SEARCH · PubMed Health

Results for “zebrafish embryos”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 505 records · Page 28Linked to original sources

PubMed2002

A floor plate enhancer of the zebrafish netrin1 gene requires Cyclops (Nodal) signalling and the winged helix transcription factor FoxA2.

The floor plate is an organising centre that controls neural differentiation and axonogenesis in the neural tube. The axon guidance molecule Netrin1 is expressed in the floor plate of zebrafish embryos. To elucidate the regulatory mechanisms underlying expression in the floor plate, we scanned the netrin1 locus for regulatory regions and identified an enhancer that drives expression in the floor plate and hypochord of transgenic embryos. The expression of the transgene is ectopically activated by Cyclops (Nodal) signals but does not respond to Hedgehog signals. The winged-helix transcription factor foxA2 (also HNF3beta, axial) is expressed in the notochord and floor plate. We show that knock-down of FoxA2 leads to loss of floor plate, while notochord and hypochord development is unaffected, suggesting a specific requirement of FoxA2 in the floor plate. The transgene is ectopically activated by FoxA2, and expression of FoxA2 leads to rescue of floor plate differentiation in mutant embryos that are deficient in Cyclops signalling. Zebrafish and mouse use different signalling systems to specify floor plate. The zebrafish netrin1 regulatory region also drives expression in the floor plate of mouse and chicken embryos. This suggests that components of the regulatory circuits controlling expression in the floor plate are conserved and that FoxA2-given its importance for midline development also in the mouse-may be one such component.

Animals↗

PubMed2005

Tead proteins activate the Foxa2 enhancer in the node in cooperation with a second factor.

The cell population and the activity of the organizer change during the course of development. We addressed the mechanism of mouse node development via an analysis of the node/notochord enhancer (NE) of Foxa2. We first identified the core element (CE) of the enhancer, which in multimeric form drives gene expression in the node. The CE was activated in Wnt/beta-catenin-treated P19 cells with a time lag, and this activation was dependent on two separate sequence motifs within the CE. These same motifs were also required for enhancer activity in transgenic embryos. We identified the Tead family of transcription factors as binding proteins for the 3' motif. Teads and their co-factor YAP65 activated the CE in P19 cells, and binding of Tead to CE was essential for enhancer activity. Inhibition of Tead activity by repressor-modified Tead compromised NE enhancer activation and notochord development in transgenic mouse embryos. Furthermore, manipulation of Tead activity in zebrafish embryos led to altered expression of foxa2 in the embryonic shield. These results suggest that Tead activates the Foxa2 enhancer core element in the mouse node in cooperation with a second factor that binds to the 5' element, and that a similar mechanism also operates in the zebrafish shield.

Animals↗

PubMed2001

The role of vascular endothelial growth factor (VEGF) in vasculogenesis, angiogenesis, and hematopoiesis in zebrafish development.

Vascular endothelial growth factor (VEGF, VEGF-A), a selective mitogen for endothelial cells is a critical factor for vascular development. Two isoforms that differ in the presence of exons 6 and 7, Vegf(165) and Vegf(121), are the dominant forms expressed in zebrafish embryo. Simultaneous overexpression of both isoforms in the embryo results in increased production of flk1, tie1, scl, and gata1 transcripts, indicating a stimulation of both endothelial and hematopoietic lineages. We also demonstrate that vegf can stimulate hematopoiesis in zebrafish by promoting the formation of terminally differentiated red blood cells. Simultaneous overexpression of both isoforms also causes ectopic vasculature and blood cells in many of the injected embryos as well as pericardial edema in later stage embryos. Overexpression of vegf also resulted in earlier onset of flk1, tie1, scl, and gata1 expression in the embryo, indicating a possible role of vegf in stimulating the differentiation of both vascular and hematopoietic lineages. Co-injection of RNAs for both isoforms results in increased expression of three of these markers over and above that observed when either RNA is singly injected and analysis of vegf expression in the notochord mutants no tail and floating head suggests that the notochord patterns the formation of the dorsal aorta by stimulating adjacent somite cells to express vegf, which in turn functions as a signal in dorsal aorta patterning. Finally, studies of vegf expression in cloche mutant indicate that vegf expression is generally independent of cloche function. These results show that in the zebrafish embryo, vegf can not only stimulate endothelial cell differentiation but also hematopoiesis. Moreover, these effects are most dramatic when both vegf isoforms are co-expressed, indicating a synergistic effect of the expression of the two forms of the VEGF protein.

