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PubMed2005

Characterization of snakehead rhabdovirus infection in zebrafish (Danio rerio).

The zebrafish, Danio rerio, has become recognized as a valuable model for the study of development, genetics, and toxicology. Recently, the zebrafish has been recognized as a useful model for infectious disease and immunity. In this study, the pathogenesis and antiviral immune response of zebrafish to experimental snakehead rhabdovirus (SHRV) infection was characterized. Zebrafish 24 h postfertilization to 30 days postfertilization were susceptible to infection by immersion in 10(6) 50% tissue culture infective doses (TCID50) of SHRV/ml, and adult zebrafish were susceptible to infection by intraperitoneal (i.p.) injection of 10(5) TCID50 of SHRV/ml. Mortalities exceeded 40% in infected fish, and clinical presentation of infection included petechial hemorrhaging, redness of the abdomen, and erratic swim behavior. Virus reisolation and reverse transcription-PCR analysis of the viral nucleocapsid gene confirmed the presence of SHRV. Histological sections of moribund embryonic and juvenile fish revealed necrosis of the pharyngeal epithelium and liver, in addition to congestion of the swim bladder by cell debris. Histopathology in adult fish injected i.p. was confined to the site of injection. The antiviral response in zebrafish was monitored by quantitative real-time PCR analysis of zebrafish interferon (IFN) and Mx expression. IFN and Mx levels were elevated in zebrafish exposed to SHRV, although expression and intensity differed with age and route of infection. This study is the first to examine the pathogenesis of SHRV infection in zebrafish. Furthermore, this study is the first to describe experimental infection of zebrafish embryos with a viral pathogen, which will be important for future experiments involving targeted gene disruption and forward genetic screens.

Animals↗

PubMed2002

Furrow-specific endocytosis during cytokinesis of zebrafish blastomeres.

Mutations affecting endocytosis, such as those in clathrin and dynamin, unexpectedly cause defects in cytokinesis in a number of organisms. To explore the relationship between endocytosis and cytokinesis, we used the relatively large cells of the transparent zebrafish embryo. Using fluorescent markers for fluid-phase as well as plasma membrane uptake, we demonstrate that cytokinesis involves furrow-specific endocytosis. Clathrin-coated pits are visible near the furrow in ultrathin sections, while immunolabeling demonstrates that clathrin and caveolin are localized to the cleavage furrow. Hence, it is likely that both clathrin- and caveolae-mediated endocytosis occurs at the furrow during cytokinesis. Dynamin II is also localized to the furrow and may mediate furrow-specific endocytosis. Treatment of embryos with chlorpromazine or with methyl-beta-cyclodextrin, both of which inhibit endocytosis, prevents the normal completion of cytokinesis. These data suggest that furrow-specific endocytosis is an integral part of cytokinesis.

Animals↗

PubMed1999

Dual specificity of activin type II receptor ActRIIb in dorso-ventral patterning during zebrafish embryogenesis.

Members of the transforming growth factor-beta (TGF-beta) superfamily are thought to regulate specification of a variety of tissue types in early embryogenesis. These effects are mediated through a cell surface receptor complex, consisting of two classes of ser/thr kinase receptor, type I and type II. In the present study, cDNA encoding zebrafish activin type II receptors, ActRIIa and ActRIIb was cloned and characterized. Overexpression of ActRIIb in zebrafish embryos caused dorsalization of embryos, as observed in activin-overexpressing embryos. However, in blastula stage embryos, ActRIIb induced formation of both dorsal and ventro-lateral mesoderm. It has been suggested that these inducing signals from ActRIIb are mediated through each specific type I receptor, TARAM-A and BMPRIA, depending on activin and bone morphogenetic protein (BMP), respectively. In addition, it was shown that a kinase-deleted form of ActRIIb (dnActRIIb) suppressed both activin- and BMP-like signaling pathways. These results suggest that ActRIIb at least has dual roles in both activin and BMP signaling pathways during zebrafish embryogenesis.

Activin Receptors, Type II↗

PubMed1993

Zebrafish primary neurons initiate expression of the LIM homeodomain protein Isl-1 at the end of gastrulation.

