Learning from Small Fry: The Zebrafish as a Genetic Model Organism for Aquaculture Fish Species

In recent years, the zebrafish has become one of the most prominent vertebrate model organisms used to study the genetics underlying development, normal body function, and disease. The growing interest in zebrafish research was paralleled by an increase in tools and methods available to study zebrafish. While zebrafish research initially centered on mutagenesis screens (forward genetics), recent years saw the establishment of reverse genetic methods (morpholino knock-down, TILLING). In addition, increasingly sophisticated protocols for generating transgenic zebrafish have been developed and microarrays are now available to characterize gene expression on a near genome-wide scale. The identification of loci underlying specific traits is aided by genetic, physical, and radiation hybrid maps of the zebrafish genome and the zebrafish genome project. As genomic resources for aquacultural species are increasingly being generated, a meaningful interaction between zebrafish and aquacultural research now appears to be possible and beneficial for both sides. In particular, research on nutrition and growth, stress, and disease resistance in the zebrafish can be expected to produce results applicable to aquacultural fish, for example, by improving husbandry and formulated feeds. Forward and reverse genetics approaches in the zebrafish, together with the known conservation of synteny between the species, offer the potential to identify and verify candidate genes for quantitative trait loci (QTLs) to be used in marker-assisted breeding. Moreover, some technologies from the zebrafish field such as TILLING may be directly transferable to aquacultural research and production.     

Acute and chronic alcohol dose: population differences in behavior and neurochemistry of zebrafish

The zebrafish has been in the forefront of developmental genetics for decades and has also been gaining attention in neurobehavioral genetics. It has been proposed to model alcohol-induced changes in human brain function and behavior. Here, adult zebrafish populations, AB and SF (short-fin wild type), were exposed to chronic treatment (several days in 0.00% or 0.50% alcohol v/v) and a subsequent acute treatment (1 h in 0.00%, 0.25%, 0.50% or 1.00% alcohol). Behavioral responses of zebrafish to computer-animated images, including a zebrafish shoal and a predator, were quantified using videotracking. Neurochemical changes in the dopaminergic and serotoninergic systems in the brain of the fish were measured using high-precision liquid chromatography with electrochemical detection. The results showed genetic differences in numerous aspects of alcohol-induced changes, including, for the first time, the behavioral effects of withdrawal from alcohol and neurochemical responses to alcohol. For example, withdrawal from alcohol abolished shoaling and increased dopamine and 3,4-dihydroxyphenylacetic acid in AB but not in SF fish. The findings show that, first, acute and chronic alcohol induced changes are quantifiable with automated behavioral paradigms; second, robust neurochemical changes are also detectable; and third, genetic factors influence both alcohol-induced behavioral and neurotransmitter level changes. Although the causal relationship underlying the alcohol-induced changes in behavior and neurochemistry is speculative at this point, the results suggest that zebrafish will be a useful tool for the analysis of the biological mechanisms of alcohol-induced functional changes in the adult brain.     

Medaka and zebrafish, an evolutionary twin study

Comparison of two related species is one of the most successful approaches to decipher general genetic principles in eukaryotes. This is best illustrated in yeast, where the model systems Saccharomyyces. cervisiae and Schizosaccharomyces. pombe have been examined. Powerful forward genetics in both species, species-specific differences in biological features and the phylogenetic distance between the two species, make them well suited for a comparative approach. Recent whole genome sequencing has also facilitated comparative genomics of these simple eukaryotes. It is now possible to go a step further using higher eukaryotes. A duplication of the genome at the base of the teleost radiation, facilitated evolution of almost 25,000 fish species, more than half of all vertebrate species together. Two teleost genetic model systems have emerged in the past few decades: zebrafish, in which large-scale mutagenesis has been successfully performed, and Medaka, a Japanese killifish with a century of history in genetics and now, as reported in this issue, many induced mutations. In this review we will illustrate how comparison of these two model species, Medaka and zebrafish, can reveal conserved and species-specific genetic and molecular mechanisms underlying vertebrate development.     

