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Results for “zebrafish embryos”

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PubMed1996

The nuclear localization sequence of the SV40 T antigen promotes transgene uptake and expression in zebrafish embryo nuclei.

We report luciferase expression in zebrafish embryos after cytoplasmic injection of low copy numbers of plasmid DNA coupled to the SV40 T antigen nuclear localization sequence (NLS). Binding of NLS to plasmid DNA (pCMVL) occurs at room temperature in 0.25 M KCl, as assayed by gel retardation at molar ratios of NLS:pCMVL of at least 100:1. Luciferase expression is induced in 35% of embryos with as low as 10(3) NLS-bound pCMVL copies. With 10(4) copies, the proportion of expression increases from 6% at 0:1 to 70% 100:1 NLS:pCMVL (p < 0.01). The beneficial effect of NLS is abolished at DNA concentrations promoting high frequencies of transgene expression without NLS. Regardless of the DNA concentration, the use of NLS does not affect embryo viability for at least up to 10 days. The specificity of NLS on luciferase expression was tested by using a nuclear import deficient reverse NLS peptide (revNLS). revNLS binds to pCMVL, causing gel retardation similarly to NLS, but does not promote transgene expression. Binding of equimolar amounts of revNLS and NLS to DNA reduces by 50% the beneficial effect of NLS on transgene expression. The results suggest efficient targeting of MLS-bound plasmid DNA to the nucleus, and subsequent enhanced uptake of DNA by the nucleus. The data suggest that the use of NLS may reduce the need for using elevated DNA copy numbers in some gene transfer applications.

Amino Acid Sequence↗

PubMed2004

Characterization of mid-spindle microtubules during furrow positioning in early cleavage period zebrafish embryos.

We report evidence to suggest that during the first few meroblastic cell divisions in zebrafish embryos a dynamic population of central-spindle microtubules serve a crucial function in positioning the cleavage furrow at the surface of the blastoderm. Originating from the mid-zone of the mitotic spindle they develop into what we term a mid-spindle 'pre-furrowing microtubule array' that expands upward and outward from the spindle mid-zone towards the blastodisc surface. We suggest that this structure transmits positional information to the blastodisc cortex that results in the correctly positioned assembly of the cytokinetic contractile apparatus. We also propose that the pre-furrowing microtubule array then develops into a furrow-ingression microtubule array that helps direct and assemble the deepening furrow as it cuts its way through the blastodisc. Due to the location of its origin, the pre-furrowing microtubule array serves to successfully separate the daughter nuclei and thus equally divide the blastoderm. Furthermore, co-localization with elements of the cortical endoplasmic reticulum and their inositol 1,4,5-trisphosphate receptors suggests that the pre-furrowing microtubule array may also play a role in organizing localized Ca2+ transients that have been shown to be essential to the furrow positioning, propagation and deepening process during cytokinesis in zebrafish embryos.

Animals↗

PubMed2001

Expression of brain subtype creatine kinase in the zebrafish embryo.

Creatine kinases (CK) play crucial roles in intracellular energy transfer. We have isolated a cDNA from zebrafish embryos, which encodes a CK highly related to the mammalian brain subtype creatine kinase (BCK). The bck mRNA is expressed maternally in the zebrafish embryo and transcripts are distributed uniformly in blastula and gastrula stages. Expression becomes restricted to the prechordal plate and the nervous system during subsequent somitogenesis stages. bck transcripts are abundant in primary neurons in the developing central nervous system of the 1-day-old embryo. While some bck expression persists in the hindbrain, expression vanishes in the spinal cord of the 2-day-old embryo. In summary, the expression pattern of bck is highly dynamic and suggests a role for bck during gastrulation and neuronal differentiation.

Amino Acid Sequence↗

PubMed2005

Expression of complex-type N-glycans in developmental periods of zebrafish embryo.

As a first step to elucidate a role of N-glycans in development of vertebrates, we analyzed structures of the glycans expressed in early stages of zebrafish embryo. N-glycans were prepared from zebrafish embryos at several developmental stages followed by tagging with a fluorophore, 2-aminopyridine. The labeled glycans were analyzed by two modes of HPLC's. The comparison of the elution profiles of HPLC's unveil the change of the oligosaccharide structure during the development. These peaks were merely detected during 4-7 h after fertilization, however, increased from 12 h, and at 15 h a fairly amount of them was appeared. Structure analysis revealed that they were bianntenary complex-type N-glycans with or without fucose and/or bisecting N-acetylglucosamine residues. These results suggest that the complex-type N-glycans are concerned in some developmental event from segmentation period downward in zebrafish.

