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PubMed2004

Expression of VE-cadherin in zebrafish embryos: a new tool to evaluate vascular development.

We have identified the zebrafish homologue of VE-cadherin and documented its expression in the developing vascular system. The zebrafish VE-cadherin gene is specifically expressed in the vascular endothelial cell lineage beginning with the differentiation and migration of angioblasts and persists throughout vasculogenesis, angiogenesis, and endocardium development. Staining zebrafish embryos by whole-mount in situ hybridization with the VE-cadherin probe provides a method to screen embryos for vascular defects. To illustrate this utility, we used VE-cadherin expression to demonstrate a conservation of vascular endothelial growth factor-A (VEGF-A) function. The morpholino antisense oligonucleotide knockdown of VEGF-A function in zebrafish embryos results in a loss of angiogenic blood vessels, as indicated by the lack of VE-cadherin expression in the intersegmental vasculature. This loss can be restored in embryos supplemented with either zebrafish or human VEGF-A, the latter indicating that genes crucial to angiogenesis have highly conserved functional activities in vertebrates.

Amino Acid Sequence↗

PubMed2004

Expression profiling and comparative genomics identify a conserved regulatory region controlling midline expression in the zebrafish embryo.

Differential gene transcription is a fundamental regulatory mechanism of biological systems during development, body homeostasis, and disease. Comparative genomics is believed to be a rapid means for the identification of regulatory sequences in genomes. We tested this approach to identify regulatory sequences that control expression in the midline of the zebrafish embryo. We first isolated a set of genes that are coexpressed in the midline of the zebrafish embryo during somitogenesis stages by gene array analysis and subsequent rescreens by in situ hybridization. We subjected 45 of these genes to Compare and DotPlot analysis to detect conserved sequences in noncoding regions of orthologous loci in the zebrafish and Takifugu genomes. The regions of homology that were scored in nonconserved regions were inserted into expression vectors and tested for their regulatory activity by transient transgenesis in the zebrafish embryo. We identified one conserved region from the connective tissue growth factor gene (ctgf), which was able to drive expression in the midline of the embryo. This region shares sequence similarity with other floor plate/notochord-specific regulatory regions. Our results demonstrate that an unbiased comparative approach is a relevant method for the identification of tissue-specific cis-regulatory sequences in the zebrafish embryo.

Animals↗

PubMed1997

Wnt5 is required for tail formation in the zebrafish embryo.

Intercellular signaling molecules, such as those encoded by the Wnt gene family, have a fundamental role in various aspects of pattern formation in the developing embryo. The zebrafish wnt5 gene encodes a member of a subfamily of Wnt molecules thought to be involved in modulating cell behavior during vertebrate development. Here, we show that the zebrafish pipetail gene is identical to wnt5. The pipetail mutant phenotype is characterized by defects in tail formation and impaired maturation of the cells that contribute to cartilaginous elements of the head skeleton. This suggests a major role for wnt5 in morphogenetic processes underlying tail outgrowth and cartilage differentiation in the head. To investigate the function of maternally derived wnt5 mRNA, we generated females that were homozygous for pipetail. The lack of a maternal effect phenotype in the progeny of these females suggests that no obvious function for the maternal wnt5 expression can be deduced.

Amino Acid Sequence↗

PubMed2003

Zebrafish embryos express an orthologue of HERG and are sensitive toward a range of QT-prolonging drugs inducing severe arrhythmia.

A wide range of drugs has been shown to prolong the QT interval of the electrocardiogram by blocking the pore-forming subunit of the rapidly activating delayed rectifier K+ channel, HERG (ether-à-go-go-related gene), sometimes leading to life-threatening arrhythmia. In this paper we describe cloning, sequence, and expression of the zebrafish orthologue of HERG, Zerg. Further, we studied effects of Zerg inhibition in zebrafish embryos caused by drugs or by an antisense approach. Zerg is expressed specifically in both heart chambers of zebrafish embryos, is composed of six transmembrane domains, and shows an especially high degree of amino acid conservation in the S6 and pore domain (99% identity). Several QT-prolonging drugs added to the bathing medium elicited bradycardia and arrhythmia in zebrafish embryos. The arrhythmia induced ranged from an atrioventricular 2:1 block, the ventricle beating half as often as the atrium, to more severe irregular arrhythmia with higher concentrations of the drugs. These effects were highly specific, reproducible, and rapid, e.g., 10 microM astemizole caused a 2:1 heartbeat within a minute after addition of the compound in all the embryos studied. Morpholino antisense oligonucleotides targeting Zerg were injected into zebrafish embryos and elicited similar dose-sensitive and specific arrhythmia as the QT-prolonging drugs, suggesting an evolutionarily conserved role for Erg in regulating heartbeat rate and rhythm. Further, we identified a mutation in the Per-Arnt-Sim domain of the Zerg channel in the breakdance mutant, also characterized by a 2:1 atrioventricular block. In conclusion, the zebrafish could be a tractable model organism for the study of Erg function and modulation but might also have a value in the field of cardiovascular pharmacology, e.g., as an early preclinical model for testing drugs under development for potential QT prolongation.

