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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↗

PubMed1998

The nieuwkoid gene characterizes and mediates a Nieuwkoop-center-like activity in the zebrafish.

BACKGROUND: In amphibians, the Nieuwkoop center--a primary inducing region--has a central role in the induction of dorsal mesodermal cells to form the Spemann organizer. In teleosts, such as the zebrafish, Danio rerio, the functional equivalent of the amphibian Spemann organizer is the dorsal shield. Historically, a small region of the teleost yolk syncytial layer (YSL), an extraembryonic tissue that underlies the entire blastoderm, has been implicated in dorsal shield specification. Difficulties in transplanting discrete regions of the YSL and the previous lack of localized expression patterns unique to the YSL have, however, hindered efforts to prove definitively that the YSL possesses Nieuwkoop-center-like activities. RESULTS: Here, we describe the isolation and analysis of a new homeobox gene, called nieuwkoid, which is first expressed immediately following the mid-blastula transition on the dorsal side of the zebrafish pregastrula embryo. We found that, by the onset of gastrulation, nieuwkoid expression becomes localized to a restricted region of the YSL, directly underlying the future dorsal shield. Mis-expression of nieuwkoid in early zebrafish embryos was found to be sufficient for the induction of ectopic organizer regions and secondary axes. Mis-expression of nieuwkoid by cell transplantation or by direct injection into the YSL led to the non-autonomous induction of ectopic organizer gene expression. CONCLUSIONS: The dynamic and restricted expression of the nieuwkoid gene, combined with its potent dorsalizing activity, suggests that nieuwkoid is an important component in the regionalization of the gastrula organizer, possibly characterizing and mediating an organizer-inducing/Nieuwkoop-center-like activity.

Amino Acid Sequence↗

PubMed1999

A role for the extraembryonic yolk syncytial layer in patterning the zebrafish embryo suggested by properties of the hex gene.

Recent studies in mouse suggest that the extraembryonic endoderm has an important role in early embryonic patterning [1]. To analyze whether similar mechanisms operate in other vertebrates, we cloned the zebrafish homologue of Hex, a homeobox gene that is expressed asymmetrically in the mouse visceral endoderm [2]. Early expression of zebrafish hex is restricted to the dorsal portion of the yolk syncytial layer (YSL), an extraembryonic tissue. By the onset of gastrulation, hex is expressed in the entire dorsal half of the YSL, which directly underlies the cells fated to form the neural plate. We show that hex expression is initially regulated by the maternal Wnt pathway and later by a Bmp-mediated pathway. Overexpression experiments of wild-type and chimeric Hex constructs indicate that Hex functions as a transcriptional repressor and its overexpression led to the downregulation of bmp2b and wnt8 expression and the expansion of chordin expression. These findings provide further evidence that the zebrafish YSL is the functional equivalent of the mouse visceral endoderm and that extraembryonic structures may regulate early embryonic patterning in many vertebrates.

Animals↗

PubMed2003

Heat- and cold-inducible regulation of HSP70 expression in zebrafish ZF4 cells.

Elevated temperature induces a rapid heat shock transcription factor (HSFs)-mediated expression of heat shock (hsp) genes. The effect of cold exposure on hsp gene expression has hardly been investigated, although ectothermic animals experience both cold and heat stress. We have previously shown in zebrafish that the expression of hsf1a and a unique isoform hsf1b vary in a tissue-specific manner upon heat stress. In the current study, using a zebrafish (Danio rerio) embryonic cell line (ZF4), we have compared the effects of heat shock (28-->37 degrees C) vs. cold shock (28-->20 degrees C) on the expression of ahsf1a, zhsf1b and hsp70. Concomitantly, the suitability of the ZF4 cells as a model system was verified. The expression pattern of HSP70 proteins following heat or cold exposure is distinct, and the total HSP70 level is upregulated or stable, respectively. Moreover, heat exposure specifically increases the ratio of zhsf1a/b expression (10-fold), whereas cold exposure decreases it to one half. These data suggest that the zhsf1a/zhsf1b ratio is regulated in a temperature-dependent manner, and the ratio may be indicative of the stressor-specific HSP70 expression. Furthermore, the response in ZF4 cells upon heat shock resembles the response observed in zebrafish liver and thus, supports the use of this cell line in stress response studies.