Animals↗

PubMed2005

Investigating chorion softening of zebrafish embryos with a microrobotic force sensing system.

The zebrafish is a model organism for addressing questions of vertebrate embryo development. In this paper, the softening phenomenon of the chorion envelope of zebrafish embryos at different developmental stages was mechanically quantitated by using a microrobotic force sensing system. The microrobotic system integrates a piezoelectric cellular force sensor to measure the required forces for penetrating the chorion envelope. Magnitude of penetration forces was found to decrease as an embryo develops. The results mechanically quantitate "chorion softening" in zebrafish embryos due to protease activities subtly modifying the chorion structure, providing an understanding of zebrafish embryo development.

Animals↗

PubMed1997

Promoter analysis in living zebrafish embryos identifies a cis-acting motif required for neuronal expression of GATA-2.

We have used zebrafish embryos to dissect the promoter activity of a gene with a complex expression pattern during embryogenesis. GATA-2 is a transcription factor required for hematopoiesis and is dynamically expressed in hematopoietic tissues and in the central nervous system. Using constructs containing zebrafish GATA-2 genomic flanking sequences and the green fluorescent protein (GFP) reporter gene, we demonstrate that distinct regulatory domains are required for hematopoietic, enveloping layer (EVL), and neuronal expression of GATA-2. During gastrulation, GFP expression is confined to the ventral ectoderm and lateral mesoderm and is lacking in the dorsal shield. Cells derived from the regions expressing GFP give rise to hematopoietic progenitors, EVL cells, and neurons. Deletion analysis of the 7.3-kb GATA-2 promoter region revealed that a 1.1-kb DNA sequence is critical for expression of GATA-2 in neurons. Fine mapping revealed that a 31-bp region is required for neuron enhancer activity, and mutagenesis showed that the DNA motif CCCTCCT is essential for GATA-2 promoter activity in the central nervous system of zebrafish. Our use of zebrafish embryos can be exploited as a whole animal system for the dissection of any developmentally regulated vertebrate promoter.

Animals↗

PubMed2006

MyD88 innate immune function in a zebrafish embryo infection model.

Innate immunity signaling mechanisms during vertebrate embryogenesis are largely unknown. To study Toll-like receptor (TLR) signaling function in the zebrafish embryo model, we designed an experimental setup for antisense morpholino knockdown under conditions of bacterial infection. Clearance of Salmonella enterica serovar Typhimurium Ra bacteria was significantly impaired after knockdown of myeloid differentiation factor 88 (MyD88), a common adaptor protein in TLR and interleukin-1 receptor signaling. Thereby, we demonstrate for the first time that the innate immune response of the developing embryo involves MyD88-dependent signaling, which further establishes the zebrafish embryo as a model for the study of vertebrate innate immunity.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development.

When aequorin is microinjected into cleavage-stage zebrafish embryos, it is largely used up by ~24 hours. Thus, it is currently not possible to image Ca(2+) signals from later stages of zebrafish development using this approach. We have, therefore, developed protocols to express apoaequorin, i.e., the protein component of aequorin, transiently in zebrafish embryos and then reconstitute intact aequorin in vivo by loading the coelenterazine co-factor into the embryos separately. Two types of apoaequorin mRNA, aeq-mRNA and aeq::EGFP-mRNA, the latter containing the enhanced green fluorescent protein (EGFP) sequence, were in vitro transcribed and when these were microinjected into embryos, they successfully translated apoaequorin and a fusion protein of apoaequorin and EGFP (apoaequorin-EGFP), respectively. We show that aeq::EGFP -mRNA was more toxic to embryos than equivalent amounts of aeq-mRNA. In addition, in an in vitro reconstitution assay, apoaequorin-EGFP produced less luminescence than apoaequorin, after reconstitution with coelenterazine and with the addition of Ca(2+). Furthermore, when imaging intact coelenterazine-loaded embryos that expressed apoaequorin, Ca(2+ )signals from ~2.5 to 48 hpf were observed, with the spatio-temporal pattern of these signals up to 24 hpf, being comparable to that observed with aequorin. This transient aequorin expression approach using aeq-mRNA provides a valuable tool for monitoring Ca(2+ )signaling during the 2448 hpf period of zebrafish development. Thus, it effectively extends the aequorin-based Ca(2+) imaging window by an additional 24 hours.

