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PubMed1997

The aquatic vertebrate embryo as a sentinel for toxins: zebrafish embryo dechorionation and perivitelline space microinjection.

Pollution of aquatic ecosystems poses a serious threat to aquatic organisms and ultimately the entire ecosystem. Understanding how a toxin affects embryonic development is key to determining the risk a pollutant represents to the environment. Extraembryonic membranes, such as the chorion of fish eggs, provide a protective barrier between the embryo and the environment. Although the fish chorion excludes many chemical pollutants, some noxious agents can still gain access to the aquatic embryo. Therefore a monitoring system that tests the effects directly upon the embryo must be established. Although exposure to a single toxin in the laboratory can determine the concentration at which a pollutant becomes a health or environmental hazard, embryos and adults in nature are not merely affected by a single chemical, but are exposed to mixtures of different pollutants. Zebrafish (Danio rerio) and medaka (Oryzias latipes) embryos were employed for the rapid observation of the effects of single chemicals and chemical mixtures on development. Using dechorionation and a perivitelline space microinjection system, the embryos were effective sentinels for low concentrations of aquatic pollutants. The developmental effects of small quantities of toxins were observed. Embryos treated during the late gastrula stage of development with hexachlorobenzene (HCB); 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); toluene; benzene; or mixtures of these chemicals developed cardiovascular abnormalities. The zebrafish dechorionation exposure technique, Micro Intrachorionic Zebrafish Embryo Live Laboratory test, was especially effective in testing the pollutant mixtures. Combinations of both TCDD and benzene (as well as the toluene and benzene combinations) were tested and the mixtures acted synergistically; the combinations were more toxic than either chemical by itself. Hexachlorobenzene- and TCDD-treated embryos tested positively for expression of cytochrome P450 1A indicating that the cytochrome metabolic pathways were already functional in these early embryos, and suggested that a product of the cytochrome system may be involved in HCB and TCDD pollution associated cardiovascular defects.

Abnormalities, Drug-Induced↗

PubMed1997

Heat shock genes and the heat shock response in zebrafish embryos.

Heat shock genes exhibit complex patterns of spatial and temporal regulation during embryonic development in a wide range of organisms. Our laboratory has initiated an analysis of heat shock protein gene expression in the zebrafish, a model system that is now utilized extensively for the examination of early embryonic development of vertebrates. We have cloned members of the zebrafish hsp47, hsp70, and hsp90 gene families and shown them to be closely related to their counterparts in higher vertebrates. Whole mount in situ hybridization and Northern blot analyses have revealed that these genes are regulated in distinct spatial, temporal, and stress-specific manners. Furthermore, the tissue-specific expression patterns of the hsp47 and hsp90 alpha genes correlate closely with the expression of genes encoding known chaperone targets of Hsp47 and Hsp90 in other systems. The data raise a number of interesting questions regarding the function and regulation of these heat shock genes in zebrafish embryos during normal development and following exposure to environmental stress.

Animals↗

PubMed1997

DNA methylation and genome imprinting in the zebrafish, Danio rerio: some evolutionary ramifications.

Although methylation has been recognized as an important component in a number of developmental processes in mammals, in zebrafish almost nothing is known about this epigenetic modification. This is despite the fact that the zebrafish is becoming increasingly popular as a developmental model system. The little work that has been done on methylation and development in fish concerns genomic imprinting. In mammals, imprinting results in an inability to reproduce parthenogenetically because a genetic contribution from both parents is necessary to successfully complete development. However, this is not true of zebrafish, and a number of the theories that have been presented to explain the evolution of imprinting are not consistent with imprinting in these fish. A new model is presented that discusses some of the potential evolutionary ramifications of methylation and imprinting and that leads to the suggestion that imprinting may actually be a simple genetic mechanism to enhance the efficient evolution of both individual genetic loci and combinations of loci with related functions, without risking the population as a whole. This model can accommodate all of the information known about imprinting, including its broad phylogenetic range, imprinting by both males and females, and the diverse nature of the genes that are known to be imprinted.

Animals↗

PubMed1997

High-resolution imaging at the cellular and subcellular levels in flattened whole mounts of early zebrafish embryos.