Animals↗

PubMed2006

Development of a zebrafish 4-day embryo-larval bioassay to assess toxicity of chemicals.

A 4-day embryo-larval zebrafish test, from blastula stage to hatching included, was developed. The observations of embryo developmental were made at different development stages, for which morphological, physiological, and behavioral endpoints were selected and quantified for unexposed and exposed embryos. The sensitivity and the ability of these endpoints to inform about mode of action (MoA) were established in testing three model toxicants with well-known toxic effects (propranolol, malathion, cadmium). Lethal, sublethal (heart rate/edema, spontaneous movements, and hatching rate/time disturbance), and teratogenic effects were detected for all the studied compounds. This bioassay allows characterization of impairments at different biological levels: neuromuscular, physiological, morphological, and behavioral, and brings useful information about the toxic MoA of the chemicals on nontarget organisms. In this sense to answers the chemical industries and international organization (EMEA) requirements for the environmental risk assessment of new chemicals and pharmaceuticals.

Animals↗

PubMed1998

Permeability of dechorionated one-cell and six-somite stage zebrafish (Brachydanio rerio) embryos to water and methanol.

The permeability of dechorionated one-cell and six-somite stage zebrafish (Brachydanio rerio) embryos to water and the cryoprotectant methanol at 22 degreesC was studied, using real-time video microscopy to determine the volumetric changes of the embryos during cryoprotectant exposure. The equilibrium volumetric behavior of the embryos and the Boyle-van't Hoff relationships were determined using sucrose as a nonpermeating compound. These showed the embryos to behave as nearly ideal osmometers over the range of 253-1724 mOsm, with osmotically inactive volumes of 72.9 and 82.6% for one-cell and six-somite stage embryos, respectively. The Boyle-van't Hoff relationship of the ovary eggs was also determined for comparison and their osmotically inactive volume was 63.9%. The Kedem-Katchalsky parameters of water permeability (Lp), cryoprotectant permeability (Ps), and reflection coefficient (sigma) were determined using DIFFCHAM software. The parameters reported in this study are phenomenological parameters referring to the overall embryo response. The mean values of these parameters were Lp = 0.34 and 0.35 (micrometer/min*atm), Ps = 0.45 and 0.04 (micrometer/s), and sigma = 0.88 and 0.93 for one-cell and six-somite stage embryos, respectively. While the water permeability of the dechorionated zebrafish embryos at different developmental stages remained relatively stable, the permeability to the cryoprotectant methanol (Ps) appeared to decrease during embryo development. The Ps and sigma values for methanol are the first reported for dechorionated fish embryos at these stages.

Animals↗

PubMed2005

Pigment pattern formation in the medaka embryo.

For the study of development of pigmentation, compared with mammalian models, fish offer the advantage of multiple chromatophore types and ready access to the developing embryo for observation and experimental manipulation. Compared with zebrafish embryos, medaka embryos have an additional unique chromatophore-type and superb properties for conditional mutation studies. The rich resources of medaka mutants, combined with data obtainable from other species, potentially offer information not otherwise readily available regarding chromatophore lineage. Here we summarize the embryonic development of normal medaka pigment pattern, based on observations using embryos of a panel of wild type and mutant fish. A more detailed description of development is available in the appendix of the on-line version of this paper (see Supplementary Material). We make some comparisons with zebrafish development to emphasize the increased power of both systems when utilized together. These two models will, in combination, be a powerful system for studies of the embryogenesis and evolution of pigmentation.

Animals↗

PubMed2002

Functional analysis of the proximal promoter regions of fish rhodopsin and myf-5 genes using transgenesis.

