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Results for “zebrafish embryos”

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At least 649 records · Page 36Linked to original sources

PubMed2005

Hematopoietic perturbation in zebrafish expressing a tel-jak2a fusion.

OBJECTIVE: Various TEL-JAK2 fusions have been identified in patients with lymphoblastic and myeloid leukemias that result in constitutive activation of the JAK2 kinase domain. Such fusions can mediate factor-independent growth of hematopoietic cell lines and induction of malignancy in mouse models. MATERIALS AND METHODS: To assess whether zebrafish could be utilized as a suitable model for the study of myeloid oncogenesis, we generated a zebrafish tel-jak2a fusion oncoprotein based on that seen in a case of chronic myeloid leukemia. This was transiently expressed in zebrafish embryos under the control of the spi1 promoter, which is strongly active in myeloid precursors. RESULTS: Visual, histological, and molecular analysis revealed disruption of normal embryonic hematopoiesis, including perturbation of the myeloid and erythroid lineages. CONCLUSION: These results indicate that the zebrafish tel-jak2a oncoprotein is functional, and suggest that this organism will be useful for the experimental study of myeloid malignancy.

Acute Disease↗

PubMed1999

Effects of lithium on pigmentation in the embryonic zebrafish (Brachydanio rerio).

Pigment cell precursors of the embryonic zebrafish give rise to melanophores, xanthophores and/or iridophores. Cell signaling mechanisms related to the development of pigmentation remain obscure. In order to examine the mechanisms involved in pigment cell signaling, we treated zebrafish embryos with various activators and inhibitors of signaling pathways. Among those chemicals tested, LiCl and LiCl/forskolin had a stimulatory effect on pigmentation, most notable in the melanophore population. We propose that the inositol phosphate (IP) pathway, is involved in pigment pattern formation in zebrafish through its involvement in the: (1) differentiation/proliferation of melanophores; (2) dispersion of melanosomes; and/or (3) synthesis/deposition of melanin. To discern at what level pigmentation was being effected we: (1) counted the number of melanophores in control and experimental animals 5 days after treatment; (2) measured tyrosinase activity and melanin content; and (3) employed immunoblotting techniques with anti-tyrosine-related protein-2 and anti-melanocyte-specific gene-1 as melanophore-specific markers. Although gross pigmentation increased dramatically in LiCl- and LiCl/forskolin treated embryos, the effect on pigmentation was not due to an increase in the proliferation of melanophores, but was possibly through an increase in melanin synthesis and/or deposition. Collectively, results from these studies suggest the involvement of an IP-signaling pathway in the stimulation of pigmentation in embryonic zebrafish through the synthesis/deposition of melanin within the neural crest-derived melanophores.

Animals↗

PubMed2006

Profiling of prostanoids in zebrafish embryonic development.

Prostanoids (PG) play important roles in vascular, pulmonary, reproductive and renal physiology. Little is known about their roles in the embryonic development. Using the oviparous zebrafish embryo as a model, we determined the temporal expression of PGs synthesized from exogenous prostaglandin H(2). Prostaglandin E(2) is the major PG throughout first 120 h post-fertilization (hpf), whereas prostaglandin F(2)(alpha) is at a lower but also a constant level. Reverse transcription-polymerase chain reaction (RT-PCR) showed that transcripts of cytosolic and membrane-bound PGE synthases were evident during the 120 hpf period. Compared with thromboxane A(2), the level of prostacyclin (PGI(2))is higher at first 24 hpf, the stage before the formation of blood vessel. RT-PCR showed that transcript of prostacyclin synthase appeared at 7 hpf whereas thromboxane synthase appeared at 48 hpf, suggesting that PGI(2) has additional functions besides hemostasis. Interestingly, level of prostaglandin D(2) (PGD(2)) followed an exponential decay over 120 hpf with a rate constant of 0.048 h(-1) and transcript of lipocalin-type PGD synthase was expressed at a higher level at early stage of development, suggesting that PGD(2) is highly regulated during embryogenesis.

Animals↗

PubMed2003

Changes in gravitational force induce alterations in gene expression that can be monitored in the live, developing zebrafish heart.