We describe a rapid and sensitive method for high-resolution imaging at the cellular and subcellular levels in the whole-mount zebrafish embryo. The procedure involves fixing and staining the embryo, followed by deyolking and flattening it under a cover slip, to produce a planar mount that is 20 to 100 microns thick. Such a flattened whole mount allows imaging with a spatial resolution of approximately 500 nm in the x-y plane and does not require the use of embedding, sectioning, confocal microscopy, or computational deblurring procedures. We can resolve all individual nuclei and chromosome sets in the embryo, up to the late gastrula stage (10,000 cell stage). In addition, older embryos (through the segmentation stage) can also be examined, with the preservation of significant morphological detail. Because of its ability to resolve subcellular detail, the flattened whole-mount method can provide significant biological information beyond what can be obtained from conventional (three-dimensional) whole mounts. We have used the flattened whole-mount method to study subcellular events related to progression through the cell cycle or to apoptosis, in cells of the early zebrafish embryo. A specific DNA-binding dye (Hoechst 33258) or an antibody against a chromosomal protein (histone H1) was used to stain the nuclei of individual cells in the embryo. This allowed us to determine the spatial positions of all the individual cells, and also their stages in the cell cycle. A terminal transferase (TUNEL) assay was used to detect apoptotic cells. This combination of specific stains allowed us to study the behaviors of groups of cells in situ, within the developing zebrafish embryo.

Animals↗

PubMed1997

Nuclear localization signals: a driving force for nuclear transport of plasmid DNA in zebrafish.

Nuclear localization signals (NLSs) are short peptides required for nuclear transport of karyophilic proteins. We review in this paper how the nuclear targeting property of NLS peptides has been taken advantage of to enhance the efficiency of nuclear uptake of transgene DNA in zebrafish and how it may improve the efficiency of transgenesis in this species. Synthetic NLS peptides can bind to plasmid DNA by ionic interactions. Cytoplasmic injection of DNA-NLS complexes in zebrafish eggs enhances the rate and the amount of plasmid DNA taken up by embryonic nuclei. Nuclear import of DNA-NLS complexes has been duplicated in vitro and exhibits energetic and cytosolic requirements similar to those for nuclear protein import. Furthermore, binding NLSs to DNA increases expression frequency of the transgene. We suggest that NLS peptides may constitute a valuable tool to improve the efficiency of transgenesis in zebrafish and other species.

Animals↗

PubMed1998

Physiological properties of the Mauthner system in the adult zebrafish.

We investigated the morphological and electrophysiological properties of the Mauthner (M-) cell and its networks in the adult zebrafish (Danio rerio) in comparison with those in the goldfish (Carassius auratus). The zebrafish M-cell has an axon cap, a high resistivity structure which surrounds the initial segment of the M-axon, and accounts for an unusual amplification of the fields generated within and around it. Second, extra- and intracellular recordings were performed with microelectrodes. The resting potential was approximately -80 mV with an input resistance of approximately 0.42 M omega. The M-cell extracellular field was large (10-20 mV), close to the axon hillock, and the latency of antidromic spikes short (approximately 0.4 milliseconds), confirming a high conduction velocity in the M-axon. The extrinsic hyperpolarizing potential (EHP), which signals firing of presynaptic cells and collateral inhibition, was markedly lower at frequencies of spinal stimulation > approximately 5/second, suggesting an organization of the recurrent collateral network similar to that in the goldfish. Inhibitory postsynaptic potentials (IPSPs) were highly voltage-dependent; their decay time constant was increased by depolarizations. The presynaptic neurons which are numerous could be identified by their passive hyperpolarizing potential (PHP) produced by the M-spike current. Auditory responses, mediated via mixed synapses (electrical and chemical), had short delays and hence are well suited to trigger the escape reaction. The similarities of their properties indicate that the wealth of information generated over decades in the goldfish can be extrapolated to the zebrafish.

Animals↗

PubMed1998

Sequence and expression of glutamic acid decarboxylase isoforms in the developing zebrafish.

