection assays were developed to quantify NET release and degranulation of neutrophil primary granules from whole zebrafish kidneys. The assays were used to measure the effects of acute crowding and handling stress on neutrophils, and to examine the potential for use of zebrafish whole kidney assays in evaluation of neutrophil function under different conditions in vivo. The whole kidney NET release and degranulation assays are quantitative, can rapidly measure a large number of samples, and are capable of detecting inhibition of neutrophil activity in stressed fish, overcoming the limitations that prevented use of zebrafish in the investigations of cellular innate immune function. The assays can be used as a new research model to study effects of stress, immunomodulators, toxicants, and diseases on fish neutrophil biology.

Animals↗