Little is known about the cis-acting elements controlling transcriptional activities of fish rhodopsin and myf-5 genes. Transgenic medaka and zebrafish were used to characterize promoters of carp (Cyprinus carpio) rhodopsin gene (cRh) and zebrafish (Danio rerio) myf-5 gene (myf-5), respectively. Transgenic medaka carrying different lengths of cRh upstream fragments fused with enhanced green fluorescence protein (EGFP) gene revealed several functional regions. Both upstream regions from -1261 to -163 bp (-1261/-163) and -163/-138 contributed to enhance the retina-specific activities of cRh. The cNRE (-75/-63) and CSE (-52/-46) motifs located at the cRh proximal promoter were sufficient to drive the transgene expressed in retinae. The -73/-68 within cNRE was also proved to be essential for the integrity of the minimal cis-regulatory elements. Regarding the myf-5, transgene expression in zebrafish embryos injected with different deletion fragments of zebrafish myf-5 upstream sequences showed that the -2776/-2456 and -1046/-844 regions contributed to the enhancer function. The -290/-154 region might be involved in controlling the translocation of progenitor muscle cells. Moreover, the upstream -82/-1 region was identified as a minimal promoter required for somite-specific expression. The -82/-62 region was the most critical sequence for myf-5 specificity.

Journal Article↗

PubMed1996

High transgene activity in the yolk syncytial layer affects quantitative transient expression assays in zebrafish Danio rerio) embryos.

For the purpose of studying the factors that cause wide variation in transient transgene expression in individual fish, a lacZ reporter gene linked to a carp beta-actin regulatory sequence was introduced into zebrafish embryos. As a general trend, a correlation between the number of transgene copies injected and the level of transgene expression was found. However, a substantial variation in the level of expression still occurred that could not be attributed to technical factors such as the difference in injected volume of the transgene. Co-injection of 32P-dCTP and transgene into the same embryo followed by detection of beta-galactosidase activity, has shown that the volume used for transgene injection, which was determined in terms of radioactivity, is not closely related to the level and location of transgene expression. Injection into the animal pole at zygote stage and the yolk cytoplasmic layer (YCL) at the 64-cell stage followed by determination of transgene expression in terms of unit injection volume, revealed that there are marked differences among tissues with regard to their capacity for transgene expression, and that the yolk syncytial layer is higher in this capacity. This high activity is assumed to be due to the high transcriptional activity or enhanced transgene replication in the syncytial layer, which is known to contain giant polyploid nuclei. The high levels of expression in the YSL may influence transient expression studies using quantitative comparative analyses and should be taken into consideration when expression data are derived from homogenates of yolk sac embryos.

Actins↗

PubMed2005

Slit-like 2, a novel zebrafish slit homologue that might involve in zebrafish central neural and vascular morphogenesis.

Nervous and vascular systems grow as parallel networks, indicating common cues in distal targets. We have identified a novel zebrafish gene slit-like 2 (slitl2) that might involve in zebrafish central neural and vascular morphogenesis. Whole-mount in situ hybridization of zebrafish embryo detected distinct signals of slitl2 transcripts in zebrafish midline structure of central nervous system similar to that of slits. Strong expression is also observed in zebrafish vasculature. Zebrafish slitl2 shares amino acid sequence identity of 41% with Homo sapiens slitl2 (vasorin) and Mus musculus slitl2, and 35%, 33% with Danio rerio slit3, slit2. Analysis of zebrafish slitl2 cripto growth factor domain, extracellular matrix protein slit domain, and putative signal peptide confirms that as a secreted and cell-surface protein slitl2 may be essential in axon guidance, vessel development, and axis patterning. These results provide evidence that slitl2 may play important roles in zebrafish central nervous system and vascular morphogenesis.

Amino Acid Sequence↗

PubMed2003

Isolation and characterisation of tilapia beta-actin promoter and comparison of its activity with carp beta-actin promoter.

