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PubMed2002

Two Na,K-ATPase beta 2 subunit isoforms are differentially expressed within the central nervous system and sensory organs during zebrafish embryogenesis.

We have identified cDNAs encoding a second zebrafish ortholog of the human Na,K-ATPase beta 2 subunit. The beta 2b cDNA encodes a 292 amino acid-long polypeptide with 74% identity to the previously characterized zebrafish beta 2a subunit. By using a zebrafish meiotic mapping panel, we determined that the beta 2b gene (atp1b2b) was tightly linked to markers on linkage group 5, whereas the beta 2a gene was located on linkage group 23. In situ hybridization analysis shows that in developing zebrafish embryos, atp1b2a and atp1b2b are predominantly expressed in the nervous system. beta 2a transcripts were abundantly expressed throughout brain as well as spinal cord neurons and lateral line ganglia. In contrast, beta 2b mRNA expression was primarily detected in sensory organs, including retina, otic vesicles, and lateral line neuromast cells. These results suggest that the beta 2a and beta 2b genes play distinct roles in developing brain and sensory organs, and raise the possibility that the functions encoded by the single mammalian beta 2 gene may be partitioned between the two zebrafish beta 2 orthologs.

Animals↗

PubMed2006

Organometallic compounds with biological activity: a very selective and highly potent cellular inhibitor for glycogen synthase kinase 3.

A chiral second-generation organoruthenium half-sandwich compound is disclosed that shows a remarkable selectivity and cellular potency for the inhibition of glycogen synthase kinase 3 (GSK-3). The selectivity was evaluated against a panel of 57 protein kinases, in which no other kinase was inhibited to the same extent, with a selectivity window of at least tenfold to more than 1000-fold at 100 microM ATP. Furthermore, a comparison with organic GSK-3 inhibitors demonstrated the superior cellular activity of this ruthenium compound: wnt signaling was fully induced at concentrations down to 30 nM. For comparison, the well-established organic GSK-3 inhibitors 6-bromoindirubin-3'-oxime (BIO) and kenpaullone activate the wnt pathway at concentrations that are higher by around 30-fold and 100-fold, respectively. The treatment of zebrafish embryos with the organometallic inhibitor resulted in a phenotype that is typical for the inhibition of GSK-3. No phenotypic change was observed with the mirror-imaged ruthenium complex. The latter does not, in fact, show any of the pharmacological properties for the inhibition of GSK-3. Overall, these results demonstrate the potential usefulness of organometallic compounds as molecular probes in cultured cells and whole organisms.

Animals↗

PubMed2006

Gene duplication and functional divergence of the zebrafish insulin-like growth factor 1 receptors.

Insulin-like growth factor (IGF) 1 receptor (IGF1R)-mediated signaling plays key roles in growth, development, and physiology. Recent studies have shown that there are two distinct ig f1r genes in zebrafish, termed ig f1ra and ig f1rb. In this study, we tested the hypothesis that zebrafish ig f1ra and ig f1rb resulted from a gene duplication event at the ig f1r locus and that this has led to their functional divergence. The genomic structures of zebrafish ig f1ra and ig f1rb were determined and their loci mapped. While zebrafish ig f1ra has 21 exons and is located on linkage group (LG) 18, zebrafish ig f1rb has 22 exons and mapped to LG 7. There is a strong syntenic relationship between the two zebrafish genes and the human IG F1R gene. Using a MO-based loss-of-function approach, we show that both Igf1ra and Igf1rb are required for zebrafish embryo viability and proper growth and development. Although Igf1ra and Igf1rb demonstrated a large degree of functional overlap with regard to cell differentiation in the developing eye, inner ear, heart, and muscle, they also exhibited functional distinction involving a greater requirement for Igf1rb in spontaneous muscle contractility. These findings suggest that the duplicated zebrafish ig f1r genes play largely overlapping but not identical functional roles in early development and provide novel insight into the functional evolution of the IGF1R/insulin receptor gene family.

Animals↗

PubMed2003

Ccd1, a novel protein with a DIX domain, is a positive regulator in the Wnt signaling during zebrafish neural patterning.

