Quantification of birefringence readily measures the level of muscle damage in zebrafish

Muscular dystrophies are a group of genetic disorders that progressively weaken and degenerate muscle. Many zebrafish models for human muscular dystrophies have been generated and analysed, including dystrophin-deficient zebrafish mutants dmd that model Duchenne Muscular Dystrophy. Under polarised light the zebrafish muscle can be detected as a bright area in an otherwise dark background. This light effect, called birefringence, results from the diffraction of polarised light through the pseudo-crystalline array of the muscle sarcomeres. Muscle damage, as seen in zebrafish models for muscular dystrophies, can readily be detected by a reduction in the birefringence. Therefore, birefringence is a very sensitive indicator of overall muscle integrity within larval zebrafish. Unbiased documentation of the birefringence followed by densitometric measurement enables the quantification of the birefringence of zebrafish larvae. Thereby, the overall level of muscle integrity can be detected, allowing the identification and categorisation of zebrafish muscle mutants. In addition, we propose that the establish protocol can be used to analyse treatments aimed at ameliorating dystrophic zebrafish models.     

The zebrafish as a model organism for the study of apoptosis

Apoptosis plays important roles in embryogenesis, tissue homeostasis, and immune system regulation. The zebrafish (Danio rerio) is a powerful vertebrate model organism that has been extensively used to study apoptotic cell death during normal development and under conditions of cellular stress. In the past 5 years, a detailed picture has begun to emerge of the molecular underpinnings of the cell-intrinsic and the cell-extrinsic apoptosis signaling pathways in zebrafish. We begin this review with an introduction to the techniques and experimental approaches that are used to study apoptosis in zebrafish. We follow with a general overview of developmental apoptosis during zebrafish embryogenesis. Finally, we present a comprehensive review of the intrinsic and extrinsic apoptosis pathways in zebrafish, focusing on the high degree of conservation with humans and other mammals. Recent publications that draw upon the unique advantages of the zebrafish system to study novel aspects of apoptosis regulation and function are highlighted throughout.     

The utility of zebrafish for studies of the comparative biology of motor systems

Although zebrafish are best known as a model for studies of development, there is now a growing role for the model in studies of the functional organization of the nervous system, including studies of a variety of sensory systems, central processing, and motor output. The zebrafish has much to offer for such work because of the unique combination of genetics, optical methods, and physiology it allows. Here I illustrate, using three examples, the broad range of avenues along which zebrafish can inform us about motor systems. The examples include efforts to understand the functional organization and evolution of spinal interneurons, the role of mutants in informing us about motor dysfunction and human disease, and the ability to use the special features of zebrafish to explore strategies to restore function after injury. The most important aspects of these studies are evident only when they are placed in a comparative context, so they serve to highlight the power of zebrafish in studies of the comparative biology of motor control.     

Building an asymmetric brain: Development of the zebrafish epithalamus

The human brain exhibits notable asymmetries. Little is known about these symmetry deviations; however scientists are beginning to understand them by employing the lateralized zebrafish epithalamus as a model. The zebrafish epithalamus consists of the pineal and parapineal organs and paired habenular nuclei located bilateral to the pineal complex. While zebrafish pineal and parapineal organs arise from a common population of cells, parapineal cells undergo a separate program that allows them to migrate left of the pineal anlage. Studying the processes that lead to brain laterality in zebrafish will allow a better understanding of how human brain laterality is established.     

Light reaches the very heart of the zebrafish clock

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early-stage zebrafish embryos contain clocks that are directly light-responsive. We describe recent experiments using single-cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light-responsiveness of early-stage zebrafish embryos.     

Expression of Dlx genes during the development of the zebrafish pharyngeal dentition: evolutionary implications

In order to investigate similarities and differences in genetic control of development among teeth within and between species, we determined the expression pattern of all eight Dlx genes of the zebrafish during development of the pharyngeal dentition and compared these data with that reported for mouse molar tooth development. We found that (i) dlx1a and dlx6a are not expressed in teeth, in contrast to their murine orthologs, Dlx1 and Dlx6; (ii) the expression of the six other zebrafish Dlx genes overlaps in time and space, particularly during early morphogenesis; (iii) teeth in different locations and generations within the zebrafish dentition differ in the number of genes expressed; (iv) expression similarities and differences between zebrafish Dlx genes do not clearly follow phylogenetic and linkage relationships; and (v) similarities and differences exist in the expression of zebrafish and mouse Dlx orthologs. Taken together, these results indicate that the Dlx gene family, despite having been involved in vertebrate tooth development for over 400 million years, has undergone extensive diversification of expression of individual genes both within and between dentitions. The latter type of difference may reflect the highly specialized dentition of the mouse relative to that of the zebrafish, and/or genome duplication in the zebrafish lineage facilitating a redistribution of Dlx gene function during odontogenesis.     

