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PubMed2003

Developmental control of Presenilin1 expression, endoproteolysis, and interaction in zebrafish embryos.

Dominant mutations in presenilin1 (PS1) and presenilin2 (PS2) are a major cause of early-onset Alzheimer's disease. In this report we analyze the expression of the zebrafish presenilin1 (Psen1) and presenilin2 (Psen2) proteins during embryogenesis. We demonstrate that Psen1 and Psen2 holoproteins are relatively abundant in zebrafish embryos and are proteolytically processed. Psen1 is maternally expressed, whereas Psen2 is expressed at later stages during development. The Psen1 C-terminal proteolytic fragment (CTF) is present at varying levels during embryogenesis, indicating the existence of developmental control mechanisms regulating its production. We examine the codependency of Psen1 and Psen2 expression during early embryogenesis. Forced overexpression of psen2 increases expression of Psen2 holoprotein, but not the N-terminal fragment (NTF), indicating that levels of Psen2 NTF are strictly controlled. Overexpression of psen2 did not alter levels of Psen1 holoprotein, CTF, or higher molecular weight complexes. Reduction of Psen1 activity in zebrafish embryos produces similar developmental defects to those seen for loss of PS1 activity in knockout mice. The relevance of these results to previous work on presenilin protein regulation and function are discussed. Our work shows that zebrafish embryos are a valid and valuable system in which to study presenilin interactions, regulation, and function.

Alzheimer Disease↗

PubMed1999

The zebrafish genome contains two distinct selenocysteine tRNA[Ser]sec genes.

The zebrafish is widely used as a model system for studying mammalian developmental genetics and more recently, as a model system for carcinogenesis. Since there is mounting evidence that selenium can prevent cancer in mammals, including humans, we characterized the selenocysteine tRNA[Ser]sec gene and its product in zebrafish. Two genes for this tRNA were isolated and sequenced and were found to map at different loci within the zebrafish genome. The encoding sequences of both are identical and their flanking sequences are highly homologous for several hundred bases in both directions. The two genes likely arose from gene duplication which is a common phenomenon among many genes in this species. In addition, zebrafish tRNA[Ser]sec was isolated from the total tRNA population and shown to decode UGA in a ribosomal binding assay.

Animals↗

PubMed2002

Cloning of zebrafish ovarian carbonyl reductase-like 20 beta-hydroxysteroid dehydrogenase and characterization of its spatial and temporal expression.

20 beta-Hydroxysteroid dehydrogenase (20 beta-HSD) is a crucial enzyme that converts 17 alpha-hydroxyprogesterone to 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (DHP), which triggers oocyte maturation in most teleost fish. A full-length cDNA for a carbonyl reductase-like 20 beta-HSD (CR/20 beta-HSD) has been cloned from the zebrafish ovary. Although the zebrafish CR/20 beta-HSD is expressed in all of the tissues tested, it is predominantly expressed in the ovary, testis, kidney, and gill. In the ovary, the enzyme was shown to be expressed in the follicle cells and its expression appeared to be constitutive. No significant difference was noticed in the level of CR/20 beta-HSD expression among follicles of different stages. Furthermore, analysis of the ovarian samples taken at different times before spawning showed no significant change of the enzyme expression. In agreement with these results, treatment of the cultured zebrafish ovarian follicle cells with gonadotropin and activin had little effect on the expression of the enzyme. Taken together, these results point to the possibility that the gonadotropin-induced DHP production and final oocyte maturation in the zebrafish may not involve significant change of CR/20 beta-HSD expression as evidenced in the salmonids, or that there might be other isoforms of 20 beta-HSD whose expression is tightly controlled by endocrine and paracrine factors.

Alcohol Oxidoreductases↗

PubMed1997

Behavioural lateralisation of the tetrapod type in the zebrafish (Brachydanio rerio).

Visual lateralisation resembling that found in a bird (domestic chick) is here demonstrated in a teleost (zebrafish, Brachydanio rerio). Zebrafish predominantly view objects with the body axis close to facing the object (0-20 degrees on either side of facing). Strange objects were viewed at first exposure chiefly with the right frontal field; so was a complex and unfamiliar scene made up of familiar components. In a second trial, using the same stimulus or scene, left frontal viewing tended to be used instead. A familiar partner (a fish of another species) was viewed left frontally. The domestic chick also uses the left eye to view familiar stimuli, shifting to the right when it has to decide what response is appropriate to the object at which it is looking. An empty scene in which nothing could be concealed (and so no response was called for) was viewed by zebrafish with the left eye from the start. In zebrafish and the chick, the right eye is used when it is necessary to inhibit premature response, in order to sustain viewing until a decision is reached, and the left is used when it is necessary to keep an eye on a familiar or clearly empty scene. The findings suggest homology of cerebral lateralisation in teleost fish and tetrapods.

