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PubMed2002

Effects of ethynylestradiol on the reproductive physiology in zebrafish (Danio rerio): time dependency and reversibility.

Environmental pollution with natural or synthetic estrogens may pose a serious threat to reproduction of wildlife species. This study describes the effects of 17-alpha-ethynylestradiol (EE2) on fish reproductive organs in a laboratory model. Adult zebrafish were semistatically exposed to nominal concentrations of 0, 10, and 25 ng/L EE2 for 24 d and then transferred to EE2-free medium. Gonadosomatic index (GSI), plasma vitellogenin concentration (VTG), and histology of the gonads (control and 10 ng/L only) were examined as a function of time. It was found that EE2 has an adverse impact on both male and female reproductive organs. Notably in females, gonadal changes were observed through histological evaluation after 3 d of exposure to 10 ng/L EE2. and this was followed by a reduction of GSI at day 6 of exposure. In males, a reduction of GSI and altered testis histology was found after 24 d of exposure to 10 ng/L. The observed effects on the ovary after EE2 exposure, combined with complete recovery after 24 d, is considered to be triggered by feedback at the level of the pituitary. In both males and females, VTG was induced in response to EE2 and normalized during the recovery period. The observed correlation between VTG and ovarian somatic index (OSI) demonstrates that excessive VTG induction may be predictive for adverse effects of EE2 on ovarian function in female zebrafish. These results indicate that long-term stimulation by synthetic estrogens such as EE2 might impair reproductive function in zebrafish in a reversible manner.

Animals↗

PubMed2002

Development of axon pathways in the zebrafish central nervous system.

The zebrafish has a number of distinct advantages as an experimental model in developmental biology. For example, large numbers of embryos can be generated in each lay, development proceeds rapidly through a very precise temporal staging which exhibits minimal batch-to-batch variability, embryos are transparent and imaging of wholemounts negates the need for tedious histological preparation while preserving three-dimensional spatial relationships. The zebrafish nervous system is proving a convenient model for studies of axon guidance because of its small size and highly stereotypical trajectory of axons. Moreover, a simple scaffold of axon tracts and nerves is established early and provides a template for subsequent development. The ease with which this template can be visualized as well as the ability to spatially resolve individual pioneer axons enables the role of specific cell-cell and molecular interactions to be clearly deciphered. We describe here the morphology and development of the earliest axon pathways in the embryonic zebrafish central nervous system and highlight the major questions that remain to be addressed with regard to axon guidance.

Animals↗

PubMedDate not supplied

Transgenic expression of a GFP-rhodopsin COOH-terminal fusion protein in zebrafish rod photoreceptors.

To facilitate the identification and characterization of mutations affecting the retina and photoreceptors in the zebrafish, a transgene expressing green fluorescent protein (GFP) fused to the C-terminal 44 amino acids of Xenopus rhodopsin (Tam et al., 2000) under the control of the 1.3-kb proximal Xenopus opsin promoter was inserted into the zebrafish genome. GFP expression was easily observed in a ventral patch of retinal cells at 4 days postfertilization (dpf). Between 45-50% of the progeny from the F1, F2, and F3 generations expressed the transgene, consistent with a single integration event following microinjection. Immunohistochemical analysis demonstrated that GFP is expressed exclusively in rod photoreceptors and not in the UV, blue, or red/green double cones. Furthermore, GFP is localized to the rod outer segments with little to no fluorescence in the rod inner segments, rod cell bodies, or rod synapse regions, indicating proper targeting and transport of the GFP fusion protein. Application of exogenous retinoic acid (RA) increased the number of GFP-expressing cells throughout the retina, and possibly the level of expressed rhodopsin. When bred to a zebrafish rod degeneration mutant, fewer GFP-expressing rods were seen in living mutants as compared to wild-type siblings. This transgenic line will facilitate the search for recessive and dominant mutations affecting rod photoreceptor development and survival as well as proper rhodopsin expression, targeting, and transport.