We describe the isolation two glutamic acid decarboxylase (GAD) cDNAs from zebrafish with over 84% identity to human GAD65 and GAD67. In situ hybridization studies revealed that both GAD65 and GAD67 were expressed in the early zebrafish embryo during the period of axonogenesis, suggesting a role for GABA prior to synapse formation. Both GAD genes were detected in the telencephalon, in the nucleus of the medial longitudinal fasciculus in the midbrain, and at the border regions of the rhombomeres in the rostral hindbrain. In the caudal hindbrain, only GAD67 was detected (in neurons with large-caliber axons). In the spinal cord, both GAD genes were detected in dorsal longitudinal neurons, commissural secondary ascending neurons, ventral longitudinal neurons, and Kolmer-Agduhr neurons. Immunohistochemistry for gamma-aminobutyric acid (GABA) revealed that GABA is produced at all sites of GAD expression, including the novel cells in the caudal hindbrain. These results are discussed in the context of the hindbrain circuitry that supports the escape response. We conclude that fish, like mammals, have two GAD genes. The zebrafish GAD65 and GAD67 are present in identified neurons in the forebrain, midbrain, hindbrain, and spinal cord, and they catalyze the production of GABA in the developing embryo.

Amino Acid Sequence↗

PubMed2026

Maternal redd1 mRNA decline triggers mTORC1 activation during the blastula-gastrula transition in zebrafish embryos.

During early metazoan development, maternal mRNAs and proteins stored in the egg sustain initial cellular functions. After the blastula stage, developmental control shifts to zygotic gene expression, and maternal transcripts are progressively degraded. Although mTORC1 is a central regulator of global mRNA translation and cell growth, its role in controlling maternal mRNA translation prior to gastrulation remains poorly understood. In zebrafish embryos, the mTORC1 inhibitor redd1 is abundantly expressed after fertilization but decreases following the maternal-to-zygotic transition (MZT), inversely correlating with mTORC1 activity. Overexpression of redd1 suppresses mTORC1, impairs gastrulation, and reduces translation of 5'TOP mRNAs and key regulatory genes, underscoring the necessity of relieving mTORC1 inhibition after the blastula stage. To investigate redd1 translation under conditions of low mTORC1 activity, we injected reporter mRNAs containing its 5' and 3' UTRs. The 3'UTR promoted polyadenylation and enhanced translation, while both UTRs enabled efficient reporter expression despite mTORC1 suppression, indicating that redd1 mRNA is translated independently of canonical mTORC1 pathways. Similarly, maternal mRNAs such as nanog, myca, pou5f3, and ccnb1, as well as the early zygotic transcript dharma, are translated through mTORC1-independent mechanisms. Together, these findings reveal a transient phase of mTORC1 suppression in early zebrafish embryos and demonstrate that select maternal and zygotic mRNAs bypass this regulation to ensure proper developmental progression.

Animals↗

PubMed1985

Filipin-sterol complexes in the plasma membrane of zebrafish spermatozoa.

The presence and distribution of filipin-sterol complexes in the plasma membrane of zebrafish (Brachydanio rerio) sperm was investigated. The zebrafish sperm plasma membrane, treated with freeze-fracture techniques, is seen to contain a multitude of intramembranous particles that, in a specific region of the posterior part of the sperm head, are organized into unusual particle arrays that appear as simple hexagons or parallelograms. The polyene antibiotic filipin forms complexes with 3-beta-OH sterols to produce characteristic protrusions and pits in membranes that are readily observable in freeze-fracture replicas. Numerous filipin-sterol complexes were found to populate the sperm plasma membrane, and the complexes exhibited variability in their distribution in different sperm. This appears to be the first illustrated example of an acrosomeless sperm that exhibits a high concentration of filipin-sterol complexes. In contrast, the unique grating formed by the intramembranous particles as well as variable amounts of membrane surrounding the unusual particle arrays were always free of the filipin-sterol complexes. Thus, while cholesterol appears to be present in the plasma membrane of the zebrafish sperm, it is not apparent in the highly differentiated region of the membrane based on the observed distribution of the filipin-sterol complexes.

Animals↗

PubMed1997

Olfaction in zebrafish: what does a tiny teleost tell us?

Zebrafish, Danio rerio, possess a well-developed sense of smell which governs a variety of behaviors. Both the number of odorant receptor genes and the number of modules in the olfactory bulb (glomeruli) are about an order of magnitude smaller than those of mammals. Nevertheless, the spatial organization of functional properties within the sensory surface and the olfactory bulb are comparable to those of mammals. The quantitatively reduced olfactory system of zebrafish, together with the suitability of this species for developmental and genetic studies, make zebrafish an interesting model system to study olfactory differentiation and neuronal representation of olfactory information.