Wnt signaling plays a crucial role in directing cell differentiation, polarity, and growth. In the canonical pathway, Wnt receptors activate Dishevelled (Dvl), which then blocks the degradation of a key signal transducer, beta-catenin, leading to the nuclear accumulation of beta-catenin and induction of Wnt target genes through TCF/LEF family transcription factors. Here we identified a novel zebrafish gene encoding Ccd1, which possesses a DIX (Dishevelled-Axin) domain. DIX domains are essential for the signal transduction of two major Wnt downstream mediators, Dvl and Axin. Ccd1 formed homomeric and heteromeric complexes with Dvl and Axin and activated TCF-dependent transcription in vitro. In addition, overexpression of ccd1 in zebrafish embryos led to a reduction in the size of the eyes and forebrain (posteriorization), as seen with wnt8 overexpression, whereas a dominant-negative ccd1 (DN-ccd1) caused the opposite phenotype. Furthermore, the Wnt activation phenotype induced by ccd1 was inhibited by the expression of axin1 or DN-ccd1, and the wnt8 overexpression phenotype was rescued by DN-ccd1, suggesting that Ccd1 functions downstream of the Wnt receptor and upstream of Axin. These results indicate that Ccd1 is a novel positive regulator in this Wnt signaling pathway during zebrafish neural patterning.

Amino Acid Sequence↗

PubMed2006

A keratin 18 transgenic zebrafish Tg(k18(2.9):RFP) treated with inorganic arsenite reveals visible overproliferation of epithelial cells.

Inorganic arsenic has strong human carcinogenic potential, but the availability of an animal model to study toxicity is extremely limited. Here, we used the transgenic zebrafish line Tg(k18(2.9):RFP) as an animal model to study arsenite toxicity. This line was chosen because the red fluorescent protein (RFP) is expressed in stratified epithelia (including skin), due to the RFP reporter driven by the promoter of the zebrafish keratin 18 gene. We titrated doses of inorganic arsenite for zebrafish embryos and found that arsenite exposure at 50 microM for 120 h was suitable for mimicking a long-term, chronic effect. When embryos derived from Tg(k18(2.9):RFP) adults were treated with this arsenite dose and time of exposure, abnormal phenotypes were not noticeable under the light microscope. However, arsenic keratosis was visible in the epithelial cells under the fluorescent microscope. Morphological defects became more severe with increased dose and exposure duration, suggesting that the severity of skin lesions was dose- and time-dependent. Histochemical examination of keratosis after 4',6'-diamidino-2-phenylindole hydrochloride (DAPI) staining showed that the epithelial cells overproliferated after treatment with arsenite. Therefore, this Tg(k18(2.9):RFP) zebrafish line is an excellent model for studying toxicity induced by inorganic arsenite and may have potential for studying other environmental pollutants.

Animals↗

PubMed2004

Endocytosis controls spreading and effective signaling range of Fgf8 protein.

Secreted signaling molecules released from a restricted source are of great importance during embryonic development because they elicit induction, proliferation, differentiation, and patterning events in target cells . Fgf8 is a member of the fibroblast growth factor family with key inductive functions during vertebrate development of, for example, the forebrain , midbrain , cerebellum , heart , inner ear , and mesoderm . However, the mechanism by which the signaling range of Fgf8 is controlled in a field of target cells is unknown. We studied Fgf8 as a potential morphogen in the nascent neuroectoderm of living zebrafish embryos. We find that spreading of epitope-tagged Fgf8 through target tissue is carefully controlled by endocytosis and subsequent degradation in lysosomes, or "restrictive clearance," from extracellular spaces. If internalization is inhibited, Fgf8 protein accumulates extracellularly, spreads further, and activates target gene expression over a greater distance. Conversely, enhanced internalization increases Fgf8 uptake and shortens its effective signaling range. Our results suggest that Fgf8 spreads extracellularly by a diffusion-based mechanism and demonstrate that target cells can actively influence, through endocytosis and subsequent degradation, the availability of Fgf8 ligand to other target cells.

Animals↗

PubMed2005

Noncanonical Wnt signaling regulates midline convergence of organ primordia during zebrafish development.

Several components of noncanonical Wnt signaling pathways are involved in the control of convergence and extension (CE) movements during zebrafish and Xenopus gastrulation. However, the complexity of these pathways and the wide patterns of expression and activity displayed by some of their components immediately suggest additional morphogenetic roles beyond the control of CE. Here we show that the key modular intracellular mediator Dishevelled, through a specific activation of RhoA GTPase, controls the process of convergence of endoderm and organ precursors toward the embryonic midline in the zebrafish embryo. We also show that three Wnt noncanonical ligands wnt4a, silberblick/wnt11, and wnt11-related regulate this process by acting in a largely redundant way. The same ligands are also required, nonredundantly, to control specific aspects of CE that involve interaction of Dishevelled with mediators different from that of RhoA GTPase. Overall, our results uncover a late, previously unexpected role of noncanonical Wnt signaling in the control of midline assembly of organ precursors during vertebrate embryo development.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Genome-wide reverse genetics framework to identify novel functions of the vertebrate secretome.

