Development and fine structure of pharyngeal replacement teeth in juvenile zebrafish (Danio rerio) (Teleostei, Cyprinidae)

Teeth are commonly used model systems for the study of epithelial-mesenchymal interactions during organogenesis. We describe here the ultrastructural characteristics of developing pharyngeal replacement teeth in juvenile zebrafish, an increasingly important model organism for vertebrate development. Replacement teeth develop in close association with the dental organ of a functional tooth. Morphogenesis is well advanced prior to the start of cytodifferentiation. Fibrillar enameloid matrix is formed first, followed by the deposition of predentine. Initial mineralization of the enameloid proceeds quickly; maturation involves the presence of ruffled-bordered ameloblasts. Dentine mineralization is inotropic and is mediated by matrix vesicles. Woven-fibred attachment bone matrix is deposited before completion of dentine mineralization. Eruption of fully ankylosed teeth is a fast process and may involve degenerative changes in the pharyngeal epithelium. Mononucleated osteoclasts and clastic cells located in the pulp cavity intervene in tooth resorption prior to shedding. Structural differences with larval, first-generation zebrafish teeth include the presence of dentinal tubules and the absence of an electron-dense covering membrane. Part of these differences may relate to size differences of the teeth. Others, like the site of the replacement tooth bud, suggest that initiation may take place in already committed epithelium from the first initiation event in the larval stage.     

The ontogeny and homology of the Weberian apparatus in the zebrafish Danio rerio (Ostariophysi: Cypriniformes)

The ontogeny of the Weberian apparatus was examined in the zebrafish, Danio rerio , using both cleared and stained specimens and histology. Over 300 individuals from four independent zebrafish lineages, ranging in size from 3 to 28 mm TL, were examined for this study. Results provide a basic understanding of the development of the Weberian apparatus in the wild-type zebrafish. Our results, in conjunction with those already published, point to substantial variation in the development of the Weberian apparatus among otophysans and new interpretations of the homology of certain ossicles (e.g. tripus and claustrum). Hypotheses of homology among various Weberian ossicles are considered and represent an important step in understanding the evolution of sound transmission in ostariophysan fishes.  © 2004 The Linnean Society of London, Zoological Journal of the Linnean Society , 2004, 140 , 241–254.     

Development of sensory systems in zebrafish (Danio rerio)

Zebrafish possess all of the classic sensory modalities: taste, tactile, smell, balance, vision, and hearing. For each sensory system, this article provides a brief overview of the system in the adult zebrafish followed by a more detailed overview of the development of the system. By far the majority of studies performed in each of the sensory systems of the zebrafish have involved some aspect of molecular biology or genetics. Although molecular biology and genetics are not major foci of the paper, brief discussions of some of the mutant strains of zebrafish that have developmental defects in each specific sensory system are included. The development of the sensory systems is only a small sampling of the work being done using zebrafish and provides a mere glimpse of the potential of this model for the study of vertebrate development, physiology, and human disease.     

Classical and molecular cytogenetics of the zebrafish, Danio rerio (Cyprinidae, Cypriniformes): an overview

The zebrafish, Danio rerio, has recently become the model system for the genetic analysis of vertebrate development. This paper reviews the advances in zebrafish cytogenetics, obtained through classical and molecular techniques, which will lead to the assignment of specific linkage groups to specific chromosome pairs in the zebrafish genome project. Several chromosome pairs of the 50-chromosome karyotype of D. rerio were differentially stained by classical staining techniques and additional information has been obtained by molecular cytogenetics. Indeed, the analysis of constitutive heterochromatin by C-banding and base-specific fluorochrome staining had suggested a differential composition of peri- and paracentromeric constitutive heterochromatin. The chromosome mapping of distinct AT- and GC-rich zebrafish satellite DNAs by means of PRINS (Primed in situ) and multicolor FISH (Fluorescence in situ Hybridization) has confirmed this hypothesis, which therefore provided the chromosome localization of 10% of the zebrafish genome. The analysis of nucleolus organizer regions (NORs) by silver staining and by FISH with 18S rDNA has also revealed the existence of variable and inactive NORs, in addition to those on the terminal regions of the long arms of the three NOR-bearing chromosome pairs. Other multicopy genes, such as minor ribosomal genes, or multicopy repeats, such as telomere specific sequences, have now been mapped on zebrafish chromosomes. The latest advancement in zebrafish molecular cytogenetics is the chromosome mapping of single locus genes. Single-copy genes from each of the 25 genetic linkage groups are now being mapped on zebrafish chromosomes by using PAC clones.     