Amblyopia↗

PubMed2003

Zebrafish: from disease modeling to drug discovery.

The study of zebrafish, a leading model organism for developmental biology, is rapidly expanding to include human disease. Zebrafish models based on known disease mechanisms have been developed in several therapeutic areas, including blood diseases, diabetes, muscular dystrophy, neurodegenerative disease, angiogenesis and lipid metabolism. This review summarizes recent progress in disease model development, and outlines the potential of zebrafish to contribute to drug discovery through the identification of novel drug targets, validation of those targets and screening for new therapeutic compounds.

Animals↗

PubMed2003

Effects of ammonium perchlorate on the reproductive performance and thyroid follicle histology of zebrafish.

Adult zebrafish were reared up to eight weeks in control water or in water containing ammonium perchlorate (AP) at measured perchlorate concentrations of 18 (environmentally relevant, high) and 677 ppm. Groups of eight females were paired with four males on a weekly basis to assess AP effects on spawned egg volume, an index of reproductive performance. All treatments were applied to four to five spawning replicates. At 677 ppm, spawn volume was reduced within one week and became negligible after four weeks. At 18 ppm, spawn volume was unaffected even after eight weeks. Also, perchlorate at 18 ppm did not affect percentage egg fertilization. Fish were collected at the end of the exposures (677 ppm, four weeks; control and 18 ppm, eight weeks) for whole-body perchlorate content and thyroid histopathological analysis. Fish perchlorate levels were about one-hundredth of those of treatment water levels, indicating that waterborne perchlorate does not accumulate in whole fish. At 677 ppm for four weeks, perchlorate caused thyroid follicle cell (nuclear) hypertrophy and angiogenesis, whereas at 18 ppm for eight weeks, its effects were more pronounced and included hypertrophy, angiogenesis, hyperplasia, and colloid depletion. In conclusion, an eight-week exposure of adult zebrafish to 18 ppm perchlorate (high environmentally relevant concentrations) affected the histological condition of their thyroid follicles but not their reproductive performance. The effect of 677 ppm perchlorate on reproduction may be due to extrathyroidal toxicity. Further research is needed to determine if AP at lower environmentally relevant concentrations also affects the thyroid follicles of zebrafish.

Animals↗

PubMedDate not supplied

Transient expression of RSVCAT in transgenic zebrafish made by electroporation.

We report the fish use of an exponential decay electroporation system to introduce foreign DNA into fertilized zebrafish embryos. The plasmid RSVCAT (Rous sarcoma viral promoter (RSV) upstream from the chloramphenicol acetyltransferase gene (CAT)) was linearized and introduced into fertile zebrafish embryos by electroporation no later than the four-cell stage. Conditions for the procedure were empirically derived, and 68% of the treated animals survived through hatching to at least 6 days after fertilization and well beyond. Dot-blot analysis on DNA extracted from individual hatching fry demonstrated that 65% of the animals tested carried the foreign construct. Enzyme assays on the soluble proteins of treated animals were positive for chloramphenicol acetyltransferase activity. These data demonstrate that the foreign construct was being transiently expressed in the developing tissues of the embryo. The simplicity of this technique will greatly enhance the ability to analyze gene promoter regulation in vivo in transgenic zebrafish. The ability of the electroporated DNA to integrate into the host genome and to generate stable lines of transgenic fish is discussed.

Animals↗

PubMed2003

Susceptibility of zebrafish (Danio rerio) to a model pathogen, spring viremia of carp virus.