The regulatory sequence including proximal promoter, untranslated exon 1 and intron 1 of the beta-actin gene from tilapia (Oreochromis niloticus) has been isolated and spliced to a beta-galactosidase reporter gene to test its activity. Comparisons of promoter activity have been carried out with three different constructs: (1) 1.6 kb tilapia beta-actin regulatory sequence, (2) 1.5 kb carp beta-actin regulatory sequence, and (3) 4.7 kb carp beta-actin regulatory sequence. Although the 1.6 kb tilapia beta-actin regulatory sequence gave slightly different expression patterns in tilapia embryos assayed by in situ X-gal staining, no difference was observed in expression level when the tilapia sequence was compared with the 4.7 kb carp beta-actin regulatory sequence by quantitative assay. In comparison with the 1.5 kb carp beta-actin regulatory sequence, the 1.6 kb tilapia beta-actin regulatory sequence gave higher expression levels in tilapia embryos, while a reverse result was observed in zebrafish embryos. In cell transfection experiments, the 1.6 kb tilapia beta-actin regulatory sequence showed three to four times better activity in blue gill cells than either the 4.7 kb carp beta-actin or the 1.5 kb carp beta-actin regulatory sequences. The 1.6 kb tilapia beta-actin regulatory sequence also drove higher reporter gene activity in somatic cells of tilapia than did the 4.7 kb carp beta-actin regulatory sequence following direct injection of constructs into muscle. Therefore, taken together, the data demonstrate that the tilapia beta-actin promoter can be used as an efficient regulatory sequence to produce autotransgenic tilapia.

Actins↗

PubMed2004

Spatio-temporal distribution of cellular retinol-binding protein gene transcripts (CRBPI and CRBPII) in the developing and adult zebrafish (Danio rerio).

We have cloned and determined the nucleotide sequence of the cDNA coding for a cellular retinol-binding protein type I (CRBPI) from zebrafish. The deduced amino acid sequence of the zebrafish CRBPI showed highest sequence identity ( approximately 59%) to the mammalian CRBPIs of the intracellular lipid-binding protein (iLBP) multigene family. Phylogenetic analysis clustered the zebrafish CRBPI to the CRBPI clade. The zebrafish CRBPI gene (rbp1) and CRBPII gene (rbp2) both consist of four exons separated by three introns, identical to all other iLBP genes in vertebrates. Two transcription start sites were identified in the rbp1 promoter and a single transcription start site was identified for rbp2. Radiation hybrid mapping assigned the zebrafish rbp1 gene to linkage group 16 and conserved syntenic genes were found by comparative analysis of mammalian orthologous rbp1 genes. RT-PCR detected mRNA transcripts in the adult intestine, liver, brain, ovary and testis for rbp1 gene and in the intestine and liver for rbp2 gene. Whole mount in situ hybridization of zebrafish embryos revealed rbp1 mRNA expression in the developing zebrafish central nervous system at specific sites that are known to have abundant retinoic acid distribution and significant retinoic acid action. Whole mount in situ hybridization also showed that the zebrafish rbp2 mRNA was localized specifically in the embryonic intestinal bulb and the developing intestine during the larval stage, implying a novel function for the rbp2 gene product during organogenesis and development of the zebrafish intestine.

Amino Acid Sequence↗

PubMed2006

Identification of multiple integrin beta1 homologs in zebrafish (Danio rerio).

BACKGROUND: Integrins comprise a large family of alpha,beta heterodimeric, transmembrane cell adhesion receptors that mediate diverse essential biological functions. Higher vertebrates possess a single beta1 gene, and the beta1 subunit associates with a large number of alpha subunits to form the major class of extracellular matrix (ECM) receptors. Despite the fact that the zebrafish (Danio rerio) is a rapidly emerging model organism of choice for developmental biology and for models of human disease, little is currently known about beta1 integrin sequences and functions in this organism. RESULTS: Using RT-PCR, complete coding sequences of zebrafish beta1 paralogs were obtained from zebrafish embryos or adult tissues. The results show that zebrafish possess two beta1 paralogs (beta1-1 and beta1-2) that have a high degree of identity to other vertebrate beta1 subunits. In addition, a third, more divergent, beta1 paralog is present (beta1-3), which may have altered ligand-binding properties. Zebrafish also have other divergent beta1-like transcripts, which are C-terminally truncated forms lacking the transmembrane and cytoplasmic domains. Together with beta1-3 these truncated forms comprise a novel group of beta1 paralogs, all of which have a mutation in the ADMIDAS cation-binding site. Phylogenetic and genomic analyses indicate that the duplication that gave rise to beta1-1 and beta1-2 occurred after the divergence of the tetrapod and fish lineages, while a subsequent duplication of the ancestor of beta1-2 may have given rise to beta1-3 and an ancestral truncated paralog. A very recent tandem duplication of the truncated beta1 paralogs appears to have taken place. The different zebrafish beta1 paralogs have varied patterns of temporal expression during development. Beta1-1 and beta1-2 are ubiquitously expressed in adult tissues, whereas the other beta1 paralogs generally show more restricted patterns of expression. CONCLUSION: Zebrafish have a large set of integrin beta1 paralogs. beta1-1 and beta1-2 may share the roles of the solitary beta1 subunit found in other vertebrates, whereas beta1-3 and the truncated beta1 paralogs may have acquired novel functions.

