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At least 829 records · Page 46Linked to original sources
PubMed2002

Unraveling the mechanisms of the vertebrate circadian clock: zebrafish may light the way.

Most organisms display oscillations of approximately 24 hours in their physiology. In higher organisms, these circadian oscillations in biochemical and physiological processes ultimately control complex behavioral rhythms that allow an organism to thrive in its natural habitat. Daily and seasonal light cycles are mainly responsible for keeping the circadian system properly aligned with the environment. The molecular mechanisms responsible for the control of the circadian clock have been explored in a number of systems. Interestingly, the circadian oscillations that are responsive to environmental stimuli are present very early during development. This review focuses on the advantages of using the zebrafish to study the development of the vertebrate circadian system and light-dependent signaling to the clock.

Animals↗
PubMed1999

Zebrafish leopard gene as a component of the putative reaction-diffusion system.

It has been suggested, on a theoretical basis, that a reaction-diffusion (RD) mechanism underlies pigment pattern formation in animals, but as yet, there is no molecular evidence for the putative mechanism. Mutations in the zebrafish gene, leopard, change the pattern from stripes to spots. Interestingly each allele gives a characteristic pattern, which varies in spot size, density and connectivity. That mutations in a single gene can generate such a variety of patterns can be understood using a RD model. All the pattern variations of leopard mutants can be generated in a simulation by changing a parameter value that corresponds to the reaction kinetics in a putative RD system. Substituting an intermediate value of the parameter makes the patterns similar to the heterozygous fish. These results suggest that the leopard gene product is a component of the putative RD mechanism.

Animals↗
PubMed2003

Windows into development: historic, current, and future perspectives on transgenic zebrafish.

The recent explosion of transgenic zebrafish lines in the literature demonstrates the value of this model system for detailed in vivo analysis of gene regulation and morphogenetic movements. The optical clarity and rapid early development of zebrafish provides the ability to follow these events as they occur in live, developing embryos. This article will review the development of transgenic technology in zebrafish as well as the current and future uses of transgenic zebrafish to explore the dynamic environment of the developing vertebrate embryo.

Animals↗
PubMed2003

[Vaccine, methadone and zebrafish].

Substances of abuse have been known since antiquity till now. Current trends in neurobiological and genetical study related to drug dependency were presented in the study. The trial of preparation a cocaine vaccine and the new hope for dependent people coming from investigation of Zebrafish were also described.

Animals↗
PubMed2004

On the relevance of genotoxicity for fish populations II: genotoxic effects in zebrafish (Danio rerio) exposed to 4-nitroquinoline-1-oxide in a complete life-cycle test.

In order to characterize the impact of genotoxic potentials on populations of aquatic organisms in surface waters, zebrafish (Danio rerio) were exposed to the model genotoxicant 4-nitroquinoline-1-oxide (NQO) in a complete life-cycle test. Fish exposed to mean NQO concentrations of 0, 0.1, 0.3, 1.1, and 2.9 microg/l were examined by several genotoxicity assays with different endpoints. Assays included the unscheduled DNA synthesis (UDS) test, the comet assay, the alkaline filter elution, and the micronucleus test. The genotoxicity assays revealed an increasing genotoxicity, ranging from induction of DNA repair (even at the lowest concentration tested) to primary and secondary DNA alterations at higher concentrations of 1.1 and 2.9 microg/l NQO. Whether the lowered reproductivity observed in the life-cycle test is caused by genotoxic pathways of NQO, remains unclear. However, the results indicate a contradiction to an earlier assumption that genotoxicants as found in the environment are likely to not impact natural populations.

4-Nitroquinoline-1-oxide↗
PubMed2004

Molecular cloning of zebrafish and medaka vitellogenin genes and comparison of their expression in response to 17beta-estradiol.

