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PubMed2000

Vasa protein expression and localization in the zebrafish.

Primordial germ cell (PGC) development in the zebrafish is poorly understood. The expression of vasa RNA, the only molecular marker so far found to be expressed in fish PGCs, suggests its function in the establishment of the germline. The protein product of vasa is present throughout the life cycle in the germline of Drosophila, Caenorhabditis and Xenopus. The expression pattern of the Vasa protein in zebrafish, is still unresolved. We generated an anti-Vasa polyclonal antibody and show that it is maternally expressed initially throughout the embryo. Interestingly, from the two-cell- to the 1000-cell stage the protein is highly concentrated in two 'dots' near the center of the blastomeres and as such remains longest detectable in the animal pole blastomeres. The first distinct cell-specific expression occurs at 60% epiboly on one side of the blastoderm margin. The Vasa protein in the PGCs is organized in a subcellular granular-like conformation which is dynamic throughout development.

Animals↗
PubMed2005

Gata4 regulates the formation of multiple organs.

We have developed a loss-of-function model for Gata4 in zebrafish, in order to examine broadly its requirement for organogenesis. We show that the function of Gata4 in zebrafish heart development is well conserved with that in mouse, and that, in addition, Gata4 is required for development of the intestine, liver, pancreas and swim bladder. Therefore, a single transcription factor regulates the formation of many organs. Gata6 is a closely related transcription factor with an overlapping expression pattern. We show that zebrafish depleted of Gata6 show defects in liver bud growth similar to mouse Gata6 mutants and zebrafish Gata4 morphants, and that zebrafish embryos depleted of both Gata4 and Gata6 display an earlier block in liver development, and thus completely lack liver buds. Therefore, Gata4 and Gata6 have distinct non-redundant functions in cardiac morphogenesis, but are redundant for an early step of liver development. In addition, both Gata4 and Gata6 are essential and non-redundant for liver growth following initial budding.

Animals↗
PubMed2006

Effects of binary mixtures of xenoestrogens on gonadal development and reproduction in zebrafish.

Previous studies exposing fish to xenoestrogens have demonstrated vitellogenin (VTG) induction, delayed gametogenesis, altered sex ratio, and decreased reproductive performance, with a majority of those studies focusing on exposure to single chemicals. The objective of this study was to determine the effects of binary mixtures of a weak estrogen receptor agonist, nonylphenol (NP) and a potent estrogen receptor agonist, 17alpha-ethinylestradiol (EE) on sex ratios, gametogenesis, VTG induction, heat shock protein 70 (HSP70) expression and reproductive capacity in zebrafish (Danio rerio). Fish were exposed from 2 to 60 days post-hatch (dph) to nominal concentrations of 10 or 100 microg/l NP (NP10 or NP100, respectively), 1 or 10 ng/l EE (EE1 or EE10, respectively), 1 ng/l EE+10 or 100 microg/l NP (EE1+NP10 or EE1+NP100, respectively), 10 ng/l EE+10 or 100 microg/l NP (EE10+NP10 or EE10+NP100, respectively) or solvent control (0.01% acetone, v/v) in a static-renewal system with replacement every 48 h. At 60 dph, fish from each treatment were euthanized for histological examination of gonads, and whole body VTG and HSP70 levels. Remaining fish were reared in clean water until adulthood (240 dph) for breeding studies. In all EE10 exposure groups (EE10, EE10+NP10 and EE10+NP100), increasing NP concentration acted antagonistically to the action of EE in terms of VTG induction at 60 dph. Similarly, non-additivity was observed with egg production, where EE1+NP100 exposure resulted in significantly more eggs produced per breeding trial than EE1 alone. Histological staging of oogenesis revealed suppressed gametogenesis in an additive fashion in females at 60 dph. There were no differences among treatment groups in whole body HSP70 expression in 60 dph fish or in gonadal HSP70 expression in adult fish. Although there was no statistical evidence of non-additivity, breeding trials in adults revealed significant reductions in egg viability, egg hatchability and/or F1 swim-up success, suggesting that developmental exposures to xenoestrogens may cause irreversible effects on egg quality and progeny even after periods of depuration. In conclusion, these results suggest that environmentally relevant mixtures of NP and EE can produce additive or non-additive effects that depend on the particular response being determined and the respective exposure concentrations of each chemical.

Animals↗
PubMed2023

Ypel5 regulates liver development and function in zebrafish.