Animals↗

PubMed2002

Expression of Zash-1a in the postembryonic zebrafish brain allows comparison to mouse Mash1 domains.

Four areas in the late embryonic murine forebrain, i.e. the subpallium (striatum), the preoptic region, the ventral thalamus, and the hypothalamus, have been described to express the basic helix-loop-helix (bHLH) gene mammalian achaete-scute homolog Mash1 (Ascl1, Mouse Genome Informatics) in a complementary fashion to another bHLH gene, neurogenin1 (ngn1) (Neurod3, Mouse Genome Informatics), which is expressed in directly adjacent forebrain regions. We report here that the four regions previously identified as subpallium, preoptic region, ventral thalamus and hypothalamus (i.e. ventral inferior lobe) in the postembryonic zebrafish brain show Zash-1a expression at 3 days postfertilization (dpf), whereas none of those areas express the bHLH gene neuroD (nrd) between 2 and 5 dpf. This indicates that two well established alternative genetic pathways involved in neurogenesis in the amniote (mammalian) brain are present in homologous phenotypic locations in the anamniote (zebrafish) brain as well and that these pathways possibly act similarly in the generation of different neuronal phenotypes (e.g. subpallial GABAergic interneurons versus pallial glutamatergic projection neurons, or dopaminergic neurons versus other neurotransmitter phenotypes). Furthermore, previous initial identification of early postembryonic brain subdivisions in the zebrafish is strongly corroborated by these expression patterns.

Animals↗

PubMed1996

Insertional mutagenesis and rapid cloning of essential genes in zebrafish.

Large-scale chemical mutagenesis screens in zebrafish have led to the isolation of thousands of lethal mutations in genes that are essential for embryonic development. However, the cloning of these mutated genes is difficult at present as it requires positional cloning methods. In Drosophila, chemical mutagenesis screens were complemented with P-element insertional mutagenesis which facilitated the cloning of many genes that had been identified by chemical lesions. To facilitate the cloning of vertebrate genes that are important during embryogenesis, we have developed an insertional mutagenesis strategy in zebrafish using a retroviral vector. Here, in a pilot screen of 217 proviral insertions, we obtained three insertional mutants with embryonic lethal phenotypes, and identified two of the disrupted genes. One of these, no arches, is essential for normal pharyngeal arch development, and is homologous to the recently characterized Drosophila zinc-finger gene, clipper, which encodes a novel type of ribonuclease. As it is easy to generate tens to hundreds of thousands of proviral transgenes in zebrafish, it should now be possible to use this screening method to mutate and then rapidly clone a large number of genes affecting vertebrate developmental and cellular processes.

Amino Acid Sequence↗

PubMed2005

The immunoglobulin heavy-chain locus in zebrafish: identification and expression of a previously unknown isotype, immunoglobulin Z.

The only immunoglobulin heavy-chain classes known so far in teleosts have been mu and delta. We identify here a previously unknown class, immunoglobulin zeta, expressed in zebrafish and other teleosts. In the zebrafish heavy-chain locus, variable (V) gene segments lie upstream of two tandem diversity, joining and constant (DJC) clusters, resembling the mouse T cell receptor alpha (Tcra) and delta (Tcrd) locus. V genes rearrange to (DJC)(zeta) or to (DJC)(mu) without evidence of switch rearrangement. The zebrafish immunoglobulin zeta gene (ighz) and mouse Tcrd, which are proximal to the V gene array, are expressed earlier in development. In adults, ighz was expressed only in kidney and thymus, which are primary lymphoid organs in teleosts. This additional class adds complexity to the immunoglobulin repertoire and raises questions concerning the evolution of immunoglobulins and the regulation of the differential expression of ighz and ighm.

Animals↗

PubMed2006

Fgf21 is essential for haematopoiesis in zebrafish.