Zebrafish (Danio rerio) as a model organism for investigating endocrine disruption

Endocrine-disrupting compounds (EDCs) are widespread in the aquatic environment and can cause alterations in development, physiological homeostasis and health of vertebrates. Zebrafish, Danio rerio, has been suggested as a model species to identify targets as well as modes of EDC action. In fact, zebrafish has been found useful in EDC screening, in EDC effects assessment and in studying targets and mechanisms of EDC action. Since many of the environmental EDCs interfere with the sex steroid system of vertebrates, most EDC studies with zebrafish addressed disruption of sexual differentiation and reproduction. However, other targets of EDCs action must not be overlooked. For using a species as a toxicological model, a good knowledge of the biological traits of this species is a pre-requisite for the rational design of test protocols and endpoints as well as for the interpretation and extrapolation of the toxicological findings. Due to the genomic resources available for zebrafish and the long experience with zebrafish in toxicity testing, it is easily possible to establish molecular endpoints for EDC effects assessment. Additionally, the zebrafish model offers a number of technical advantages including ease and cost of maintenance, rapid development, high fecundity, optical transparency of embryos supporting phenotypic screening, existence of many mutant strains, or amenability for both forward and reverse genetics. To date, the zebrafish has been mainly used to identify molecular targets of EDC action and to determine effect thresholds, while the potential of this model species to study immediate and delayed physiological consequences of molecular interactions has been instrumentalized only partly. One factor that may limit the exploitation of this potential is the still rather fragmentary knowledge of basic biological and endocrine traits of zebrafish. Information on species-specific features in endocrine processes and biological properties, however, need to be considered in establishing EDC test protocols using zebrafish, in extrapolating findings from zebrafish to other vertebrate species, and in understanding how EDC-induced gene expression changes translate into disease.     

Zebrafish as a model organism for nutrition and growth: towards comparative studies of nutritional genomics applied to aquacultured fishes

Zebrafish (Danio rerio) is a common research model in fish studies of toxicology, developmental biology, neurobiology and molecular genetics; it has been proposed as a possible model organism for nutrition and growth studies in fish. The advantages of working with zebrafish in these areas are their small size, short generation time (12–14 weeks) and their capacity to produce numerous eggs (100–200 eggs/clutch). Since a wide variety of molecular tools and information are available for genomic analysis, zebrafish has also been proposed as a model for nutritional genomic studies in fish. The detailed study of every species employed as a model organism is important because these species are used to generalize how several biological processes occur in related organisms, and contribute considerably toward improving our understanding of the mechanisms involved in nutrition and growth. The objective of this review is to show the relevant aspects of the nutrition and growth in zebrafish that support its utility as a model organism for nutritional genomics studies. We made a particular emphasis that gene expression and genetic variants in response to zebrafish nutrition will shed light on similar processes in aquacultured fish.     

DNA分子标记技术及其在水产动物遗传上的应用研究

随着DNA分子标记技术的发展,其在动物遗传上发挥了重大作用,使用DNA分子标记可以观察到整个基因组的遗传多样性。目前,在水产养殖种类中使用的遗传标记主要包括线粒体DNA、RFLP、RAPD、AFLP、微卫星、SNP和EST标记。DNA分子标记的应用使得人们对水产养殖动物的遗传多样性、近亲繁殖、种类和品系鉴定以及遗传连锁图谱建立的研究都取得了很大进展,也加快了数量性状位点(QTL)基因的鉴定作为分子标记辅助选择(MAS)的研究。将这些标记技术在水产动物上的应用进行了论述,以及如何从人类基因组工程和斑马鱼这种模式鱼的研究中得到启发,更好的应用于水产动物基因组学和遗传学研究做一讨论。     

模式动物斑马鱼在神经系统疾病研究中的应用

近年来,斑马鱼作为一种新型模式动物被广泛地应用于发育学、遗传学、行为学和分子生物学等研究领域。其具有繁殖能力强、发育迅速且同步、体外受精和幼体透明等生物学和形态学特点,经广泛培养和筛选突变品种,目前斑马鱼品系资源丰富。与其他非脊椎模式动物相比,它与人类有更高的同源性。本文主要介绍斑马鱼作为一种理想的模式动物,结合其特殊的行为学检测手段和分子生物学特点,在研究神经系统疾病的发病机制、构建疾病模型和相应药物筛选等方面的应用。     

Zebrafish transgenic Enhancer TRAP line database (ZETRAP)

Background  

The zebrafish, Danio rerio, is used as a model organism to study vertebrate genetics and development. An effective enhancer trap (ET) in zebrafish using the Tol2 transposon has been demonstrated. This approach could be used to study embryogenesis of a vertebrate species in real time and with high resolution.     