Isl-1 has previously been established as the earliest marker of developing chicken spinal motor neurons where it is regulated by inductive signals from the floorplate and notochord. We now report that, in zebrafish, the expression of Isl-1 is initiated in Rohon-Beard cells, primary motor neurons, interneurons and cranial ganglia, hours before the neural tube itself is formed. The expression is initiated simultaneously in the Rohon-Beard cells and the primary motor neurons, at the axial level of the presumptive first somite. The Isl-1-expressing motor neurons appear on either side of the ventral midline whereas the interneurons and Rohon-Beard cells initiate expression while located at the edge of the germinal shield. Isl-1 expression is initiated in these cells before the formation of a differentiated notochord. Isl-1 is expressed in the various functional classes of primary neurons at 24 hours postfertilization. This selective expression of a homeodomain protein in the primary neurons implies that these neurons share a common program of early development and that they have evolved and been selected for as a coordinated system. One of the functions of the primary neurons is to send long axons which pioneer the major axon tracts in the zebrafish embryo. An evolutionary conserved functional role for Isl-1 in the expression of the pioneering phenotype of the primary neurons is suggested.

Animals↗

PubMed2006

Coordination of symmetric cyclic gene expression during somitogenesis by Suppressor of Hairless involves regulation of retinoic acid catabolism.

Vertebrate embryos faithfully produce bilaterally symmetric somites that give rise to repetitive body structures such as vertebrae and skeletal muscle. Body segmentation is regulated by a cyclic gene expression system, containing the Delta-Notch pathway and targets, which generates bilaterally symmetric oscillations across the Pre-Somitic Mesoderm (PSM). The position of the forming somite boundary is controlled by interaction of this oscillator with a determination front comprised of opposing gradients of FGF and retinoic acid (RA) signalling. Disruption of RA production leads to asymmetries in cyclic gene expression, but the link between RA and the oscillator is unknown. In somitogenesis, Notch signalling activates target genes through the transcription factor Suppressor of Hairless (Su(H)). Here, we report that two Su(H) genes coordinate bilaterally symmetric positioning of somite boundaries in the zebrafish embryo. Combined Su(H) gene knockdown caused defects in visceral left/right asymmetry, neurogenic lateral inhibition, and symmetrical failure of the segmentation oscillator. However, by selectively down-regulating Su(H)2 or Su(H)1 function using specific antisense morpholinos, we observed asymmetric defects in anterior or posterior somite boundaries, respectively. These morphological abnormalities were reflected by underlying asymmetric cyclic gene expression waves in the presomitic mesoderm, indicating a key role for Su(H) in coordinating the left-right symmetry of this process. Strikingly, expression of the RA-degrading enzyme cyp26a1 in the tailbud was controlled by Su(H) activity, and morpholino knockdown of cyp26a1 alone caused asymmetric cyclic dlc expression, suggesting that excess RA in the tailbud may contribute to the cyclic asymmetries. Indeed, exogenous RA was sufficient to generate asymmetric expression of all cyclic genes. Our observations indicate that one element of the Notch signalling pathway, Su(H), is required for control of RA metabolism in the tailbud and that this regulation is involved in bilateral symmetry of cyclic gene expression and somitogenesis.

Amino Acid Sequence↗

PubMed2006

Egr1 gene knockdown affects embryonic ocular development in zebrafish.

