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.     

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.     

Axonal outgrowth within the abnormal scaffold of brain tracts in a zebrafish mutant

The role of specific axonal tracts for the guidance of growth cones was investigated by examining axonal outgrowth within the abnormal brain tracts of zebrafish cyclops mutants. Normally, the earliest differentiating neurons in the zebrafish brain establish a simple scaffold of axonal tracts. Later-developing axons follow cell-specific pathways within this axonal scaffold. In Cyclops embryos, this scaffold is perturbed due to the deletion of some ventromedial neurons that establish parts of the axonal scaffold and the development of an abnormal crease in the brain. In these mutant embryos, the growth cones projected by the neurons of the nucleus of the posterior commissure (nur PC) are deprived of the two tracts of axons that they sequentially follow to first extend ventrally, then posteriorly. These growth cones respond to the abnormal scaffold in several interesting ways. First, nuc PC growth cones initially always extend ventrally as in wild-type embryos. This suggests that for the first portion of their pathway the axons they normally follow are not required for proper navigation. Second, approximately half of the nuc PC growth cones follow aberrant longitudinal pathways after the first portion of their pathway. This suggests that for the longitudinal portion of the pathway, specific growth cone/axon interactions are important for guiding growth cones. Third, although approximately half of the nuc PC growth cones follow aberrant longitudinal pathways, the rest follow normal pathways despite the absence of the axons that they normally follow. This suggests that cues independent of these axons may be capable of guiding nuc PC growth cones as well. These results suggest that different guidance cues or combinations of cues guide specific growth cones along different portions of their pathway. 1994 John Wiley & Sons, Inc.     

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     

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.     

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     

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.     

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.     

Relationships between annulate lamellae and filament bundles in oocytes of the zebrafish,Brachydanio rerio

Summary Bundles of filaments have been observed in the vitellogenic oocyte of the zebrafish, Brachydanio rerio; and these filaments illustrate a close spatial and structural relationship to annulate lamellae. The filaments range from 6–8 nm in diameter, and the annulate lamellae may cap both rounded ends of the bundle as well as extend parallel to the surface of the filament bundles. The ends of the filaments can be observed to exhibit an apparent termination in close relation to pore margins of the annulate lamellae, the membrane of the interpore regions of the annulate lamellae, as well as many nearby polyribosomes. The possible functional significance of this unique relationship is discussed in reference to a recent hypothesis regarding the function of annulate lamellae.     

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).     

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.     

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).     

Novel guidance cues during neuronal pathfinding in the early scaffold of axon tracts in the rostral brain

A scaffold of axons consisting of a pair of longitudinal tracts and several commissures is established during early development of the vertebrate brain. We report here that NOC-2, a cell surface carbohydrate, is selectively expressed by a subpopulation of growing axons in this scaffold in Xenopus. NOC-2 is present on two glycoproteins, one of which is a novel glycoform of the neural cell adhesion molecule N-CAM. When the function of NOC-2 was perturbed using either soluble carbohydrates or anti-NOC-2 antibodies, axons expressing NOC-2 exhibited aberrant growth at specific points in their pathway. NOC-2 is the first-identified axon guidance molecule essential for development of the axon scaffold in the embryonic vertebrate brain.     

Characterization of a zebrafish (Danio rerio) sphingosine 1-phosphate receptor expressed in the embryonic brain

Sphingosine 1-phosphate elicits a variety of responses in mammals via at least five G protein-coupled Edg receptors. We cloned zebrafish edg1 and expressed it in Rh7777 cells. In these cultures, S1P inhibited forskolin-driven rises in cAMP and this response was eliminated by pretreatment of the cultures with pertussis toxin. In Rh7777 membranes, S1P stimulated GTPgamma[(35)S] binding 2-3 fold. Zebrafish edg1 is expressed in embryonic brain, particularly ventral diencephalon, optic stalks, and anterior hindbrain. Our findings suggest that nonmammalian vertebrates use S1P to signal during embryogenesis and that the properties of Edg1 receptor have been conserved for 400 million years.     

Ultrastructure and lipid content of the liver of the zebrafish,Brachydanio rerio,related to vitellogenin synthesis

The female zebrafish is capable of producing mature eggs on the fifth day of each reproductive cycle. During this five-day period the ultrastructure of hepatocytes undergoes several changes. The number of nuclear pores increases rapidly during spawning, followed by a proliferation of RER within 24 h. Two days after spawning, glycogen has disappeared and the liver contains large amounts of lipids. The lipid droplets are closely surrounded by elongated mitochondria. Golgi complexes are abundant, secreting dense bodies. Four days after spawning the hepatocytes tend to regain their pre-spawning appearance. It is suggested that the changes in the hepatocytes, which coincide with special phases of ovarian activity, are related to vitellogenin synthesis. Steroids, especially estradiol-17beta, may trigger this process in the liver.