Development of zebrafish (Danio rerio) pectoral fin musculature

During posthatching development the fins of fishes undergo striking changes in both structure and function. In this article we examine the development of the pectoral fins from larval through adult life history stages in the zebrafish (Danio rerio), describing in detail their pectoral muscle morphology. We explore the development of muscle structure as a way to interpret the fins' role in locomotion. Genetic approaches in the zebrafish model are providing new tools for examining fin development and we take advantage of transgenic lines in which fluorescent protein is expressed in specific tissues to perform detailed three-dimensional, in vivo fin imaging. The fin musculature of larval zebrafish is organized into two thin sheets of fibers, an abductor and adductor, one on each side of an endoskeletal disk. Through the juvenile stage the number of muscle fibers increases and muscle sheets cleave into distinct muscle subdivisions as fibers orient to the developing fin skeleton. By the end of the juvenile period the pectoral girdle and fin muscles have reoriented to take on the adult organization. We find that this change in morphology is associated with a switch of fin function from activity during axial locomotion in larvae to use in swim initiation and maneuvering in adults. The examination of pectoral fins of the zebrafish highlights the yet to be explored diversity of fin structure and function in subadult developmental stages. J. Morphol. (c) 2005 Wiley-Liss, Inc.     

Liver development in zebrafish （Danio rerio）

Liver is one of the largest internal organs in the body and its importance for metabolism, detoxification and homeostasis has been well established. In this review, we summarized recent progresses in studying liver initiation and development during embryogenesis using zebrafish as a model system. We mainly focused on topics related to the specification of hepatoblasts from endoderm, the formation and growth of liver bud, the differentiation of hepatocytes and bile duct cells from hepatoblasts, and finally the role of mesodermal signals in controlling liver development in zebrafish.     

Biophysics of zebrafish (Danio rerio) sperm

In the past two decades, laboratories around the world have produced thousands of mutant, transgenic, and wild-type zebrafish lines for biomedical research. Although slow-freezing cryopreservation of zebrafish sperm has been available for 30 years, current protocols lack standardization and yield inconsistent post-thaw fertilization rates. Cell cryopreservation cannot be improved without basic physiological knowledge, which was lacking for zebrafish sperm. The first goal was to define basic cryobiological values for wild-type zebrafish sperm and to evaluate how modern physiological methods could aid in developing improved cryopreservation protocols. Coulter counting methods measured an osmotically inactive water fraction (Vb) of 0.37 ± 0.02 (SEM), an isosmotic cell volume (V_o) of 12.1 ± 0.2 μm³ (SEM), a water permeability (L_p) in 10% dimethyl sulfoxide of 0.021 ± 0.001(SEM) μm/min/atm, and a cryoprotectant permeability (P_s) of 0.10 ± 0.01 (SEM) × 10⁻³ cm/min. Fourier transform infrared spectroscopy indicated that sperm membranes frozen without cryoprotectant showed damage and lipid reorganization, while those exposed to 10% glycerol demonstrated decreased lipid phase transition temperatures, which would stabilize the cells during cooling. The second goal was to determine the practicality and viability of shipping cooled zebrafish sperm overnight through the mail. Flow cytometry demonstrated that chilled fresh sperm can be maintained at 92% viability for 24 h at 0 °C, suggesting that it can be shipped and exchanged between laboratories. Additional methods will be necessary to analyze and improve cryopreservation techniques and post-thaw fertility of zebrafish sperm. The present study is a first step to explore such techniques.     

Acridine mutagenesis of zebrafish (Danio rerio)

Mutagenesis screening, in which heritable traits are isolated following damage to the genome, is a powerful approach for investigating gene function. Among vertebrate model organisms, the zebrafish (Danio rerio) is ideally suited to mutagenesis screens. The success of large-scale screens is dependent on the way in which changes are identified. The type of damage induced is also pivotal. Single base coding region deletions and insertions are suited to abolition of gene function whilst inducing a small physical alteration to the genome. Such mutations are not commonly found following mutagenesis schemes reported to date. Here, we show that an acridine mutagen, ICR191, which in other model organisms frequently induces single base deletions and insertions, is mutagenic in zebrafish. ICR191 induces hallmark phenotypes associated with genetic damage in treated embryos. Alterations are heritable. Offspring of mutagenised fish had mutations in a marker gene and were found to produce offspring with abnormal development. Using an adaptation of a molecular mutation detection method, fluorescent arbitrary primed PCR, we identified an induced alteration directly. The estimated frequency of induced mutations was sufficiently high to make it feasible to employ this approach for mutagenesis screening.     