To improve our understanding of the genetic basis of fish disease, we developed a pathogen model, using zebrafish (Danio rerio) and spring virema of carp virus (SVCV). Replicate groups of 10 fish were acclimated to 20 or 24 degrees C, then were exposed to SVCV concentrations of 10(3) to 10(5) plaque-forming units per milliliter (PFU/ml) of water and observed daily. In a second trial, fish were acclimated to 15 degrees C, and replicate groups of 10 fish were exposed to SVCV at a concentration of 10(5) PFU/ml; however, the temperature was raised 1 degrees C/wk. Moribund fish were collected for histologic examination, and dead fish were assayed for virus by use of cell culture and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Mortality exceeded 50% in fish exposed to 10(5) PFU of SVCV/ml at the lower temperatures. Clinical signs of disease became evident seven days after viral exposure and were observed most consistently in fish of the 10(5) PFU/ml groups. Affected zebrafish were anorectic and listless, with epidermal petechial hemorrhages followed by death. Use of plaque assays and RT-PCR analysis confirmed presence of SVCV at titers > or = 10(4) PFU/g of tissue. Histologic lesions included multifocal brachial necrosis and melanomacrophage proliferation in gills, liver, and kidneys. These results indicate that zebrafish are susceptible to infection by SVCV under conditions that mimic a natural route of exposure.

Animals↗

PubMed2004

[The shh promoter of zebrafish directs the expression of GFP in notochord].

In vitro experiment showed that HNF3beta was the direct regulator of sonic hedgehog (shh) promoter. To investigate the activity of zebrafish shh promoter in vivo, we constructed the expression vector pShh-EGFP with ligating a 538 bp zebrafish shh promoter,which contained two HNF3beta binding sites, to EGFP. The pShh-EGFP DNA was microinjected into one-cell stage embryos of the zebrafish and the embryos were observed for GFP expression with fluorescent microscopy. GFP expression started during gastrulation in the axial hypoblast layer. During segmentation, GFP was detected in the notochord but not in the foor plate. Our experiment demonstrated that the 538 bp shh promoter containing two HNF3beta binding sites is able to confer the notochord-expressing activity.

Animals↗

PubMedDate not supplied

Studies on cryoprotectant toxicity to zebrafish (Danio rerio) oocytes.

Cryopreservation of fish germ cells is an important measure in conservation of fish genetic material. Although investigations on cryopreservation of fish sperm and embryos have been carried out extensively, cryopreservation of fish oocytes has not been studied systematically. In the present study the toxicity of cryoprotectants to zebrafish (Danio rerio) oocytes was investigated. Commonly used cryoprotectants dimethyl sulfoxide (DMSO), methanol, ethylene glycol (EG), propylene glycol (PG), sucrose and glucose were studied. Stage III (vitellogenic), stage IV (maturation) and stage V (mature egg) zebrafish oocytes were incubated in Hank's medium containing different concentrations of cryoprotectants (0.25-4M) for 30 min at room temperature. Three different tests were used to assess oocyte viability: trypan blue (TB) staining, thiazolyl blue (MTT) staining and in vitro maturation followed by observation of germinal vesicle breakdown (GVBD). Results showed that the toxic effect of cryoprotectant on oocytes generally increased with increasing concentration. MTT test was shown to be the least sensitive testing method and gave poor correlation to subsequent GVBD results. Sensitivity of vital tests increases in the order of MTT, TB and GVBD. GVBD test showed that cryoprotectant toxicity to stage III zebrafish oocytes increased in the order of methanol, PG, DMSO, EG, glucose and sucrose. No Observed Effect Concentrations (NOECs) for stage III oocytes were 2M, 1M, 1M, 0.5M, less than 0.25M and less than 0.25M for methanol, PG, DMSO, EG, glucose and sucrose respectively. TB test also showed that the toxicity of tested cryoprotectants increased in the same order. The sensitivity of oocytes to cryoprotectants appeared to increase with development stage with stage V oocytes being the most sensitive.

Alcohols↗

PubMed2006

Egr1 gene knockdown affects embryonic ocular development in zebrafish.