Aequorin↗

PubMed1998

Modulation of plasmid DNA methylation and expression in zebrafish embryos.

Gene expression is under the influence of DNA methylation and assembly of chromatin structure. This paper reports the modulation of transgene expression in zebrafish embryos by altering DNA methylation with 5-azacytidine and heterochromatin formation with sodium butyrate, an inhibitor of histone deacetylation. A CMV promoter-luciferase fusion gene construct (pCMVL) microinjected into zebrafish eggs becomes gradually methylated during development, starting at approximately 12 h post-injection. When methylated in vitro by Hpa II methylase prior to injection, the construct is rapidly demethylated in vivo before being de novo methylated. Demethylation is independent of DNA replication, indicating that it is an active DNA repair process. Demethylating activity has been characterized in zebrafish embryo nuclear extracts, in which this activity is heat-labile, sensitive to protease and RNase and requires ATP hydrolysis. Demethylating activity in vitro is dependent on the developmental stage of the embryo from which extracts are prepared. In vivo , luciferase transcripts are detected prior to de novo plasmid methylation. Furthermore, incubation of pCMVL-injected embryos with 5-azacytidine or butyrate immediately after injection inhibits plasmid methylation and extends the period of luciferase expression. When applied after de novo methylation has occurred, both inhibitors prevent methylation of newly replicated DNA and promote transgene expression. These data suggest that methylation of the injected construct during early development induces repression of the transgene, perhaps by converting the construct to a repressive chromatin structure.

Animals↗

PubMed2001

Photo-mediated gene activation using caged RNA/DNA in zebrafish embryos.

We report a new and simple technique for photo-mediated temporal and spatial control of gene activation in zebrafish embryos as an alternative to the gene 'knockdown' approach using antisense, morpholino-modified oligonucleotides (morpholinos). The synthetic compound 6-bromo-4-diazomethyl-7-hydroxycoumarin (Bhc-diazo) forms a covalent bond with the phosphate moiety of the sugar-phosphate backbone of RNA, a process known as caging. The 6-bromo-7-hydroxycoumarin-4-ylmethyl (Bhc) group binds to approximately 30 sites on the phosphate moieties per 1 kb of RNA sequence. Bhc-caged mRNA undergoes photolysis (uncaging) when exposed to long-wave ultraviolet light (350 to 365 nm). We show that Bhc-caged green fluorescent protein (Gfp) mRNA has severely reduced translational activity in vitro, whereas illumination of Bhc-caged mRNA with ultraviolet light leads to partial recovery of translational activity. Bhc-caged mRNA is highly stable in zebrafish embryos. In embryos injected with Bhc-caged Gfp mRNA at the one-cell stage, GFP protein expression and fluorescence is specifically induced by ultraviolet light. We also show that, consistent with results obtained using other methods, uncaging eng2a (which encodes the transcription factor Engrailed2a) in the head region during early development causes a severe reduction in the size of the eye and enhanced development of the midbrain and the midbrain-hindbrain boundary at the expense of the forebrain.

Animals↗

PubMed1997

Effects of hirudin (thrombin specific inhibitor) in zebrafish embryos: a developmental role for thrombin.

To address the role of thrombin in early embryogenesis, hirudin, a thrombin specific inhibitor was microinjected into developing zebrafish embryos to inhibit the temporal activity of thrombin during early embryonic development. We found that the fibrin forming activity is inhibited by the presence of hirudin. We also found that hirudin affects development in zebrafish embryos suggesting thrombin's role in early embryogenesis. This ability to inhibit thrombin activity in developing embryos should facilitate studies on identifying signal transduction pathways affected by thrombin during embryogenesis.

Animals↗

PubMed2003

Multiple regulatory elements with spatially and temporally distinct activities control neurogenin1 expression in primary neurons of the zebrafish embryo.