Animals↗

PubMed2003

Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections.

Bacterial virulence is best studied in animal models. However, the lack of possibilities for real time analysis and the need for laborious and invasive sample analysis limit the use of experimental animals. In the present study 28 h-old zebrafish embryos were infected with DsRed-labelled cells of Salmonella typhimurium. Using multidimensional digital imaging microscopy we were able to determine the exact location and fate of these bacterial pathogens in a living vertebrate host during three days. A low dose of wild-type S. typhimurium resulted in a lethal infection with bacteria residing and multiplying both in macrophage-like cells and at the epithelium of blood vessels. Lipopolysaccharide (LPS) mutants of S. typhimurium, known to be attenuated in the murine model, proved to be non-pathogenic in the zebrafish embryos and were partially lysed in the bloodstream or degraded in macrophage-like cells. However, injection of LPS mutants in the yolk of the embryo resulted in uncontrolled bacterial proliferation. Heat-killed, wild-type bacteria were completely lysed extracellularly within minutes after injection, which shows that the blood of these zebrafish embryos does already contain lytic activity. In conclusion, the zebrafish embryo model allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host.

Animals↗

PubMed1990

Axonal outgrowth by identified neurons in the spinal cord of zebrafish embryos.

The spinal cord of early zebrafish embryos contains a small number of neurons per hemisegment. The earliest neurons are identifiable as individual neurons or small groups of homogeneous neurons and project growth cones that follow stereotyped, cell-specific pathways to their targets. These growth cones appear to bypass some axons but follow others during pathfinding, suggesting that they can distinguish among the different axons they normally encounter. Furthermore, identified growth cones exhibit cell-specific behaviors in apparent contact with the floor plate cells, which are found at the ventral midline of the early cord. These observations suggest the testable hypothesis that the floor plate may mediate multiple, cell-specific actions on identified growth cones in the zebrafish cord. One hypothesized action is inhibition of specific growth cones to prevent them from crossing the ventral midline.

Animals↗

PubMed2006

A novel contact assay for testing genotoxicity of chemicals and whole sediments in zebrafish embryos.

Broad consensus exists that whole-sediment exposure protocols represent the most realistic scenario to simulate in situ exposure conditions. So far, however, several endpoints including genotoxicity in vertebrate-based systems could be tested only after transfer of particle-bound substances into the aqueous phase. The present study was carried out to develop a protocol for generating a suspension of single cells from sediment-exposed zebrafish embryos that is suitable for detecting particle-bound genotoxicity in the alkaline single cell gel electrophoresis (comet assay). In this solid-phase genotoxicity assay, a whole-body cell suspension derived from zebrafish embryos exposed to native (whole) sediments is assayed in the comet assay. Several chemical and mechanical isolation procedures were compared to optimize cell yield and minimize DNA damage by the method itself. If compared to collagenase isolation, mechanical cell dissociation gave less DNA damage; trypsinization resulted in similarly low DNA damage but significantly lower cell yield. In order to test the optimized protocol, effects of well-known genotoxicants (4-nitroquinoline-N-oxide, nitrofurantoin, hydrogen peroxide, benzo[a]pyrene) and of two sediments from the upper Rhine River (Germany) on zebrafish embryos were investigated. Results documented clear-cut genotoxicity for all four substances and for one of the two whole-sediment samples. An ultraviolet (UV) light exposure of whole embryos and primary cultures from embryos elucidated only minor effects for the whole embryos compared to the primary cells. Consequently, UV irradiation cannot be suggested as a positive control in intact zebrafish embryos. In conclusion, the newly developed sediment contact assay can be recommended for the detection of both single substances but also the bioavailable fraction of the total hazard potential of sediments.

Animals↗

PubMed2003

Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo.