BACKGROUND: Understanding the functional role(s) of the more than 20,000 proteins of the vertebrate genome is a major next step in the post-genome era. The approximately 4,000 co-translationally translocated (CTT) proteins - representing the vertebrate secretome - are important for such vertebrate-critical processes as organogenesis. However, the role(s) for most of these genes is currently unknown. RESULTS: We identified 585 putative full-length zebrafish CTT proteins using cross-species genomic and EST-based comparative sequence analyses. We further investigated 150 of these genes (Figure 1) for unique function using morpholino-based analysis in zebrafish embryos. 12% of the CTT protein-deficient embryos resulted in specific developmental defects, a notably higher rate of gene function annotation than the 2%-3% estimate from random gene mutagenesis studies. CONCLUSION: This initial collection includes novel genes required for the development of vascular, hematopoietic, pigmentation, and craniofacial tissues, as well as lipid metabolism, and organogenesis. This study provides a framework utilizing zebrafish for the systematic assignment of biological function in a vertebrate genome.

Amino Acid Sequence↗

PubMed2003

Sequence and embryonic expression of collagen XVIII NC1 domain (endostatin) in the zebrafish.

Endostatin, located in the NC1 domain of the collagen XVIII, is believed to inhibit the migration and proliferation of endothelial cells (Fed. Am. Soc. Exp. Biol. J. 15 (2001) 1044) and to play a role in axon guidance in Caenorhabditis elegans (J. Cell Biol. 152 (2001) 1219). Zebrafish is an attractive vertebrate model to determine the role of endostatin and the entire molecule of collagen XVIII during vertebrate development. Therefore, we have investigated the expression pattern of COL18A1 in zebrafish embryos from the segmentation to the hatching period stages.

Amino Acid Sequence↗

PubMed1996

Early developmental expression and experimental axis determination by the chicken Vg1 gene.

BACKGROUND: Genes of the transforming growth factor beta (TGF beta) superfamily have been implicated in the earliest steps of developmental patterning in vertebrates. In Xenopus, the Vg1 gene is a candidate for the initiator of axis formation: its RNA and protein are broadly but appropriately localized at the start of development, and processed Vg1 protein is a powerful inducer of organized axial tissue in blastular animal caps in vitro and when locally produced in vivo after injection of Vg1 mRNA into blastomeres. Site-specific proteolytic processing occurs ubiquitously for most TGF beta members, producing the active peptide ligand, but is tightly restricted, by unknown mechanisms, for endogenous Vg protein in Xenopus and zebrafish embryos. This restriction may be involved in the spatial localization of activity required for an organizing role. RESULTS: We have characterized an amniote (chick) orthologue of Vg1, cVg1, and examined its developmental expression. The early expression of cVg1 includes a phase broadly related to the known time and site of axis (primitive streak) initiation; the initial transcription of cVg1 is centred in the posterior marginal zone (PMZ), a region of the blastoderm known to contain the axial organizing activity at this stage. We also observed later neural and paraxial mesodermal expression of cVg1, which has not been described previously for Vg homologues in other vertebrates. We have grafted transfected COS cells, producing processed cVg1 protein, to peripheral positions around the chick early blastoderm. Such grafts initiate formation of morphologically complete primitive streaks, simulating the properties of grafts from the PMZ. CONCLUSIONS: In vertebrate development, Vg genes may be required for an evolutionarily conserved early step in positioning or induction of the axis.

Amino Acid Sequence↗

PubMed2006

Ontogeny of the immune system of fish.

