On the formation of the neural keel and neural tube in the zebrafishDanio (Brachydanio) rerio

Morphogenetic movements accompanying formation of the neural keel and neural tube in the zebrafishDanino (Brachydanio) rerio were studied by labelling single neural plate cells with fluoresceinated dextran (FDA) during late gastrula stages (95–100% epiboly) and localizing their progeny with an anti-fluorescein antibody on histological sections throughout neurulation. The mediolateral extent of the neural plate correlates directly with the dorso-ventral extent of the neural tube. That is to say, the progeny of cells located medially in the neural plate come to lie ventrally in the neural tube; cells located laterally in the neural plate give rise to progeny that populate dorsal levels in the neural tube. Fixation of labelled cells at various stages reveals that neural keel and nerve rod are organized as monostratified epithelia and that they maintain this organization during neurulation. These observations strongly suggest that the neural keel in the zebrafish forms by way of infolding of the neural plate and, therefore, utilizes a mechanism similar to primary neurulation in other vertebrates. The folding process juxtaposes the apical surfaces of both flanks of the neural plate at the midline. Mitoses occur preferentially in this zone, leading very frequently to formation of bilaterally symmetrical clones of progeny cells. The size of the clones that develop from injected cells suggests that neural plate cells divide an 1.5 times on average between late gastrula and the end of neurulation. Correspondence to: J.A. Campos-Ortega     

Dorso-ventral polarity of the zebrafish embryo is distinguishable prior to the onset of gastrulation

We describe a set of observations on developing zebrafish embryos and discuss the main conclusions they allow:(1) the embryonic dorso-ventral polarity axis is morphologically distinguishable prior to the onset of gastrulation; and (2) the involution of deep layer cells starts on the prospective dorsal side of the embryo. An asymmetry can be distinguished in the organization of the blastomeres in the zebrafish blastula at the 30% epiboly stage, in that one sector of the blastoderm is thicker than the other. Dye-labelling experiments with DiI and DiO and histological analysis allow us to conclude that the embryonic shield will form on the thinner side of the blastoderm. Therefore, this side corresponds to the prospective dorsal side of the embryo. Simultaneous injections of dyes on the thinner side of the blastoderm and on the opposite side show that involution of deep layer cells during gastrulation starts at the site at which the embryonic shield will form and extends from here to the prospective ventral regions of the germ ring.     

多氯联苯(PCB1254)对斑马鱼(Brachydanio rerio)毒性及其组织结构的影响

本文报道了多氯联苯(PCB₁₂₅₄)对斑马鱼(Brachydanio rerio)的亚急性毒性试验及其组织结构的影响。结果表明,PCB₁₂₅₄对斑马鱼存活最大无影响浓度(NOEC)为0.40μg·L^-1,最低有影响浓度(LOEC)为2.0μg·L^-1, PCB₁₂₅₄对斑马鱼30d半致死剂量为5.09μg·L^-1,95% 可信区间范围为3.79～6.52μg·L^-1。在不同浓度的PCB₁₂₅₄作用30d后,斑马鱼的组织结构发生明显变化, PCB₁₂₅₄浓度大于2μg·L^-1时,对斑马鱼鳃组织呼吸上皮细胞结构产生损伤而影响其呼吸代谢;高浓度PCB₁₂₅₄(50μg·L^-1)对斑马鱼肝脏细胞结构产生明显损害,肝脏细胞结构表现出异常,肝细胞核变形萎缩,并有大量脂褐素沉积。     

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.     

Daily shoaling patterns in the zebrafish Danio rerio

Shoaling intensity in zebrafish Danio rerio is believed to vary throughout subjective day and night hours. This experiment examines long term variations in shoaling behavior. Adult zebrafish Dan io rerio were maintained under a 12：12 LD cycle （with dim red light serving as reduced visibility during subjective dark hours）, and their shoaling behavior was monitored every hour for a three-day period of time. Our results show that zebrafish perform shoaling behavior throughout subjective day and under reduced visibility conditions, although mean shoaling times during the light phase were significantly higher than mean shoaling times during the dark phase. However, on the 3^rd day of the experiment, mean shoaling times during the subjective night had increased and mean shoaling times during the subjective day had decreased. This shift in intensity was not seen on the first two days of the study, and may represent the influence of experience on the behavior of the test fish. We believe this study shows that shoaling behavior changes with light/dark cycles and that fish shoal even during reduced visibility conditions.     