In the present study, both zebrafish and medaka vitellogenin genes have been isolated and used as a biomarker to compare the two small aquarium fish in response to estrogen treatment and thus to evaluate the two fish models in development of a biomonitoring system for environmental estrogens. The isolated zebrafish vitellogenin gene, zvtg1, is the most abundantly expressed vitellogenin gene in zebrafish and its complete protein sequence of 1360 amino acids was deduced from a genomic and a cDNA clone. The isolated medaka vitellogenin (mvtg1) genomic clone covers 1053 amino acids in the N-terminal. Both zebrafish zvtg1 and medaka mvtg1 are specifically expressed in female liver and their expression can be induced by 17beta-estradiol (E2) in male fish both by intramuscular injection and immersion treatment. A real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for quantification of vitellogenin mRNA level in both control fish and E2-treated fish. The lowest-observed-effect concentrations of E2 for the induction of vitellogenin mRNAs were observed at 1 microg/l for zebrafish and 0.1 microg/l for medaka in a 2-day exposure experiment. Further kinetics studies of the two fish models indicated that medaka was able to respond much faster to E2 treatment than zebrafish, while the zebrafish can attain a much higher level of vitellogenin mRNAs than medaka after a long-term E2 treatment. The implication of these observations may be that the medaka system is better in monitoring acute treatment while the zebrafish system is better in monitoring chronic exposure.

Amino Acid Sequence↗
PubMed2002

Progress towards cell-mediated gene transfer in zebrafish.

Although the zebrafish possesses several favourable characteristics that make it an ideal model for genetic studies of vertebrate development, one disadvantage of this model system is the absence of methods for the production of gene knockouts. The authors' laboratory, and others, are working to develop zebrafish pluripotent embryonic stem (ES) and primordial germ cell (PGC) cultures that can be used for cell-mediated gene transfer and the production of knockout mutant lines of fish. Progress has been made in developing short-term cell cultures that possess the ability to contribute to multiple tissues, including the germ line of a host embryo, and transgenic lines of zebrafish have been established using the embryo cell cultures. Work is in progress to extend the length of time that the embryo cells can be maintained in culture without losing their ability to generate germ-line chimeras.

Animals↗
PubMed2005

Zebrafish bandoneon mutants display behavioral defects due to a mutation in the glycine receptor beta-subunit.

Bilateral alternation of muscle contractions requires reciprocal inhibition between the two sides of the hindbrain and spinal cord, and disruption of this inhibition should lead to simultaneous activation of bilateral muscles. At 1 day after fertilization, wild-type zebrafish respond to mechanosensory stimulation with multiple fast alternating trunk contractions, whereas bandoneon (beo) mutants contract trunk muscles on both sides simultaneously. Similar simultaneous contractions are observed in wild-type embryos treated with strychnine, a blocker of the inhibitory glycine receptor (GlyR). This result suggests that glycinergic synaptic transmission is defective in beo mutants. Muscle voltage recordings confirmed that muscles on both sides of the trunk in beo are likely to receive simultaneous synaptic input from the CNS. Recordings from motor neurons revealed that glycinergic synaptic transmission was missing in beo mutants. Furthermore, immunostaining with an antibody against GlyR showed clusters in wild-type neurons but not in beo neurons. These data suggest that the failure of GlyRs to aggregate at synaptic sites causes impairment of glycinergic transmission and abnormal behavior in beo mutants. Indeed, mutations in the GlyR beta-subunit, which are thought to be required for proper localization of GlyRs, were identified as the basis for the beo mutation. These data demonstrate that GlyRbeta is essential for physiologically relevant clustering of GlyRs in vivo. Because GlyR mutations in humans lead to hyperekplexia, a motor disorder characterized by startle responses, the zebrafish beo mutant should be a useful animal model for this condition.

Animals↗
PubMed2026

A User-Friendly Protocol for Microinjection into Teleost Embryos to Study Gene Function.

Zebrafish (Danio rerio) and medaka (Oryzias latipes) are popular teleost models used in developmental biology and functional genomics. To achieve high-quality and reproducible microinjections, it is essential to have robust protocols for breeding, egg collection, and the precise delivery of genetic material. In this protocol, we present a comprehensive and optimized methodology for setting up breeding tanks under controlled photoperiod conditions to maximize egg yield while minimizing contamination. We provide detailed procedures for sex identification, pair selection, the use of grated breeding inserts, and methods to increase egg collection efficiency. We outline procedures for making injection gel beds, pulling needles, and calibration using one-microliter microcapillaries to achieve consistent nanoliter-scale injections. Our protocol outlines settings for the pico-liter injector that are optimized to deliver a precise amount per pulse with minimal variability. Finally, we demonstrate the application of these methods for gene knockdown using morpholino antisense oligonucleotides, gene knockout using CRISPR-Cas9, and gain-of-function mRNA overexpression experiments. Phenotypic assessments conducted at various developmental stages to evaluate gene-specific effects reveal consistent phenotypic outcomes between the morpholino and CRISPR-Cas9 approaches. This easy and comprehensive protocol enables efficient, precise, and scalable genetic manipulation of zebrafish and medaka embryos, thereby supporting advanced functional studies in developmental biology and disease modeling. To our knowledge, this is the first unified protocol for both zebrafish and medaka microinjection systems achieving 97.7% phenotype penetrance in CRISPR-Cas9 knockouts with precision together with a triple validation approach that confirms gene function across multiple techniques.