Fibroblast growth factors (Fgfs) function as key secreted signalling molecules in many developmental events. The zebrafish is a powerful model system for the investigation of embryonic vertebrate haematopoiesis. Although the effects of Fgf signalling on haematopoiesis in vitro have been reported, the functions of Fgf signalling in haematopoiesis in vivo remain to be explained. We identified Fgf21 in zebrafish embryos. Fgf21-knockdown zebrafish embryos lacked erythroid and myeloid cells but not blood vessels and lymphoid cells. The knockdown embryos had haemangioblasts and haematopoietic stem cells. However, the knockdown embryos had significantly fewer myeloid and erythroid progenitor cells. In contrast, Fgf21 had no significant effect on cell proliferation and apoptosis in the intermediate cell mass. These results indicate that Fgf21 is a newly identified factor essential for the determination of myelo-erythroid progenitor cell fate in vivo.

Animals↗

PubMed2004

The 'definitive' (and 'primitive') guide to zebrafish hematopoiesis.

Progressive advances using zebrafish as a model organism have provided hematologists with an additional genetic system to study blood cell formation and hematological malignancies. Despite extensive evolutionary divergence between bony fish (teleosts) and mammals, the molecular pathways governing hematopoiesis have been highly conserved. As a result, most (if not all) of the critical hematopoietic transcription factor genes identified in mammals have orthologues in zebrafish. As in other vertebrates, all of the teleost blood lineages are believed to originate from a pool of pluripotent, self-renewing hematopoietic stem cells. Here, we provide a detailed review of the timing, anatomical location, and transcriptional regulation of zebrafish 'primitive' and 'definitive' hematopoiesis as well as discuss a model of T-cell leukemia and recent advances in blood cell transplantation. Given that many of the regulatory genes that control embryonic hematopoiesis have been implicated in oncogenic pathways in adults, an understanding of blood cell ontogeny is likely to provide insights into the pathophysiology of human leukemias.

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↗

PubMed2001

Gli2 mediation of hedgehog signals in slow muscle induction in zebrafish.

Zebrafish skeletal muscles are composed of two major types of muscle fibers, broadly classified as fast or slow fibers. Recent studies have demonstrated that members of the Hedgehog (Hh) family induce the formation of slow muscle fibers. Hedgehog signals are secreted proteins that function through the transcription factor Glis. We report here the characterization of a zebrafish Gli2 expression in slow and fast muscle cells and the study of the roles of Hedgehogs and Gli2 in zebrafish muscle development using two mutant strains; sonic-you (syu) and you-too (yot), respective for sonic hedgehog (shh) and Gli2 mutation. We have demonstrated that Shh and Gli2 mutation causes similar defects in slow muscle formation. There is, however, a difference in the degree of defect between these two mutants. In yot mutant embryos, development of slow muscles was completely blocked, whereas in syu mutant embryos, a small number of slow muscle cells could still form, suggesting that other Hhs were also involved in slow muscle induction. Induction of slow muscles by other Hhs appeared to require Gli2, because ectopic expression of Echidna hedgehog (Ehh) and Tiggy-winkle hedgehog (Twhh) failed to induce slow muscles in yot mutant embryos. Together, these data suggest that further Hhs, other than Shh, are also involved in the induction and differentiation of slow muscle cells and that Gli2 is required by Shh, Twhh, and Ehh, thus playing a key role in the induction and differentiation of slow muscle cells.

Animals↗

PubMed2002

Treatment with Myf5-morpholino results in somite patterning and brain formation defects in zebrafish.