Marking zebrafish, Danio rerio (cyprinidae), using scale regeneration

Tagging or marking small laboratory-bred fish species is not an easy task. This also holds for the zebrafish, Danio rerio, which is widely used throughout the world as a model organism for genetics, developmental biology, etc. We present a simple marking technique based on scale regeneration. A comparative morphological study of various types of zebrafish scales indeed shows that regenerated scales are easily distinguishable from nonregenerated ones. We propose to take advantage of this typical morphology to mark a single or several individuals. This technique, based on a natural biological process, is easy to perform and does not enhance fish mortality in laboratory breeding conditions. It permits assembly of several specimens in a single tank with the possibility of identifying each of them by regenerated-scale coding. Nevertheless, a prerequisite is that the species does not lose and regenerate scales in large numbers in laboratory breeding conditions. To check this, 5,200 scales were removed from a large region of the left flank in 100 zebrafish and the number and position of regenerated scales were statistically analysed. Our results indicate that (1) laboratory-bred zebrafish have only a few regenerated scales (7.48%), (2) the probability of finding a regenerated scale is similar whatever its position in a row (antero-posterior axis), but (3) it differs from one row to another (scales from the back are more frequently lost than those from the pectoral region). This paper presents a procedure to mark small breeding colonies of zebrafish using scale regeneration with the number and position of the scales to be removed with high probability of marking success. J. Exp. Zool. 286:297-304, 2000.     

Considering the zebrafish in a comparative context

This article introduces a special issue on zebrafish biology that attempts to integrate developmental genetics with comparative studies of other fish species. For zebrafish researchers, comparative work offers a better understanding of the evolutionary history of their model system. Comparative biologists can gain many insights from the developmental and genetic mechanisms revealed in zebrafish that have contributed to the huge range of morphological variation among fishes that has arisen over millions of years. These ideas are considered here in various contexts, including systematics, genome organization and the development of the nervous system, pigmentation, craniofacial skeleton and dentition. Studies of the zebrafish in phylogenetic context provide an opportunity for synergy between communities using these two fundamentally different approaches.     

The zebrafish genome contains two distinct selenocysteine tRNA[Ser]sec genes.

The zebrafish is widely used as a model system for studying mammalian developmental genetics and more recently, as a model system for carcinogenesis. Since there is mounting evidence that selenium can prevent cancer in mammals, including humans, we characterized the selenocysteine tRNA[Ser]sec gene and its product in zebrafish. Two genes for this tRNA were isolated and sequenced and were found to map at different loci within the zebrafish genome. The encoding sequences of both are identical and their flanking sequences are highly homologous for several hundred bases in both directions. The two genes likely arose from gene duplication which is a common phenomenon among many genes in this species. In addition, zebrafish tRNA[Ser]sec was isolated from the total tRNA population and shown to decode UGA in a ribosomal binding assay.     

Androgen response to social competition in a shoaling fish

Androgens respond to social challenges and this response has been interpreted as a way for males to adjust androgen-dependent behavior to social context. However, the androgen responsiveness to social challenges varies across species and a conceptual framework has been developed to explain this variation according to differences in the mating system and parental care type, which determines the regimen of challenges males are exposed to, and concomitantly the scope (defined as the difference between the physiological maximum and the baseline levels) of response to a social challenge. However, this framework has been focused on territorial species and no clear predictions have been made to gregarious species (e.g. shoaling fish), which although tolerating same-sex individuals may also exhibit intra-sexual competition. In this paper we extend the scope of this conceptual framework to shoaling fish by studying the endocrine response of zebrafish (Danio rerio) to social challenges. Male zebrafish exposed to real opponent agonistic interactions exhibited an increase in androgen levels (11-ketotestosterone both in Winners and Losers and testosterone in Losers). This response was absent in Mirror-fighters, that expressed similar levels of aggressive behavior to those of winners, suggesting that this response is not a mere reflex of heightened aggressive motivation. Cortisol levels were also measured and indicated an activation of the hypothalamic–pituitary–interrenal axis in Winners of real opponent fighters, but not Losers or in Mirror-fighters. These results confirm that gregarious species also exhibit an endocrine response to an acute social challenge.     

