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PubMed2003

Coordinated temporal and spatial control of motor neuron and serotonergic neuron generation from a common pool of CNS progenitors.

Neural progenitor cells often produce distinct types of neurons in a specific order, but the determinants that control the sequential generation of distinct neuronal subclasses in the vertebrate CNS remain poorly defined. We examined the sequential generation of visceral motor neurons and serotonergic neurons from a common pool of neural progenitors located in the ventral hindbrain. We found that the temporal specification of these neurons varies along the anterior-posterior axis of the hindbrain, and that the timing of their generation critically depends on the integrated activities of Nkx- and Hox-class homeodomain proteins. A primary function of these proteins is to coordinate the spatial and temporal activation of the homeodomain protein Phox2b, which in turn acts as a binary switch in the selection of motor neuron or serotonergic neuronal fate. These findings assign new roles for Nkx, Hox, and Phox2 proteins in the control of temporal neuronal fate determination, and link spatial and temporal patterning of CNS neuronal fates.

Animals↗
PubMed2006

Evolution and comparative analysis of the MHC Class III inflammatory region.

BACKGROUND: The Major Histocompatibility Complex (MHC) is essential for immune function. Historically, it has been subdivided into three regions (Class I, II, and III), but a cluster of functionally related genes within the Class III region has also been referred to as the Class IV region or "inflammatory region". This group of genes is involved in the inflammatory response, and includes members of the tumour necrosis family. Here we report the sequencing, annotation and comparative analysis of a tammar wallaby BAC containing the inflammatory region. We also discuss the extent of sequence conservation across the entire region and identify elements conserved in evolution. RESULTS: Fourteen Class III genes from the tammar wallaby inflammatory region were characterised and compared to their orthologues in other vertebrates. The organisation and sequence of genes in the inflammatory region of both the wallaby and South American opossum are highly conserved compared to known genes from eutherian ("placental") mammals. Some minor differences separate the two marsupial species. Eight genes within the inflammatory region have remained tightly clustered for at least 360 million years, predating the divergence of the amphibian lineage. Analysis of sequence conservation identified 354 elements that are conserved. These range in size from 7 to 431 bases and cover 15.6% of the inflammatory region, representing approximately a 4-fold increase compared to the average for vertebrate genomes. About 5.5% of this conserved sequence is marsupial-specific, including three cases of marsupial-specific repeats. Highly Conserved Elements were also characterised. CONCLUSION: Using comparative analysis, we show that a cluster of MHC genes involved in inflammation, including TNF, LTA (or its putative teleost homolog TNF-N), APOM, and BAT3 have remained together for over 450 million years, predating the divergence of mammals from fish. The observed enrichment in conserved sequences within the inflammatory region suggests conservation at the transcriptional regulatory level, in addition to the functional level.

Animals↗
PubMed1999

Heat shock produces periodic somitic disturbances in the zebrafish embryo.

Environmental influences are known to produce segmental defects in a variety of organisms. In this paper we report upon segmental aberrations produced by brief heat shocks delivered to developing zebrafish embryos. The initial defects in the segmental pattern of somitic boundaries and motoneuron axon outgrowth were usually observed five somites caudal to the somite which was forming at the time of heat shock application. Segmental defects in zebrafish embryos exposed to a single heat shock treatment can occur in a periodic pattern similar to the multiple disturbances observed to occur in chick embryos. These data are discussed with regard to models involving cell cycle synchrony or 'clock and wavefront' schemes in the process of somitogenesis.

Animals↗
PubMed2001

Enhanced expression and stable transmission of transgenes flanked by inverted terminal repeats from adeno-associated virus in zebrafish.

Mosaic expression of transgenes in the F0 generation severely hinders the study of transient expression in transgenic fish. To avoid mosaicism, enhanced green fluorescent protein (EGFP) gene cassettes were constructed and introduced into one-celled zebrafish embryos. These EGFP gene cassettes were flanked by inverted terminal repeats (ITRs) from adeno-associated virus (AAV) and driven by zebrafish alpha-actin (palpha-actin-EGFP-ITR) or medaka beta-actin promoters (pbeta-actin-EGFP-ITR). EGFP was expressed specifically and uniformly in the skeletal muscle of 56% +/- 8% of the palpha-actin-EGFP-ITR-injected survivors and in the entire body of 1.3% +/- 0.8% of the pbeta-actin-EGFP-ITR-injected survivors. Uniform transient expression never occurred in zebrafish embryos injected with EGFP genes that were not flanked by AAV-ITRs. In the F0 generation, uniformly distributed EGFP could mimic the stable expression in transgenic lines early in development. We established five transgenic lines derived from palpha-actin-EGFP-ITR-injected embryos crossed with wild-type fish and 11 transgenic lines derived from pbeta-actin-EGFP-ITR-injected embryos crossed with wild-type fish. None of these transgenic lines failed to express the transgene, a result confirmed by polymerase chain reaction analysis. Stable mendelian transmission of the transgenes was achieved in both alpha-actin and beta-actin transgenic lines without changing the patterns of expression and integration. Progeny inheritance test and Southern blot analysis results strongly suggest that transgenes flanked by AAV-ITRs were integrated randomly into the genome at a single locus with a concatamerized multiplier. Thus, incorporating AAV-ITRs into transgenes results in uniform gene expression in the F0 generation and stable transmission of transgenes in zebrafish.