PURPOSE: To identify the changes in zebrafish embryonic ocular development after early growth response factor 1 (Egr1) gene knockdown by Egr1-specific translation inhibitor, morpholino oligonucleotides (MO). METHODS: Two kinds of Egr1-MO were microinjected separately with various dosages into one to four celled zebrafish embryos to find an optimal dose generating an acceptable mortality rate and high frequency of specific phenotype. Chordin-MO served as the positive control; a 5 mismatch MO of Egr1-MO1 and a nonspecific MO served as negative controls. We graded the Egr1 morphants according to their gross abnormalities, and measured their ocular dimensions accordingly. Western blot analysis and synthetic Egr1 mRNA rescue experiments confirmed whether the deformities were caused by Egr1 gene knockdown. Histological examination and three kinds of immunohistochemical staining were applied to identify glutamate receptor one expression in retinal ganglion cells and amacrine cells, to recognize acetylated alpha-tubulin expression which indicated axonogenesis, and to label photoreceptor cells with zpr-1 antibody. RESULTS: After microinjection of 8 ng Egr1-MO1 or 2 ng Egr1-MO2, 81.8% and 97.3% of larvae at 72 h postfertilization had specific defects, respectively. The gross phenotype included string-like heart, flat head, and deformed tail. The more severely deformed larvae had smaller eyes and pupils. Co-injection of 8 ng Egr1-MO1 and supplementary 12 pg synthetic Egr1 mRNA reduced the gross abnormality rate from 84.4% to 29.7%, and decreased the severity of deformities. Egr1 protein appeared in the wildtype and rescued morphants, but was lacking in the Egr1 morphants with specific deformities. Lenses of Egr1 morphants were smaller and had some residual nucleated lens fiber cells. Morphants' retinal cells arranged disorderly and compactly with thin plexiform layers. Immunohistochemical studies showed that morphants had a markedly decreased number of mature retinal ganglion cells, amacrine cells, and photoreceptor cells. Retinal axonogenesis was prominently reduced in morphants. CONCLUSIONS: The Egr1 gene plays an important role in zebrafish embryonic oculogenesis. Ocular structures including lens and retina were primitive and lacked appropriate differentiation. Such arrested retinal and lenticular development in Egr1 morphants resulted in microphthalmos.

Animals↗

PubMed1990

Development of the retinotectal projection in zebrafish embryos under TTX-induced neural-impulse blockade.

The influence of neural activity on the morphology of retinal-axon-terminal arbors and the precision of the developing retinotectal projection in zebrafish embryos was explored. Terminal-arbor morphology and their distribution in the tectum was determined with anatomical fiber-tracing methods using the fluorescent dyes dil and diO. To allow development under activity-deprived conditions, TTX was injected into the eyes of 30-38-hr-old zebrafish embryos at concentrations that effectively blocked neural activity both in retinal ganglion cells and throughout the CNS. Much like axons with normal neural-activity patterns, activity-deprived axons from dorsal and ventral and from temporal and nasal regions in the retina terminated over retinotopically appropriate and nonoverlapping regions of the tectum. Even after ablation of 1 hemiretina at the time of axonal outgrowth, activity-deprived axons from the remaining hemiretina grew directed toward and arborized selectively within their retinotopically appropriate tectal half in the same way as would nondeprived axons. Besides being retinotopic, the area over which small populations of activity-deprived axons from neighboring ganglion cells arborize is as small as that of active axons. The size of terminal arbors of retinal ganglion cell axons was unaffected by blockade of neural activity. The mean terminal-arbor size was 27 x 18 microns for the TTX-injected and 31 x 22 microns for the control embryos. The tectal coverage of TTX-blocked and control axons was equally small, with values of 1.4% and 1.6%, respectively. These data show that a precisely organized retinotopic map in developing zebrafish forms independent of neural-impulse activity.

Animals↗

PubMed1995

Ultrastructural in situ hybridization and autoradiographic detection of foreign DNA in zebrafish spermatozoa.

Conditions have been established which permit specific hybridization of foreign DNA to complimentary DNA in ultra thin sections of zebrafish sperm prepared for electron microscopy (EM)1 after fixation in 2% paraformaldehyde, 2.5% glutaraldehyde and embedded in spurs resin. Zebrafish sperm incubated with foreign DNA were subjected to ultrastructural in situ hybridization and autoradiographic detection to determine if the sperm could retain and internalize the foreign DNA sequences. Specific and intense signals were detected from the sperm head indicating that the zebrafish sperm are capable of internalizing foreign DNA.

