Identification of two families of satellite-like repetitive DNA sequences from the zebrafish (Brachydanio rerio).

To further our understanding of the structure and organization of the zebrafish genome, we have undertaken the analysis of highly and middle-repetitive DNA sequences. We have cloned and sequenced two families of tandemly repeated DNA fragments. The monomer units of the Type I satellite-like sequence are 186 bp long, A+T-rich (65%), and exhibit a high degree of sequence conservation. The Type I satellite-like sequence constitutes 8% of the zebrafish genome, or approximately 8 x 10(5) copies per haploid genome. Southern analysis of genomic DNA, digested with several restriction endonucleases, shows a ladder of hybridizing bands, consistent with a tandem array, and suggests longer range periodic variations in the sequence of the tandem repeats. The Type II satellite has a monomer length of 165 bp, is also A+T-rich (68%), and constitutes 0.2% of the zebrafish genome (22,000 copies per haploid genome). Southern analysis reveals a complex pattern rather than a ladder of regularly spaced hybridizing bands.     

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

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

Gill morphology in the zebrafish, Brachydanio rerio (Hamilton- Buchanan)

A light and electron microscopic study of the gills of the zebrafish, Brachydanio rerio , were made to serve as a morphological basis for future investigations. It was found that for fixation of B. rerio gills, a mixture of 1·5% gluturaldehyde and 1·5% paraformaldehyde gives a mucus-free surface. Morphometric measurements of structural components of the gill secondary lamellae were made. Observations at SEM were correlated with those made at TEM. The different cell types in the branchial epithelium were characterized. Chloride cells were mainly located in the interlamellar regions and on the afferent side of the primary lamellae. Two morphologically different chloride cells were seen. The first type communicates with the external environment through a reservoir-like lumen, which is normally absent in freshwater fishes. The second type of chloride cell has more direct contact with the ambient water, resembling chloride cells from other freshwater fishes. Another cell type with features similar to those of the rodlet cell was frequently observed. This cell is interposed between other types of cells in the epithelium, and sometimes junctional complexes were present between the rodlet cell and surrounding cells.     

Stages of oocyte development in the zebrafish,Brachydanio rerio

Oocyte development has been divided into five stages in the zebrafish Brachydanio rerio, based on morphological criteria and on physiological and biochemical events. In stage I (primary growth stage), oocytes reside in nests with other oocytes (Stage IA) and then within a definitive follicle (Stage IB), where they greatly increase in size. In stage II (cortical alveolus stage), oocytes are distinguished by the appearance of variably sized cortical alveoli and the vitelline envelope becomes prominent. In stage III (vitellogenesis), yolk proteins appear in oocytes and yolk bodies with crystalline yolk accrue during this major growth stage. Ooctes develop the capacity to respond in vitro to the steroid 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) by undergoing oocyte maturation. In stage IV (oocyte maturation), oocytes increase slightly in size, become translucent, and their yolk becomes non-crystalline as they undergo final meiotic maturation in vivo (and in response to DHP in vitro). In stage V (mature egg), eggs (approx. 0.75 mm) are ovulated into the ovarian lumen and are capable of fertilization. This staging series lays the foundation for future studies on the cellular processes occurring during oocyte development in zebrafish and should be useful for experimentation that requires an understanding of stage-specific events. © 1993 Wiley-Liss, Inc.     

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.[/ p]Abbreviations TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin - EROD, 7-ethyoxyresorufin - HDPDS, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid - EDTA, ethylanediaminetetraacetic acid - FBS, fetal bovine serum - LDF, limit dilution factor - DMSO, dimethyl sulfoxide - ES, embryonal stem - PAH, polycylic aromatic hydrocarbons - ZG, zebrafish gill - ZBF, zebrafish pelvic fin - ZV, Zebrafish viscera - ZCF, zebrafish caudal fin - ZEM, diploid blastula-derived     

Effects of lithium on pigmentation in the embryonic zebrafish (Brachydanio rerio)