Amino Acid Sequence↗

PubMed2006

Functional dissection of the Tol2 transposable element identified the minimal cis-sequence and a highly repetitive sequence in the subterminal region essential for transposition.

The Tol2 element is a naturally occurring active transposable element found in vertebrate genomes. The Tol2 transposon system has been shown to be active from fish to mammals and considered to be a useful gene transfer vector in vertebrates. However, cis-sequences essential for transposition have not been characterized. Here we report the characterization of the minimal cis-sequence of the Tol2 element. We constructed Tol2 vectors containing various lengths of DNA from both the left (5') and the right (3') ends and tested their transpositional activities both by the transient excision assay using zebrafish embryos and by analyzing chromosomal transposition in the zebrafish germ lineage. We demonstrated that Tol2 vectors with 200 bp from the left end and 150 bp from the right end were capable of transposition without reducing the transpositional efficiency and found that these sequences, including the terminal inverted repeats (TIRs) and the subterminal regions, are sufficient and required for transposition. The left and right ends were not interchangeable. The Tol2 vector carrying an insert of >11 kb could transpose, but a certain length of spacer, <276 but >18 bp, between the left and right ends was necessary for excision. Furthermore, we found that a 5-bp sequence, 5'-(A/G)AGTA-3', is repeated 33 times in the essential subterminal region. Mutations in the repeat sequence at 13 different sites in the subterminal region, as well as mutations in TIRs, severely reduced the excision activity, indicating that they play important roles in transposition. The identification of the minimal cis-sequence of the Tol2 element and the construction of mini-Tol2 vectors will facilitate development of useful transposon tools in vertebrates. Also, our study established a basis for further biochemical and molecular biological studies for understanding roles of the repetitive sequence in the subterminal region in transposition.

Animals↗

PubMed2006

Three types of cilia including a novel 9+4 axoneme on the notochordal plate of the rabbit embryo.

Motile monocilia play a pivotal role in left-right axis determination in mouse and zebrafish embryos. Cilia with 9+0 axonemes localize to the distal indentation of the mouse egg cylinder ("node"), while Kupffer's vesicle cilia in zebrafish show 9+2 arrangements. Here we studied cilia in a prototype mammalian embryo, the rabbit, which develops via a flat blastodisc. Transcription of ciliary marker genes Foxj1, Rfx3, lrd, polaris, and Kif3a initiated in Hensen's node and persisted in the nascent notochord. Cilia emerged on cells leaving Hensen's node anteriorly to form the notochordal plate. Cilia lengthened to about 5 mum and polarized from an initially central position to the posterior pole of cells. Electron-microscopic analysis revealed 9+0 and 9+2 cilia and a novel 9+4 axoneme intermingled in a salt-and-pepper-like fashion. Our data suggest that despite a highly conserved ciliogenic program, which initiates in the organizer, axonemal structures may vary widely within the vertebrates.

Animals↗

PubMed2005

Cloning and biological analysis of the zebrafish (Danio rerio) insulin-like growth factor binding protein-2 proximal promoter region.