YPEL5 is a member of the Yippee-like (YPEL) gene family that is evolutionarily conserved in eukaryotic species. To date, the physiological function of YPEL5 has not been assessed due to a paucity of genetic animal models. Here, using CRISPR/Cas9-mediated genome editing, we generated a stable ypel5-/- mutant zebrafish line. Disruption of ypel5 expression leads to liver enlargement associated with hepatic cell proliferation. Meanwhile, hepatic metabolism and function are dysregulated in ypel5-/- mutant zebrafish, as revealed by metabolomic and transcriptomic analyses. Mechanistically, Hnf4a is identified as a crucial downstream mediator that is positively regulated by Ypel5. Zebrafish hnf4a overexpression could largely rescue ypel5 deficiency-induced hepatic defects. Furthermore, PPARα signaling mediates the regulation of Hnf4a by Ypel5 through directly binding to the transcriptional enhancer of the Hnf4a gene. Herein, this work demonstrates an essential role of Ypel5 in hepatocyte proliferation and function and provides the first in vivo evidence for a physiological role of the ypel5 gene in vertebrates.

Animals↗
PubMed2004

The pu.1 promoter drives myeloid gene expression in zebrafish.

PU.1 is a member of the Ets family of transcription factors and plays an essential role in the development of both myeloid and lymphoid cells. To examine zebrafish pu.1 (zpu.1) expression in subpopulations of blood cells during zebrafish development, we linked a 9-kb zebrafish genomic fragment upstream of the zpu.1 initiator codon to green fluorescent protein (GFP) and microinjected this construct to generate stable transgenic lines. GFP-positive fluorescent myeloid precursors were observed migrating from the anterolateral mesoderm in living embryos from 16 to 28 hours after fertilization (hpf) in a pattern that overlaps the expression pattern of endogenous zpu.1 mRNA. Analysis of larval histologic sections revealed GFP-expressing hematopoietic cells in the developing zebrafish kidney. Flow cytometric analysis of cells from adult whole kidney marrow revealed 2 discrete subpopulations of GFP-positive cells, which after cell sorting exhibited either myeloid or early lymphoid morphology. Thus, the zebrafish zpu.1 promoter fragment used here is capable of driving reporter gene expression in subsets of embryonic and adult hematopoietic cells. These transgenic lines will be useful to dissect the cellular and molecular control of myeloid cell differentiation, and this promoter fragment may prove useful in the development of zebrafish models of acute myeloid leukemia.

Animals↗
PubMed2005

Development of oxygen sensing in the gills of zebrafish.

Previous studies have described the morphology, innervation and O(2)-chemoreceptive properties of neuroepithelial cells (NECs) of the zebrafish gill filaments. The present work describes the ontogenesis of these cells, and the formation of functional O(2)-sensing pathways in developing zebrafish. Confocal immunofluorescence was performed on whole-mount gill preparations using antibodies against serotonin (5-HT) and a zebrafish-derived neuronal marker (zn-12) to identify the appearance and innervation of gill NECs during larval stages. NECs were first expressed in gill filament primordia of larvae at 5 days postfertilization (d.p.f.) and were fully innervated by 7 d.p.f. In vivo ventilation frequency analysis revealed that a behavioural response to hypoxia (11.2+/-2.8 min(-1)) developed in embryos as early as 2 d.p.f., and a significant increase (P<0.05) in the ventilatory response to hypoxia (200.8+/-23.0 min(-1)) coincided with innervation of NECs of the filaments. In addition, exogenous application of quinidine, a blocker of O(2)-sensitive background K(+) channels in NECs, induced hyperventilation in adults in a dose-dependent manner and revealed the development of a quinidine-sensitive ventilatory response in 7 d.p.f. larvae. This study shows that NEC innervation in the gill filaments may account for the development of a functional O(2)-sensing pathway and the hyperventilatory response to hypoxia in zebrafish larvae. At earlier stages, however, O(2)-sensing must occur through another pathway. The possibility that a new type of 5-HT-positive NEC of the gill arches may account for this earlier hypoxic response is discussed.

Analysis of Variance↗
PubMed2006

Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio.