Pigment cell precursors of the embryonic zebrafish give rise to melanophores, xanthophores and/or iridophores. Cell signaling mechanisms related to the development of pigmentation remain obscure. In order to examine the mechanisms involved in pigment cell signaling, we treated zebrafish embryos with various activators and inhibitors of signaling pathways. Among those chemicals tested, LiCl and LiCl/forskolin had a stimulatory effect on pigmentation, most notable in the melanophore population. We propose that the inositol phosphate (IP) pathway, is involved in pigment pattern formation in zebrafish through its involvement in the: (1) differentiation/proliferation of melanophores; (2) dispersion of melanosomes; and/or (3) synthesis/deposition of melanin. To discern at what level pigmentation was being effected we: (1) counted the number of melanophores in control and experimental animals 5 days after treatment; (2) measured tyrosinase activity and melanin content; and (3) employed immunoblotting techniques with anti-tyrosine-related protein-2 and anti-melanocyte-specific gene-1 as melanophore-specific markers. Although gross pigmentation increased dramatically in LiCl- and LiCl/forskolin treated embryos, the effect on pigmentation was not due to an increase in the proliferation of melanophores, but was possibly through an increase in melanin synthesis and/or deposition. Collectively, results from these studies suggest the involvement of an IP-signaling pathway in the stimulation of pigmentation in embryonic zebrafish through the synthesis/deposition of melanin within the neural crest-derived melanophores.     

Neurulation in the anterior trunk region of the zebrafish Brachydanio rerio

We have studied the process of neurulation within the anterior trunk region of the zebrafish by means of serial sectioning of staged embryos and labelling cells by applications of the dye Dil and intracellular injections of fluoresceine dextran amine. The first morphological manifestation of the prospective neural plate is a dorsomedial ectodermal thickening which becomes visible immediately after gastrulation. Within 1–2 h, by the time somatogenesis begins, two bilaterally symmetrical thickenings have appeared more laterally, which eventually fuse with the medial thickening to form the neural keel. The central canal forms next by separation of the cells on either side of the midline of the neural keel, beginning ventrally at the 17-somite stage and progressing towards dorsal levels. By means of fluorescent dye labelling in the late gastrula, we found that both the medial and lateral thickenings contribute to the nerve cord. The medial thickening was found to contain, exclusively, neural progenitor cells from the 90–100% epiboly stage on, whereas the adjacent regions contained a mixture of neural and epidermal progenitor cells, as well as prospective neural crest cells. Between the 90–100% epiboly and 2-somite stages, this heterogeneity of developmental capabilities is resolved into territories, with epidermogenic and neurogenic cells clearly separated from each other. To achieve this segregation into neural and epidermal anlagen, cells from the lateral thickenings have to move over a distance of roughly 400 m within 1–2 h. Epidermal overgrowth of the nerve cord occurs during the morphogenetic movements that accompany nerve cord formation. Correspondence to: J.A. Campos-Ortega     

Chromosomal localization of zebrafish AluI repeats by primed in situ (PRINS) labeling.

Two zebrafish AluI repeats were localized in metaphase chromosomes by means of the primed in situ (PRINS) labeling technique, using oligonucleotide primers based on published sequences. An AT-rich, tandemly repeated, long AluI restriction fragment (RFAL1) labeled the (peri)centromeric regions of all chromosomes. The GC-rich short fragment (RFAS) was found to be localized in the paracentromeric regions of 17 chromosome pairs, which were mostly subtelocentric. The RFAS labeling pattern generally fits the previously described chromomycin A3 (CMA3) staining pattern. The differential composition of heterochromatin in zebrafish chromosomes is discussed.     

Overcoming a permeability barrier by microinjecting cryoprotectants into zebrafish embryos (Brachydanio rerio)