Animals↗
PubMed2000

On the mechanism of ooplasmic segregation in single-cell zebrafish embryos.

It has been previously shown that localized elevations of free cytosolic calcium are associated with a morphological contraction in the forming blastodisc and animal hemisphere cortex during ooplasmic segregation in zebrafish zygotes. It was subsequently proposed, in a hypothetical model, that these calcium transients might be linked to the contraction of a cortically located actin microfilament network as a potential driving force for segregation. Here, by labeling single-cell embryos during the major phase of segregation with rhodamine-phalloidin, direct evidence is presented to indicate that the surface contraction was generated by an actin-based cortical network. Furthermore, while zygotes incubated with colchicine underwent normal ooplasmic segregation, those incubated with cytochalasin B did not generate a constriction band or segregate to form a blastodisc. During segregation at the single-cell stage, ooplasm simultaneously moved in two directions: toward the blastodisc within the so-called axial streamers, and toward the vegetal pole in the peripheral ooplasm. The velocities of both axial and peripheral streaming movements are reported. By injection of a fluorescein isothiocyanate (FITC)-labeled 2000 kDa dextran into the peripheral ooplasm it was demonstrated that a portion of it feeds into the bases of the extending streamers, which helps to explain the lack of accumulation of ooplasm at the vegetal pole. These new data were incorporated into the original model to link the bipolar ooplasmic movements with the calcium-modulated, actin-mediated contraction of the animal hemisphere cortex as a means of establishing and driving ooplasmic segregation in zebrafish.

Actins↗
PubMed2004

[Mechanotransduction of hemodynamic forces regulates organogenesis].

Endothelial cells (EC) of the vertebrate cardiovascular system (CVS) are bona fide, yet enigmatic mechanoreceptors. When cultured in vitro and exposed to fluid forces, EC modify their physiological behaviour at the structural, metabolical and gene expression levels in response to the mechanical stimulus. However, as a direct consequence of the hypoxic bias (and often the lethality) that results from blocking blood flow in most animal systems, the in vivo role of EC mechanosensation (ECMS) remains poorly understood. The zebrafish has recently emerged as an alternative genetic model for the study of vertebrate development. Its striking ability to survive until larval stages in the absence of blood circulation circumveys the usual caveats that are inherent to CVS research, and offers the exciting opportunity to dissect the function of ECMS in vivo. Two groups have already uncovered an essential role for ECMS in zebrafish organogenesis, particularly in heart morphogenesis. In embryos in which intracardiac blood flow is genetically or physically compromised, several features of the normally developing heart, including valve formation, are specifically disrupted. In addition, impressive imaging studies of zebrafish hemodynamics demonstrate that the shear stress exerted upon the cardiac endothelium is largely in the range of the stimulus that in vitro activates cytoskelettal remodeling and gene expression changes in EC. Hence the cardiac phenotypes observed in vivo may indeed directly result from a lack of ECMS-dependent EC activity. These data shed first light on the role of ECMS in vivo. Notably, they also suggest that a number of human congenital cardiomyopathies may arise through abnormal fetal hemodynamics and/or EC sensory activity. Finally, these discoveries reinforce the too often neglected role of epigenetic factors (in this case, fluid forces) in the regulation of animal development.

Animals↗
PubMed2002

In vivo electroporation: a new frontier for gene delivery and embryology.