A class of recessive lethal zebrafish mutations has been identified in which normal skeletal muscle differentiation is followed by a tissue-specific degeneration that is reminiscent of the human muscular dystrophies. Here, we show that one of these mutations, sapje, disrupts the zebrafish orthologue of the X-linked human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology through disconnecting the cytoskeleton from the extracellular matrix in skeletal muscle by reducing the level of dystrophin protein at the sarcolemma. This is thought to allow tearing of this membrane, which in turn leads to cell death. Surprisingly, we have found that the progressive muscle degeneration phenotype of sapje mutant zebrafish embryos is caused by the failure of embryonic muscle end attachments. Although a role for dystrophin in maintaining vertebrate myotendinous junctions (MTJs) has been postulated previously and MTJ structural abnormalities have been identified in the Dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure has thus far been lacking. This zebrafish mutation may therefore provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases.

Amino Acid Sequence↗

PubMed1992

Growth cone guidance by floor plate cells in the spinal cord of zebrafish embryos.

The spinal cord of early zebrafish embryos contains a small number of neuronal classes whose growth cones all follow stereotyped, cell-specific pathways to their targets. Two classes of spinal neurons make cell-specific turns at or near the ventral midline of the spinal cord, which is occupied by a single row of midline floor plate cells. We tested whether these cells guide the growth cones by examining embryos missing the midline floor plate cells due either to laser ablation of the cells or to a mutation (cyc-1). In these embryos the growth cones followed both normal and aberrant pathways once near the ventral midline. This suggests that normally the midline floor plate cells do provide guidance cues, but that these cues are not obligatory.

Animals↗

PubMed1997

High-resolution imaging at the cellular and subcellular levels in flattened whole mounts of early zebrafish embryos.

We describe a rapid and sensitive method for high-resolution imaging at the cellular and subcellular levels in the whole-mount zebrafish embryo. The procedure involves fixing and staining the embryo, followed by deyolking and flattening it under a cover slip, to produce a planar mount that is 20 to 100 microns thick. Such a flattened whole mount allows imaging with a spatial resolution of approximately 500 nm in the x-y plane and does not require the use of embedding, sectioning, confocal microscopy, or computational deblurring procedures. We can resolve all individual nuclei and chromosome sets in the embryo, up to the late gastrula stage (10,000 cell stage). In addition, older embryos (through the segmentation stage) can also be examined, with the preservation of significant morphological detail. Because of its ability to resolve subcellular detail, the flattened whole-mount method can provide significant biological information beyond what can be obtained from conventional (three-dimensional) whole mounts. We have used the flattened whole-mount method to study subcellular events related to progression through the cell cycle or to apoptosis, in cells of the early zebrafish embryo. A specific DNA-binding dye (Hoechst 33258) or an antibody against a chromosomal protein (histone H1) was used to stain the nuclei of individual cells in the embryo. This allowed us to determine the spatial positions of all the individual cells, and also their stages in the cell cycle. A terminal transferase (TUNEL) assay was used to detect apoptotic cells. This combination of specific stains allowed us to study the behaviors of groups of cells in situ, within the developing zebrafish embryo.

Animals↗

PubMed1998

Characterization of a major permeability barrier in the zebrafish embryo.

Fish embryos represent a class of multicompartmental biological systems that have not been successfully cryopreserved, primarily because of the lack of understanding of how water and cryoprotectants permeate the compartments. We are using the zebrafish embryo as a model to understand these kinetics. Zebrafish embryos have two major compartments, the blastoderm and the yolk, which is surrounded by the multinucleated yolk syncytial layer (YSL). We determined the water and cryoprotectant permeability in these compartments using two methods. First, we measured shrink/swell dynamics in optical volumetric experiments. Zebrafish embryos shrank over time and did not re-expand while immersed in dimethyl sulfoxide (DMSO) or propylene glycol. Second, we measured DMSO uptake with diffusion-weighted nuclear magnetic resonance spectroscopy. DMSO uptake was rapid during the first few minutes, then gradual thereafter. We used one- and two-compartment models to analyze the data and to determine the permeability parameters. We found that the two-compartment model provided a better fit to the data. On the basis of this model and in the presence of DMSO, the yolk and blastoderm had very similar water permeabilities (i.e., 0.01 and 0. 005 micron x min-1atm-1, respectively), but they had different DMSO permeabilities separated by three orders of magnitude (i.e., </= 5 x 10(-6) and 1.5 x 10(-3) cm/min, respectively). The low solute permeability of the yolk predicted that the yolk/YSL compartment should be more susceptible to cryodamage. To test this, the yolk, blastoderm, and YSL were examined at the ultrastructural level after vitrification. Only the YSL incurred significant damage after freezing and thawing (p </= 0.05).

Animals↗

PubMed2006

Uptake and elimination of ethanol by young zebrafish embryos.