PURPOSE: To identify the changes in zebrafish embryonic ocular development after early growth response factor 1 (Egr1) gene knockdown by Egr1-specific translation inhibitor, morpholino oligonucleotides (MO). METHODS: Two kinds of Egr1-MO were microinjected separately with various dosages into one to four celled zebrafish embryos to find an optimal dose generating an acceptable mortality rate and high frequency of specific phenotype. Chordin-MO served as the positive control; a 5 mismatch MO of Egr1-MO1 and a nonspecific MO served as negative controls. We graded the Egr1 morphants according to their gross abnormalities, and measured their ocular dimensions accordingly. Western blot analysis and synthetic Egr1 mRNA rescue experiments confirmed whether the deformities were caused by Egr1 gene knockdown. Histological examination and three kinds of immunohistochemical staining were applied to identify glutamate receptor one expression in retinal ganglion cells and amacrine cells, to recognize acetylated alpha-tubulin expression which indicated axonogenesis, and to label photoreceptor cells with zpr-1 antibody. RESULTS: After microinjection of 8 ng Egr1-MO1 or 2 ng Egr1-MO2, 81.8% and 97.3% of larvae at 72 h postfertilization had specific defects, respectively. The gross phenotype included string-like heart, flat head, and deformed tail. The more severely deformed larvae had smaller eyes and pupils. Co-injection of 8 ng Egr1-MO1 and supplementary 12 pg synthetic Egr1 mRNA reduced the gross abnormality rate from 84.4% to 29.7%, and decreased the severity of deformities. Egr1 protein appeared in the wildtype and rescued morphants, but was lacking in the Egr1 morphants with specific deformities. Lenses of Egr1 morphants were smaller and had some residual nucleated lens fiber cells. Morphants' retinal cells arranged disorderly and compactly with thin plexiform layers. Immunohistochemical studies showed that morphants had a markedly decreased number of mature retinal ganglion cells, amacrine cells, and photoreceptor cells. Retinal axonogenesis was prominently reduced in morphants. CONCLUSIONS: The Egr1 gene plays an important role in zebrafish embryonic oculogenesis. Ocular structures including lens and retina were primitive and lacked appropriate differentiation. Such arrested retinal and lenticular development in Egr1 morphants resulted in microphthalmos.

Animals↗

PubMed2006

Different words, same meaning: understanding the languages of the genome.

There is undeniable value in using sequence comparison to identify putative regulatory sequences. However, a recent report has demonstrated that not all regulatory sequences are evolutionarily conserved. Cis-acting sequences around the RET gene, conserved in mammals but not in fish, are able to reproduce patterns of RET expression in zebrafish embryos. It is as yet unclear whether these sequences are 'below the radar' of current sequence alignment tools or whether their functional homology is not sequence based.

Animals↗

PubMed1995

A novel gene expressed during zebrafish gastrulation identified by differential RNA display.

Vertebrate gastrulation is a dynamic period of development characterized by extensive cell migrations. This stage of development is likely to require the expression of a new genetic repertoire to initiate and direct these dramatic changes. The differential RNA display has been used to identify genes specifically expressed during the gastrula stage of a model vertebrate, the zebrafish. One of the genes isolated by the differential display technique has been sequenced and characterized for its spatial and temporal expression. This gene, called G12, is expressed during a narrow window of time during gastrulation and is restricted to a single cell type. At this time of development the zebrafish embryo consists of three cell types: the yolk cell, EVL cells and deep cells. Interestingly, both EVL and deep cells derive early in development from common progenitor cells but G12 expression is restricted only to the EVL lineage. Comparison of the amino acid sequence from this gene with the Genbank database indicates similarity to two previously reported mammalian genes. The similarity between these three genes suggests that they may serve a common function. The G12 gene is the first example of restricted gene expression in EVL cells of the zebrafish. The G12 gene should prove to be a useful model for the study of regulated gene expression during gastrulation.

Amino Acid Sequence↗

PubMed2003

The prickle-related gene in vertebrates is essential for gastrulation cell movements.