Hematologic and serum biochemical values for zebrafish (Danio rerio)

The zebrafish (Danio rerio) has proven an excellent model for study of vertebrate development and genetics. Mutagenesis studies have produced many blood mutants with defects ranging from hematopoiesis to coagulation. The overwhelming majority of zebrafish studies have focused on development and mutational effects in embryos, whereas effects in mature zebrafish have gone largely unexplored. We believe that zebrafish will prove a valuable model for study of aging and age-related diseases, and we have sought to characterize some of the basic features of mature zebrafish. Accordingly, blood was collected from adult zebrafish and was analyzed to determine reference hematologic and biochemical parameters. White blood cell differential counts indicated predominantly lymphocytes, with mean proportion of 82.95%. Total red blood cell counts averaged 3.02 x 10(6) cells/microl. Except for increases in alanine transaminase (ALT), amylase, and phosphorus values, serum biochemical analytes were within the range of reported values for mammals and other species of fish. Accurate analysis of the many zebrafish mutants generated requires determination of normal characteristics of zebrafish. We believe results such as these will help define normal adult zebrafish, which have a tremendous potential for use in the study of human disease and aging.     

In vivo cell biology: following the zebrafish trend

A deeper understanding of the mechanisms of cell behavior is essential if we want to comprehend how an organism develops and functions. Changes in cellular processes, including the orientation of cell divisions, cell shape, polarity, differentiation and migration, account for tissue rearrangements during development and homeostasis. The in vivo relevance of in vitro findings is being constantly debated and the need for in vivo systems becoming more pressing. The zebrafish (Danio rerio) might become the vertebrate system of choice for a wide spectrum of biological questions that need to be investigated in vivo at cellular and subcellular resolutions. Here, we discuss some recent studies in which the zebrafish was used to gain insight into cell-biological mechanisms. Although this model system has been predominantly appreciated for its amenability to forward genetics, current advances in imaging technology and an increasing number of transgenic lines are bringing it closer to its full potential.     

Xenopus as a model organism in developmental chemical genetic screens

Chemical genetics is a potentially powerful tool for studying developmental processes in vertebrate systems. We present data showing Xenopus laevis as a model organism in which systematic chemical genetic screens can be carried out. Previous forward chemical genetic screens, including those with developing zebrafish embryos, have demonstrated the nature and value of biological information gained with this approach. We show how amenable Xenopus is to chemical genetics by investigating a series of compounds either with known biochemical effects, or previously identified to give developmental phenotypes, on a range of biological functions, including the development of pigmentation, the heart and the central nervous system in zebrafish. We have found that the compounds give comparable phenotypes when applied to developing Xenopus embryos. We have also studied the penetrance and expressivity of these chemical genetic phenotypes in relation to genetic variation and the developmental window during which the compound is present. Finally, we assess the feasibility and the potential throughput of a screen in this vertebrate species.     

nanos1 is required to maintain oocyte production in adult zebrafish

Development of the germline requires the specification and survival of primordial germ cells (PGCs) in the embryo as well as the maintenance of gamete production during the reproductive life of the adult. These processes appear to be fundamental to all Metazoans, and some components of the genetic pathway regulating germ cell development and function are evolutionarily conserved. In both vertebrates and invertebrates, nanos-related genes, which encode RNA-binding zinc finger proteins, have been shown to play essential and conserved roles during germ cell formation. In Drosophila, maternally supplied nanos is required for survival of PGCs in the embryo, while in adults, nanos is required for the continued production of oocytes by maintaining germline stem cells self-renewal. In mice and zebrafish, nanos orthologs are required for PGC survival during embryogenesis, but a role in adults has not been explored. We show here that nanos1 in zebrafish is expressed in early stage oocytes in the adult female germline. We have identified a mutation in nanos1 using a reverse genetics method and show that young female nanos mutants contain oocytes, but fail to maintain oocyte production. This progressive loss of fertility in homozygous females is not a phenotype that has been described previously in the zebrafish and underlines the value of a reverse genetics approach in this model system.     