Among animal models being explored to understand ethanol-induced teratogenesis, the zebrafish (Danio rerio) is attracting attention because its embryonic development is well characterized and readily visualized. Despite the potential of the zebrafish embryo in research on developmental anomalies produced by ethanol exposure, little is known about the relationship between embryonic ethanol content and the nature/severity of ethanol-mediated deficits. Here, using gas chromatography and radiometry of labeled ethanol carbon, we examine accumulation and clearance of ethanol by dechorionated zebrafish embryos during blastulation/gastrulation. Our data indicate that: (a) rates of uptake and loss of ethanol are directly proportional to the extra-/intra-embryonic ethanol concentration gradient and (b) ethanol in the water fraction of embryos reaches near equimolarity with ethanol in the exposure medium. It appears that, within a wide range of exposure concentrations, embryonic ethanol content can be predicted accurately according to exposure time. Furthermore, it appears that embryonic ethanol can be adjusted rapidly to and maintained at a targeted concentration.

Analysis of Variance↗

PubMed2001

Analysis of Cx43alpha1 promoter function in the developing zebrafish embryo.

The Cx43alpha1 gap junctions play an important role in cardiovascular development. Studies using transgenic mouse models have indicated that this involves an essential role for Cx43alpha1 in modulating neural crest cell motility. We previously showed that a 6.8 kb mouse genomic sequence containing the promoter and upstream regulatory sequences of the Cx43alpha1 gene can drive lacZ reporter gene expression in all neural crest cell lineages in the mouse embryo. To obtain further insights into the sequence motifs and regulatory pathways involved in targeting Cx43alpha1 gene expression in neural crest cells, we assayed the activity of the mouse Cx43alpha1 promoter in evolutionarily distantly related zebrafish embryos. For these studies, the 6.8kb Cx43alpha1 genomic sequence and various deletion derivatives were used to generate GFP or lacZ expression vectors. The transcriptional activities of these constructs were analyzed in vivo after microinjection into one- or two- cell stage zebrafish embryos. These studies indicated that the mouse Cx43alpha1 promoter can drive lacZ expression in neural crest cells in the zebrafish embryos. Analysis by whole mount in situ hybridization showed that the endogenous zebrafish Cx43alpha1 gene is expressed maternally and zygotically, and expression is observed in regions where neural crest cells are found. To further elucidate the developmental regulation of Cx43alpha1 gene expression, we screened a zebrafish BAC library and identified a clone containing the entire zebrafish Cx43alpha1 gene and flanking upstream and downstream sequences. The upstrean Cx43alpha1 promoter sequences from zebrafish, mouse, and human were analyzed for evolutionarily conserved DNA motifs. Overall these studies suggest that the sequence motifs and transcriptional regulation involved in the targeting Cx43alpha1 expression to neural crest cells are evolutionarily conserved in zebrafish and mouse embryos.

Animals↗

PubMed1991

Elimination of a brain tract increases errors in pathfinding by follower growth cones in the zebrafish embryo.

The early zebrafish brain contains a simple axon scaffold of longitudinal tracts connected by commissures. Neurons in the nucleus of the posterior commissure (nuc PC) project growth cones along a specific route in this axonal scaffold, raising the possibility that specific axons in the early scaffold guide nuc PC growth cones. We tested this possibility by analyzing the behavior of nuc PC growth cones in embryos in which a portion of the scaffold, normally traversed by nuc PC growth cones, was surgically prevented from forming. Under these conditions nuc PC growth cones extended along both normal and aberrant pathways. This suggests that specific axons do provide guidance cues, since their removal leads to errors. However, these cues are not obligatory, since some growth cones still followed normal pathways.

Animals↗

PubMed2002

Molecular characterization of Calymmin, a novel notochord sheath-associated extracellular matrix protein in the zebrafish embryo.

During the screening of a zebrafish postsomitogenesis embryo cDNA library, we have identified a cDNA corresponding to a novel type of protein localized to the notochordal sheath-associated extracellular matrix (ECM) of the embryo. The 4.049-kb mRNA encodes a predicted polypeptide of 1,207 amino acids (122 kDa, pI 10.50) with a potential signal peptide of 20 amino acids. After the signal peptide, the mature protein consists of 1,187 amino acids (119 kDa, pI 10.46), for which the name "Calymmin" (from Greek chialphalambdanumumualpha, to envelop, to cover) is proposed. The Calymmin mRNA is highly and transiently expressed by the notochord cells of the embryo from the 10- to 12-somite stage to the pharyngula period (13 and 24 hours postfertilization, respectively), and light and electron microscopical immunolocalization analysis revealed that the protein was specifically localized within a granular and filamentous layer of the ECM compartment surrounding the notochord. In zebrafish no tail mutants (ntl(tc41)), in which the notochord precursor cells are present but fail to differentiate, the Calymmin protein was not detected, confirming the notochord origin of Calymmin. These results indicate that Calymmin is a novel constitutive protein of the ECM compartment associated to the perinotochordal sheath in the zebrafish embryo, which is specifically expressed by the differentiating notochord cells.