In vitro Oocyte Maturation in the Zebrafish Brachydanio rerio and Fertilization and Development of the Mature Egg

Maturation process of zebrafish oocyte was investigated using in vitro incubation.In medium EM-199 containing 0.5 μg/ml of 17α-hydroxyprogesterone incubated under 80％ O_2 and at 25°C,germinal vesicles(GV)of oocytes in stage Ⅳ migrated from midway between the center and theperiphery ofoocytes to the periphery in 40 minutes and the oocytes went into stage V.Half an hourlater,the oocytes underwent germinel vesicle breakdown(GVBD)with a breakdown rate of 59％.Two more hours were needed for such oocytes to complete their final maturation.The mature eggscould not come off from the follicle layer surrounding them by themselves(ovulation).By removingthe follicle and adding active sperms for insemination,we could make the mature eggs fertilized.Thechorion was elevated and blastoderm formed on the animal pole.The cleavage and development ofthese fertilized eggs followed the same course as the naturally matured and fertilized eggs.Usingblastula formation as a marker of successful fertilization of the in vitro matured egg,the fertilizationrate was 78％.This is the first report on the successful in vitro incubation of mature oocytes inzebrafish.The establishment of this in vitro oocyte maturation technology has laid the foundationfor further investigation of the transfer of foreign genes in the germinal vesicles of oocytes.     

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.     

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.     

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.     

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.     

Retinoic acid in the anteroposterior patterning of the zebrafish trunk

Retinoic acid has been linked to pattern formation in the vertebrate anteroposterior axis. This report describes the spatial and temporal distributions of both endogenous retinoic acid and retinoic acid synthase activity along the anteroposterior axis of neurulating zebrafish embryos, as detected by a transient transgenic assay and by a zymography bioassay. Both retinoic acid levels and synthase activity were found to be highest in anterior regions of the trunk at all of the stages which were analysed. The drug disulfiram inhibited retinoic acid synthase activity in the zebrafish trunk both in vitro and in vivo, and reduced retinoic acid levels in vivo. Disulfiram treatment of neurulating embryos resulted in larvae with hypertrophic wavy notochords, shortened spinal cords and deformed pectoral fins. The results support the hypothesis that retinoic acid plays a role in the coordination of axial patterning at the developing node/zone of involution, as well as in the subsequent development of anterior trunk structures such as the fins.     

Polar effects of lithium in the heart of the zebrafish Danio rerio

 The molecular signalling mechanisms that are believed to govern the patterning of the heart early in embryonic development are not well understood. We have investigated the events which occur during patterning of the vertebrate heart by exposing gastrula stage zebrafish embryos to lithium, which is known to affect the phosphoinositol signalling pathway. Treatment of embryos at 50% epiboly (5.25 h after fertilization at 28.5°C) with 0.3 m LiCl for 5–15 min, results in embryos with defects which range from mild to severe, depending on the length of time the embryos are exposed to lithium. In the heart, defects appear progressively in the inflow tract, the sinus venosus and atrium. By using an antibody that recognizes an atrium-specific isoform of myosin, our results show that lithium treatment at gastrulation specifically affects the atrium and sinus venosus, and has little obvious effect on the ventricle. Defects induced by lithium differ from those induced by retinoic acid (RA) treatment of similarly staged embryos, and suggest that lithium and RA may affect the patterning signals important for establishment of the vertebrate heart by acting on different populations of cells or by influencing different patterning pathways. Received: 8 December 1995 / Accepted: 11 April 1996     