Little is known about the effect of microgravity on gene expression, particularly in vivo during embryonic development. Using transgenic zebrafish that express the gfp gene under the influence of a beta-actin promoter, we examined the affect of simulated-microgravity on GFP expression in the heart. Zebrafish embryos, at the 18-20 somite-stage, were exposed to simulated-microgravity for 24 hours. The intensity of GFP fluorescence associated with the heart was then determined using fluorescence microscopy. Our measurements indicated that simulated-microgravity induced a 23.9% increase in GFP-associated fluorescence in the heart. In contrast, the caudal notochord showed a 17.5% increase and the embryo as a whole showed only an 8.5% increase in GFP-associated fluorescence. This suggests that there are specific effects on the heart causing the more dramatic increase. These studies indicate that microgravity can influence gene expression and demonstrate the usefulness of this in vivo model of 'reporter-gene' expression for studying the effects of microgravity.

Animals↗

PubMed2006

Regulation of primary cilia formation and left-right patterning in zebrafish by a noncanonical Wnt signaling mediator, duboraya.

Primary cilia are microtubule-based organelles that project from the surface of nearly every animal cell. Although important functions of primary cilia in morphogenesis and tissue homeostasis have been identified, the mechanisms that control the formation of primary cilia are not understood. Here we characterize a zebrafish gene, termed duboraya (dub), that is essential for ciliogenesis. Knockdown of dub in zebrafish embryos results in both defects in primary cilia formation in Kupffer's vesicle and randomization of left-right organ asymmetries. We show that, at the molecular level, the function of dub in ciliogenesis is regulated by phosphorylation, which in turn depends on Frizzled-2-mediated noncanonical Wnt signaling. We also provide evidence that, at the cellular level, dub function is essential for actin organization in the cells lining Kupffer's vesicle. Taken together, our findings identify a molecular factor that links noncanonical Wnt signaling with the control of left-right axis specification, and provide an entry point for analyzing the mechanisms that regulate primary cilia formation.

Animals↗

PubMed2002

Zebrafish mitotic kinesin-like protein 1 (Mklp1) functions in embryonic cytokinesis.

To understand the functions of microtubule motors in vertebrate development, we are investigating the kinesin-like proteins (KLPs) of the zebrafish, Danio rerio. Here we describe the structure, intracellular distribution, and function of zebrafish mitotic KLP1 (Mklp1). The zebrafish mklp1 gene that encodes this 867-amino acid protein maps to a region of zebrafish linkage group 18 that is syntenic with part of human chromosome 15. In zebrafish AB9 fibroblasts and in COS-7 cells, the zebrafish Mklp1 protein decorates spindle microtubules at metaphase, redistributes to the spindle midzone during anaphase, and becomes concentrated in the midbody during telophase and cytokinesis. The motor is detected consistently in interphase nuclei of COS cells and occasionally in those of AB9 cells. Nuclear targeting of Mklp1 is conferred by two basic motifs located in the COOH terminus of the motor. In cleaving zebrafish embryos, green fluorescent protein (GFP)-tagged Mklp1 is found in the nucleus in interphase and associates with microtubules of the spindle midbody in cytokinesis. One- or two-cell embryos injected with synthetic mRNAs encoding dominant-negative variants of GFP-Mklp1 frequently fail to complete cytokinesis during cleavage, resulting in formation of multinucleated blastomeres. Our results indicate that the zebrafish Mklp1 motor performs a critical function that is required for completion of embryonic cytokinesis.

Amino Acid Sequence↗

PubMed2003

Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation.

Epithelial-to-mesenchymal transition (EMT) is fundamental to both embryogenesis and tumor metastasis. The Notch intercellular signaling pathway regulates cell fate determination throughout metazoan evolution, and overexpression of activating alleles is oncogenic in mammals. Here we demonstrate that Notch activity promotes EMT during both cardiac development and oncogenic transformation via transcriptional induction of the Snail repressor, a potent and evolutionarily conserved mediator of EMT in many tissues and tumor types. In the embryonic heart, Notch functions via lateral induction to promote a selective transforming growth factor-beta (TGFbeta)-mediated EMT that leads to cellularization of developing cardiac valvular primordia. Embryos that lack Notch signaling elements exhibit severely attenuated cardiac snail expression, abnormal maintenance of intercellular endocardial adhesion complexes, and abortive endocardial EMT in vivo and in vitro. Accordingly, transient ectopic expression of activated Notch1 (N1IC) in zebrafish embryos leads to hypercellular cardiac valves, whereas Notch inhibition prevents valve development. Overexpression of N1IC in immortalized endothelial cells in vitro induces EMT accompanied by oncogenic transformation, with corresponding induction of snail and repression of VE-cadherin expression. Notch is expressed in embryonic regions where EMT occurs, suggesting an intimate and fundamental role for Notch, which may be reactivated during tumor metastasis.