One of the key techniques in developmental biology is introducing transgenes into tissues and analyzing their subsequent effects on morphogenesis and organogenesis. In mammals, the transgenic approach is a way to misexpress foreign genes in various tissues and organs. However, targeting expression to certain tissues is totally dependent on the availability of specific promoters. Hence, it is not an easy task to control transgene expression temporally and spatially during embryogenesis. Further, if the transgene is toxic, embryonic development can be disrupted, resulting in premature death before the desired stages of development. As alternative systems, Xenopus and zebrafish are used frequently. In these vertebrate models, overexpression of genes can be carried out by injecting synthetic RNAs into eggs. However, genetic techniques in these systems are limited only to early development, prohibiting the precise analysis of gene effects on organogenesis in later stages. In contrast, the chick embryo has long served as a powerful and useful model system, holding a unique position in the field of developmental biology. Although trials of transgenic chicks have never been successful, easy accessibility to the developing embryo through a window opened in an eggshell enables performance of a variety of techniques, such as time-lapse cinephotomatography, microsurgical manipulations (including chick/quail chimeras), transplantation of cells and tissues, New's in vitro culture, etc. (Bortier et al., 1996; Douarin et al., 1996; Selleck, 1996). In addition to these experimental advantages, retrovirus-mediated gene delivery, and recently, adenovirus-mediated misexpression have been employed routinely in chick embryos (Leber et al., 1996; Morgan and Fekete, 1996).

Animals↗
PubMed2005

Central connection of the optic, oculomotor, trochlear and abducens nerves in medaka, Oryzias latipes.

Medaka (Oryzias latipes) is one of the few vertebrate experimental animals in which inbred lines have been established. It is also a species that has advanced in genetic studies in a manner comparable to zebrafish. This fish is therefore a good model for studying functional organization of the nervous system, but anatomical analysis of its nervous system has been limited to embryonic stages. In the present study, we investigated anatomy of cranial nerves in adult fish focusing on the visual function, using an inbred strain of medaka. Cranial nerves of medaka were labeled using biocytin, revealing a central distribution of retinofugal terminals, retinopetal neurons, and oculomotor, trochlear and abducens motor neurons. The optic nerve of the adult medaka was of a complete decussation type. Retinofugal terminals were located in 8 brain nuclei, the suprachiasmatic nucleus, nucleus pretectalis superficialis, nucleus dorsolateralis thalami, area pretectalis pars dorsalis (APd), area pretectalis pars ventralis (APv), nucleus of the posterior commissure (NPC), accessory optic nucleus, and the tectum opticum. Retinopetal neurons were identified in 6 brain nuclei, the ganglion of the terminal nerve, preoptic retinopetal nucleus, nucleus dorsolateralis thalami, APd, APv, and NPC. The oculomotor neurons were mostly labeled ipsilaterally and were located dorsomedially, abutting the fasciculus longitudinalis medialis in the mesencephalon. The trochlear nucleus was located contralaterally and dorsolaterally adjacent to the fasciculus longitudinalis medialis in the mesencephalon. The abducens nucleus was located ipsilaterally in a ventrolateral part of the rhombencephalic reticular formation. These results, generally similar to those in other teleosts, provide the basis for future behavioral and genetic studies in medaka.

Abducens Nerve↗
PubMed2002

Removal of dystroglycan causes severe muscular dystrophy in zebrafish embryos.

Muscular dystrophy is frequently caused by disruption of the dystrophin-glycoprotein complex (DGC), which links muscle cells to the extracellular matrix. Dystroglycan, a central component of the DGC, serves as a laminin receptor via its extracellular alpha subunit, and interacts with dystrophin (and thus the actin cytoskeleton) through its integral membrane beta subunit. We have removed the function of dystroglycan in zebrafish embryos. In contrast to mouse, where dystroglycan mutations lead to peri-implantation lethality, dystroglycan is dispensable for basement membrane formation during early zebrafish development. At later stages, however, loss of dystroglycan leads to a disruption of the DGC, concurrent with loss of muscle integrity and necrosis. In addition, we find that loss of the DGC leads to loss of sarcomere and sarcoplasmic reticulum organisation. The DGC is required for long-term survival of muscle cells in zebrafish, but is dispensable for muscle formation. Dystroglycan or the DGC is also required for normal sarcomere and sarcoplasmic reticulum organisation. Because zebrafish embryos lacking dystroglycan share several characteristics with human muscular dystrophy, they should serve as a useful model for the disease. In addition, knowing the dystroglycan null phenotype in zebrafish will facilitate the isolation of other molecules involved in muscular dystrophy pathogenesis.