Actins↗
PubMed2004

Reconsidering the evolution of eukaryotic selenoproteins: a novel nonmammalian family with scattered phylogenetic distribution.

While the genome sequence and gene content are available for an increasing number of organisms, eukaryotic selenoproteins remain poorly characterized. The dual role of the UGA codon confounds the identification of novel selenoprotein genes. Here, we describe a comparative genomics approach that relies on the genome-wide prediction of genes with in-frame TGA codons, and the subsequent comparison of predictions from different genomes, wherein conservation in regions flanking the TGA codon suggests selenocysteine coding function. Application of this method to human and fugu genomes identified a novel selenoprotein family, named SelU, in the puffer fish. The selenocysteine-containing form also occurred in other fish, chicken, sea urchin, green algae and diatoms. In contrast, mammals, worms and land plants contained cysteine homologues. We demonstrated selenium incorporation into chicken SelU and characterized the SelU expression pattern in zebrafish embryos. Our data indicate a scattered evolutionary distribution of selenoproteins in eukaryotes, and suggest that, contrary to the picture emerging from data available so far, other taxa-specific selenoproteins probably exist.

Amino Acid Sequence↗
PubMed2001

Cytoplasmic expression of ribozyme in zebrafish using a T7 autogene system.

A cytoplasmic ribozyme expression system, based on codelivery of a ribozyme vector, a T7 autogene vector, and T7 RNA polymerase (RNAP), has been developed and used to generate a specific phenotype in zebrafish by targeting a no tail (ntl) mRNA. The expression of the no tail ribozyme sequence is under the control of a tandem of two promoters: The T7 promoter and an adenoviral va 1 (pol III) promoter. The coinjection of the ribozyme vector pT7vaRz, the T7 autogene vector pT7T7, and the T7 RNAP resulted in rapid synthesis of the ribozyme against the ntl mRNA in the cytoplasm of the injected zebrafish embryos, generating no tail phenotypes in up to 10-20% of the injected embryos. The phenotypic change rates have been found to be related to the concentrations of the plasmid vectors and T7 RNAP injected and to the ratios of the three injected components. This cytoplasmic ribozyme expression system may be useful for efficiently targeting other mRNA and for various biomedical applications. These potential applications may include rapid identification of biological functions of novel genes from zebrafish and humans based on partial gene sequence information and gene therapy of genetic and acquired diseases.

Adenoviridae↗
PubMed1999

A radiation hybrid map of the zebrafish genome.

Recent large-scale mutagenesis screens have made the zebrafish the first vertebrate organism to allow a forward genetic approach to the discovery of developmental control genes. Mutations can be cloned positionally, or placed on a simple sequence length polymorphism (SSLP) map to match them with mapped candidate genes and expressed sequence tags (ESTs). To facilitate the mapping of candidate genes and to increase the density of markers available for positional cloning, we have created a radiation hybrid (RH) map of the zebrafish genome. This technique is based on somatic cell hybrid lines produced by fusion of lethally irradiated cells of the species of interest with a rodent cell line. Random fragments of the donor chromosomes are integrated into recipient chromosomes or retained as separate minichromosomes. The radiation-induced breakpoints can be used for mapping in a manner analogous to genetic mapping, but at higher resolution and without a need for polymorphism. Genome-wide maps exist for the human, based on three RH panels of different resolutions, as well as for the dog, rat and mouse. For our map of the zebrafish genome, we used an existing RH panel and 1,451 sequence tagged site (STS) markers, including SSLPs, cloned candidate genes and ESTs. Of these, 1,275 (87.9%) have significant linkage to at least one other marker. The fraction of ESTs with significant linkage, which can be used as an estimate of map coverage, is 81.9%. We found the average marker retention frequency to be 18.4%. One cR3000 is equivalent to 61 kb, resulting in a potential resolution of approximately 350 kb.