Involving dynamic and coordinated cell movements that cause drastic changes in embryo shape, gastrulation is one of the most important processes of early development. Gastrulation proceeds by various types of cell movements, including convergence and extension, during which polarized axial mesodermal cells intercalate in radial and mediolateral directions and thus elongate the dorsal marginal zone along the anterior-posterior axis [1,2]. Recently, it was reported that a noncanonical Wnt signaling pathway, which is known to regulate planar cell polarity (PCP) in Drosophila [3,4], participates in the regulation of convergent extension movements in Xenopus as well as in the zebrafish embryo [5-8]. The Wnt5a/Wnt11 signal is mediated by members of the seven-pass transmembrane receptor Frizzled (Fz) and the signal transducer Dishevelled (Dsh) through the Dsh domains that are required for the PCP signal [6-8]. It has also been shown that the relocalization of Dsh to the cell membrane is required for convergent extension movements in Xenopus gastrulae. Although it appears that signaling via these components leads to the activation of JNK [9,10] and rearrangement of microtubules, the precise interplay among these intercellular components is largely unknown. In this study, we show that Xenopus prickle (Xpk), a Xenopus homolog of a Drosophila PCP gene [11-13], is an essential component for gastrulation cell movement. Both gain-of-function and loss-of-function of Xpk severely perturbed gastrulation and caused spina bifida embryos without affecting mesodermal differentiation. We also demonstrate that XPK binds to Xenopus Dsh as well as to JNK. This suggests that XPK plays a pivotal role in connecting Dsh function to JNK activation.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Nicalin and its binding partner Nomo are novel Nodal signaling antagonists.

Nodals are signaling factors of the transforming growth factor-beta (TGFbeta) superfamily with a key role in vertebrate development. They control a variety of cell fate decisions required for the establishment of the embryonic body plan. We have identified two highly conserved transmembrane proteins, Nicalin and Nomo (Nodal modulator, previously known as pM5), as novel antagonists of Nodal signaling. Nicalin is distantly related to Nicastrin, a component of the Alzheimer's disease-associated gamma-secretase, and forms a complex with Nomo. Ectopic expression of both proteins in zebrafish embryos causes cyclopia, a phenotype that can arise from a defect in mesendoderm patterning mediated by the Nodal signaling pathway. Accordingly, downregulation of Nomo resulted in an increase in anterior axial mesendoderm and the development of an enlarged hatching gland. Inhibition of Nodal signaling by ectopic expression of Lefty was rescued by reducing Nomo levels. Furthermore, Nodal- as well as Activin-induced signaling was inhibited by Nicalin and Nomo in a cell-based reporter assay. Our data demonstrate that the Nicalin/Nomo complex antagonizes Nodal signaling during mesendodermal patterning in zebrafish.

Amyloid Precursor Protein Secretases↗

PubMed1997

Novel Eph-family receptor tyrosine kinase is widely expressed in the developing zebrafish nervous system.

In a search for novel tyrosine kinases involved in vertebrate development, we have isolated cDNAs corresponding to three distinct members of the Eph-family of receptor tyrosine kinases. Whole mount RNA in situ hybridization analysis showed all three genes were most abundantly expressed in the developing nervous system. zek1 (zebrafish Eph-like kinase1) encodes a 981 amino acid polypeptide closely related to the murine Sek1 and Bsk receptors. Cos-1 cells transfected with zek1 produce a 141 kilodalton tyrosine phosphorylated protein which is recognized by antibodies raised against two predicted Zek1 peptides. These antibodies also recognized a protein of the same apparent molecular weight in lysates from zebrafish embryos and adults. Widespread expression of zek1 in the developing brain and neural tube suggested a generalized function of the Zek1 receptor in neuronal cell ontogeny.

Amino Acid Sequence↗

PubMed2001

Molecular cloning of a human Vent-like homeobox gene.

We have isolated a previously unknown human homeobox-containing cDNA, VENT-like homeobox-2 (VENTX2), using PCR with a bone marrow cDNA library and primers designed from the VENTX1 (alias HPX42) homeobox sequence. Here we describe the molecular cloning, chromosomal localization to 10q26.3, and functional analysis of this gene. The 2.4-kb human VENTX2 cDNA encoded a protein with a predicted molecular weight of 28 kDa containing a homeodomain with 65% identity to the Xenopus laevis ventralizing gene Xvent2B. VENTX2 antisera detected a 28-kDa protein in cells transfected with a VENTX2 expression construct, in a human erythroleukemic cell line and in bone marrow samples obtained from patients in recovery phase after chemotherapy. The similarity of the homeodomains from VENTX2 and the X. laevis Vent gene family places them in the same homeodomain class. Consistent with this structural classification, overexpression of VENTX2 in zebrafish embryos led to anterior truncations and failure to form a notochord, which are characteristics of ventralization.