Myf-5 is a stage-dependent transcription factor associated with somitogenesis. To study its biological functions in zebrafish, we injected the Myf5-morpholinos ZMF-MO (antisense nucleotides 28 to 52) and ZMF-OTHER (antisense nucleotides 3 to 27) into zebrafish embryos to establish a myf-5 gene knockdown. No phenotypic abnormalities were observed following injection with 0.2 ng of ZMF-MO, but defects were displayed in 2 of 118 (1.7%) surviving embryos injected with 1 ng ZMF-MO. Morphological defects became more severe with increased dosages: 105 of 270 (38.9%) surviving embryos injected with 4.5 ng of ZMF-MO displayed such abnormalities as the absence of eyes or brains in addition to the following low-dosage defects in 24 hpf embryos: longitudinal yolk sacs, incomplete epiboly coverage, abnormal and suspended tail buds, diffused somite boundaries, and head shrinkage. Similar results were observed in the 4.5 ng ZMF-OTHER injection group. However, when fish were co-injected with 4.5 ng ZMF-MO and 4.5 ng myf-5 mRNA, abnormality rates decreased from 49.6% to 5.5%. Our results show that the brain krox20 gene was down-regulated at rhombomere 3; the pax2.1 gene was completely down-regulated; myoD was expressed normally; myogenin was substantially down-regulated in whole somites; and desmin was partly inhibited in newly forming somites. Our conclusion is that zebrafish Myf-5 may play important roles in brain formation and in the convergence and extension of shield epiblasts and tail buds during early embryogenesis, in addition to its well-understood role as a muscle regulatory factor in somites.

Animals↗

PubMed2004

Mutation of the zebrafish choroideremia gene encoding Rab escort protein 1 devastates hair cells.

To identify genes important for hair-cell function, we conducted a mutagenic screen in zebrafish. Larvae from one mutant line, ru848, were unresponsive to acoustic stimuli and unable to balance. The mutation results in a 90% reduction in hair-cell number and partial retinal degeneration by 5 days postfertilization. We localized the recessive ru848 mutation by positional cloning to the zebrafish homolog of the human Choroideremia gene, which encodes Rab escort protein 1. This protein is essential for the normal prenylation of Rabs. Mutations in the human gene induce choroideremia, a disease marked by slow-onset degeneration of rod photoreceptors and retinal pigment epithelial cells. The degenerative phenotype resulting from a null mutation in the zebrafish gene indicates that hair cells and retinal cells require Rab escort protein 1 for survival.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Common pathways in circadian and cell cycle clocks: light-dependent activation of Fos/AP-1 in zebrafish controls CRY-1a and WEE-1.

The cell cycle and the circadian clock are endogenous pacemakers, which coexist in most eukaryotic cells and share a number of conceptual features. In the zebrafish, light directly regulates the timing of both clocks, although the signaling and transcriptional pathways that convey photic information to essential nuclear regulators have yet to be deciphered. We have previously established the Z3 cell line, which recapitulates the features of zebrafish circadian clock and represents an ideal system to study light-dependent signaling and gene regulation. We conducted a search for light-responsive transcription factors and found that AP-1 DNA binding is highly induced. Light induces the expression of zWee1, a cell cycle gene essential for G2/M transition, and zCry1a, a clock gene of the feedback regulatory loop. We have found consensus AP-1 sites in the regulatory regions of both zWee1 and zCry1a genes, and we show that light inducibility of both genes is abrogated by inhibition of AP-1 function. Light also elicits chromatin remodeling by stimulating hyperacetylation at Lys-14 of histone H3 at both zWee1 and zCry1a promoters, as assessed by chromatin immunoprecipitation assays by using anti-Fos antibody. These findings provide strong evidence that circadian and cell cycle clocks share unique light-responsive pathways in zebrafish.

Animals↗

PubMed2006

Diversity of zebrafish peripheral oscillators revealed by luciferase reporting.

In various multicellular organisms, circadian clocks are present not only in the central nervous system, but also in peripheral organs and tissues. In mammals peripheral oscillators are not directly responsive to light, but are entrained by the central oscillator in the suprachiasmatic nucleus. These individual oscillators are diverse in their free-running periods and phases. In contrast, cultured peripheral tissues and cell lines from zebrafish are not only rhythmic, but can also be directly entrained by light. Because of the convenience of studying rhythms in cultured cells, however, little has been known about properties of individual oscillators in intact zebrafish. Here, we show the remarkable diversity and consistency of oscillator properties in various peripheral organs and tissues from the period3-luciferase (per3-luc) transgenic zebrafish. Tissue-dependent differences were found in free-running period, phase, response to light, and temperature compensation. Furthermore, cycling amplitudes were reduced at lower temperatures in some, but not all, of the organs tested. Finally, we found that per3-luc rhythms can free run in both constant dark and constant light with remarkably similar amplitudes, phases, and periods, despite the fact that the mRNA of per2 and per1 has been shown not to oscillate in constant light.

