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PubMed2000

Function for Hedgehog genes in zebrafish retinal development.

The hedgehog (hh) genes encode secreted signaling proteins that have important developmental functions in vertebrates and invertebrates. In Drosophila, expression of hh coordinates retinal development by propagating a wave of photoreceptor differentiation across the eye primordium. Here we report that two vertebrate hh genes, sonic hedgehog (shh) and tiggy-winkle hedgehog (twhh), may perform similar functions in the developing zebrafish. Both shh and twhh are expressed in the embryonic zebrafish retinal pigmented epithelium (RPE), initially in a discrete ventral patch which then expands outward in advance of an expanding wave of photoreceptor recruitment in the subjacent neural retina. A gene encoding a receptor for the hedgehog protein, ptc-2, is expressed by retinal neuroepithelial cells. Injection of a cocktail of antisense (alphashh/alphatwhh) oligonucleotides reduces expression of both hh genes in the RPE and slows or arrests the progression of rod and cone photoreceptor differentiation. Zebrafish strains known to have mutations in Hh signaling pathway genes similarly exhibit retardation of photoreceptor differentiation. We propose that hedgehog genes may play a role in propagating photoreceptor differentiation across the developing eye of the zebrafish.

Animals↗

PubMed2002

In vivo imaging of embryonic vascular development using transgenic zebrafish.

In this study we describe a model system that allows continuous in vivo observation of the vertebrate embryonic vasculature. We find that the zebrafish fli1 promoter is able to drive expression of enhanced green fluorescent protein (EGFP) in all blood vessels throughout embryogenesis. We demonstrate the utility of vascular-specific transgenic zebrafish in conjunction with time-lapse multiphoton laser scanning microscopy by directly observing angiogenesis within the brain of developing embryos. Our images reveal that blood vessels undergoing active angiogenic growth display extensive filopodial activity and pathfinding behavior similar to that of neuronal growth cones. We further show, using the zebrafish mindbomb mutant as an example, that the expression of EGFP within developing blood vessels permits detailed analysis of vascular defects associated with genetic mutations. Thus, these transgenic lines allow detailed analysis of both wild type and mutant embryonic vasculature and, together with the ability to perform large scale forward-genetic screens in zebrafish, will facilitate identification of new mutants affecting vascular development.

Animals↗

PubMed2002

Radar is required for the establishment of vascular integrity in the zebrafish.

The precise assembly of an integrated network of blood vessels is essential for the survival of vertebrate embryos. However, the processes by which primitive endothelial cells form mature vessels capable of supplying oxygen and nutrients to developing tissues remain incompletely understood. Here, we propose a role for Radar, one of the zebrafish orthologues of gdf6, in establishing integrity of the trunk vasculature in zebrafish embryos. We show that radar expression is appropriately placed, both spatially and temporally, to perform such a role. Transcripts for radar are detected in the hypochord and the primitive gut endoderm. These tissues intimately flank developing axial vessels in the trunk and have been previously implicated in the regulation of vascular development. Morpholino-based targeted gene knock-down has generated a Radar-specific loss-of-function zebrafish model. These embryos display normal initiation of vascular patterning and commencement of circulation. However, by day 2 of development, the integrity of the axial vasculature is compromised with hemorrhages and circulation short-circuits throughout the developing trunk. We show that this aberrant vascular development is specific to a reduction of the radar gene product. These results suggest that Radar is involved in a signaling pathway required for establishing the integrity of the axial vessels during zebrafish development.

Animals↗

PubMed2004

Diversity of guanylate cyclase-activating proteins (GCAPs) in teleost fish: characterization of three novel GCAPs (GCAP4, GCAP5, GCAP7) from zebrafish (Danio rerio) and prediction of eight GCAPs (GCAP1-8) in pufferfish (Fugu rubripes).