Information on the ontogeny of the fish immune system is largely restricted to a few species of teleosts (e.g., rainbow trout, catfish, zebrafish, sea bass) and has previously focused on morphological features. However, basic questions including the identification of the first lympho-hematopoietic sites, the origin of T- and B-lymphocytes and the acquisition of full immunological capacities remain to be resolved. We review these three main topics with special emphasis on recent results obtained from the zebrafish, a new experimental model particularly suitable for study of the ontogeny of the immune system because of its rapid development and easy manipulation. This species also provides an easy way of creating mutations that can be detected by various types of screens. In some teleosts (i.e., angelfish) the first blood cells are formed in the yolk sac. In others, such as zebrafish, the first hematopoietic site is an intraembryonic locus, the intermediate cell mass (ICM), whereas in both killifish and rainbow trout the first blood cells appear for a short time in the yolk sac but later the ICM becomes the main hematopoietic area. Erythrocytes and macrophages are the first blood cells to be identified in zebrafish embryos. They occur in the ICM, the duct of Cuvier and the peripheral circulation. Between 24 and 30 hour post-fertilization (hpf) at a temperature of 28 degrees C a few myeloblasts and myelocytes appear between the yolk sac and the body walls, and the ventral region of the tail of 1-2 day-old zebrafish also contains developing blood cells. The thymus, kidney and spleen are the major lymphoid organs of teleosts. The thymus is the first organ to become lymphoid, although earlier the kidney can contain hematopoietic precursors but not lymphocytes. In freshwater, but not in marine, teleosts the spleen is the last organ to acquire that condition. We and other authors have demonstrated an early expression of Rag-1 in the zebrafish thymus that correlates well with the morphological identification of lymphoid cells. On the other hand, the origins and time of appearance of B lymphocytes in teleosts are a matter of discussion and recent results are summarized here. The functioning rather than the mere morphological evidence of lymphocytes determines when the full immunocompetence in fish is attained. Information on the histogenesis of fish lymphoid organs can also be obtained by analysing zebrafish mutants with defects in the development of immune progenitors and/or in the maturation of non-lymphoid stromal elements of the lymphoid organs. The main characteristics of some of these mutants will also be described.

Animals↗

PubMed2005

Identification of the F1F0 mitochondrial ATPase as a target for modulating skin pigmentation by screening a tagged triazine library in zebrafish.

A triazine-based combinatorial library of small molecules was screened in zebrafish to identify compounds that produced interesting phenotypes. One compound (of 1536 screened) induced a dramatic increase in the pigmentation of early stage zebrafish embryos. This compound, PPA, was also found to increase pigmentation in cultured mammalian melanocytes. The cellular target was identified as the mitochondrial F1F0-ATP synthase (ATPase) by affinity chromatography. Oligomycin, a small molecule known to inhibit the mitochondrial ATPase, competed with PPA for its cellular target in melanocytes. In addition, PPA was shown to alter the membrane potential of mitochondria, consistent with inhibition of the mitochondrial ATPase. Thus, PPA has been successfully used as a chemical probe in a forward chemical genetic approach to establish a link between the phenotype and the protein. The results attest to the power of screening small molecule libraries in zebrafish as a means of identifying mammalian targets and suggest the mitochondrial ATPase as a target for modulating pigmentation in both melanocytes and melanoma cells.

Amino Acid Sequence↗

PubMed2006

Imaging macrophage chemotaxis in vivo: studies of microtubule function in zebrafish wound inflammation.

The inflammatory response is one of the most dramatic examples of directed cell movement in nature. Inflammation is triggered at the site of injury and results in the migration of immune cells to the site to protect the host from infection. We have devised an in vivo inflammation assay using translucent zebrafish embryos, which allow live imaging and pharmacological manipulation of macrophage chemotaxis to wounds inflicted with a laser. Using this assay, we test the role of the microtubule cytoskeleton in macrophage chemotaxis in vivo using nocodazole to disrupt microtubule polymerization. We find that de-stabilisation of microtubules with nocodazole impairs macrophage recruitment to wounds, but that addition of the Rho kinase inhibitor Y-27632 suppresses these effects and restores the recruitment of macrophages to wounds. Taken together, these results suggest that destabilizing microtubules activates Rho kinase and that this increase in Rho kinase activity interferes with leukocyte recruitment in vivo.

Amides↗

PubMed2000

Characterisation of the promoter region of the zebrafish six7 gene.

The Drosophila homeobox gene sine oculis and its murine homologue Six3 have both been shown to have regulatory functions in eye and brain development. In zebrafish, three Six3-related genes with conserved expression during early eye and head formation have been identified. One of these, six7, is first expressed at the gastrula stage in the involuting axial mesoderm, and later in the overlying neuroectoderm from which the forebrain and optic primordium develop. To elucidate the mechanisms regulating six7 expression, we isolated a 2.7-kb fragment of the 5'-flanking region. Three sequentially deleted fragments of this upstream region were used to produce GFP reporter constructs for analysis of tissue-specific expression in zebrafish embryos. The results show that a 625-bp upstream fragment is sufficient to direct strong expression of the reporter during gastrulation and early neurulation. The proximal part of the promoter contains binding sites for various constitutive transcription factors and an additional upstream element that was shown to be critical in directing expression to the anterior region of the zebrafish brain.

Animals↗

PubMed2004

The basic helix-loop-helix transcription factor, Tal2, marks the lateral floor plate of the spinal cord in zebrafish.