虚拟视觉刺激引起幼年斑马鱼捕食行为放弃的研究

目的 当动物重复某种行为以逃避危险或获取奖励而无法成功时，会产生放弃。放弃是一种常见且基本的行为，在小鼠等模式动物中已经被广泛研究，但是其部分神经机制仍未被阐明。幼年斑马鱼适合进行全脑光学成像，是神经科学领域的重要模式生物。已经有研究者通过持续电击等消极刺激诱发斑马鱼放弃行为，然而奖励刺激能否引起斑马鱼放弃尚无报道。本文对奖励刺激引起的斑马鱼放弃行为进行了探究。方法 通过给予斑马鱼虚拟的食物视觉刺激，检验斑马鱼对虚拟食物的捕食情况，比较斑马鱼捕食频率和单次捕食时长随时间的变化。结果 虚拟的食物视觉刺激可以引起斑马鱼的捕食行为，接受25 min虚拟刺激后，8日龄以上斑马鱼的捕食频率和单次捕食时长均出现显著下降。结论 此研究丰富了斑马鱼放弃行为的研究范式，实验结果表明，缺失真实奖励的虚拟食物刺激可以诱导斑马鱼放弃捕食行为，这将进一步加深对动物放弃行为的理解，推动对其神经机制的研究。     

Functional characterization of visual opsin repertoire in Medaka (Oryzias latipes)

A variety of visual pigment repertoires present in fish species is believed due to the great variation under the water of light environment. A complete set of visual opsin genes has been isolated and characterized for absorption spectra and expression in the retina only in zebrafish. Medaka (Oryzias latipes) is a fish species phylogenetically distant from zebrafish and has served as an important vertebrate model system in molecular and developmental genetics. We previously isolated a medaka rod opsin gene (RH1). In the present study we isolated all the cone opsin genes of medaka by genome screening of a lambda-phage and bacterial artificial chromosome (BAC) libraries. The medaka genome contains two red, LWS-A and LWS-B, three green, RH2-A, RH2-B and RH2-C, and two blue, SWS2-A and SWS2-B, subtype opsin genes as well as a single-copy of the ultraviolet, SWS1, opsin gene. Previously only one gene was believed present for each opsin type as reported in a cDNA-based study. These subtype opsin genes are closely linked and must be the products of local gene duplications but not of a genome-wide duplication. Peak absorption spectra (lambda(max)) of the reconstituted photopigments with 11-cis retinal varied greatly among the three green opsins, 452 nm for RH2-A, 516 nm for RH2-B and 492 nm for RH2-C, and between the two blue opsins, 439 nm for SWS2-A and 405 nm for SWS2-B. Zebrafish also has multiple opsin subtypes, but phylogenetic analysis revealed that medaka and zebrafish gained the subtype opsins independently. The lambda and BAC DNA clones isolated in this study could be useful for investigating the regulatory mechanisms and evolutionary diversity of fish opsin genes.     

Cerebroventricular microinjection (CVMI) into adult zebrafish brain is an efficient misexpression method for forebrain ventricular cells

The teleost fish Danio rerio (zebrafish) has a remarkable ability to generate newborn neurons in its brain at adult stages of its lifespan-a process called adult neurogenesis. This ability relies on proliferating ventricular progenitors and is in striking contrast to mammalian brains that have rather restricted capacity for adult neurogenesis. Therefore, investigating the zebrafish brain can help not only to elucidate the molecular mechanisms of widespread adult neurogenesis in a vertebrate species, but also to design therapies in humans with what we learn from this teleost. Yet, understanding the cellular behavior and molecular programs underlying different biological processes in the adult zebrafish brain requires techniques that allow manipulation of gene function. As a complementary method to the currently used misexpression techniques in zebrafish, such as transgenic approaches or electroporation-based delivery of DNA, we devised a cerebroventricular microinjection (CVMI)-assisted knockdown protocol that relies on vivo morpholino oligonucleotides, which do not require electroporation for cellular uptake. This rapid method allows uniform and efficient knockdown of genes in the ventricular cells of the zebrafish brain, which contain the neurogenic progenitors. We also provide data on the use of CVMI for growth factor administration to the brain--in our case FGF8, which modulates the proliferation rate of the ventricular cells. In this paper, we describe the CVMI method and discuss its potential uses in zebrafish.