The guanylate cyclase-activating proteins (GCAPs) are Ca(2+)-binding proteins of the calmodulin (CaM) gene superfamily that function in the regulation of photoreceptor guanylate cyclases (GCs). In the mammalian retina, two GCAPs (GCAP 1-2) and two transmembrane GCs have been identified as part of a complex regulatory system responsive to fluctuating levels of free Ca(2+). A third GCAP, GCAP3, is expressed in human and zebrafish (Danio rerio) retinas, and a guanylate cyclase-inhibitory protein (GCIP) has been shown to be present in frog cones. To explore the diversity of GCAPs in more detail, we searched the pufferfish (Fugu rubripes) and zebrafish (Danio rerio) genomes for GCAP-related gene sequences (fuGCAPs and zGCAPs, respectively) and found that at least five additional GCAPs (GCAP4-8) are predicted to be present in these species. We identified genomic contigs encoding fuGCAPl-8, fuGCIP, zGCAPl-5, zGCAP7 and zGCIP. We describe cloning, expression and localization of three novel GCAPs present in the zebrafish retina (zGCAP4, zGCAP5, and zGCAP7). The results show that recombinant zGCAP4 stimulated bovine rod outer segment GC in a Ca(2+)-dependent manner. RT-PCR with zGCAP specific primers showed specific expression of zGCAPs and zGCIP in the retina, while zGCAPl mRNA is also present in the brain. In situ hybridization with anti-sense zGCAP4, zGCAP5 and zGCAP7 RNA showed exclusive expression in zebrafish cone photoreceptors. The presence of at least eight GCAP genes suggests an unexpected diversity within this subfamily of Ca(2+)-binding proteins in the teleost retina, and suggests additional functions for GCAPs apart from stimulation of GC. Based on genome searches and EST analyses, the mouse and human genomes do not harbor GCAP4-8 or GCIP genes.

Amino Acid Sequence↗

PubMed2004

The Irx gene family in zebrafish: genomic structure, evolution and initial characterization of irx5b.

Genes of the iroquois ( Iro/Irx) family are highly conserved from Drosophila to mammals and they have been implicated in a number of developmental processes. In flies, the Iro genes participate in patterning events in the early larva and in imaginal disk specification. In vertebrates, the Irx genes regulate developmental events during gastrulation, nervous system regionalization, activation of proneural genes and organ patterning. The Iro genes in Drosophila and the Irx genes of mammals show a clustered organization in the genome. Flies have a single cluster comprising three genes while mammals have two clusters also having three genes each. Moreover, experimental evidence in flies shows that transcriptional regulatory elements are shared among genes within the Iro cluster, suggesting that the same may be true in vertebrates. To date, the genomic organization of the Irx genes in non-mammalian species has not been studied. In this work, we have isolated the irx5b gene from zebrafish, Danio rerio, and have characterized its expression pattern. Furthermore, we have identified the complete set of Irx genes in two fish species, the zebrafish and pufferfish, Takifugu rubripes, and have determined the genomic organization of these genes. Our analysis indicates that early in fish evolutionary history, the Irx gene clusters have been duplicated and that subsequent events have maintained the clustered organization for some of the genes, while others have been lost. In total there are 11 existing Irx genes in zebrafish and 10 in pufferfish. We propose a new nomenclature for the zebrafish Irx genes based on the analysis of their sequences and their genomic relationships.

Animals↗

PubMed2006

Alternatively spliced Robo2 isoforms in zebrafish and rat.

Robo2, a member of the robo gene family, functions as a repulsive axon guidance receptor as well as a regulator of cell migration and tissue morphogenesis in different taxa. In this study, a novel isoform of the zebrafish robo2 (robo2_tv2), which included an otherwise alternatively spliced exon (CAE), has been characterized. Robo2_tv2 is expressed differentially in most non-neuronal tissues of adult zebrafish whereas robo2_tv1 expression to a great extent is restricted to the brain and eye. In zebrafish, robo2_tv2 exhibits a very-low-level basal expression starting from 1 day post fertilization until the mid-larval stages, at which time its expression increases dramatically and could be detected throughout adulthood. Our findings demonstrate that the amino acid sequence coded by CAE of the robo2 gene is highly conserved between zebrafish and mammals, and also contains conserved motifs shared with robo1 and robo4 but not with robo3. Furthermore, we provide an account of differential transcription of the CAE homolog in various tissues of the adult rat. These results suggest that the alternatively spliced robo2 isoforms may exhibit tissue specificity.

Alternative Splicing↗

PubMed2006

Zebrafish orthopedia (otp) is required for isotocin cell development.

Several behavioral and physiological processes such as social, sexual, and maternal behaviors, learning and memory, and parturition are influenced by the neurohypophysial peptide oxytocin. Studies in knockout mice have identified four transcriptional regulatory genes that are required for oxytocin neuronal development in the hypothalamus. These are the basic helix-loop-helix PAS genes Single-minded 1 (Sim1) and Arylhydrocarbon receptor nuclear translocator 2 (Arnt2), the POU homeobox gene Pou3f2, and the paired homeobox gene Orthopedia (Otp). Overall, however, the molecular control of oxytocin cell development is poorly understood. Studies in zebrafish provide a complementary view to mouse knockout experiments and facilitate understanding of neuroendocrine cell development. Isotocin, which is orthologous to oxytocin, is expressed early in the developing zebrafish brain. In this paper we show that zebrafish otp mRNA expression in the embryonic forebrain is dynamic and complex, and that it overlaps with isotocin expression in the dorsal preoptic area. Additionally, these studies demonstrate that otp is required for isotocin cell development. Evidence is also provided that otp and sim1 function in parallel to direct the differentiation of isotocin cells, and that otp is unlikely to affect brain patterning. Overall, these studies support the hypothesis that the role of otp in zebrafish neuroendocrine cell development is evolutionarily conserved with that of mammals.