Amino Acid Sequence↗

PubMed2003

Developmental control of Presenilin1 expression, endoproteolysis, and interaction in zebrafish embryos.

Dominant mutations in presenilin1 (PS1) and presenilin2 (PS2) are a major cause of early-onset Alzheimer's disease. In this report we analyze the expression of the zebrafish presenilin1 (Psen1) and presenilin2 (Psen2) proteins during embryogenesis. We demonstrate that Psen1 and Psen2 holoproteins are relatively abundant in zebrafish embryos and are proteolytically processed. Psen1 is maternally expressed, whereas Psen2 is expressed at later stages during development. The Psen1 C-terminal proteolytic fragment (CTF) is present at varying levels during embryogenesis, indicating the existence of developmental control mechanisms regulating its production. We examine the codependency of Psen1 and Psen2 expression during early embryogenesis. Forced overexpression of psen2 increases expression of Psen2 holoprotein, but not the N-terminal fragment (NTF), indicating that levels of Psen2 NTF are strictly controlled. Overexpression of psen2 did not alter levels of Psen1 holoprotein, CTF, or higher molecular weight complexes. Reduction of Psen1 activity in zebrafish embryos produces similar developmental defects to those seen for loss of PS1 activity in knockout mice. The relevance of these results to previous work on presenilin protein regulation and function are discussed. Our work shows that zebrafish embryos are a valid and valuable system in which to study presenilin interactions, regulation, and function.

Alzheimer Disease↗

PubMed2000

Development of the hypochord and dorsal aorta in the zebrafish embryo (Danio rerio).

The hypochord of the zebrafish embryo (Danio rerio) emerges at the 9-somite stage as a single row of cells in the dorsomedial endoderm immediately ventral to the notochord. It is recognizable from the 2(nd) or 3(rd) somite and extends along the trunk to the same extent as the somites. A basal lamina surrounds the hypochord and its cells are slightly larger than the nearby endoderm cells. TEM studies have shown that the hypochord cells contain, in addition to mitochondria, well-developed rough endoplasmic reticula and Golgi networks, indicating synthetic activity. Once formed, the hypochord will stay in close association with the notochord, and this axial complex gradually moves dorsally, separating the hypochord from the endoderm as a one-cell-wide, rod-like structure that is bean-shaped in transverse section. This is the situation in the 15-somite embryo, at the level of the 4-5(th) somites. In the gap between the hypochord and the endoderm, angioblast cells aggregate and start to form the dorsal aorta, which becomes intimately associated with the hypochord. In the 17-somite embryo the aortic rudiment is established just ventral to the hypochord as a tube with a lumen. As development proceeds, the size of the hypochord decreases. In the pec fin embryo the hypochord is still recognizable in the posterior trunk, but has apparently vanished in anterior regions. The temporal correlation between the appearance of the hypochord and the formation of the dorsal aorta, coupled with the intimate relationship between these structures, suggest that the hypochord may play a role in the positioning of the dorsal aorta.

Animals↗

PubMed2006

Zebrafish embryo proteins induce apoptosis in human colon cancer cells (Caco2).

Previous studies have shown that proteins extracted from Zebrafish embryo share some cytostatic characteristics in cancer cells. Our study was conducted to ascertain the biological properties of this protein network. Cancer cell growth and apoptosis were studied in Caco2 cells treated with embryonic extracts. Cell proliferation was significantly inhibited in a dose-dependent manner. Cell-cycle analysis in treated cells revealed a marked accumulation in the G(2)/M phase preceding induction of apoptosis. Embryo proteins induced a significant reduction in FLIP levels, and increased caspase-3 and caspase-8 activity as well as the apoptotic rate. Increased phosphorylated pRb values were obtained in treated Caco2 cells: the modified balance in pRb phosphorylation was associated with an increase in E2F1 values and c-Myc over-expression. Our data support previous reports of an apoptotic enhancing effect displayed by embryo extracts, mainly through the pRb/E2F1 apoptotic pathway, which thus suggests that Zebrafish embryo proteins have complex anti-cancer properties.

Animals↗