Development of the pteridine pathway in the zebrafish, Danio rerio

In the zebrafish, the peripheral neurons and the pigment cells are derived from the neural crest and share the pteridine pathway, which leads either to the cofactor tetrahydrobiopterin or to xanthophore pigments. The components of the pteridine pattern were identified as tetrahydrobiopterin, sepiapterin, 7-oxobiopterin, isoxanthopterin, and 2,4,7-trioxopteridine. The expression of GTP cyclohydrolase I activity during the first 24-h postfertilization, followed by 6-pyruvoyl-5,6,7,8-tetrahydropterin synthase and sepiapterin reductase, suggest an early supply of tetrahydrobiopterin for neurotransmitter synthesis in the neurons and for tyrosine supply in the melanophores. At 48-h postfertilization, sepiapterin formation branches off the de novo pathway of tetrahydrobiopterin synthesis. Sepiapterin, via 7,8-dihydrobiopterin and biopterin, serves as a precursor for the formation of 7-oxobiopterin, which may be further catabolized to isoxanthopterin and 2,4,7-trioxopteridine. Neither 7, 8-dihydrobiopterin nor biopterin is a substrate for xanthine oxidoreductase. In contrast, both of these compounds are oxidized at C-7 by a xanthine oxidase variant form, which is inactivated by KCN, but is insensitive to allopurinol. The oxidase and the dehydrogenase form of xanthine oxidoreductase as well as the xanthine oxidase variant have specific developmental patterns. It follows that GTP cyclohydrolase I, the formation of sepiapterin, and the xanthine oxidoreductase family control the pteridine pathway in the zebrafish.     

Mycobacteriosis in zebrafish (Danio rerio) research facilities

The Zebrafish International Resource Center was established to support the zebrafish research community, and includes a diagnostic service. One of the most common diseases that we have diagnosed is mycobacteriosis, which represented 18% of the diagnostic cases submitted from November 1999 to June 2003. We describe here the severity of the disease and associated pathological changes of 24 diagnostic cases from 14 laboratories. Identifications of the bacteria are provided for seven of these cases. For two cases in which culture of the organism was not successful, these identifications were based on ribosomal DNA (rDNA) sequence analysis obtained directly from infected tissues. Biochemical characteristics and rDNA sequence analysis from cultures are reported for the other isolates. Two severe outbreaks from different facilities on different continents were associated with an organism identified as Mycobacterium haemophilum based on rDNA sequence from tissues. Another severe outbreak was associated with an organism most closely related to Mycobacterium peregrinum. These species are recognized pathogens of humans, but this is the first report of them from fish. Bacteria identified as Mycobacterium chelonae or M. abscessus were recovered from fish in cases categorized as moderate disease or as an incidental finding. These findings indicate that species of Mycobacterium previously undescribed from fish (i.e., M. haemophilum and M. peregrinum) may pose significant health problems in zebrafish research facilities, whereas species and strains that are already recognized as common in fish usually cause limited disease on a population basis in zebrafish.     

Locomotor behaviors in zebrafish (Danio rerio) larvae

Locomotor behaviors were examined in two experiments using zebrafish (Danio rerio) larvae at 4, 5, 6 and 7 days post fertilization (dpf). Larvae were observed in individual wells of a 12-well plate for 1 h a day. In Experiment 1, the same larvae were observed for four consecutive days beginning on post-fertilization day 4; in Experiment 2, different groups of larvae from the same egg collection were observed at 4, 5, 6 and 7 dpf. Automated images collected every 6 s were analyzed for information about larval location, orientation and general activity. In both experiments, 4 dpf larvae rested significantly more, used a smaller area of the well more frequently, and were generally less active than older larvae. All larvae exhibited a preference for facing away from the center of the well and for the edge of the well. However, prolonged exposure to the well influenced overall activity, orientation, and preference for the edge region. The implications of these results for understanding the development of larval behavior and for the design of procedures to measure the effects of experience in zebrafish larvae are discussed.     