Circadian rhythms in zebrafish (Danio rerio) behaviour and the sources of their variability

Over recent decades, changes in zebrafish (Danio rerio) behaviour have become popular quantitative indicators in biomedical studies. The circadian rhythms of behavioural processes in zebrafish are known to enable effective utilization of energy and resources, therefore attracting interest in zebrafish as a research model. This review covers a variety of circadian behaviours in this species, including diurnal rhythms of spawning, feeding, locomotor activity, shoaling, light/dark preference, and vertical position preference. Changes in circadian activity during zebrafish ontogeny are reviewed, including ageing-related alterations and chemically induced variations in rhythmicity patterns. Both exogenous and endogenous sources of inter-individual variability in zebrafish circadian behaviour are detailed. Additionally, we focus on different environmental factors with the potential to entrain circadian processes in zebrafish. This review describes two principal ways whereby diurnal behavioural rhythms can be entrained: (i) modulation of organismal physiological state, which can have masking or enhancing effects on behavioural endpoints related to endogenous circadian rhythms, and (ii) modulation of period and amplitude of the endogenous circadian rhythm due to competitive relationships between the primary and secondary zeitgebers. In addition, different peripheral oscillators in zebrafish can be entrained by diverse zeitgebers. This complicated orchestra of divergent influences may cause variability in zebrafish circadian behaviours, which should be given attention when planning behavioural studies.     

Development of the Gill System in Early Ontogenesis of the Zebrafish and Ninespine Stickleback

We studied specific features of development of the gill system during ontogenesis of the zebrafish Danio rerio and ninespine stickleback Pungitius pungitius, which differ in the rates of gill system, development. Although the development of sticklebacks proceeds in nature at lower temperatures than for zebrafish, the rate of gill formation in the former is higher. These differences are related to the specific conditions in which these fish develop: embryonic and larval development of the stickleback proceeds in bodies of water with a lower oxygen content than for zebrafish, and this results in the adaptive alteration of the rate of gill system development. Differentiation of gills in the zebrafish is accompanied by a manifold increase in the oxygen consumption rate. At different developmental stages, the incremental rates of oxygen consumption and increase in the body mass of the zebrafish larvae and fry differed significantly.     

Identification and Expression of the Family of Classical Protein-Tyrosine Phosphatases in Zebrafish

Protein-tyrosine phosphatases (PTPs) have an important role in cell survival, differentiation, proliferation, migration and other cellular processes in conjunction with protein-tyrosine kinases. Still relatively little is known about the function of PTPs in vivo. We set out to systematically identify all classical PTPs in the zebrafish genome and characterize their expression patterns during zebrafish development. We identified 48 PTP genes in the zebrafish genome by BLASTing of human PTP sequences. We verified all in silico hits by sequencing and established the spatio-temporal expression patterns of all PTPs by in situ hybridization of zebrafish embryos at six distinct developmental stages. The zebrafish genome encodes 48 PTP genes. 14 human orthologs are duplicated in the zebrafish genome and 3 human orthologs were not identified. Based on sequence conservation, most zebrafish orthologues of human PTP genes were readily assigned. Interestingly, the duplicated form of ptpn23, a catalytically inactive PTP, has lost its PTP domain, indicating that PTP activity is not required for its function, or that ptpn23b has lost its PTP domain in the course of evolution. All 48 PTPs are expressed in zebrafish embryos. Most PTPs are maternally provided and are broadly expressed early on. PTP expression becomes progressively restricted during development. Interestingly, some duplicated genes retained their expression pattern, whereas expression of other duplicated genes was distinct or even mutually exclusive, suggesting that the function of the latter PTPs has diverged. In conclusion, we have identified all members of the family of classical PTPs in the zebrafish genome and established their expression patterns. This is the first time the expression patterns of all members of the large family of PTP genes have been established in a vertebrate. Our results provide the first step towards elucidation of the function of the family of classical PTPs.     

