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PubMed2000

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.

Amelogenesis↗
PubMed2004

Transgenic zebrafish produced by retroviral infection of in vitro-cultured sperm.

Transgenic modification of sperm before fertilization has distinct advantages over conventional transgenic methods. The primary advantage is that the mosaicism inherent in those other techniques is avoided. A culture system using primary cultures of zebrafish male germ cells, in which the differentiation from spermatogonia to functional sperm can occur in vitro, provides the opportunity for genetic modification of sperm in vitro. Here, we report the production of transgenic zebrafish from cultured sperm. The sperm were differentiated from premeiotic germ cells infected with a pseudotyped retrovirus in vitro. The collected sperm were used to perform successful in vitro fertilizations, and transgenic embryos were identified. The transgenic fish transmitted the proviral integration to the next generation in a Mendelian fashion. We report the generation of a transgenic animal by cultured sperm and open the door to many exciting possibilities for the rapid generation of transgenic lines in model organisms such as zebrafish or other animal systems that are otherwise intractable to transgenesis.

Animals↗
PubMed2002

In vivo imaging of embryonic vascular development using transgenic zebrafish.

In this study we describe a model system that allows continuous in vivo observation of the vertebrate embryonic vasculature. We find that the zebrafish fli1 promoter is able to drive expression of enhanced green fluorescent protein (EGFP) in all blood vessels throughout embryogenesis. We demonstrate the utility of vascular-specific transgenic zebrafish in conjunction with time-lapse multiphoton laser scanning microscopy by directly observing angiogenesis within the brain of developing embryos. Our images reveal that blood vessels undergoing active angiogenic growth display extensive filopodial activity and pathfinding behavior similar to that of neuronal growth cones. We further show, using the zebrafish mindbomb mutant as an example, that the expression of EGFP within developing blood vessels permits detailed analysis of vascular defects associated with genetic mutations. Thus, these transgenic lines allow detailed analysis of both wild type and mutant embryonic vasculature and, together with the ability to perform large scale forward-genetic screens in zebrafish, will facilitate identification of new mutants affecting vascular development.

Animals↗
PubMed2006

Regulation of pigmentation in zebrafish melanophores.

In comparison with the molecular genetics of melanogenesis in mammals, the regulation of pigmentation in poikilothermic vertebrates is poorly understood. Mammals undergo morphological colour change under hormonal control, but strikingly, many lower vertebrates display a rapid physiological colour change in response to the same hormones. The recent provision of extensive genome sequencing data from teleost zebrafish, Danio rerio, provides the opportunity to define the genes and proteins mediating this physiological pigment response and characterise their function biologically. Here, we illustrate the background adaptation process in adults and larvae and describe a novel assay to visualize and directly quantify the rate of zebrafish melanophore pigment translocation in unprecedented detail. We demonstrate the resolution of this assay system; quantifying the zebrafish melanophore response to melanin-concentrating and melanocyte-stimulating hormones. Furthermore, we investigate the intracellular signalling downstream of hormone stimulation and the biomechanical processes involved in zebrafish pigment translocation, confirming the importance of cyclic adenosine monophosphate (cAMP) as a mediator of pigment translocation and finding intact microtubules are essential for both melanin dispersion and aggregation in zebrafish, but that microfilament disruption affects aggregation only. In conclusion, we propose these data establish the zebrafish as an experimental model for studying both physiological colour change and the molecular basis of pigment translocation.

Actin Cytoskeleton↗
PubMed2003

Fishing for novel angiogenic therapies.

The zebrafish has recently emerged as an important model for the study of vascular embryogenesis. Its genetic accessibility, external development, and optically clear embryo are just a few of the features that set the zebrafish apart as a particularly well-suited model for studying vascular development. However, there is little precedent for its use as a tool for the experimental study of therapeutic angiogenesis. Here, we review the use of the zebrafish for studying vascular development and patterning, and discuss how the zebrafish might be used more directly as a model for developing and testing effective therapeutic angiogenesis approaches.

Angiogenesis Inducing Agents↗
PubMed2003

Imaging brain development and organogenesis in zebrafish using immobilized embryonic explants.