Animals↗

PubMed2002

A zebrafish sox9 gene required for cartilage morphogenesis.

The molecular genetic mechanisms of cartilage construction are incompletely understood. Zebrafish embryos homozygous for jellyfish (jef) mutations show craniofacial defects and lack cartilage elements of the neurocranium, pharyngeal arches, and pectoral girdle similar to humans with campomelic dysplasia. We show that two alleles of jef contain mutations in sox9a, one of two zebrafish orthologs of the human transcription factor SOX9. A mutation induced by ethyl nitrosourea changed a conserved nucleotide at a splice junction and severely reduced splicing of sox9a transcript. A retrovirus insertion into sox9a disrupted its DNA-binding domain. Inhibiting splicing of the sox9a transcript in wild-type embryos with splice site-directed morpholino antisense oligonucleotides produced a phenotype like jef mutant larvae, and caused sox9a transcript to accumulate in the nucleus; this accumulation can serve as an assay for the efficacy of a morpholino independent of phenotype. RNase-protection assays showed that in morpholino-injected animals, the percent of splicing inhibition decreased from 80% at 28 hours post fertilization to 45% by 4 days. Homozygous mutant embryos had greatly reduced quantities of col2a1 message, the major collagen of cartilage. Analysis of dlx2 expression showed that neural crest specification and migration was normal in jef (sox9a) embryos. Confocal images of living embryos stained with BODIPY-ceramide revealed at single-cell resolution the formation of precartilage condensations in mutant embryos. Besides the lack of overt cartilage differentiation, pharyngeal arch condensations in jef (sox9a) mutants lacked three specific morphogenetic behaviors: the stacking of chondrocytes into orderly arrays, the individuation of pharyngeal cartilage organs and the proper shaping of individual cartilages. Despite the severe reduction of cartilages, analysis of titin expression showed normal muscle patterning in jef (sox9a) mutants. Likewise, calcein labeling revealed that early bone formation was largely unaffected in jef (sox9a) mutants. These studies show that jef (sox9a) is essential for both morphogenesis of condensations and overt cartilage differentiation.

Alleles↗

PubMed2006

TBX1, a DiGeorge syndrome candidate gene, is inhibited by retinoic acid.

Both retinoic acid (RA) and Tbx1 are definitively indispensable for the development of the pharyngeal arches. The defects produced by a loss of Tbx1 highly resemble those induced by hyper- and hypo-RA. Based on these similarities, the effects of RA on Tbx1 expression pattern were explored during pharyngeal arch development in zebrafish. Whole-mount in situ hybridization and real-time quantitative PCR were used. Zebrafish embryos were treated with 5 x 10(-8)mol/L and 10(-7)mol/L RA at 12.5 hours post fertilization for 1.5 hours, respectively. Whole-mount in situ hybridization showed that Tbx1 was expressed in the cardiac region, pharyngeal arch and otic vesicle between 24 hpf and 72 hpf in zebrafish. Tbx1 expression was obviously reduced, even lost, in the pharyngeal arch and outflow tract in RA treated groups. Real-time quantitative PCR analysis showed that Tbx1 expression rose to a peak level at 36 hpf in wild type group. Repression of Tbx1 expression was most evident at 36 hpf, 24 hours after RA treatment. 10(-7 )mol/L RA caused a more severe effect on the Tbx1 expression level than 5 x 10(-8)mol/L RA. The results suggested that RA could produce an altered Tbx1 expression pattern in zebrafish. In addition, RA could repress Tbx1 expression in a dose-dependent manner.

Animals↗

PubMed2005

Analysis of the very large G-protein coupled receptor gene (Vlgr1/Mass1/USH2C) in zebrafish.

Very Large G-protein coupled Receptor-1 (VLGR1/Mass1/USH2C) is the largest known cell surface protein in vertebrates. Mutations in VLGR1 are associated with audiogenic epilepsy in mice and Usher syndrome (sensorineural deafness and retinitis pigmentosa) in humans. We characterized the zebrafish VLGR1 gene (vlgr1). It is 51% identical to human VLGR1 in amino acid sequence, but is 64% identical in the 7-transmembrane and cytoplasmic domains. It is 6199 amino acids in size and is encoded by a 19.2 kb mRNA. All introns correspond in location and phase to those of the human and mouse genes. In situ hybridization studies of zebrafish embryos demonstrate vlgr1 expression in the developing central nervous system, particularly in the hypothalamus, epiphysis and in the rhombic lips. Expression in the eye is associated with the optic nerve. Further studies using zebrafish may help ascertain the role of Vlgr1 in neural development.