Oxidative stress in zebrafish (Danio rerio) sperm

Laboratories around the world have produced tens of thousands of mutant and transgenic zebrafish lines. As with mice, maintaining all of these valuable zebrafish genotypes is expensive, risky, and beyond the capacity of even the largest stock centers. Because reducing oxidative stress has become an important aspect of reducing the variability in mouse sperm cryopreservation, we examined whether antioxidants might improve cryopreservation of zebrafish sperm. Four experiments were conducted in this study. First, we used the xanthine-xanthine oxidase (X-XO) system to generate reactive oxygen species (ROS). The X-XO system was capable of producing a stress reaction in zebrafish sperm reducing its sperm motility in a concentration dependent manner (P<0.05). Second, we examined X-XO and the impact of antioxidants on sperm viability, ROS and motility. Catalase (CAT) mitigated stress and maintained viability and sperm motility (P>0.05), whereas superoxide dismutase (SOD) and vitamin E did not (P<0.05). Third, we evaluated ROS in zebrafish spermatozoa during cryopreservation and its effect on viability and motility. Methanol (8%) reduced viability and sperm motility (P<0.05), but the addition of CAT mitigated these effects (P>0.05), producing a mean 2.0 to 2.9-fold increase in post-thaw motility. Fourth, we examined the effect of additional cryoprotectants and CAT on fresh sperm motility. Cryoprotectants, 8% methanol and 10% dimethylacetamide (DMA), reduced the motility over the control value (P<0.5), whereas 10% dimethylformamide (DMF) with or without CAT did not (P>0.05). Zebrafish sperm protocols should be modified to improve the reliability of the cryopreservation process, perhaps using a different cryoprotectant. Regardless, the simple addition of CAT to present-day procedures will significantly improve this process, assuring increased and less variable fertilization success and allowing resource managers to dependably plan how many straws are needed to safely cryopreserve a genetic line.     

Sex-specific recombination rates in zebrafish (Danio rerio)

In many organisms, the rate of genetic recombination is not uniform along the length of chromosomes or between sexes. To compare the relative recombination rates during meiosis in male and female zebrafish, we constructed a genetic map based on male meiosis. We developed a meiotic mapping panel of 94 androgenetic haploid embryos that were scored for genetic polymorphisms. The resulting male map was compared to female and sex-average maps. We found that the recombination rate in male meiosis is dramatically suppressed relative to that of female meiosis, especially near the centromere. These findings have practical applications for experimental design. The use of exclusively female meiosis in a positional cloning project maximizes the ratio of genetic map distance to physical distance. Alternatively, the use of exclusively male meiosis to localize a mutation initially to a linkage group or to maintain relationships of linked alleles minimizes recombination, thereby facilitating some types of analysis.     

Visual discrimination learning in zebrafish (Danio rerio)

Three experiments demonstrated visual discrimination learning in zebrafish (Danio rerio). In each experiment, zebrafish were given a choice between two visually distinct arms of a T-maze. Choice of one stimulus was always followed by a food reward, but choice of the other stimulus was not rewarded. Different colored sleeves fitted around the arms of the T-maze were used in Experiments 1 (green and purple) and 2 (red and blue). The stimuli used in Experiment 3 were white sleeves lined with horizontal or vertical black stripes. In all three experiments, zebrafish acquired a significant preference for the stimulus that led to a food reward. Experiments 1 and 2 also showed that zebrafish could learn a reversal of the discrimination. Finally, the effect of discontinuing food rewards was examined after reversal training in Experiment 2 and after initial discrimination training in Experiments 1 and 3. Non-reinforcement led to a decrease in correct responding in Experiments 2 and 3 independent of stimulus identity, but to an asymmetrical pattern of responding in Experiment 1. The median latency to make a choice response decreased over the course of acquisition in all three experiments; during extinction, median response times did not change at all in Experiment 1 and increased only very slightly in Experiment 2, but showed a substantial increase in Experiment 3. The implications of these results for the zebrafish as a model system for genetic studies of learning and memory are discussed.     

Exercise-induced lordosis in zebrafish Danio rerio (Hamilton, 1822)

The anabolic effect of exercise on muscles and bones is well documented. In teleost fish, exercise has been shown to accelerate skeletogenesis, to increase bone volume, and to change the shape of vertebral bodies. Still, increased swimming has also been reported to induce malformations of the teleost vertebral column, particularly lordosis. This study examines whether zebrafish (Danio rerio) develops lordosis as a result of continuous physical exercise. Zebrafish were subjected, for 1 week, to an increased swimming exercise of 5.0, 6.5 or 8.0 total body lengths (TL) per second. Control and exercise group zebrafish were examined for the presence of vertebral abnormalities, by in vivo examination, whole mount staining for bone and cartilage and histology and micro-computed tomography (CT) scanning. Exercise zebrafish developed a significantly higher rate of lordosis in the haemal part of the vertebral column. At the end of the experiment, the frequency of lordosis in the control groups was 0.5 ± 1.3% and that in the exercise groups was 7.5 ± 10.6%, 47.5 ± 10.6% and 92.5 ± 6.0% of 5.0, 6.5 and 8.0 TL∙s⁻¹, respectively. Histological analysis and CT scanning revealed abnormal vertebrae with dorsal folding of the vertebral body end plates. Possible mechanisms that trigger lordotic spine malformations are discussed. This is the first study to report a quick, reliable and welfare-compatible method of inducing skeletal abnormalities in a vertebrate model during the post-embryonic period.     