Owing to its optical clarity and rapid rate of development, the zebrafish embryo is an ideal model system for studying the cellular mechanics of organogenesis. Unfortunately, extended time-lapse recordings of zebrafish embryos are often disrupted by the extension and straightening of the embryonic axis, as well as movement artifacts associated with developing musculature. In addition, the embryo's massive yolk cell often prevents optical access to tissues of interest. To circumvent these imaging problems, we have developed a procedure to deflate and mechanically remove the yolk cell. A "paralyzing" agent, AMP-PNP (a membrane-impermeant nonhydrolyzable analog of ATP), is first injected into the embryo's contractile yolk cell. The yolk cell is then removed using sharpened tungsten needles. Deyolked embryos, or organ rudiments explanted from them, are then immobilized on a microscope coverslip using a thin plasma clot. This plasma clot immobilization allows novel mountings of the explants so that ventral, lateral, and even cross-sectional fields of views are possible using high numerical aperture objectives. We show that isolated head rudiments undergo normal morphogenesis and gene expression for at least 1 day after being explanted into organotypic culture. These procedures can be used to study the cellular mechanics of organogenesis in "deyolked" embryos, as well as in tissues explanted from green fluorescent protein transgenic animals.

Animals↗
PubMed2002

Developmental expression of functional cyclooxygenases in zebrafish.

Study of the cyclooxygenases (COXs) has been limited by the role of COX-2 in murine reproduction and renal organogenesis. We sought to characterize COX expression and function in zebrafish (z). Full-length cDNAs of zCOX-1 and zCOX-2 were cloned and assigned to conserved regions of chromosomes 5 and 2, respectively. The deduced proteins are 67% homologous with their human orthologs. Prostaglandin (PG) E(2) is the predominant zCOX product detected by mass spectrometry. Pharmacological inhibitors demonstrate selectivity when directed against heterologously expressed zCOX isoforms. Zebrafish thrombocyte aggregation ex vivo and hemostasis in vivo are sensitive to inhibition of zCOX-1, but not zCOX-2. Both zCOXs were widely expressed during development, and knockdown of zCOX-1 causes growth arrest during early embryogenesis. zCOX-1 is widely evident in the embryonic vasculature, whereas zCOX-2 exhibits a more restricted pattern of expression. Both zCOX isoforms are genetically and functionally homologous to their mammalian orthologs. The zebrafish affords a tractable model system for the study of COX biology and development.

Amino Acid Sequence↗
PubMed2005

The genetics of hearing and balance in zebrafish.

The zebrafish is an excellent model system for studying the molecular basis of inner ear development and function. The eggs develop ex utero and the ear is transparent for the first few weeks of life. Forward genetic screens and antisense technology have helped to elucidate the signaling pathways and molecules required for inner ear development and function. This review addresses the most recent advances in our understanding of how the ear forms and discusses the molecules in hair cells that are essential for sensing sound and movement in the zebrafish.

Animals↗
PubMedDate not supplied

Genetic analysis of hemostasis and thrombosis using vascular occlusion.

The zebrafish is an excellent model for mammalian hemostasis and thrombosis since it possesses coagulation factors, thrombocyte receptors and responds to anti-coagulant and anti-platelet drugs commonly used in clinical treatment. In this study, exposure of larvae to FeCl(3) or laser irradiation produced a vessel injury that caused a visible vascular occlusion as a result of thrombus formation. Using the time to vascular occlusion as an assay, two screening strategies were tested for their utility in identifying novel genes involved in thrombosis. Morpholino knockdown studies of zebrafish factor VII showed a prolongation of the time to occlusion of the vessel whereas knockdown of the recently discovered factor VIIi resulted in a shortening of the time. Genetic screening of a population of zebrafish identified mutants that showed a prolongation of the time to occlusion. Bulk segregant analysis showed linkage of one mutant to a locus, victoria, on linkage group 7. Thus, the vascular occlusion assay developed in this report measures in vivo thrombus formation and is a powerful tool for identifying novel genes involved in thrombosis.

Animals↗
PubMed1998

Calcium transients accompany ooplasmic segregation in zebrafish embryos.

Through the injection of f-aequorin (a calcium-specific luminescent reporter), and the use of an imaging photon detector, transient localized elevations of free cytosolic calcium in the forming blastodisc (BD) and animal hemisphere cortex were visualized that correlated with ooplasmic segregation. The introduction of an appropriate concentration of the weak (KD = 1.5 micromol/L) calcium buffer 5,5'-dibromo-BAPTA results in the dissipation of these calcium domains, and inhibits cytoplasmic streaming and the subsequent formation of a BD at the animal pole. These inhibitory actions are dependent on the final cytosolic concentration of buffer within the egg: > or = 1.3 mmol/L blocks ooplasmic streaming; < 1.3 mmol/L eggs segregate normally. Injection of 5,5'-dimethyl-BAPTA (KD = 0.15 micromol/L) to a final concentration of 1.5 mmol/L as a control has no effect on ooplasmic streaming. These results suggest that localized domains of elevated free cytosolic calcium are essential for ooplasmic segregation in zebrafish. Furthermore, a hypothetical model is presented linking these calcium transients to the contraction of a cortically located actin microfilament network as a possible mechanism providing the driving force for segregation.