The thymidylate synthase (TS), an important target for many anticancer drugs, has been cloned from different species. But the cDNA property and function of TS in zebrafish are not well documented. In order to use zebrafish as an animal model for screening novel anticancer agents, we isolated TS cDNA from zebrafish and compared its sequence with those from other species. The open reading frame (ORF) of zebrafish TS cDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular mass of 36.15 kDa. The deduced amino acid sequence of zebrafish TS was similar to those from other organisms, including rat, mouse and humans. The zebrafish TS protein was expressed in Escherichia coli and purified to homogeneity. The purified zebrafish TS showed maximal activity at 28 degrees C with similar K(m) value to human TS. Western immunoblot assay confirmed that TS was expressed in all the developmental stages of zebrafish with a high level of expression at the 1-4 cell stages. To study the function of TS in zebrafish embryo development, a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV/TS, was constructed which targeted the protein-coding region of zebrafish TS mRNA. Significant change in the development of tail and epiboly was found in zebrafish embryos microinjected pSilencer4.1-CMV/TS siRNA expression vector.

Amino Acid Sequence↗
PubMed2002

Spontaneous activity-independent intracellular calcium signals in the developing spinal cord of the zebrafish embryo.

Calcium signals play an important role in a variety of processes necessary for neuronal development. Whilst the characteristics and function of calcium signals have been comprehensively examined in vitro, the significance of these signals during development in an intact embryo remains unclear. In this study, we have examined the spatial and temporal patterns of intracellular calcium signals in precursor cells (cells without processes) within the spinal cord of the intact zebrafish embryo aged between 17 and 27 h. In total, approximately one-third of cells displayed spontaneous intracellular calcium transients. The calcium transients had an average peak amplitude of 33.3 (+/-2.8%) above baseline, a duration of 52.2 (+/-6.3 s) and occurred with an average frequency of 4.6 (+/-0.4 per hour). Calcium transients were observed in precursor cells located throughout the spinal cord, with the highest percentage of active cells (35.1+/-8%) occurring at a developmental time of 21-22 h. Furthermore these intracellular calcium signals were observed in the presence of tricaine, indicating that they are not generated via sodium-dependent action potentials. In precursor cells loaded with the calcium buffer BAPTA both the frequency and the amplitude of the calcium transients was significantly reduced. The intracellular calcium transients may represent a common activity-independent calcium-mediated mechanism that contributes to the regulation of neuronal development in the spinal cord of the zebrafish embryo during the segmentation and early pharyngula period.

Action Potentials↗
PubMed2004

Melatonin stimulates cell proliferation in zebrafish embryo and accelerates its development.

All vertebrates show a dramatic circadian rhythm in circulating melatonin with high levels at night and very low levels during daytime. In adults, melatonin is thought to synchronize other circadian rhythms and regulate seasonal rhythms in photoperiodic animals by acting on specific G-protein coupled receptors. The role of melatonin in development is unknown, even though melatonin receptors appear to be more highly expressed in developing embryos and neonates than in adults. In this study on zebrafish embryos, we describe a role for melatonin in increasing cell proliferation and accelerating development. We propose that melatonin has a role in extending the safe limit of proliferation rate at night to allow more rapid development when potentially damaging ultraviolet light is absent.

Animals↗
PubMed2002

Clock mechanisms in zebrafish.

Recent research on the circadian system of the zebrafish is reviewed. This teleost has become an attractive model system because of its advantages for genetic analyses. Circadian rhythms of zebrafish behavior, visual system function, and pineal melatonin synthesis have been described, and behavioral and pineal rhythms are being used to identify and characterize clock mutants. Zebrafish heart, kidney, and embryonic cell lines contain circadian oscillators and phototransduction mechanisms for entrainment, suggesting that circadian pacemaking functions may be distributed throughout the animal. Studies of circadian system development in zebrafish have found that a molecular circadian oscillation in unfertilized oocytes persists through embryonic development with its phase intact, but that the pacemakers that drive rhythms of melatonin synthesis and behavior require environmental entraining signals late in development for initial synchronization. Zebrafish homologs of several of the core clock genes identified in other animals have been cloned. Transcripts for most of these are rhythmically expressed in multiple tissues. The interactions of clock gene products are for the most part similar to their interactions in mammals, although there are some potentially interesting differences.

Animals↗
PubMed2006

Zebrafish assays for drug toxicity screening.

Zebrafish are vertebrate organisms that are of growing interest for preclinical drug discovery applications. Zebrafish embryos develop most of the major organ systems present in mammals, including the cardiovascular, nervous and digestive systems, in < 1 week. Additional characteristics that make them advantageous for compound screening are their small size, transparency and ability to absorb compounds through the water. Furthermore, gene function analysis with antisense technology is now routine procedure. Thus, it is relatively simple to assess whether compounds or gene knockdowns cause toxic effects in zebrafish. Assays are being developed to exploit the unique characteristics of zebrafish for pharmacological toxicology. This review discusses assays that may be used to assess in vivo toxicity and provides examples of compounds known to be toxic to humans that have been demonstrated to function similarly in zebrafish.