Animals↗
PubMed2004

Adenomatous polyposis coli control of retinoic acid biosynthesis is critical for zebrafish intestinal development and differentiation.

Mutations in the APC (adenomatous polyposis coli) tumor suppressor gene cause uncontrolled proliferation and impaired differentiation of intestinal epithelial cells. Recent studies indicate that human colon adenomas and carcinomas lack retinol dehydrogenases (RDHs) and that APC regulates the expression of human RDHL. These data suggest a model wherein APC controls enterocyte differentiation by controlling retinoic acid production. However, the importance of APC and retinoic acid in mediating control of normal enterocyte development and differentiation remains unclear. To examine the relationship between APC and retinoic acid biosynthesis in normal enterocytes, we have identified two novel zebrafish retinol dehydrogenases, termed zRDHA and zRDHB, that show strong expression within the gut of developing zebrafish embryos. Morpholino knockdown of either APC or zRDHB in zebrafish embryos resulted in defects in structures known to require retinoic acid. These defects included cardiac abnormalities, pericardial edema, failed jaw and pectoral fin development, and the absence of differentiated endocrine and exocrine pancreas. In addition, APC or zRDHB morphant fish developed intestines that lacked columnar epithelial cells and failed to express the differentiation marker intestinal fatty acid-binding protein. Treatment of either APC or zRDHB morphant embryos with retinoic acid rescued the defective phenotypes. Downstream of retinoic acid production, we identified hoxc8 as a retinoic acid-induced gene that, when ectopically expressed, rescued phenotypes of APC- and zRDHB-deficient zebrafish. Our data establish a genetic link supporting a critical role for retinoic acid downstream of APC and confirm the importance of retinoic acid in enterocyte differentiation.

Adenomatous Polyposis Coli Protein↗
PubMed1998

Promoting notochord fate and repressing muscle development in zebrafish axial mesoderm.

Cell fate decisions in early embryonic cells are controlled by interactions among developmental regulatory genes. Zebrafish floating head mutants lack a notochord; instead, muscle forms under the neural tube. As shown previously, axial mesoderm in floating head mutant gastrulae fails to maintain expression of notochord genes and instead expresses muscle genes. Zebrafish spadetail mutant gastrulae have a nearly opposite phenotype; notochord markers are expressed in a wider domain than in wild-type embryos and muscle marker expression is absent. We examined whether these two phenotypes revealed an antagonistic genetic interaction by constructing the double mutant. Muscle does not form in the spadetail;floating head double mutant midline, indicating that spadetail function is required for floating head mutant axial mesoderm to transfate to muscle. Instead, the midline of spadetail;floating head double mutants is greatly restored compared to that of floating head mutants; the floor plate is almost complete and an anterior notochord develops. In addition, we find that floating head mutant cells can make both anterior and posterior notochord when transplanted into a wild-type host, showing that enviromental signals can override the predisposition of floating head mutant midline cells to make muscle. Taken together, these results suggest that repression of spadetail function by floating head is critical to promote notochord fate and prevent midline muscle development, and that cells can be recruited to the notochord by environmental signals.

Animals↗
PubMed2005

The odorant receptor repertoire of teleost fish.

BACKGROUND: Vertebrate odorant receptors comprise three types of G protein-coupled receptors: the OR, V1R and V2R receptors. The OR superfamily contains over 1,000 genes in some mammalian species, representing the largest gene superfamily in the mammalian genome. RESULTS: To facilitate an informed analysis of OR gene phylogeny, we identified the complete set of 143 OR genes in the zebrafish genome, as well as the OR repertoires in two pufferfish species, fugu (44 genes) and tetraodon (42 genes). Although the genomes analyzed here contain fewer genes than in mammalian species, the teleost OR genes can be grouped into a larger number of major clades, representing greater overall OR diversity in the fish. CONCLUSION: Based on the phylogeny of fish and mammalian repertoires, we propose a model for OR gene evolution in which different ancestral OR genes or gene families were selectively lost or expanded in different vertebrate lineages. In addition, our calculations of the ratios of non-synonymous to synonymous codon substitutions among more recently expanding OR subgroups in zebrafish implicate residues that may be involved in odorant binding.

Algorithms↗
PubMed2000

Fertilization triggers activation of Fyn kinase in the zebrafish egg.