Amino Acid Sequence↗

PubMed2026

Defining active and repressive chromatin states in neural crest cells using low-input CUT&RUN.

The transition of neural crest cells (NCCs) from a multipotent state to lineage-restricted derivatives, including melanocytes, is governed by tightly regulated epigenetic mechanisms that orchestrate cell type specific gene expression programs. Histone post-translational modifications (PTMs), in particular, play an important role in modulating chromatin accessibility, enhancer activation, and transcription factor occupancy, thereby facilitating dynamic chromatin and transcriptional reprogramming required during development. However, profiling such chromatin states in rare and transient Neural Crest Cell (NCC) populations in vivo remains technically challenging. To address this, we present an optimized low-input Cleavage Under Targets and Release Using Nuclease (CUT&RUN) workflow tailored for fluorescence-activated cell sorting (FACS) isolated NCCs from zebrafish embryos. This approach enables high-resolution and low-background mapping of key histone modifications, including H3K27ac, H3K4me3, and H3K27me3, from limited cell numbers. Collectively, these methodologies provide a robust framework for dissecting chromatin state dynamics in developmental systems and can also offer insights into epigenetic dysregulation associated with disease.

Animals↗

PubMed2002

Expression of the FGF receptor 2 gene (fgfr2) during embryogenesis in the zebrafish Danio rerio.

We isolated a full-length cDNA clone for the zebrafish homologue of fibroblast growth factor receptor (FGFR) 2. The deduced protein sequence is typical of vertebrate FGFRs in that it has three Ig-like domains in the extracellular region. The expression of fgfr2 is initiated during epiboly in the paraxial mesoderm. During early somitogenesis, fgfr2 expression was noted in the anterior neural plate as well as in newly formed somites. Whereas fgfr2 expression in somites is transient, it increases in the central nervous system (CNS), i.e. in the ventral telencephalon, anterior diencephalon, midbrain, and respective rhombomeres of the hindbrain, from the mid-somitogenesis stage. The dorsal telencephalon and the region around the midbrain-hindbrain boundary are devoid of fgfr2 expression. Essentially the same expression pattern is observed until 48 h post-fertilization in the CNS, although rhombomeric expression in the hindbrain is progressively confined to narrower stripes. After somitogenesis, fgfr2 expression was also observed in the lens, hypochord, endoderm, and fin mesenchyme. We compared the expression of the four fgfr genes (fgfr1-4) in the CNS of zebrafish embryos and show that fgfr1 is the only fgfr gene that is expressed in the dorsal telencephalon and isthmic region from which expression of fgfr2-4 is absent.

Animals↗

PubMed1997

Expression of contact, a new zebrafish DVR member, marks mesenchymal cell lineages in the developing pectoral fins and head and is regulated by retinoic acid.

Contact, a new zebrafish transforming growth factor-beta (TGF-beta) member is most closely related to mouse GDF5 and to human CDMP-1 responsible, when mutated, for limb brachypodism phenotype and Hunter-Thompson syndrome, respectively. Contact exhibits a dynamic spatial expression pattern in the pharyngeal arches and the pectoral fin buds that much prefigures cartilage formation. Within the fin buds, contact expression is detected in the proximal mesenchyme from which the endoskeleton will develop. Exogeneously applied retinoic acid (RA) induces duplication of the pectoral fin rudiment in zebrafish embryos as well as contact expression along the proximal margin of the fin mesenchyme showing that both endoskeleton and exoskeleton can be duplicated.

Amino Acid Sequence↗

PubMed2002

Signaling of rat Frizzled-2 through phosphodiesterase and cyclic GMP.