Animals↗
PubMed2004

Functionally distinctive testicular cell lines of zebrafish to support male germ cell development.

Here we describe two testicular cell lines of zebrafish with distinct functions to support the development of male germ cells. Twelve cell lines were established by single-colony isolation from tumor-like testis-derived ZtA6 cells, and the features characteristic of Sertoli cells such as phagocytic activity and transcription both of their specific genes, sox9a and Wilms' tumor suppressor WT1, and of the vas gene of germ cells were analyzed in the lines. Six lines exhibited these three characteristics of Sertoli cells. Despite the single-colony isolation, low levels of vas expression were detected in three lines. Two lines, ZtA6-2 and ZtA6-12, showed almost the same characteristics as Sertoli cells, but exhibited distinctive features when male germ cells were cocultured with each line as feeders. The in vitro fertilization by the culture of germ cells with ZtA6-12 produced more embryos than that with ZtA6-2. In contrast, ZtA6-2 gave rise to large clumps of germ cells after a 12-day culture, while ZtA6-12 still produced only small clumps. Expression of vas, strongly expressed in spermatogonia and premeiotic spermatocytes, was prolonged in the culture with ZtA6-2, while it reduced in that with ZtA6-12. Compared with the previous results obtained on the original ZtA6 cells, these results suggested that the function of the ZtA6-2 cells was directed to stimulate the proliferation of spermatogonia, and ZtA6-12 to stimulate the differentiation into sperm. These cell lines will facilitate investigation of Sertoli cell molecules that contribute to the proliferation and differentiation of spermatogonia, and the longer culture time of male germ cells by using these lines successively.

Animals↗
PubMed1999

Gata5 is required for the development of the heart and endoderm in zebrafish.

The mechanisms regulating vertebrate heart and endoderm development have recently become the focus of intense study. Here we present evidence from both loss- and gain-of-function experiments that the zinc finger transcription factor Gata5 is an essential regulator of multiple aspects of heart and endoderm development. We demonstrate that zebrafish Gata5 is encoded by the faust locus. Analysis of faust mutants indicates that early in embryogenesis Gata5 is required for the production of normal numbers of developing myocardial precursors and the expression of normal levels of several myocardial genes including nkx2.5. Later, Gata5 is necessary for the elaboration of ventricular tissue. We further demonstrate that Gata5 is required for the migration of the cardiac primordia to the embryonic midline and for endodermal morphogenesis. Significantly, overexpression of gata5 induces the ectopic expression of several myocardial genes including nkx2.5 and can produce ectopic foci of beating myocardial tissue. Together, these results implicate zebrafish Gata5 in controlling the growth, morphogenesis, and differentiation of the heart and endoderm and indicate that Gata5 regulates the expression of the early myocardial gene nkx2.5.

Amino Acid Sequence↗
PubMed1996

Early ear development in the embryo of the zebrafish, Danio rerio.

The zebrafish provides an important model for vertebrate inner ear development. The otic placode becomes visible at approximately 16 hours (at 28.5 degrees C) and forms a vesicle with a lumen by cavitation at approximately 18 hours. Two otoliths appear in the lumen by 19.5 hours, and at about 24 hours the first sensory hair cells are seen, grouped in two small patches, one beneath each otolith, corresponding to future maculae. Staining with fluorescent phalloidin reveals 10-20 hair cells in each macula by 42 hours; between 3 days and 7 days the numbers grow to approximately 80 per macula. Neurons of the statoacoustic ganglion are first visible by staining with HNK-1 antibody at about 24 hours. Serial sections and time-lapse films show that the neuronal precursors originate by delamination from the ventral face of the otocyst; the peak period of delamination is from 22 hours to 30 hours. The system of semicircular canals forms between 42 hours and 72 hours by outgrowth of protrusions from the walls of the otocyst to form pillars of tissue spanning the lumen. Three further clusters of hair cells also become visible in this period, forming the three cristae. Thus, by the end of the first week, all key components of the ear are present. Subsequent growth produces thousands more hair cells; additional neurons probably derive from proliferation of neuronal precursors within the ganglion. Although the timetable is species-specific, the principles of inner ear development in the zebrafish seem to be the same as in other vertebrates.

Animals↗