Animals↗

PubMed2006

Dose-dependent effects of chemical immobilization on the heart rate of embryonic zebrafish.

The small size and optical transparency of zebrafish embryos and larvae greatly facilitate modern intravital microscopic phenotyping of these experimentally tractable laboratory animals. Neither the experimentally derived dose-response relationships for chemicals commonly used in the mounting of live fish larvae, nor their effect on the stress of the animal, are currently available in the research literature. This is particularly problematic for IACUCs attempting to maintain the highest ethical standards of animal care in the face of a recent spate in investigator-initiated requests to use embryonic zebrafish as experimental models. The authors address this issue by describing the dose-dependent efficacy of several commonly used chemical mounting treatments and their effect on one stress parameter, embryo heart rate. The results of this study empirically define, for the first time, effective, minimally stressful treatments for immobilization and in vivo visualization during early zebrafish development.

Aminobenzoates↗

PubMed2004

Slow degeneration of zebrafish Rohon-Beard neurons during programmed cell death.

Rohon-Beard cells are large, mechanosensory neurons located in the dorsal spinal cord of anamniote vertebrates. In most species studied to date, these cells die during development. We followed labeled Rohon-Beard cells in living zebrafish embryos and found that they degenerate slowly, over many days. During degeneration, the soma shrinks and finally disappears, and the processes become beady in appearance and finally break apart, but they do not retract. Zebrafish Rohon-Beard cells apparently fragment their DNA, as revealed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) labeling, before undergoing degenerative morphologic changes. We also followed the development of labeled dorsal root ganglion neurons, as they are developing at the same stages that Rohon-Beard cells are degenerating. We found that, although axons of both cell types extend into similar regions, Rohon-Beard cells degenerate normally in mutants lacking dorsal root ganglia, providing evidence that interactions between the two cell types are not responsible for Rohon-Beard cell degeneration. Developmental Dynamics 229:30-41,2004.

Animals↗

PubMed2001

The perplexed and confused mutations affect distinct stages during the transition from proliferating to post-mitotic cells within the zebrafish retina.

To identify and study genes essential for vertebrate retinal development, we are screening zebrafish embryos for mutations that disrupt retinal histogenesis. Key steps in retinogenesis include withdrawal from mitosis by multipotent neuroepithelial cells, specification to particular cell types, migration to the appropriate laminar positions, and molecular and morphological differentiation. In this study, we have identified two recessive mutations that affect the transition of proliferating neuroepithelial cells to postmitotic retinal cells. Both the perplexed and confused mutant phenotypes were initially detectable when the first retinal neuroepithelial cells began to leave the cell cycle. At this time, each mutant retina showed increased cell death and a lack of morphological differentiation. Cell death was found to be apoptotic in both perplexed and confused retinas based on TUNEL analysis and activation of caspase-3. TUNEL-phosphoRb-BrdU colocalization studies indicated that the perplexed mutation caused death in cells transitioning from a proliferative to postmitotic state. For the confused mutation, TUNEL-phosphoRb-BrdU analysis revealed that only a subset of postmitotic cells were induced to activate apoptosis. Mosaic analysis demonstrated that within the retina the perplexed mutation functions noncell-autonomously. Furthermore, whole lens or eye cup transplantations indicated that the retinal defect was intrinsic to the retina. Mosaic analysis with confused embryos showed this mutation acts cell-autonomously. From these studies, we conclude that the perplexed and confused genes are essential at distinct stages during the transition from proliferating to postmitotic cells within the zebrafish retina.

Animals↗

PubMed2002

Construction of a zebrafish cDNA microarray: gene expression profiling of the zebrafish during development.

Vertebrate embryogenesis is a complex process controlled by a transcriptional hierarchy that coordinates the action of thousands of genes. To identify and analyze the expression patterns of these genes, we constructed a zebrafish cDNA microarray containing 4512 unique genes identified from zebrafish embryonic heart, adult hearts, and skeletal muscle cDNA libraries. We examined the patterns of gene expression during development in the zebrafish between five time points relative to 12h post-fertilization (hpf). Differentially expressed genes can be grouped into two categories, early genes that are expressed at 5hpf and genes expressed at 48/72/120hpf. Furthermore, we report the utilization of cDNA microarray technology to investigate the adaptive molecular responses of zebrafish to hypoxia during development. Our study provides the first utilization of cDNA microarray in the zebrafish and reveals dynamic changes in levels of gene expression in relation to development and survival of the zebrafish embryos under hypoxic stress.