The basic Helix-Loop-Helix gene neurogenin1 (ngn1) is expressed in a complex pattern in the neural plate of zebrafish embryos, demarcating the sites of primary neurogenesis. We have dissected the ngn1 locus to identify cis-regulatory regions that control this expression. We have isolated two upstream elements that drive expression in precursors of Rohon-Beard sensory neurons and hindbrain interneurons and in clusters of neuronal precursors in the anterior neural plate, respectively. A third regulatory region mediates later expression. Thus, regulatory sequences with temporally and spatially distinct activities control ngn1 expression in primary neurons of the zebrafish embryo. These regions are highly similar to 5' sequences in the mouse and human ngn1 gene, suggesting that amniote embryos, despite lacking primary neurons, utilize related mechanism to control ngn1 expression.

Amino Acid Sequence↗

PubMed2006

A chemical genetic screen for cell cycle inhibitors in zebrafish embryos.

Chemical genetic screening is an effective strategy to identify compounds that alter a specific biological phenotype. As a complement to cell line screens, multicellular organism screens may reveal additional compounds. The zebrafish embryo is ideal for small molecule studies because of its small size and the ease of waterborne treatment. We first examined a broad range of known cell cycle compounds in embryos using the mitotic marker phospho-histone H3. The majority of the known compounds exhibited the predicted cell cycle effect in embryos. To determine whether we could identify novel compounds, we screened a 16 320-compound library for alterations of pH3. This screen revealed 14 compounds that had not been previously identified as having cell cycle activity despite numerous mitotic screens of the same library with mammalian cell lines. With six of the novel compounds, sensitivity was greater in embryos than cell lines, but activity was still detected in cell lines at higher doses. One compound had activity in zebrafish embryos and cell lines but not in mammalian cell lines. The remaining compounds exhibited activity only in embryos. These findings demonstrate that small molecule screens in zebrafish can identify compounds with novel activity and thus may be useful tools for chemical genetics and drug discovery.

Animals↗

PubMed1995

Green fluorescent fusion proteins: powerful tools for monitoring protein expression in live zebrafish embryos.

The recent development of transgenic technology in zebrafish has opened an exciting new avenue in which to explore vertebrate development. However, as in other species, the inability to easily identify live transgenic fish severely limits the potential of this promising technology. To determine whether the recently described green fluorescent protein (GFP) might provide a convenient live staining method in zebrafish, we constructed a glutathione S-transferase/GFP fusion protein (GST-GFP). GST-GFP cRNA, when injected into individual blastomeres of early zebrafish embryos, resulted in the rapid development (3 hr) of easily detectable green fluorescence which persisted for up to 4 days. GFP fluorescence was restricted to progeny of the injected cell and appeared to have no adverse effects on embryonic development despite widespread expression. Our findings demonstrate that GFP fusion proteins will provide a simple yet powerful means of monitoring production of heterologous proteins in live zebrafish.

Animals↗

PubMed1998

Time course of the development of motor behaviors in the zebrafish embryo.

The development and properties of locomotor behaviors in zebrafish embryos raised at 28.5 degrees C were examined. When freed from the chorion, embryonic zebrafish showed three sequential stereotyped behaviors: a transient period of alternating, coiling contractions followed by touch-evoked rapid coils, then finally, organized swimming. The three different behaviors were characterized by video microscopy. Spontaneous, alternating contractions of the trunk appeared suddenly at 17 h postfertilization (hpf), with a frequency of 0.57 Hz, peaked at 19 hpf at 0.96 Hz, and gradually decreased to <0.1 Hz by 27 hpf. Starting at 21 hpf, touching either the head or the tail of the embryos resulted in vigorous coils. The coils accelerated with development, reaching a maximum speed of contraction before 48 hpf, which is near the time of hatching. After 27 hpf, touching the embryos, particularly on the tail, could induce partial coils (instead of full coils). At this time, embryos started to swim in response to a touch, preferentially to the tail. The swim cycle frequency gradually increased with age from 7 Hz at 27 hpf to 28 Hz at 36 hpf. Lesions of the central nervous system rostral to the hindbrain had no effect on the three behaviors. Lesioning the hindbrain eliminated swimming and touch responses, but not the spontaneous contractions. Our observations suggest that the spontaneous contractions result from activation of a primitive spinal circuit, while touch and swimming require additional hindbrain inputs to elicit mature locomotor behaviors.

Aging↗

PubMed1998

Preparation and imaging of nuclear spreads from cells of the zebrafish embryo. Evidence for large degradation intermediates in apoptosis.