Basic helix-loop-helix (bHLH) transcription factors play key roles in the development of the central nervous system. Here we report the isolation of a zebrafish gene that encodes a homologue of the mammalian bHLH transcription factor, Tal2. In zebrafish embryos, tal2, like its mammalian homologue, is strongly expressed in the diencephalon and the mesencephalon, with the latter expression located in post-mitotic cells of the tectum. However, in addition to this conserved brain expression, we also detect expression in the floor plate of the spinal cord. By the location of this expression relative to other genes expressed in the floor plate and by analysing expression in a selection of midline mutants, we reveal that tal2 is expressed within the lateral floor plate as opposed to the medial floor plate, and also in more dorsal cells which are distinct from motorneurons and depend on either sonic hedgehog signalling or a signal coming from the lateral floor plate. This is to our knowledge the first report of a gene expressed specifically in lateral cells of the floor plate in the spinal cord.

Amino Acid Sequence↗

PubMed2003

Double labeling of neurons by retrograde axonal tracing and non-radioactive in situ hybridization in the CNS of adult zebrafish.

A number of genes affecting axonal projections are currently being identified in zebrafish mutant screens. Analyzing the expression of these genes in the adult brain in relation to specific neuronal populations could yield insights into new functional contexts, such as the successful axonal regeneration in adult zebrafish. Here, we provide a relatively simple procedure for non-radioactive in situ hybridization in sections of adult zebrafish brains in combination with retrograde axonal tracing using the fluorescent neuronal tracer rhodamine dextran amine (RDA). A lesion is inflicted on the spinal cord of adult zebrafish and a crystal of RDA is then applied to the lesion site resulting in retrograde labeling of neurons in the brain through their spinal axons. Six to eighteen days later fish are perfusion-fixed, and in situ hybridization is carried out on vibratome-cut floating sections using a protocol simplified from that used for whole-mounted zebrafish embryos. This procedure leads to robust double labeling of axotomized neurons with RDA and an in situ hybridization signal for the growth-associated protein 43 (GAP-43). This method can be used to identify gene expression in specific populations of projection neurons and to detect changes in gene expression in axotomized neurons in the CNS of adult zebrafish.

Animals↗

PubMed2006

The KLHL12-Cullin-3 ubiquitin ligase negatively regulates the Wnt-beta-catenin pathway by targeting Dishevelled for degradation.

Dishevelled is a conserved protein that interprets signals received by Frizzled receptors. Using a tandem-affinity purification strategy and mass spectrometry we have identified proteins associated with Dishevelled, including a Cullin-3 ubiquitin ligase complex containing the Broad Complex, Tramtrack and Bric à Brac (BTB) protein Kelch-like 12 (KLHL12). This E3 ubiquitin ligase complex is recruited to Dishevelled in a Wnt-dependent manner that promotes its poly-ubiquitination and degradation. Functional analyses demonstrate that regulation of Dishevelled by this ubiquitin ligase antagonizes the Wnt-beta-catenin pathway in cultured cells, as well as in Xenopus and zebrafish embryos. Considered with evidence that the distinct Cullin-1 based SCF(beta-TrCP)complex regulates beta-catenin stability, our data on the stability of Dishevelled demonstrates that two distinct ubiquitin ligase complexes regulate the Wnt-beta-catenin pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Multiple pathways in the midline regulate concordant brain, heart and gut left-right asymmetry.

The embryonic midline in vertebrates has been implicated in left-right development, but the mechanisms by which it regulates left-right asymmetric gene expression and organ morphogenesis are unknown. Zebrafish embryos have three domains of left-right asymmetric gene expression that are useful predictors of organ situs. cyclops (nodal), lefty1 and pitx2 are expressed in the left diencephalon; cyclops, lefty2 and pitx2 are expressed in the left heart field; and cyclops and pitx2 are expressed in the left gut primordium. Distinct alterations of these expression patterns in zebrafish midline mutants identify four phenotypic classes that have different degrees of discordance among the brain, heart and gut. These classes help identify two midline domains and several genetic pathways that regulate left-right development. A cyclops-dependent midline domain, associated with the prechordal plate, regulates brain asymmetry but is dispensable for normal heart and gut left-right development. A second midline domain, associated with the anterior notochord, is dependent on no tail, floating head and momo function and is essential for restricting asymmetric gene expression to the left side. Mutants in spadetail or chordino give discordant gene expression among the brain, heart and gut. one-eyed pinhead and schmalspur are necessary for asymmetric gene expression and may mediate signaling from midline domains to lateral tissues. The different phenotypic classes help clarify the apparent disparity of mechanisms proposed to explain left-right development in different vertebrates.

Animals↗