Aequorin↗
PubMed2005

Fictive swimming motor patterns in wild type and mutant larval zebrafish.

Larval zebrafish provide a unique model for investigating the mechanisms involved in generating rhythmic patterns of behavior, such as swimming, due to the array of techniques available including genetics, optical imaging, and conventional electrophysiology. Because electrophysiological and imaging studies of rhythmic motor behaviors in paralyzed preparations depend on the ability to monitor the central motor pattern, we developed a fictive preparation in which the activity of axial motor neurons was monitored using extracellular recordings from peripheral nerves. We examined spontaneous and light induced fictive motor patterns in wild type and mutant larval zebrafish (4-6 days post-fertilization) paralyzed with curare. All spontaneous and light-induced preparations produced alternation of motor activity from side-to-side (mean contralateral phase = 50.7 +/- 7.0%; mean burst frequency = 35.6 +/- 4.7 Hz) and a progression of activity from head-to-tail (mean ipsilateral rostrocaudal delay = 0.8 +/- 0.5 ms per segment), consistent with lateral undulation and forward propulsion during swimming, respectively. The basic properties of the motor pattern were similar in spontaneous and light-induced swimming. This fictive preparation can be used in combination with conventional electrophysiological and imaging methods to investigate normal circuit function as well as to elucidate functional deficits in mutant lines. Toward this end, we show that two accordion class mutants, accordion and bandoneon, have alternating activity on opposite sides of the body, contradicting the hypothesis that their deficit results from the absence of the reciprocal glycinergic inhibition that is typically found in the spinal cord of swimming vertebrates.

Action Potentials↗
PubMed2002

Nicotinic receptors mediate changes in spinal motoneuron development and axonal pathfinding in embryonic zebrafish exposed to nicotine.

We show that transient exposure of embryonic zebrafish to nicotine delays the development of secondary spinal motoneurons. Furthermore, there is a long-lasting alteration in axonal pathfinding in secondary motoneurons that is not ameliorated by drug withdrawal. These effects of nicotine were reversed by mammalian nicotinic receptor antagonists. Coupled with these changes is a long-term alteration in swimming behavior. Our results show that transient embryonic exposure to nicotine leads to long-lasting effects on the vertebrate nervous system. These results also demonstrate that the zebrafish is a useful model to examine the effects of nicotine specifically, and drugs of abuse in general, on the development of the CNS in vertebrates.

Animals↗
PubMed2004

[Imaging the development of the zebrafish line].

The lateral line system of the zebrafish offers the qualities of simplicity, accessibility and reproducibility which are most appropriate for the analysis of neural development and pattern formation in vertebrates. Furthermore, the transparency of the fish embryos allows the application of a variety of optical and molecular methods in the living animal, which is essential for a more complete analysis of the system.

Animals↗
PubMedDate not supplied

Neoplasia in zebrafish (Danio rerio) treated with 7,12-dimethylbenz[a]anthracene by two exposure routes at different developmental stages.

Using zebrafish, Danio rerio, initial pioneering work in the 1960s revealed carcinogen responsiveness of fish, yet very few subsequent tumorigenesis investigations have utilized this species. We exposed embryos (60 hours postfertilization) and fry (3 week posthatch) to 7,12-dimethylbenz[a]anthracene (DMBA) by immersion in aqueous solutions for 24 hours, at concentrations of 0-1 or 0-5 ppm (mg/L), respectively. Juvenile zebrafish 2 months posthatch were fed a diet containing 0-1,000 ppm DMBA for 4 months. Fish were sampled for histologic evaluation at 7-12 months after the onset of carcinogen treatment. Fry were most responsive to DMBA and showed the widest diversity of target tissues and histologic types of neoplasia, having several types of epithelial, mesenchymal, and neural neoplasia. The principal target tissues for carcinogenic response were liver following embryo or fry exposure, with gill and blood vessel the second and third most responsive tissues in fry. Intestine was the primary target and gill a secondary target in fish that received dietary DMBA as juveniles. These studies indicate that young zebrafish are most responsive to DMBA, showing a greater diversity of neoplasm types than rainbow trout. Thus, zebrafish are a valuable model system in which to study mechanistic aspects of the carcinogenesis process.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2006

Genetic analysis of dopaminergic system development in zebrafish.