Animals↗
PubMed2002

HrT is required for cardiovascular development in zebrafish.

The recently identified zebrafish T-box gene hrT is expressed in the developing heart and in the endothelial cells forming the dorsal aorta. Orthologs of hrT are expressed in cardiovascular cells from Drosophila to mouse, suggesting that the function of hrT is evolutionarily conserved. The role of hrT in cardiovascular development, however, has not thus far been determined in any animal model. Using morpholino antisense oligonucleotides, we show that zebrafish embryos lacking hrT function have dysmorphic hearts and an absence of blood circulation. Although the early events in heart formation were normal in hrT morphant embryos, subsequently the hearts failed to undergo looping, and late onset defects in chamber morphology and gene expression were observed. In particular, we found that the loss of hrT function led to a dramatic upregulation of tbx5, a gene required for normal heart morphogenesis. Conversely, we show that overexpression of hrT causes a significant downregulation of tbx5, indicating that one key role of hrT is to regulate the levels of tbx5. Secondly, we found that HrT is required to inhibit the expression of the blood lineage markers gata1 and gata2 in the most posterior lateral plate mesoderm. Finally, we show that HrT is required for vasculogenesis in the trunk, leading to similar vascular defects to those observed in midline mutants such as floating head. hrT expression in the vascular progenitors depends upon midline mesoderm, indicating that this expression is one important component of the response to a midline-derived signal during vascular morphogenesis.

Animals↗
PubMed2006

Discovery of multiple beta-defensin like homologues in teleost fish.

Using a database mining approach, multiple defensin like genes have been discovered for the first time in fish, in species including zebrafish Danio rerio and the pufferfish, Takifugu rubripes and Tetraodon nigroviridis. They share the common features of vertebrate defensins, including small size, net cationic charge, and six conserved cysteines in the mature region. Based on their cysteine arrangement, the identified fish defensin like peptides resemble beta-defensin family members in birds and mammals. Computing modelling detected three beta-strands in all three zebrafish defensins and an extra N-terminal alpha-helix in one of the peptides. The coding regions of the fish genes contain three exons and two introns, the same as avian defensin genes. In zebrafish and tetraodon, two defensin genes identified are located in the same chromosome. An additional locus containing a third defensin gene has also been found in a different chromosome in zebrafish, demonstrating that multiple defensin loci may be present in fish. Comparative studies suggest that beta-defensins may represent the primitive form of the defensin family, which expanded during evolution by gene or genome duplication. In healthy zebrafish, constitutive expression of defensins was detected by RT-PCR in gill, gonad, gut, kidney, muscle, skin and spleen but the levels and patterns varied for individual defensin genes.

Amino Acid Sequence↗
PubMed1997

Mutations affecting eye morphology in the developing zebrafish (Danio rerio).

The zebrafish (Danio rerio) has received considerable attention as a mainstream model for the molecular and genetic study of vertebrate development. In our laboratory, we have conducted a third-generation screen of chemically mutagenized zebrafish for recessive mutations affecting the visual system. This report describes the visible phenotypes and number of morphological mutants so far observed and presents a more detailed histological analysis of six of these mutations. Through analysis of mutant larvae, it was determined that several of the subtle morphological mutations resulted in degeneration of specific cellular layers of the retina. Other mutations resulted in some degeneration distributed diffusely across the entire retina or concentrated at the retinal margin. A single mutation affecting invagination of the optic cup and lens vesicle formation resulted in a failure to develop an anterior chamber. These results demonstrate the utility of a small-scale, highly focused screen for uncovering novel loci involved in retinal and eye development.

Animals↗
PubMed2002

Primordial germ cell development in zebrafish.

In sexually reproducing organisms, primordial germ cells (PGCs) give rise to gametes that are responsible for the development of a new organism in the next generation. These cells follow a characteristic developmental path that is manifested in specialized regulation of basic cell functions and behavior making them an attractive system for studying cell fate specification, differentiation and migration. This review summarizes studies aimed at understanding the development of this cell population in zebrafish and compares these results with those obtained in other model organisms.

Animals↗