Animals↗

PubMed2005

Generation of aberrant transcripts of and free DNA ends in zebrafish no tail gene.

The zebrafish no tail gene (ntl) is indispensable for the formation of the notochord and the tail structure. In a wild-type zebrafish population, we occasionally observed adult zebrafish with a narrow or no tailfin. This led us to examine the hypothesis that the activity of ntl was somehow genetically unstable. Here we present two findings regarding the gene. First, approximately 3% of ntl transcripts were aberrant; most of them carried deletions at various positions. Second, free, DNA double-stranded ends (DSEs) were formed at an AT dinucleotide repeat in ntl. DSEs were also generated in another zebrafish gene, noggin2 (nog2). DSEs in ntl and nog2 had common characteristics, which suggested that the AT repeats in these genes elicited DSEs by blocking progression of the replication.

Alternative Splicing↗

PubMed1996

The zebrafish Fgf-3 gene: cDNA sequence, transcript structure and genomic organization.

We report the isolation and characterization of genomic and cDNA clones encoding zebrafish fibroblast growth factor 3 (FGF3). An initial cDNA clone was generated by PCR amplification using degenerate oligo primers corresponding to a conserved region of protein found in the mouse and human homologues. Screening a cDNA library made from 30-33-h-old zebrafish embryos with this PCR product led to the isolation of two cDNA clones. Sequence analysis of the longest cDNA insert (1810 bp) revealed a 256-amino-acid (aa) orf. The central region, composed of approx. 155 aa, shares 78% identity with the analogous region of Xenopus laevis FGF3 and 72% identity with the product of the more distantly related human gene. However, the N-and C-terminal domains of zebrafish FGF3 are very different from those of other known homologues. The cDNA was used as a probe on genomic DNA to create a physical map of the locus and to isolate a genomic clone encompassing the entire coding region and 5' sequences. DNA sequencing and RNase protection analyses indicate that zebrafish Fgf-3 (ZFgf-3) is structurally analogous to the mouse gene and regulated through two different promoters. The transcription start point of the proximal promoter aligns to that of mouse promoter P3 and lies within a conserved region of sequence.

Amino Acid Sequence↗

PubMed1995

Induction of a secondary embryonic axis in zebrafish occurs following the overexpression of beta-catenin.

Formation of the vertebrate axis may involve a Wnt signaling cascade similar to the Drosophila wingless pathway. Zebrafish wnt8 is a candidate for involvement in axis specification insofar as it is expressed maternally and when overexpressed it can induce goosecoid, a transcription factor normally expressed in the embryonic shield. In this study we demonstrate that beta-catenin, a cadherin associated protein in the Wnt pathway, is expressed maternally in zebrafish and is widely distributed in the early embryo. Overexpressing beta-catenin in early zebrafish embryos induces goosecoid and ntl, ultimately leading to a duplication of a complete secondary axis. These data are consistent with the involvement of beta-catenin in a Wnt signaling pathway which is involved in mesoderm induction in zebrafish.

Amino Acid Sequence↗

PubMed2005

Duplicate aryl hydrocarbon receptor repressor genes (ahrr1 and ahrr2) in the zebrafish Danio rerio: structure, function, evolution, and AHR-dependent regulation in vivo.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The recently identified AHR repressor (AHRR) forms a negative feedback loop with the AHR. We investigated AHRR structure, function, evolution, and regulation in zebrafish, a powerful model in developmental biology and toxicology. We identified and cloned two distinct AHRR cDNAs that encode predicted proteins of 550 (AHRR1) and 573 (AHRR2) amino acids. The ahrr1 and ahrr2 genes map to zebrafish chromosomes 24 and 2, respectively, both of which share conserved synteny with human chromosome 5, the location of human AHRR. Mapping and phylogenetic analysis show that AHRR1 and AHRR2 are co-orthologs of the mammalian AHRR. In transient transfection assays, AHRR1 and AHRR2 repressed constitutive and TCDD-inducible transactivation by AHR2. Expression of both AHRR mRNAs was induced in ZF-L cells by AHR agonists but not by non-agonists. TCDD induced AHRR1 and AHRR2 expression in a dose-dependent manner in ZF-L cells, with EC50 values similar to those for induction of CYP1A. Both AHRRs were expressed and induced by TCDD in zebrafish embryos. Thus, zebrafish possess duplicate AHR-regulated AHRR paralogs that act in a negative feedback loop to repress the AHR signaling pathway.