Animals↗

PubMed2003

Activation of Notch signaling pathway precedes heart regeneration in zebrafish.

Several vertebrates display the ability to regenerate parts of their body after amputation. During this process, differentiated cells reenter the cell cycle and proliferate to generate a mass of undifferentiated cells. Repatterning mechanisms act on these cells to eventually shape a regenerated tissue or organ that replaces the amputated one. Experiments with regenerating limbs/fins in newts and zebrafish have shown that members of the Msx family of homeodomain-containing transcription factors play key roles during blastema formation and patterning. Here we show that adult zebrafish have a remarkable capacity to regenerate the heart in a process that involves up-regulation of msxB and msxC genes. We present evidence indicating that heart regeneration involves the execution of a specific genetic program, rather than redeployment of a cardiac development program. Preceding Msx activation, there is a marked increase in the expression of notch1b and deltaC, which we show are also up-regulated during fin regeneration. These data suggest a role for the Notch pathway in the activation of the regenerative response. Taken together, our results underscore the use of zebrafish as a model for investigating the process of regeneration in particular and the biology of stem cells in general. Advances in these fields will undoubtedly aid in the implementation of strategies for regenerative medicine.

Animals↗

PubMed2002

Caspy, a zebrafish caspase, activated by ASC oligomerization is required for pharyngeal arch development.

The pyrin domain was identified recently in multiple proteins that are associated with apoptosis and/or inflammation, but the physiological and molecular function of these proteins remain poorly understood. We have identified Caspy and Caspy2, two zebrafish caspases containing N-terminal pyrin domains. Expression of Caspy and Caspy2 induced apoptosis in mammalian cells that were inhibited by general caspase inhibitors. Biochemical analysis revealed that both Caspy and Caspy2 are active caspases, but they exhibit different substrate specificity. Caspy, but not Caspy2, interacted with the zebrafish orthologue of ASC (zAsc), a pyrin- and caspase recruitment domain-containing protein identified previously in mammals. The pyrin domains of both Caspy and zAsc were required for their interaction. Furthermore, zAsc and Caspy co-localized to the "speck" when co-transfected into mammalian cells. Enforced oligomerization of zAsc, but not simple interaction with zAsc, induced specific proteolytic activation of Caspy and enhanced Caspy-dependent apoptosis. Injection of zebrafish embryos with a morpholino antisense oligonucleotide corresponding to caspy resulted in an "open mouth" phenotype associated with defective formation of the cartilaginous pharyngeal skeleton. These studies suggest that zAsc mediates the activation of Caspy, a caspase that plays an important role in the morphogenesis of the jaw and gill-bearing arches.

Amino Acid Sequence↗

PubMedDate not supplied

Isolation and phenogenetics of a novel circadian rhythm mutant in zebrafish.

Widespread use of zebrafish (Danio rerio) in genetic analysis of embryonic development has led to rapid advances in the technology required to generate, map and clone mutated genes. To identify genes involved in the generation and regulation of vertebrate circadian rhythmicity, we screened for dominant mutations that affect the circadian periodicity of larval zebrafish locomotor behavior. In a screen of 6,500 genomes, we recovered 8 homozygous viable, semi-dominant mutants, and describe one of them here. The circadian period of the lager and lime (lag(dg2)) mutant is shortened by 0.7 h in heterozygotes,and 1.3 h in homozygotes. This mutation also shortens the period of the melatonin production rhythm measured from cultured pineal glands, indicating that the mutant gene product affects circadian rhythmicity at the tissue level, as well as at the behavioral level. This mutation also alters the sensitivity of pineal circadian period to temperature, but does not affect phase shifting responses to light. Linkage mapping with microsatellite markers indicates that the lag mutation is on chromosome 7. A zebrafish homolog of period1(per1) is the only known clock gene homolog that maps near the lag locus. However, all sequence variants found in per1 cDNA from lag(dg2) mutants are also present in wild type lines, and we were unable to detect any defect in per1 mRNA splicing, so this mutation may identify a novel clock gene.

Animals↗