Journal Article↗

PubMed1982

Effects of immobilization and partial denervation on the differentiation of muscle fiber types in the Zebrafish, Brachydanio rerio.

In this study we tested the effects of immobilization and of spinal cord lesion on the differentiation of muscle fiber types in the myotomal muscle of the zebrafish. We found that the differentiation of embryonic red and white muscle fibers occurs in immobilized as well as in denervated myotomes of zebrafish embryos. Intermediate and adult red muscle fibers are not formed in denervated myotomes of larvae. Adult zebrafishes with spinal cord lesions in the anal region show abnormal undulating movements in the rostral part of the body. This change in muscle activity correlates with an increase in the number of intermediate scattered fibers. We conclude that direct contact with spinal cord motorneurons is not necessary for the differentiation of embryonic red and white fibers. The differentiation of intermediate muscle fibers is under extrinsic control. The factors that determine the formation of adult red fibers are not yet clear.

Animals↗

PubMed1999

Isolation of Complementary DNAs Coding for a Receptor for Activated C Kinase (RACK) from Zebrafish (Danio rerio) and Tilapia (Oreochromis niloticus): Constitutive Developmental and Tissue Expression.

: We have cloned and sequenced complementary DNAs coding for a receptor for activated C kinase (RACK) from two species of teleost fishes, zebrafish (Danio rerio) and tilapia (Oreochromis niloticus). The tilapia clone is 1063 nucleotides long, and the zebrafish clone is 1069 nucleotides long. Both clones contain an open reading frame coding for the complete RACK protein of 317 amino acids. Northern hybridization analysis using these clones as probes detected a 1.2-kb band, indicating that these are nearly full-length cDNA clones. In tilapia, RACK messenger RNA was expressed in all tissues examined. In situ hybridization detected the presence of mRNA for this RACK sequence in unfertilized eggs and embryos (development up to 24 hours) from zebrafish.

Journal Article↗

PubMed2001

Bioaccumulation of selected PCBs in zebrafish, three-spined stickleback, and arctic char after three different routes of exposure.

The uptake and elimination of 20 structurally diverse tetra- to heptachlorinated biphenyls were studied in zebrafish (Danio rerio), three-spined stickleback (Gasterosteus aculeatus), and Arctic char (Salvelinus alpinus). The polychlorinated biphenyls (PCBs) were administered to the fish through food, intraperitoneal injection of peanut oil, or intraperitoneal implantation of silicone capsules. The retention of the PCBs in fish exposed through their diet was related with the substitution patterns of the compounds. Ortho-substituted congeners with no unsubstituted meta-para positions had high biomagnification potential. PCBs with low biomagnification all had adjacent vicinal hydrogens, indicating that congeners with this feature may have been metabolically eliminated. The retention characteristics of the PCBs in the diet-exposed and the injected zebrafish were similar. The pattern of congeners in Arctic char indicates that they have a lower capacity to metabolize PCBs compared to three-spined sticklebacks and zebrafish. The levels in the fish exposed to the PCBs through a silastic implant were negatively correlated with the hydrophobicity of the congeners. Most probably congener-specific release rates of the PCBs from the implants mask their retention characteristics. It is suggested that food, mimicking the natural intake route, should be used in PCB exposure studies to validate extrapolations to natural situations.

Administration, Oral↗

PubMed2001

Fibroblast growth factor inhibits expression of neural markers in cultures of zebrafish early embryo cells.

Basic fibroblast growth factor (FGF) regulation of developmental markers in cell cultures derived from early zebrafish embryos was examined with the goal of in vitro culture of zebrafish embryonic stem cells and gaining an understanding of extracellular influences on early embryonic development. Markers were stem/primordial germ cell markers pou-2 and vas, neural markers zp-50, pax[zf-a], en-3, and wnt-1, and mesodermal markers gsc and myoD. Previously we had shown that FGF prevents the development of zebrafish pigment cells in vitro. In our culture system, FGF reduced expression of neural-specific markers, possibly implicating the FGF family in suppression of early neural cell development. Exposure to FGF for 24 hours at the time of seeding the cells was sufficient to suppress neural marker expression for a subsequent 4 days of culture, while absence of FGF for the first 24 hours of culture nullified the effect of FGF added subsequently. FGF predictably increased expression of gsc and myoD. Vas expression was unaffected, while pou-2 expression decreased with time in culture in the presence or absence of FGF. However, in situ hybridization identified a subpopulation of cells expressing pou-2, suggesting the possible continued existence of undifferentiated stem cells in the cultures.