Zinc transporter expression in zebrafish (Danio rerio) during development

Zinc is a micronutrient important in several biological processes including growth and development. We have limited knowledge on the impact of maternal zinc deficiency on zinc and zinc regulatory mechanisms in the developing embryo due to a lack of in vivo experimental models that allow us to directly study the effects of maternal zinc on embryonic development following implantation. To overcome this barrier, we have proposed to use zebrafish as a model organism to study the impact of zinc during development. The goal of the current study was to profile the mRNA expression of all the known zinc transporter genes in the zebrafish across embryonic and larval development and to quantify the embryonic zinc concentrations at these corresponding developmental time points. The SLC30A zinc transporter family (ZnT) and SLC39A family, Zir-,Irt-like protein (ZIP) zinc transporter proteins were profiled in zebrafish embryos at 0, 2, 6, 12, 24, 48 and 120 h post fertilization to capture expression patterns from a single cell through full development. We observed consistent embryonic zinc levels, but differential expression of several zinc transporters across development. These results suggest that zebrafish is an effective model organism to study the effects of zinc deficiency and further investigation is underway to identify possible molecular pathways that are dysregulated with maternal zinc deficiency.     

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     

15-zinc finger protein Bloody Fingers is required for zebrafish morphogenetic movements during neurulation

A novel zebrafish gene bloody fingers (blf) encoding a 478 amino acid protein containing fifteen C(2)H(2) type zinc fingers was identified by expression screening. As determined by in situ hybridization, blf RNA displays strong ubiquitous early zygotic expression, while during late gastrulation and early somitogenesis, blf expression becomes transiently restricted to the posterior dorsal and lateral mesoderm. During later somitogenesis, blf expression appears only in hematopoietic cells. It is completely eliminated in cloche, moonshine but not in vlad tepes (gata1) mutant embryos. Morpholino (MO) knockdown of the Blf protein results in the defects of morphogenetic movements. Blf-MO-injected embryos (morphants) display shortened and widened axial tissues due to defective convergent extension. Unlike other convergent extension mutants, blf morphants display a split neural tube, resulting in a phenotype similar to the human open neural tube defect spina bifida. In addition, dorsal ectodermal cells delaminate in blf morphants during late somitogenesis. We propose a model explaining the role of blf in convergent extension and neurulation. We conclude that blf plays an important role in regulating morphogenetic movements during gastrulation and neurulation while its role in hematopoiesis may be redundant.     

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.     

Ultrastructure of the distal retina of the adult zebrafish, Danio rerio

The organization, morphological characteristics, and synaptic structure of photoreceptors in the adult zebrafish retina were studied using light and electron microscopy. Adult photoreceptors show a typical ordered tier arrangement with rods easily distinguished from cones based on outer segment (OS) morphology. Both rods and cones contain mitochondria within the inner segments (IS), including the large, electron-dense megamitochondria previously described (Kim et al.) Four major ultrastructural differences were observed between zebrafish rods and cones: (1) the membranes of cone lamellar disks showed a wider variety of relationships to the plasma membrane than those of rods, (2) cone pedicles typically had multiple synaptic ribbons, while rod spherules had 1-2 ribbons, (3) synaptic ribbons in rod spherules were ∼2 times longer than ribbons in cone pedicles, and (4) rod spherules had a more electron-dense cytoplasm than cone pedicles. Examination of photoreceptor terminals identified four synaptic relationships at cone pedicles: (1) invaginating contacts postsynaptic to cone ribbons forming dyad, triad, and quadrad synapses, (2) presumed gap junctions connecting adjacent postsynaptic processes invaginating into cone terminals, (3) basal junctions away from synaptic ribbons, and (4) gap junctions between adjacent photoreceptor terminals. More vitread and slightly farther removed from photoreceptor terminals, extracellular microtubule-like structures were identified in association with presumed horizontal cell processes in the OPL. These findings, the first to document the ultrastructure of the distal retina in adult zebrafish, indicate that zebrafish photoreceptors have many characteristics similar to other species, further supporting the use of zebrafish as a model for the vertebrate visual system.     

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