The goal of this research was to examine the developmental effects on zebrafish embryos (Brachydanio rerio) when cryoprotectants were directly microinjected into the yolk. Our objectives were to: (i) determine the final concentration of propylene glycol (PG) and dimethyl sulfoxide (Me(2)SO) that the embryos could tolerate without causing teratogenic effects; (ii) determine if the toxicity of Me(2)SO could be reduced by the simultaneous presence of various proportions of amides; and (iii) examine whether this intracellular cryoprotectant incorporation could reduce the cryodamage to the yolk syncytial layer (YSL) after vitrification trials. The rationale for conducting these microinjection experiments was to overcome the permeability barrier of the YSL. Intracellular PG produced better survival than Me(2)SO (P < 0.05). Embryos tolerated both 10- and 30-nl microinjections of PG, yielding final concentrations of 2.3 and 5.0 M within the yolk, resulting in 70 +/- 3 and 35 +/- 4% survival at day 5, respectively. In similar experiments with Me(2)SO, survival was lower than PG at 60 +/- 4 and 14 +/- 4% at 2.4 and 5.2 M. Unlike other cellular systems, the presence of amides, specifically acetamide or formamide, did not reduce the toxicity of Me(2)SO in zebrafish embryos (P > 0.05). During vitrification trials, we estimated a 25% dehydration of the yolk, yielding an effective PG concentration of 5.9 M. However, the incorporation of this vitrifiable concentration of PG was not sufficient to improve the postthaw morphology of the YSL (P > 0.05). Clearly, other factors need to be examined in establishing a successful vitrification protocol for zebrafish embryos.     

High mortality due to Tetrahymena sp. infection in laboratory-maintained zebrafish (Brachydanio rerio)

A large colony of laboratory zebrafish (Brachydanio rerio) used in the study of early vertebrate embryogenesis began experiencing acute, unexplained mortality that approached 100% among approximately 30-day-old resident fry. The initial differential diagnosis included ammonia, nitrite, or chlorine toxicosis, as well as iatrogenically induced toxicosis associated with improper sanitation procedures of laboratory equipment. Necropsy of dead and moribund fry prior to fixation revealed swarms of ovoid-shaped, motile, ciliated protozoa with a "spiraling football" motion. Wet mount preparations of various water samples also contained high numbers of similar protozoa. Histologic examination of affected fry revealed numerous, periodic acid-Schiff-positive forms within the body coelom, and epithelial and muscle tissues. The protozoa were consistent morphologically with members of the genus Tetrahymena, which is usually a free-living, nonpathogenic ciliated protozoa in fresh and saltwater environments. Relevant disease associated with Tetrahymena spp. in viviparous fish has been reported as a result of concurrent disease, immunosuppression, or poor water quality conditions. To the authors' knowledge, this is the first report of an epizootic involving laboratory maintained zebrafish, and the diagnostic course and therapeutic interventions undertaken to alleviate Tetrahymena species-associated clinical disease.     

Fish embryo culture: Migration and spreading of zebrafish (Brachydanio rerio) pigmented retinal epithelium

Summary Blastoderm explants fromBrachydanio rerio (Teleostei: Cyprinidae) high blastulas exhibited limited differentiation of optic structures in culture. A number of explants showed migration of pigmented retinal epithelial cells and formation of monolayers. The findings permit comparative studies in vitro on phenomena pertaining to pigmented retinal epithelial cell morphology, function, and differentiation. This investigation was supported by a grant from the Natural Sciences and Engineering Research Council of Canada.     

The biology and use of zebrafish, Brachydanio rerio in fisheries research.

PREFACE TO THE REVIEW BY PROFESSOR H. LAALE ON THE BIOLOGY AND USE OF THE ZEBRAFISH, BRACHYDANIO RERIO IN FISHERIES RESEARCH
The growing demand for increasingly sophisticated information on the toxic hazards of potential water pollutants has focused attention on the need for a suitable 'standard' animal model which could be accepted internationally. The Zebrafish, Brachydanio rerio (Hamilton-Buchanan, 1822, 1823) is considered to be the most likely candidate. It is relatively easy to maintain and breed in laboratory aquaria and it has proved to be responsive to a wide range of mutagens, carcinogens and teratogens, as well as direct toxicants. B. rerio has been the subject of a considerable number of investigations involving a diverse spectrum of disciplines in a number of countries. Professor Hans Laale, who has himself contributed to our knowledge of the embryopathology of B. rerio , has summarized and collated the findings of 450 publications, a number of which are unlikely to have become available to fishery scientists. We hope the publication of this review will aid those who are working with B. rerio and provide comprehensive data on the advantages and limitations of B. rerio as a contender for the standard laboratory fish for the safety evaluation of aquatic pollutants.
T he E ditor     

Scanning electron microscope studies on blastodisc formation in the zebrafish,Brachydanio rerio