Effects of arsenic (As) exposure on the antioxidant status of gills of the zebrafish Danio rerio (Cyprinidae)

In fishes, arsenic (As) is absorbed via the gills and is capable of causing disturbance to the antioxidant system. The objective of present study was to evaluate antioxidant responses after As exposure in gills of zebrafish (Danio rerio, Cyprinidae). Fish were exposed for 48 h to three concentration of As, including the highest As concentration allowed by current Brazilian legislation (10 μg As/L). A control group was exposed to tap water (pH 8.0; 26 °C; 7.20 mg O₂/L). As exposure resulted in (1) an increase (p < 0.05) of glutathione (GSH) levels after exposure to 10 and 100 μg As/L, (2) an increase of the glutamate cysteine ligase (GCL) activity in the same concentrations (p < 0.05), (3) no significant differences in terms of glutathione reductase, glutathione-S-transferase and catalase activities; (4) a significantly lower (p < 0.05) oxygen consumption after exposure to 100 μg As/L; (4) no differences in terms of oxygen reactive species generation and lipid peroxidation content (p > 0,05). In the gills, only inorganic As was detected. Overall, it can be concluded that As affected the antioxidant responses increasing GCL activity and GSH levels, even at concentration considered safe by Brazilian legislation.     

Development of the hypochord and dorsal aorta in the zebrafish embryo (Danio rerio)

The hypochord of the zebrafish embryo (Danio rerio) emerges at the 9-somite stage as a single row of cells in the dorsomedial endoderm immediately ventral to the notochord. It is recognizable from the 2(nd) or 3(rd) somite and extends along the trunk to the same extent as the somites. A basal lamina surrounds the hypochord and its cells are slightly larger than the nearby endoderm cells. TEM studies have shown that the hypochord cells contain, in addition to mitochondria, well-developed rough endoplasmic reticula and Golgi networks, indicating synthetic activity. Once formed, the hypochord will stay in close association with the notochord, and this axial complex gradually moves dorsally, separating the hypochord from the endoderm as a one-cell-wide, rod-like structure that is bean-shaped in transverse section. This is the situation in the 15-somite embryo, at the level of the 4-5(th) somites. In the gap between the hypochord and the endoderm, angioblast cells aggregate and start to form the dorsal aorta, which becomes intimately associated with the hypochord. In the 17-somite embryo the aortic rudiment is established just ventral to the hypochord as a tube with a lumen. As development proceeds, the size of the hypochord decreases. In the pec fin embryo the hypochord is still recognizable in the posterior trunk, but has apparently vanished in anterior regions. The temporal correlation between the appearance of the hypochord and the formation of the dorsal aorta, coupled with the intimate relationship between these structures, suggest that the hypochord may play a role in the positioning of the dorsal aorta.     

Long-term repeatability of behaviours in zebrafish (Danio rerio)

Animal personality is defined as individual variation in behaviour that is consistent over time and/or across contexts. Animal personality is now considered a fundamental aspect in the fields of animal behaviour and behavioural ecology, yet the majority of studies assess repeatability of behaviours over only relatively short time periods (e.g. a week) using just two measures. Understanding whether behavioural traits are repeatable over longer periods is important for the assessment of individual differences in behaviour. Using zebrafish (Danio rerio), we investigated the repeatability of activity and exploratory behaviours, including distance travelled, time spent in the bottom of the arena, stationary time and overall exploration of the novel arena over a 28-week period, using five intervals. All measures were repeatable over 28 weeks, but the repeatability estimates were much lower when comparing the initial week one and week two behaviours. There were clear sex differences in aspects of activity, with males more active than females. Importantly, our behavioural assays suggest that zebrafish require an initial “tank experience,” prior to the main phenotyping session, to ensure that behaviours being measured are repeatable—these effects are often not considered, but have implications for the many studies that measure behaviour at a single time point only.