Animals↗

PubMed2003

Knockdown of zebrafish Fancd2 causes developmental abnormalities via p53-dependent apoptosis.

Mechanisms underlying the multiple developmental defects observed in Fanconi anemia (FA) patients are not well defined. We have identified the zebrafish homolog of human FANCD2, which encodes a nuclear effector protein that is monoubiquitinated in response to DNA damage, targeting it to nuclear foci where it preserves chromosomal integrity. Fancd2-deficient zebrafish embryos develop defects similar to those found in children with FA, including shortened body length, microcephaly, and microophthalmia, which are due to extensive cellular apoptosis. Developmental defects and increased apoptosis in Fancd2-deficient zebrafish were corrected by injection of human FANCD2 or zebrafish bcl2 mRNA, or by knockdown of p53, indicating that in the absence of Fancd2, developing tissues spontaneously undergo p53-dependent apoptosis. Thus, Fancd2 is essential during embryogenesis to prevent inappropriate apoptosis in neural cells and other tissues undergoing high levels of proliferative expansion, implicating this mechanism in the congenital abnormalities observed in human infants with FA.

Abnormalities, Multiple↗

PubMed1993

Pathway selection by growth cones in the zebrafish central nervous system.

The accessibility and simplicity of the zebrafish embryo have led to a detailed characterization of pathfinding by identifiable growth cones in both the spinal cord and brain. These growth cones follow precise, cell-specific pathways to their targets. Analyses of pathfinding in mutant embryos and wild-type embryos following laser ablation or transplantation of specific cells demonstrate that growth cones accomplish this by interacting with specific cellular cues in their environment, many of which are likely to be redundant. These experiments suggest that a combination of separate pathway and directionality cues are required for pathfinding by growth cones. Growth cones distinguish different pathways by sensing specific pathway cues and know what direction they should extend on a pathway by sensing widely distributed directionality cues.

Animals↗

PubMed1998

Cloning and characterization of vascular endothelial growth factor (VEGF) from zebrafish, Danio rerio.

We have cloned and sequenced a zebrafish (Danio rerio) Vascular Endothelial Growth Factor (vegf) cDNA. It encodes a precursor protein of 188 amino acids with a putative 23 amino acids signal peptide. Sequence comparison analysis indicates that the zebrafish vegf cDNA corresponds to the human VEGF165 isoform and shows about 52% identity to human VEGF165 at the amino acid level. A 2.8 kb vegf message RNA was detected in adult zebrafish by Northern blot analysis. Expression of vegf165 is also detected by RT-PCR in adult fish and throughout the zebrafish embryonic development. Whole mount in situ hybridization of zebrafish embryos indicates strong expression in four areas of the 18-19 h post-fertilization (hpf) embryo: within the anterior central nervous system in the prospective optic stalk, in mesoderm overlapping the bilaterally located merging heart fields, in mesoderm underlying and flanking the hindbrain posterior to rhombomere 4, and in medial regions of the somites. The study of vegf function in zebrafish embryonic vascular development will contribute to our understanding of the mechanisms of vertebrate endothelial cell differentiation and vasculature formation.

Amino Acid Sequence↗

PubMed2004

Immunoanalysis of antiviral Mx protein expression in Japanese flounder (Paralichthys olivaceus) cells.

Mx proteins are interferon-inducible GTPases that possess antiviral properties in vertebrates. Japanese flounder (Paralichthys olivaceus) Mx protein has previously been shown to possess some antiviral activity against rhabdoviruses. A polyclonal antibody was generated against a purified peptide fragment of Japanese flounder Mx protein that had been produced in an Escherichia coli expression system. The PAb detected the approximately 71 kDa Mx protein from Japanese flounder (hirame) natural embryo (HINAE) cells that had been cultured with poly I:C, an interferon inducer, but not in unstimulated cells. The polyclonal antibody did not cross react with Mx protein from carp epithelial, grouper fin and zebrafish embryo cell lines that had been similarly induced or transfected with poly I:C. By immunofluorescence cytochemistry, Japanese flounder Mx protein was localized to the cell cytoplasm. Hirame rhabdovirus stimulated expression of Mx protein in the infected and surrounding HINAE cells. Within virus-infected cells, there was some indication of Mx protein colocalizing with viral proteins. Poly I:C stimulation of HINAE cells induced an early increase in Mx protein mRNA transcripts, but maximum Mx mRNA transcript and protein expression was reached after 48 h. Both Mx mRNA transcripts and protein levels were maintained till at least 72 h.

Amino Acid Sequence↗