The Frizzled-2 receptor (Rfz2) from rat binds Wnt proteins and can signal by activating calcium release from intracellular stores. We show that wild-type Rfz2 and a chimeric receptor consisting of the extracellular and transmembrane portions of the beta2-adrenergic receptor with cytoplasmic domains of Rfz2 also signaled through modulation of cyclic guanosine 3',5'-monophosphate (cGMP). Activation of either receptor led to a decline in the intracellular concentration of cGMP, a process that was inhibited in cells treated with pertussis toxin, reduced by suppression of the expression of the heterotrimeric GTP-binding protein (G protein) transducin, and suppressed through inhibition of cGMP-specific phosphodiesterase (PDE) activity. Moreover, PDE inhibitors blocked Rfz2-induced calcium transients in zebrafish embryos. Thus, Frizzled-2 appears to couple to PDEs and calcium transients through G proteins.

Amino Acid Sequence↗

PubMed2002

Dual origin of the floor plate in the avian embryo.

Molecular analysis carried out on quail-chick chimeras, in which quail Hensen's node was substituted for its chick counterpart at the five- to six-somite stage (ss), showed that the floor plate of the avian neural tube is composed of distinct areas: (1) a median one (medial floor plate or MFP) derived from Hensen's node and characterised by the same gene expression pattern as the node cells (i.e. expression of HNF3beta and Shh to the exclusion of genes early expressed in the neural ectoderm such as CSox1); and (2) lateral regions that are differentiated from the neuralised ectoderm (CSox1 positive) and form the lateral floor plate (LFP). LFP cells are induced by the MFP to express HNF3beta transiently, Shh continuously and other floor-plate characteristic genes such as NETRIN: In contrast to MFP cells, LFP cells also express neural markers such as Nkx2.2 and Sim1. This pattern of avian floor-plate development presents some similarities to floor-plate formation in zebrafish embryos. We also demonstrate that, although MFP and LFP have different embryonic origins in normal development, one can experimentally obtain a complete floor plate in the neural epithelium by the inductive action of either a notochord or a MFP. The competence of the neuroepithelium to respond to notochord or MFP signals is restricted to a short time window, as only the posterior-most region of the neural plate of embryos younger than 15 ss is able to differentiate a complete floor plate comprising MFP and LFP. Moreover, MFP differentiation requires between 4 and 5 days of exposure to the inducing tissues. Under the same conditions LFP and SHH-producing cells only induce LFP-type cells. These results show that the capacity to induce a complete floor plate is restricted to node-derived tissues and probably involves a still unknown factor that is not SHH, the latter being able to induce only LFP characteristics in neuralised epithelium.

Animals↗

PubMed2005

Antiangiogenic and vascular-targeting activity of the microtubule-destabilizing trans-resveratrol derivative 3,5,4'-trimethoxystilbene.

Neovascularization plays an important role in neoplasia and angioproliferative diseases. Two major modalities have been developed so far to affect neovascularization: its prevention by antiangiogenic compounds, and immature vessel disruption by vascular-targeting agents. trans-Resveratrol, found in grapes and wine, exerts antioxidant, antineoplastic, and antiangiogenic activities. Here, among various synthetic trans-resveratrol derivatives tested, 3,5,4'-trimethoxystilbene was an antiangiogenic agent 30 to 100 times more potent than parent compound in inhibiting endothelial cell proliferation, sprouting, collagen gel invasion, and morphogenesis (ID50 = 0.3-3.0 microM). In addition, 3,5,4'-trimethoxystilbene acts as a vascular-targeting agent by causing microtubule disassembling and tubulin depolymerization and by impairing the repositioning of the microtubule organization center and the formation of membrane ruffles in migrating endothelial cells. In keeping with a vascular-targeting ability, 3,5,4'-trimethoxystilbene induced apoptosis only in subconfluent endothelial cells and apoptotic regression of immature vessels in the ex vivo rat aorta ring assay. In vivo, 3,5,4'-trimethoxystilbene caused the rapid stasis of blood flow and regression of intersegmental vessels in the trunk of zebrafish embryos. In addition, it inhibited blood vessel growth and caused the disappearance of pre-existing blood vessels in the area vasculosa of the chick embryo. In conclusion, 3,5,4'-trimethoxystilbene associates an antiangiogenic profile to a significant vascular-targeting activity.

Angiogenesis Inhibitors↗