Light Reaches the Very Heart of the Zebrafish Clock

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early-stage zebrafish embryos contain clocks that are directly light-responsive. We describe recent experiments using single-cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light-responsiveness of early-stage zebrafish embryos.     

Micromechanical function of myofibrils isolated from skeletal and cardiac muscles of the zebrafish

The zebrafish is a potentially important and cost-effective model for studies of development, motility, regeneration, and inherited human diseases. The object of our work was to show whether myofibrils isolated from zebrafish striated muscle represent a valid subcellular contractile model. These organelles, which determine contractile function in muscle, were used in a fast kinetic mechanical technique based on an atomic force probe and video microscopy. Mechanical variables measured included rate constants of force development (k(ACT)) after Ca(2+) activation and of force decay (τ(REL)(-1)) during relaxation upon Ca(2+) removal, isometric force at maximal (F(max)) or partial Ca(2+) activations, and force response to an external stretch applied to the relaxed myofibril (F(pass)). Myotomal myofibrils from larvae developed greater active and passive forces, and contracted and relaxed faster than skeletal myofibrils from adult zebrafish, indicating developmental changes in the contractile organelles of the myotomal muscles. Compared with murine cardiac myofibrils, measurements of adult zebrafish ventricular myofibrils show that k(ACT), F(max), Ca(2+) sensitivity of the force, and F(pass) were comparable and τ(REL)(-1) was smaller. These results suggest that cardiac myofibrils from zebrafish, like those from mice, are suitable contractile models to study cardiac function at the sarcomeric level. The results prove the practicability and usefulness of mechanical and kinetic investigations on myofibrils isolated from larval and adult zebrafish muscles. This novel approach for investigating myotomal and myocardial function in zebrafish at the subcellular level, combined with the powerful genetic manipulations that are possible in the zebrafish, will allow the investigation of the functional primary consequences of human disease-related mutations in sarcomeric proteins in the zebrafish model.     

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.     

Flounder and fugu have a single lefty gene that covers the functions of lefty1 and lefty2 of zebrafish during L-R patterning

The lefty gene encodes a member of the TGF-beta superfamily that regulates L-R axis formation during embryogenesis via antagonistic activity against Nodal, another TGF-beta superfamily member. Both mouse and zebrafish have two lefty genes, lefty1 and lefty2. Interestingly, the expression domains of mouse and zebrafish lefty are different from one another. At present, the orthology and functional diversity of the mouse and zebrafish lefty genes are not clear. Here, we report that flounder and two fugu species, Takifugu and Tetraodon, have a single lefty gene in their genomes. In addition, we provide evidence that the mouse lefty genes were duplicated on a single chromosome but the zebrafish lefty genes arose from a whole-genome duplication that occurred early in the divergence of ray-finned fishes. These independent origins likely explain the difference in the expression domains of the mouse and zebrafish lefty gene pairs. Furthermore, we found that the duplication corresponding to the zebrafish lefty2 gene was lost from the fugu genome, suggesting that loss of lefty2 in the fugu/flounder lineage occurred after its divergence from the zebrafish lineage. During L-R patterning, the single lefty gene of flounder covers two expression domains, the left side of the dorsal diencephalon and the left LPM, which are regulated separately by lefty1 and lefty2 in zebrafish. We infer that the lefty genes of the ray-finned fishes and mammals underwent independent gene duplication events that resulted in independent regulation of lefty expression.     

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.     

Development of the gill system in early ontogenesis of Danio and nine spike stickleback

We studied specific features of development of the gill system during ontogenesis of the zebrafish Danio rerio and ninespine stickleback Pungitius pungitius, which differ in the rates of gill system, development. Although the development of sticklebacks proceeds in nature at lower temperatures than for zebrafish, the rate of gill formation in the former is higher. These differences are related to the specific conditions in which these fish develop: embryonic and larval development of the stickleback proceeds in bodies of water with a lower oxygen content than for zebrafish, and this results in the adaptive alteration of the rate of gill system development. Differentiation of gills in the zebrafish is accompanied by a manifold increase in the oxygen consumption rate. At different developmental stages, the incremental rates of oxygen consumption and increase in the body mass of the zebrafish larvae and fry differed significantly.     

Light Reaches the Very Heart of the Zebrafish Clock

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early‐stage zebrafish embryos contain clocks that are directly light‐responsive. We describe recent experiments using single‐cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light‐responsiveness of early‐stage zebrafish embryos.     

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.