Fertilization results in the tyrosine phosphorylation of several egg proteins and studies have shown that tyrosine protein kinase activity is required for successful fertilization. The Fyn protein kinase has been detected in eggs of the sea urchin, frog and rat, although measurement of fertilization-induced changes in Fyn kinase activity have only been successful in the sea urchin system. The present study demonstrates the presence of Fyn kinase in the zebrafish egg and the stimulation of this enzyme at fertilization. Activation of Fyn was detected as early as 30 seconds post-fertilization and increased approximately six-fold by 2 minutes post-insemination. The activation of Fyn in the zebrafish egg required sperm and was not observed in spontaneously activated eggs.

Animals↗
PubMed2005

The zebrafish progranulin gene family and antisense transcripts.

BACKGROUND: Progranulin is an epithelial tissue growth factor (also known as proepithelin, acrogranin and PC-cell-derived growth factor) that has been implicated in development, wound healing and in the progression of many cancers. The single mammalian progranulin gene encodes a glycoprotein precursor consisting of seven and one half tandemly repeated non-identical copies of the cystine-rich granulin motif. A genome-wide duplication event hypothesized to have occurred at the base of the teleost radiation predicts that mammalian progranulin may be represented by two co-orthologues in zebrafish. RESULTS: The cDNAs encoding two zebrafish granulin precursors, progranulins-A and -B, were characterized and found to contain 10 and 9 copies of the granulin motif respectively. The cDNAs and genes encoding the two forms of granulin, progranulins-1 and -2, were also cloned and sequenced. Both latter peptides were found to be encoded by precursors with a simplified architecture consisting of one and one half copies of the granulin motif. A cDNA encoding a chimeric progranulin which likely arises through the mechanism of trans-splicing between grn1 and grn2 was also characterized. A non-coding RNA gene with antisense complementarity to both grn1 and grn2 was identified which may have functional implications with respect to gene dosage, as well as in restricting the formation of the chimeric form of progranulin. Chromosomal localization of the four progranulin (grn) genes reveals syntenic conservation for grna only, suggesting that it is the true orthologue of mammalian grn. RT-PCR and whole-mount in situ hybridization analysis of zebrafish grns during development reveals that combined expression of grna and grnb, but not grn1 and grn2, recapitulate many of the expression patterns observed for the murine counterpart. This includes maternal deposition, widespread central nervous system distribution and specific localization within the epithelial compartments of various organs. CONCLUSION: In support of the duplication-degeneration-complementation model of duplicate gene retention, partitioning of expression between grna and grnb was observed in the intermediate cell mass and yolk syncytial layer, respectively. Taken together these expression patterns suggest that the function of an ancestral grn gene has been devolved upon four paralogues in zebrafish.

Amino Acid Motifs↗
PubMed2006

Characterisation of duplicate zinc finger like 2 erythroid precursor genes in zebrafish.

In separate expression pattern and micro-array screens the zinc finger containing factor, znfl2, has been previously implicated in hematopoiesis. Here we analysed znfl2 expression in detail and performed genetic epistatic analysis in a series of hematopoietic mutants and transient gain-of-function models. znfl2 expression in the hematopoietic intermediate mesoderm and derived erythrocytes required early genes cloche and spadetail, but not gata1. Expression was up-regulated in scl gain-of-function embryos, identifying znfl2 as an early erythroid factor that is regulated upstream or independently of gata1. Furthermore, we identified a duplicate znfl2 gene in the genome (znfl2b) which was expressed in early mesendoderm and weakly in the lateral plate mesoderm, overlapping in expression with znfl2. The production of loss-of-function models for znfl2, znfl2b and znfl2/znfl2b together suggested that these erythrocyte specific zinc finger genes are dispensible for erythropoiesis.

Animals↗
PubMed1996

Cloning, expression and properties of the alpha' subunit of casein kinase 2 from zebrafish (Danio rerio).