Journal Article↗

PubMed1988

In situ hybridization patterns of zebrafish homeobox genes homologous to Hox-2.1 and En-2 of mouse.

Vertebrate homeobox genes have been identified on the basis of sequence homology to Drosophila segmentation and homeotic genes. We have analyzed the spatial distribution patterns in hatching larvae of transcripts from two different types of zebrafish (Brachydanio rerio) homeobox genes. Tritiated DNA fragments from one zebrafish gene (ZF-21) of the Antennapedia-class and one (ZF-EN) of the engrailed-type were used as probes to detect transcripts in larval tissue sections. Both zebrafish genes are expressed mainly in spatially restricted regions of the central nervous system. The transcripts from ZF-21 was detected in the posterior part of the hindbrain, and ZF-EN was found to be most intensively expressed in a restricted domain located in the region of the junction between the mid- and hindbrain. These neural expression patterns are very similar to those of the homologous murine genes Hox-2.1 and En-2 and clearly suggest a strong functional conservation.

Animals↗

PubMed1999

Boron is required for zebrafish embryogenesis

Boron is the only element known to be essential for plants, but only circumstantial evidence for essentiality exists in animals. We report here that boron is essential for the embryonic development of zebrafish (Danio rerio). Zebrafish were maintained in water with a boron concentration of 0.1 micromol l-1 or supplemented to a concentration of 45 micromol l-1 using ultrapure boric acid. Both groups were fed boron-depleted brine shrimp. This procedure depleted the boron content of the blastulas from low-boron parents to only 5 % of the boron content of the blastulas from boron-supplemented parents. Sperm from low-boron males successfully fertilized eggs from low-boron females, but 92 % of the embryos died within 10 days. The early cleavage stage of development was the most sensitive to boron deficiency. Of the fertilized embryos, 46% did not live to complete the blastula stage. Repletion of low-boron embryos during the first hour after fertilization rescued them from death. These observations provide strong evidence that boron is essential for zebrafish development.

Journal Article↗

PubMed1998

Homologous recombination and DNA-end joining reactions in zygotes and early embryos of zebrafish (Danio rerio) and Drosophila melanogaster.

A linear DNA with partial sequence redundancy can be recircularized in cells by either nonhomologous end joining (NEJ) or by homologous recombination (HR). We have studied the relative contributions of these processes in zygotes or early embryos of species that serve as model organisms for developmental genetics. Thus, we have microinjected a linearized plasmid substrate into zygotes of zebrafish (Danio rerio) or into the posterior end of Drosophila melanogaster early embryos before pole cell formation. Similar to the situation observed previously in Xenopus zygotes/early embryos, we detected a large preponderance of DNA-end joining over homologous recombination. A comparison of end-joined junctions revealed that from the three species tested, zebrafish introduced the least number of sequence distortions upon DNA-end joining, while Drosophila produced the largest deletions (average 14 bp) with occasional nucleotide patch insertions, reminiscent of the N nucleotides at V(D)J junctions in mammalian immune receptor genes. Double-strand gap repair by homologous sequences ('homologous recombination') involving a bimolecular reaction was readily detectable in both zebrafish and Drosophila. This involved specifically designed recombination substrates consisting of a mutagenized linear plasmid and DNA fragments carrying the wild-type sequence. Our results show that the basic machinery for homologous recombination is present at early developmental stages of these two genetic model organisms. However, it seems that for any experimental exploitation, such as targeted gene disruption, one would have to inhibit or bypass the overwhelming DNA-end joining activity.

Animals↗

PubMed1998

Zebrafish Genomic Library in Yeast Artificial Chromosomes

We have constructed a zebrafish yeast artificial chromosome (YAC) library using genomic DNA isolated from the inbred AB zebrafish strain. The average insert size is 470 kb, estimated from analysis of 155 random selected YACs. The library consists of 17,000 clones, providing about a 4.7-fold coverage of zebrafish genome. The YAC clones have been arrayed in individual wells of 96-well microplates and also pooled to permit rapid polymerase chain reaction screening of the entire library. We have also found that the YAC ends can be easily rescued and sequenced from pRML1/pRML2-based mini-YAC clones. Copyright 1998 Academic Press.

Journal Article↗