Upon fertilization, the zebrafish egg undergoes marked physiological and structural changes, one of which involves blastodisc formation. Before fertilization, yolk globules are rounded and the endoplasm extends throughout the oocyte. During blastodisc formation, the yolk globules become angular and the endoplasm is restricted to streamers among the yolk globules. The streamers are oriented in an anterior-posterior axis of the egg. During blastodisc formation the cytoskeleton consists of an extensive array of filamentous structures of variable width in both the cortex as well as within elongate endoplasmic streamers. Although the filamentous components in the cortex and endoplasmic streamers probably include both microfilaments and microtubules, frequently they are somewhat wider than the usual dimensions, and possible reasons for this are suggested. From their arrangement in both the cortex and endoplasm, it seems likely that the components of the cytoskeleton (e.g., microfilaments and microtubules) may provide, through contraction, the major force responsible for the streaming of the endoplasm into the forming blastodisc. It is assumed that the surface tension of the vegetal hemisphere exceeds that of the animal hemisphere, thus forcing, through differential contraction, the endoplasm to flow in the direction of the forming blastodisc. No distinct barrier between the yolk and forming blastodisc was observed. The compressed condition of the larger and many-sided yolk globules could prevent their movement into the blastodisc. Scanning electron microscopy is limited in the resolution with which it can depict the cytoskeleton, but nonetheless it provides useful information about structural interrelationships.     

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

The zebrafish (Brachydanio rerio) offers many advantages for immunological and immunogenetic research and has the potential for becoming one of the most important nonmammalian vertebrate research models. With this in mind, we initiated a systematic study of the zebrafish major histocompatibility complex (Mhc) genes. In this report, we describe the cloning and characteristics of the zebrafish class I A genes coding for the chains of the heterodimer and thus complete the identification of all four classes and subclasses of the Mhc in this species. We describe the full class I cDNA sequence as well as the exon-intron organization of the class I A genes, including intron sequences. We identify three families of class I A genes which we designate Bree-UAA,-UBA, and -UCA. The three families originated about the time of the divergence of cyprinid and salmonid fishes. All three families are members of an ancient lineage that diverged from another, older lineage also represented in cyprinid fishes before the radiation of teleost orders. The fish class I A genes therefore evolve differently from mammalian class I A genes, in which the establishment of lineages and families mostly postdates the divergence of orders.The nucleotide sequence data reported in this Papershave been submitted to the EMBL/GenBank nucleotide sequence databases and have been assigned the accession numbers Z46776–Z46779     

Attraction of zebrafish, Brachydanio rerio, to alanine and its suppression by copper

Preference responses of zebrafish to 10⁻³, 10⁻⁴ and 10⁻⁵M alanine (Ala) were concentration- dependent. Behavioural responses to copper (Cu) and Cu + Ala mixtures were also assessed. Zebrafish avoided 100 and 10 μg Cu l⁻¹, but not 1 μg l⁻¹. Mixtures of 10⁻³ m Ala+ 100 μg Cu l⁻¹ and 10 ⁴ M Ala + 10 μg Cu 1⁻¹ were avoided as intensely as was Cu alone. Responses to 10⁻³ M Ala + 10 or 1 μg Cu l⁻¹ and 10 ⁴ M Ala +1 μg Cu l⁻¹ did not differ statistically from controls (no detectable preference or avoidance). These results demonstrate, firstly, that a concentration of a pollutant avoided by itself (10 μg Cu l⁻¹) may not be avoided when encountered with an attractant chemical stimulus (Ala) and may suppress the preference for an attractant stimulus, and secondly, that a concentration of a pollutant not avoided by itself and not considered deleterious (1 μg Cu l⁻¹) suppresses attraction to Ala (an important constituent of prey odours for many fishes).     

Attraction of zebrafish,Brachydanio rerio,to isolated and partially purified chromatographic fractions

Synopsis Water from donor fish of either sex maintained in tank systems for 16 days was tested to determine intrasexual responses in a T-maze apparatus. Only donor water attracted fish, suggesting the presence of intrasexual pheromone(s). The sexual attractant(s) was removed by methylchloroform extraction. The residue from this extraction elicited positive responses in test fish. Dried residues showed 5 bands in thin-layer chromatograms, but only one band (R_f 0.94), identified as cholesterol ester. contained the sexual attractant(s).     

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.