The protein kinase casein kinase 2 (CK2) is ubiquitous in eukaryotic cells and is apparently involved in the control of cell division. The holoenzyme is a tetramer composed of two catalytic subunits (alpha and/or alpha') and regulatory subunits (beta 2). The alpha and alpha' subunits are encoded by different genes but are very similar in amino acid sequence, except that alpha' is normally considerably shorter. There have been extensive biochemical studies with recombinant alpha and beta subunits of many species, but only one previous description of the activity of an isolated recombinant alpha' subunit from human CK2 (Bodenbach, L., Fauss, J., Robitzki, A., Krehan, A., Lorenz, P., Lozeman, F. J. & Pyerin, W. (1994) Recombinant human casein kinase II. A study with the complete set of subunits (alpha, alpha', and beta), site-directed autophosphorylation mutants and a bicistronically expressed holoenzyme, Eur. J. Biochem. 220, 263-273). In the present work, the isolation and bacterial expression of a cDNA coding for the alpha' subunit of zebrafish (Danio rerio) is reported. The clone covers the complete coding region that generates a protein of 348 amino acids that is 86% identical to the alpha' subunits of human and chicken, and 82% identical to the sequenced portion of the CK2 alpha subunit of zebrafish. The recombinant alpha' subunit has apparent K(m) values for ATP (6 microM), GTP (20 microM), casein (2.0 mg/ml) and the model peptide RRRDDDSEDD (0.3 mM) which are very similar to those of the recombinant alpha subunit of Xenopus laevis. The alpha' subunit kcat was 7.2 min-1 which is again similar to that of Xenopus laevis alpha subunit (7.5 min-1). The alpha' subunit also behaved similarly to CK2 alpha with regard to optimal concentrations for Mg+2 or Mn+2 and to the inhibition by heparin and the poly(Glu80Tyr20) peptide. However alpha' kinase activity was less sensitive to poly(U) inhibition than alpha, it was more heat stable than alpha, and alpha' was slightly more sensitive to KCl inhibition than alpha. The difference in salt sensitivity, however, was enhanced by the presence of the regulatory beta subunit which shifted the optimal salt concentration of the phosphorylating activity. The alpha' 2 beta 2 holoenzyme was inhibited by KCl concentrations above 100 mM, while the alpha 2 beta 2 enzyme was stimulated by KCl concentrations up to 150 mM and required 180 mM for inhibition. Another important difference between alpha and alpha' is seen in the degree of the stimulation of casein phosphorylation activity in the presence of the regulatory beta subunit. When assayed at 100 mM KCl stoichiometric amounts of CK2 beta produced maximal stimulation of both alpha' (D. rerio) and alpha (X. laevis), however the activity levels with alpha' were stimulated 20-fold by beta while the addition of beta stimulated alpha (X. laevis) only 7-8-fold.

Amino Acid Sequence↗
PubMed1999

Development and evolution of melanophore patterns in fishes of the genus Danio (Teleostei: Cyprinidae).

Pigmentation patterns in vertebrates have become an important model for those interested in mechanisms of pattern determination. I present detailed information on the development of melanophore patterns in the zebrafish, Danio rerio, five close relatives of that species, and an outgroup. The comparison of the ontogeny of melanophore patterns in this group is an important first step towards understanding the developmental basis of the interspecific variation. Pigment patterns in this group range from no distinct patterning at all to stripes of differing numbers and widths to reticulated stripes. Species examined form identical larval patterns and follow a common sequence of events from which different elements are eliminated or altered to produce the variety of patterns seen in the group. As flexion is completed, melanophores move from larval positions onto the flanks of the fish. In D. rerio, D. rerio 'leo,' D. kerri, and D. malabaricus, xanthophores become established on the body of the fish as the melanophores move; erythrophores become established on the flanks of D. albolineatus and D. sp. cf. aequipinnatus. An increase in melanophore number, begun at this time, continues at a higher rate in D. rerio, D. kerri, D. sp. cf. aequipinnatus and Tanichthys albonubes than in the other three species. This results in a greater number of melanophores on adults in those species with a higher rate of melanophore increase. No distinct pattern forms, except on the caudal peduncle, in D. albolineatus. In all other Danio species, melanophore stripes form first below then above the horizontal myoseptum. Additional stripes are added first below then above these initial two stripes. D. kerri develops fewer, wider melanophore stripes than D. rerio. After initial stripe formation, D. malabaricus and D. sp. cf. aequipinnatus both developed vertical pattern elements and reticulations in the melanophore pattern. Differences in patterns between species are similar in several cases to described mutants of the zebrafish, suggesting that some aspects of interspecific pigmentation pattern variation may be under relatively simple genetic control. Copyright 1999 Wiley-Liss, Inc.

Journal Article↗
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↗
PubMed2006

Netrins promote developmental and therapeutic angiogenesis.

Axonal guidance and vascular patterning share several guidance cues, including proteins in the netrin family. We demonstrate that netrins stimulate proliferation, migration, and tube formation of human endothelial cells in vitro and that this stimulation is independent of known netrin receptors. Suppression of netrin1a messenger RNA in zebrafish inhibits vascular sprouting, implying a proangiogenic role for netrins during vertebrate development. We also show that netrins accelerate neovascularization in an in vivo model of ischemia and that they reverse neuropathy and vasculopathy in a diabetic murine model. We propose that the attractive vascular and neural guidance functions of netrins offer a unique therapeutic potential.

Angiogenesis Inducing Agents↗