Zebrafish have become an important model organism to study the genetic control of vertebrate nervous system development. Here, we present an overview on the formation of dopaminergic neuronal groups in zebrafish and compare the positions of DA neurons in fish and mammals using the neuromere model of the vertebrate brain. Based on mutant analysis, we evaluate the role of several signaling pathways in catecholaminergic neuron specification. We further discuss the prospect of identifying novel genes involved in dopaminergic development through forward genetics mutagenesis screens.

Animals↗
PubMed2005

Characterization of snakehead rhabdovirus infection in zebrafish (Danio rerio).

The zebrafish, Danio rerio, has become recognized as a valuable model for the study of development, genetics, and toxicology. Recently, the zebrafish has been recognized as a useful model for infectious disease and immunity. In this study, the pathogenesis and antiviral immune response of zebrafish to experimental snakehead rhabdovirus (SHRV) infection was characterized. Zebrafish 24 h postfertilization to 30 days postfertilization were susceptible to infection by immersion in 10(6) 50% tissue culture infective doses (TCID50) of SHRV/ml, and adult zebrafish were susceptible to infection by intraperitoneal (i.p.) injection of 10(5) TCID50 of SHRV/ml. Mortalities exceeded 40% in infected fish, and clinical presentation of infection included petechial hemorrhaging, redness of the abdomen, and erratic swim behavior. Virus reisolation and reverse transcription-PCR analysis of the viral nucleocapsid gene confirmed the presence of SHRV. Histological sections of moribund embryonic and juvenile fish revealed necrosis of the pharyngeal epithelium and liver, in addition to congestion of the swim bladder by cell debris. Histopathology in adult fish injected i.p. was confined to the site of injection. The antiviral response in zebrafish was monitored by quantitative real-time PCR analysis of zebrafish interferon (IFN) and Mx expression. IFN and Mx levels were elevated in zebrafish exposed to SHRV, although expression and intensity differed with age and route of infection. This study is the first to examine the pathogenesis of SHRV infection in zebrafish. Furthermore, this study is the first to describe experimental infection of zebrafish embryos with a viral pathogen, which will be important for future experiments involving targeted gene disruption and forward genetic screens.

Animals↗
PubMed2006

The homeodomain transcription factor drg11 is expressed in primary sensory neurons and their putative CNS targets during embryonic development of the zebrafish.

The drg11 gene is a member of the vertebrate aristaless-related gene family and encodes a paired homeodomain transcription factor. Its expression is largely restricted to PNS neurons subserving somatosensory functions and their CNS targets in rodents. The phenotype of drg11 null mice suggests that it is crucial for the proper development in the embryo of nociceptive circuits. To allow functional studies in the zebrafish, a simple vertebrate model organism, we have cloned the homologous gene and studied its expression throughout embryonic development. drg11 transcripts are first detected at neurula stage in the developing trigeminal ganglion, where it persists throughout development. This is followed by transient expression in spinal cord mechanosensory Rohon-Beard neurons shortly before axogenesis. Expression is later evident in neuronal populations of the dorsal spinal cord and in the dorsal root ganglia. In the developing brain, drg11 expression is mainly restricted to sensory neuron populations of the midbrain and hindbrain, in cranial sensory ganglia and in the habenula. Unlike rodents, however, trochlear motor neurons transiently express drg11. Our results suggest that drg11 expression in the developing zebrafish is, in common with its mammalian homologous gene, predominantly localised to neurons in sensory processing areas of the embryonic nervous system and is both spatially and temporally regulated.

Amino Acid Sequence↗
PubMed2005

Insulin-like growth factor signaling in fish.

The insulin-like growth factor (IGF) system plays a central role in the neuroendocrine regulation of growth in all vertebrates. Evidence from studies in a variety of vertebrate species suggest that this growth factor complex, composed of ligands, receptors, and high-affinity binding proteins, evolved early during vertebrate evolution. Among nonmammalian vertebrates, IGF signaling has been studied most extensively in fish, particularly teleosts of commercial importance. The unique life history characteristics associated with their primarily aquatic existence has fortuitously led to the identification of novel functions of the IGF system that are not evident from studies in mammals and other tetrapod vertebrates. Furthermore, the emergence of the zebrafish as a preferred model for development genetics has spawned progress in determining the requirements for IGF signaling during vertebrate embryonic development. This review is intended as a summary of our understanding of IGF signaling, as revealed through research into the expression, function, and evolution of IGF ligands, receptors, and binding proteins in fish.

Animals↗