Animals↗

PubMed1995

Caudal fin regeneration in wild type and long-fin mutant zebrafish is affected by retinoic acid.

Zebrafish (Danio rerio) represents an ideal experimental model to tackle fundamental issues concerned with organogenesis during development and regeneration of complex body structures. We discuss here the development of the skeleton in zebrafish caudal fins, their regenerative ability in wild type and long-fin mutant adult fish, and how retinoic acid (RA), which induces duplications along the proximodistal axis in regenerating limbs, affects regeneration of the caudal fin. The dorsal and ventral lobes of zebrafish caudal fins are apparently symmetrical along the dorsoventral axis, but all of the skeletal elements and most of the soft tissues of both lobes originate from the ventral part of the embryo, as demonstrated by whole-mount staining of developing fish. Analysis of caudal fin regenerates in wild type adults does not reveal any difference in the regenerative ability of the two lobes, and in the length of the regenerate in comparison with the amputated part. In contrast, in the long-fin mutant the regenerated caudal fin is always somehow defective in that the original asymmetry in the length of the two lobes observed in this mutant is not reproduced in the regenerate. Furthermore, in the majority of the batches studied the regenerate is much smaller in size than the amputated part. This suggests that this mutant may be valuable to further our understanding of the mechanisms underlying growth control and patterning during regeneration. Finally, we show that the regenerating caudal fin is sensitive to RA-treatment, and clear teratogenic effects on the dorso-ventral axis are observed under many of the experimental conditions investigated both in wild type and long-fin mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Combinatorial expression of three zebrafish genes related to distal-less: part of a homeobox gene code for the head.

We describe analysis of zebrafish distal-less-related homeobox genes that may serve as specifiers of positional information in anterior regions of the CNS and in peripheral structures. We isolated three zebrafish genes, dlx2, dlx3, and dlx4, by screening embryonic cDNA libraries. Comparisons of the predicted sequences of the Dlx2, Dlx3, and Dlx4 proteins with distal-less proteins from other species suggest that vertebrate distal-less genes can be divided into four orthologous groups. We observed similarities but also unique features of the expression patterns of the zebrafish dlx genes. Among the three genes, dlx3 alone is expressed during gastrulation. Shortly after gastrulation, cells in the ventral forebrain rudiment express dlx2 and dlx4, but not dlx3, and hindbrain neural crest cells express only dlx2. Presumptive precursor cells of the olfactory placodes express dlx3 and dlx4 but not dlx2. Transcripts of dlx3 and dlx4 are present in overlapping subsets of cells in the auditory vesicle and in cells of the median fin fold, whereas dlx2 is never expressed in the auditory vesicle and only at low levels in localized regions of the median fin fold. Cells of the visceral arches and their primordia express all three dlx genes, but with different developmental time courses. We suggest that combinatorial expression of the dlx genes is part of a homeobox gene code specifying pattern formation or cell fate determination in the forebrain, in peripheral structures of the head, and in the fins.

Amino Acid Sequence↗

PubMed1993

[Ventral and posterior expression of the homeo box gene eve1 in zebrafish (Brachydanio rerio) is repressed in dorsalized embryos].

We have identified and characterized the zebrafish eve1 homeo box gene, a member of the Drosophila even-skipped (eve) gene family. eve1 is expressed in the most posterior part of the tail bud during somitogenesis. During gastrulation, transcripts are confined to ventral and lateral cells of the marginal zone of the embryo. We show that LiCl, known to dorsalize Xenopus embryos, has the same effect in zebrafish embryos. In LiCl-treated embryos, eve1 transcripts are completely absent, suggesting that eve1 marks the ventral specification of mesoderm in zebrafish gastrulae.

Animals↗

PubMed1994

Olfactory glomeruli in the zebrafish form an invariant pattern and are identifiable across animals.