Insulin-like growth factor binding protein 2 (IGFBP-2) plays an important role in the regulation of IGF's action and endocrinology in fish. To understand the molecular mechanism which controls transcription of the IGFBP-2 gene, we cloned and sequenced the IGFBP-2 proximal promoter region of the zebrafish IGFBP-2 gene and characterized its activity by firefly luciferase transient transfection expression assays. Different fragments of the zebrafish IGFBP-2 5'-flanking region were transfected into Hela and ZFL cells. In these cell lines, maximum promoter activity was located in the 900 base pairs (bp) of the zebrafish IGFBP-2 5' flanking region in the ZFL cell line and 318 bp of the zebrafish IGFBP-2 5' flanking region in the Hela cell line. The in vivo actions of the IGFBP-2 promoter on developmental stage expression were further investigated in transgenic zebrafish in which an IGFBP-2 (900-bp) promoter-driven green fluorescent protein encoding the GFP cDNA transgene was microinjected into zebrafish embryos. Morphological and RT-PCR studies of transgenic zebrafish indicated that the IGFBP-2 promoter-driven GFP transcripts appeared for the first time in the 32-cell stage. These results indicate that the IGFBP-2 promoter is active in a development-specific manner. These results suggest that the IGFBP-2 promoter plays an important role in teleost embryo growth.

Animals↗

PubMed1996

Vertebrate heart development.

This review summarizes recent studies of the cellular and molecular events involved in the determination and differentiation of cardiac myocytes in vertebrate embryos. Fate-mapping studies in mouse, chick, amphibian and zebrafish embryos suggest that cardiac muscle precursors are specified shortly before or at the time of gastrulation. Nuclear factors, such as dHAND, aryl hydrocarbon receptor, GATA-6, Nkx-2.3, growth arrest homeobox (Gax) and cardiac adriamycin responsive protein (CARP), which have recently been described as playing a role in the commitment and/or differentiation of cardiac myocytes are discussed.

Amphibians↗

PubMed2004

Tol2 transposon-mediated enhancer trap to identify developmentally regulated zebrafish genes in vivo.

We have used the Tol2 transposable element to design and perform effective enhancer trapping in zebrafish. Modified transposon DNA and transposase RNA were delivered into zebrafish embryos by microinjection to produce heritable insertions in the zebrafish genome. The enhancer trap construct carries the EGFP gene controlled by a partial epithelial promoter from the keratin8 gene. Enhanced green fluorescent protein (EGFP) is used as a marker to select F1 transgenic fish and as a reporter to trap enhancers. We have isolated 28 transgenic lines that were derived from the 37 GFP-positive F0 founders and displayed various specific EGFP expression patterns in addition to basal expression from the modified keratin 8 promoter. Analyses of expression by whole-mount RNA in situ hybridization demonstrated that these patterns could recapitulate the expression of the tagged genes to a variable extent and, therefore, confirmed that our construct worked effectively as an enhancer trap. Transgenic offspring from the 37 F0 EGFP-positive founders have been genetically analyzed up to the F2 generation. Flanking sequences from 65 separate transposon insertion sites were identified by thermal asymmetric interlaced polymerase chain reaction. Injection of the transposase RNA into transgenic embryos induced remobilization of genomic Tol2 copies producing novel insertions including some in the germ line. The approach has great potential for developmental and anatomical studies of teleosts.

Animals↗

PubMed2025

The snakehead retrovirus promoter functions independently of the 3'ORF protein and its products are maternally inherited in transgenic zebrafish.

The exogenous snakehead retrovirus (SnRV) is an unclassified member of the Orthoretrovirinae subfamily, discovered in cell lines derived from several fish species. SnRV resembles complex lentiviruses and potentially encodes accessory proteins, including the product of the 3' open reading frame (3'ORF). The 3'ORF protein was suggested to function as a transactivator of transcription (Tat). Here, we constructed an infectious molecular clone for SnRV and tested the effects of 3'ORF mutations on SnRV transcription. Although replacing 3'ORF with foreign sequences strongly reduced virus expression and production, an out-of-frame point mutation in 3'ORF had only a minimal effect on SnRV replication. This latter result suggests that the 3'ORF protein does not function as Tat and that SnRV transcription is largely independent of the product of this ORF. We also show that in vitro, the SnRV promoter is versatile and robustly functioning in both fish and mammalian cultured cells. Finally, the SnRV promoter was transiently active in injected zebrafish embryos as early as the blastula stage. In transgenic zebrafish, this promoter drives enhanced expression in sensory organs and gonads, and its generated products are maternally inherited. Considering these characteristics, the SnRV promoter emerges as a promising candidate for developing versatile expression vectors applicable to research and biotechnological applications.

Animals↗