Amino Acid Sequence↗

PubMed2003

Cloning and expression analysis of a Parkinson's disease gene, uch-L1, and its promoter in zebrafish.

Three genes, alpha-synuclein, parkin, and ubiquitin C-terminal hydrolase L1 (UCH-L1), have been associated with inherited forms of Parkinson's disease (PD), although their in vivo functions have remained largely unknown. To develop an animal model for the molecular study of PD, we cloned zebrafish uch-L1 cDNA and its gene promoter. Sequence analysis revealed that the zebrafish Uch-L1 is highly homologous (79%) to the human UCH-L1, which is a member of the deubiquitinating enzymes. By whole-mount in situ hybridization, we examined the spatiotemporal expression of uch-L1 mRNA in developing zebrafish embryos. The uch-L1 mRNAs are detected in neuronal cells at the first day of embryo development. The expression domain of uch-L1 overlaps with that of tyrosine hydroxylase, a molecular marker for dopaminergic neurons, in the ventral diencephalon, an equivalent structure to the substantia nigra where PD progresses in human. To further analyze the tissue-specific regulation of uch-L1 gene expression, we also tested its gene promoter activity and showed a preferential neuronal expression in transient transgenic zebrafish embryos. These results suggest that uch-L1 may have an important role in the development of neuronal cells in early embryos as well as in the degeneration and disease of neuronal cells in late adult brain.

Amino Acid Sequence↗

PubMed2004

MafT, a new member of the small Maf protein family in zebrafish.

Small Maf proteins play critical roles on morphogenesis and homeostasis through associating with CNC proteins. To date, three small Maf proteins, MafF, MafG, and MafK, have been reported in vertebrates, which share redundant functions. In this study, we tried to identify and characterize small Maf proteins in zebrafish to elucidate their conservation and diversity in the fish kingdom. We identified homolog genes of MafG and MafK but not MafF in zebrafish, indicating the former two are conserved among vertebrates. In addition, a novel type of small Maf protein MafT was identified. MafT protein bound MARE sequence as a homodimer or heterodimers with zebrafish Nrf2 or p45 Nfe2. Co-overexpression of MafT and Nrf2 synergistically activated MARE-mediated gene expression in zebrafish embryos. These results indicated that MafT is a new member of small Maf proteins and involved in the Nrf2-dependent gene regulation in cellular defense system.

Amino Acid Sequence↗

PubMed2006

Requirement of nuclear localization and transcriptional activity of p53 for its targeting to the yolk syncytial layer (YSL) nuclei in zebrafish embryo and its use for apoptosis assay.

We expressed zebrafish p53 protein fused to GFP by a neuron-specific HuC promoter in zebrafish embryos. Instead of displaying neuronal expression patterns, p53-GFP was targeted to zebrafish YSL nuclei. This YSL targeting is p53 sequence-specific because GFP fusion proteins of p63 and p73 displayed neuronal-specific patterns. To dissect the underlying mechanisms, various constructs encoding a series of p53 mutant proteins under the control of different promoters were generated. Our results showed that expression of p53, in early zebrafish embryo, is preferentially targeted to the nuclei of YSL, which is mediated by importin. Similarly, this targeting is abrogated when p53 nuclear localization signal is disrupted. In addition, the transcriptional activity of p53 is required for this targeting. We further showed that fusion of pro-apoptotic BAD protein to p53-GFP led to apoptosis of YSL cells, and subsequent imperfect microtubule formation and abnormal blastomere movements.

Animals↗

PubMed2006

Delta-sarcoglycan is necessary for early heart and muscle development in zebrafish.

Delta-sarcoglycan, one member of the sarcoglycan complex, is a very conservative muscle-specific protein exclusively expressed in the skeletal and cardiac muscles of vertebrates. Mutations in sarcoglycans are known to be involved in limb-girdle muscular dystrophy (LGMD) and dilated cardiomyopathy (DCM) in humans. To address the role of delta-sarcoglycan gene in zebrafish development, we have studied expression pattern of delta-sarcoglycan in zebrafish embryos and examined the role of delta-sarcoglycan in zebrafish embryonic development by morpholino. Strong expression of delta-sarcoglycan was observed in various muscles including those of the segment, heart, eye, jaw, pectoral fin, branchial arches, and swim bladder in zebrafish embryo. Delta-sarcoglycan was also expressed in midbrain and retina. Knockdown of delta-sarcoglycan resulted in severe abnormality in both the cardiac and skeletal muscles. Some severe ones displayed serious morphological abnormality such as hypoplastic head, linear heart, very weak heartbeats, and runtish trunk, all dead within 5 dpf. Whole-mount in situ hybridization analysis showed that adaxial cells and muscle pioneers were affected in delta-sarcoglycan knockdown embryos. In addition, absence of delta-sarcoglycan protein severely delayed the cardiac development and influenced the differentiation of cardiac muscle, and the cardiac left-right asymmetry was dramatically changed in morpholino-treated embryos. These data together suggest that delta-sarcoglycan plays an important role in early heart and muscle development.