Glomeruli are anatomical and possibly functional modules in the vertebrate olfactory bulb. We investigated the spatial arrangement of glomeruli in the olfactory bulbs of adult zebrafish (Brachydanio rerio). A solution of the lipophilic tracer Dil was injected into the nasal cavities. Axons of sensory neurons projecting from the olfactory epithelium into the bulb were traced anterogradely, thus labeling the whole population of glomeruli. The glomerular distribution was analyzed in detail by confocal laser-scanning microscopy. We find that a typical olfactory bulb contains a small number of about 80 glomeruli that have a stereotyped configuration in all animals investigated. All glomeruli exhibit bilateral symmetry. Twenty-two single glomeruli could be identified from animal to animal by their characteristic position and morphology. The remaining glomeruli either are embedded in glomerular plexus and therefore cannot be delineated reliably, or belong to a densely clustered subpopulation of on average 49 glomeruli in the dorsal olfactory bulb. No sexually dimorphic glomeruli were identified. To test whether glomerular constancy is specific for the zebrafish, we performed similar tracing experiments in the goldfish and found several indications for a similar invariance of glomeruli in this species. The remarkable stereotypy of this pattern is reminiscent of the insect olfactory system and has been demonstrated here for the first time in a vertebrate. It will now be possible to examine whether these identifiable glomeruli are functionally specialized in terms of odor processing. If so, zebrafish may emerge as a tractable model system for studies on olfactory coding.

Animals↗

PubMed1996

Comparative virulence between different strains of Listeria in zebrafish (Brachydanio rerio) and mice.

Listeriosis is a disease found in most animal species but is relatively uncommon in fish. We studied the relationship between Listeria and zebrafish by injecting Brachydanio rerio intraperitoneally with different Listeria strains having pathological or food-stuff origins. We then compared these results with those obtained in Swiss mice. Experimental Listeriosis in Zebrafish differs greatly from that observed in mice. The 50% lethal dose (LD50) previously determined was much higher than that observed in mice. In fish, a good correlation exists between infection found in renal tissue, an important lymphoïd organ and that present in whole fish (p < 0.001). Infection kinetics showed that, in contrast with mice, L. monocytogenes was unable to multiply in fish. Differential blood counts showed the development of an immune response in fish. The difference in the expression of Listeria virulence between Zebrafish and mice was also seen in their reactions to different wild strains inoculate i.p. Strains belonging the innocua, ivanovii, seeligeri and welshimeri were weakly or not virulent in mice but virulent in fish. Nevertheless, as in mice, differences in virulence existed between strains of L. monocytogenes belonging to serovars 4b, 1/2a, 1/2b and 1/2c.

Animals↗

PubMed1997

Characterization of adult alpha- and beta-globin genes in the zebrafish.

Developmental switching of hemoglobins (Hbs) occurs in most vertebrates, yet the cellular and molecular basis for this process remains elusive. The zebrafish is a new genetic and developmental system that can be used to study embryogenesis, and mutants with a variety of defects in hematopoiesis have recently been derived. To initiate our studies on Hb switching in this organism, we have characterized the globins expressed in the adult. Reversed-phase high performance liquid chromatography and mass spectrometric analyses of adult peripheral blood hemolysates showed that there are three major alpha globins and two beta globins in circulating erythroid cells. In addition, we have isolated and characterized zebrafish adult alpha- and beta-globin cDNA clones that encode some of these globins. High levels of alpha- and beta-globin gene expression were detected in adult erythroid cells, whereas embryonic erythroid cells expressed little, if any, of these RNAs. We have also shown that the alpha- and beta-globin genes are tightly linked on the same chromosome and are arrayed in a 3'-5' to 5'-3' configuration, respectively. The characterization of these genes and regulatory elements in this globin locus will provide insight into the process of globin gene transcription. With these reagents, future studies of Hb switching in zebrafish mutants with defective hematopoiesis will be possible.

Amino Acid Sequence↗