We describe a method for preparing nuclear spreads from cells of live, unfixed zebrafish embryos at the late-gastrula (approximately 8000 cell) stage of development. The method consists of a sequence of four steps: (1) a slow, gentle lysis, in low to moderate salt concentration, of cells and then nuclei, to release DNA-containing fibres; (2) spreading of the released fibres by a transverse fluid flow; (3) electrostatic, and possibly also covalent, attachment of the spread fibers to poly(L-lysine)-coated glass microscope slides; and (4) continued incubation to produce periodic cleavage of the DNA within the fibres, apparently through activation of endogenous nucleases. The nuclear spreads are imaged with epifluorescence, at a spatial resolution approaching the Rayleigh limit (approximately 230 nm for blue light). The epifluorescent signal is provided from Hoechst 33,258 bound specifically to the DNA, from a dye-coupled antibody conjugate bound specifically to histone H1 in the fibres, or from a DNA nick end-labelling assay. The spontaneous cleavage of DNA-containing fibres in step (4) of the above procedure can be blocked by the chelating agents EGTA and EDTA, by the caspase-2,3,7 inhibitor N-acetyl-Asp-Glu-Val-Asp-aldehyde, and by the caspase-1,4,5 inhibitors N-acetyl-Tyr-Val-Ala-Asp-aldehyde and N-acetyl-Tyr-Val-Ala-Asp-chloromethyl ketone. These data suggest that the spontaneous cleavage of fibres is catalysed by nucleases that become activated through a caspase-mediated mechanism. The involvement of caspase-dependent nucleases would suggest that an apoptosis pathway is activated in the spreads during their prolonged incubation. If bona fide apoptosis is induced in living zebrafish embryos by treatment with camptothecin (a topoisomerase I poison), and then nuclear spreads are prepared, we observe a similar fragmentation of the spread fibres. However, in this case the fragmentation is more rapid and complete. We hypothesize that, during the early phase of apoptosis, one or more endogenous nucleases are activated by a caspase-mediated mechanism. The nuclease(s) then specifically recognize and cleave a susceptible, periodically repeating feature of interphase chromatin.

Animals↗

PubMed1993

Commitment of cell fate in the early zebrafish embryo.

When do single cells in the early zebrafish embryo become irreversibly committed to a specific fate? Work with lineage tracing and fate mapping has shown that the marginal cells of the blastoderm give rise to hypoblast-derived fates (mesoderm and endoderm). However, experiments described here show that these marginal blastoderm cells remain pluripotent and uncommitted throughout the late blastula and early gastrula stages. Embryonic cells become committed to a hypoblast-derived fate at mid-gastrulation. Time-lapse photographic analysis reveals that committed cells, when transplanted heterotopically and heterochronically, can migrate along atypical pathways to reposition themselves within a more correct environment.

Animals↗

PubMed1997

Effect of inhibitors of DNA replication on early zebrafish embryos: evidence for coordinate activation of multiple intrinsic cell-cycle checkpoints at the mid-blastula transition.

We address the developmental activation, in the zebrafish embryo, of intrinsic cell-cycle checkpoints which monitor the DNA replication process and progression through the cell cycle. Eukaryotic DNA replication is probably carried out by a multiprotein complex containing numerous enzymes and accessory factors that act in concert to effect processive DNA synthesis (Applegren, N. et al. (1995) J. Cell. Biochem. 59, 91-107). We have exposed early zebrafish embryos to three chemical agents which are predicted to specifically inhibit the DNA polymerase alpha, topoisomerase I and topoisomerase II components of the DNA replication complex. We present four findings: (1) Before mid-blastula transition (MBT) an inhibition of DNA synthesis does not block cells from attempting to proceed through mitosis, implying the lack of functional checkpoints. (2) After MBT, the embryo displays two distinct modes of intrinsic checkpoint operation. One mode is a rapid and complete stop of cell division, and the other is an 'adaptive' response in which the cell cycle continues to operate, perhaps in a 'repair' mode, to generate daughter nuclei with few visible defects. (3) The embryo does not display a maximal capability for the 'adaptive' response until several hours after MBT, which is consistent with a slow transcriptional control mechanism for checkpoint activation. (4) The slow activation of checkpoints at MBT provides a window of time during which inhibitors of DNA synthesis will induce cytogenetic lesions without killing the embryo. This could be useful in the design of a deletion-mutagenesis strategy.

Animals↗