Animals↗

PubMed2006

Molecular analysis of Dec1 and Dec2 in the peripheral circadian clock of zebrafish photosensitive cells.

To elucidate the roles of DEC1 and DEC2, basic helix-loop-helix transcription factors, in the circadian clock of photosensitive zebrafish peripheral cells, zebrafish Dec1 and Dec2 (zDec1 and zDec2) were cloned and their functions and expression patterns were examined in BRF41, a zebrafish cell line. zDEC1 and zDEC2 have high sequence similarity to mammalian counterparts and the molecular phylogenetic analysis of the zDEC1 and zDEC2 sequences reflected the predicted pattern of species classification. zDEC1 and zDEC2 inhibited zCLOCK1:zBMAL3 mediated transcription as CRY1a. zDec1 and zDec2 mRNA showed robust circadian oscillation in BRF41 cells. However, zDec1 and zDec2 mRNA was not strongly induced by exposure to light. These results indicate that zDec1 and zDec2 are involved in the circadian clock mechanism in photosensitive zebrafish peripheral cells by suppressing CLOCK/BMAL-induced gene expression and that the feedback loops of zDEC1 and zDEC2 may be interlocked with the PER/CRY core circadian feedback loops.

Animals↗

PubMedDate not supplied

Knockdown of prothrombin in zebrafish.

Thrombin is a serine protease generated from its zymogen, prothrombin, and plays a central role in the coagulation cascade. It is also important for mammalian development. The zebrafish has now been established as an excellent genetic model for studies on mammalian hemostasis and development. In this report, we used prothrombin-specific antisense morpholinos to knock down the levels of prothrombin to characterize the effects of prothrombin deficiency in the zebrafish embryo. Prothrombin morpholino-injected zebrafish embryos yielded an early phenotype exhibiting severe abnormalities that later showed occasional bleeding. In a second late phenotype, the embryos had no observable morphological abnormalities in early stages, but showed occasional bleeding at later stages. These phenotypes resembled characteristics shown by prothrombin knockout mice. Laser-induced vascular injury on some of the normal appearing phenotypic larvae showed a prolonged time to occlusion, and recombinant zebrafish prothrombin injected into these larvae restored a normal time to occlusion thus showing the specificity of the morpholino effect. The system developed here should be useful for investigation of the role of thrombin in vertebrate development.

Animals↗

PubMed2006

Cloning of zebrafish (Danio rerio) heat shock factor 2 (HSF2) and similar patterns of HSF2 and HSF1 mRNA expression in brain tissues.

The transcriptional activation of heat shock protein (HSP) genes is initiated by the binding of heat shock factors (HSFs) to heat shock elements (HSEs) located at the promotor regions. Multiple HSFs exist in larger eukaryotic organisms in order to sense various types of stress signals. Here we report the cloning of zebrafish (Danio rerio) HSF2 (zHSF2) cDNA (GenBank accession number AF412833 ) that has an open reading frame of 1470 nucleotides, encoding a polypeptide of 489 amino acids. Domain architecture analysis of the deduced zHSF2 sequence revealed the presence of a DNA-binding domain at the N-terminal end, an adjacent oligomerization domain and a vertebrate heat shock transcription factor domain. Amino acid alignment showed a 70% sequence identity between zHSF2 and human or mouse HSF2, while only a 45% identity was found between zHSF1a and zHSF2. Recombinant zHSF2 bound with a very high specificity to HSEs arranged as inverted arrays of 5'-nGAAn-3', as replacing one GAA with GTA almost abolished the formation of HSE-binding complex. Similar patterns of zHSF1a and zHSF2 mRNA expression in the brain regions of developing zebrafish were detected by whole mount in situ hybridization and paraffin sectioning, suggesting that most of the two HSF gene activities were controlled by a common mechanism during the embryonic development of zebrafish. The levels of both zHSF1a and zHSF2 mRNA in zebrafish tissues were moderately increased after heat stress.

Amino Acid Sequence↗