Aggression↗

PubMed1998

Circadian rhythms of locomotor activity in zebrafish.

As part of an effort to characterize the circadian system of the zebrafish, we examined the circadian regulation of locomotor activity in adult males and females. Gross locomotor activity was measured using infrared movement detectors. The effects of light, dark, and temperature on the amplitude, phase, and free-running periods of locomotor rhythms were determined. When zebrafish were maintained in a 12-h light:12 h dark cycle at 25 degrees C, 86% of the fish were most active during the light phase of the cycle. The phases of free-running rhythms measured after transfer of fish from light cycles to constant conditions indicate that this diurnal activity profile reflects entrained circadian rhythmicity. When animals were maintained in constant conditions, the proportion that expressed significant circadian rhythmicity depended on ambient temperature. At 21 degrees C, 73% of the animals were rhythmic in constant darkness, and 65% were rhythmic in constant light. Fewer (28-59%) were rhythmic at 18 degrees, 25 degrees, and 28.5 degrees C. The free-running period of rhythmic animals was not affected by temperature within this range. The average period was shorter in constant light (LL; 12 lx) than in constant darkness (DD) in all but one experiment, and the difference was statistically significant for animals held at 21 degrees C. These data indicate that zebrafish locomotor activity is regulated by a circadian clock that is temperature compensated. Because rhythmicity is most robust at 21 degrees C, this would be the optimal temperature for future studies of the physiological basis of zebrafish behavioral rhythms.

Analysis of Variance↗

PubMed2002

Trophic transfer of heavy metals from freshwater zooplankton Daphnia magna to zebrafish Danio reiro.

The trophic transfer of metals along the food chain has been recognized as an important issue in the study of water quality in recent years. Feeding experiments were conducted to examine the assimilation of three metals (Cd, Cr and Zn) by the zebrafish Danio reiro feeding on the freshwater zooplankton Daphnia magna. The zooplankton were exposed to radiotracers from both the aqueous and dietary phases for different duration, and then pulse-fed to the zebrafish for measurements of metal assimilation efficiency (AE). The calculated AEs were 3-8% for Cd, 2-39% for Cr, and 17-36% for Zn in the zebrafish. For Cd and Zn, there was no statistically significant difference between the two different radiolabeling routes (aqueous and dietary exposure). For Cr, the AEs were higher when it was accumulated by D. magna from the dietary source than when it was accumulated from the aqueous phase. The gut passage time (GPT) was 6-10 h for all metals, with less variation for Zn among the different treatments. There was no obvious relationship between metal GPT and metal AE, presumably due to the narrow range of variation of metal gut passage. About 5-36%, 20-31%, and 8-30% of the total Cd, Cr and Zn was found in the soft tissue of D. magna after the radiolabeling. A much higher fraction of Cd and Zn was found in the soft tissue of D. magna when the metals were accumulated from the dietary phase. No significant relationship between the metal AE and the metal distribution in the soft tissue of D. magna was however documented in this study. Our results demonstrated that there was major difference in metal AE in freshwater fish among different metals. Metal localization in prey organisms and GPT appear to have little influence on metal assimilation by the zebrafish.

Animals↗

PubMed2004

Transgene manipulation in zebrafish by using recombinases.

Although much remains to be done, our results to date suggest that efficient and precise genome engineering in zebrafish will be possible in the future by using Cre recombinase and SB transposase in combination with their respective target sites. In this study, we provide the first evidence that Cre recombinase can mediate effective site-specific deletion of transgenes in zebrafish. We found that the efficiency of target site utilization could approach 100%, independent of whether the target site was provided transiently by injection or stably within an integrated transgene. Microinjection of Cre mRNA appeared to be slightly more effective for this purpose than microinjection of Cre-expressing plasmid DNA. Our work has not yet progressed to the point where SB-mediated mobilization of our transgene constructs would be observed. However, a recent report has demonstrated that SB can enhance transgenesis rates sixfold over conventional methods by efficiently mediating multiple single-copy insertion of transgenes into the zebrafish genome (Davidson et al., 2003). Therefore, it seems likely that a combined system should eventually allow both SB-mediated transgene mobilization and Cre-mediated transgene modification. Our goal is to validate methods for the precise reengineering of the zebrafish genome by using a combination of Cre-loxP and SB transposon systems. These methods can be used to delete, replace, or mobilize large pieces of DNA or to modify the genome only when and where required by the investigator. For example, it should be possible to deliver particular RNAi genes to well-expressed chromosomal loci and then exchange them easily with alternative RNAi genes for the specific suppression of alternative targets. As a nonviral vector for gene therapy, the transposon component allows for the possibility of highly efficient integration, whereas the Cre-loxP component can target the integration and/or exchange of foreign DNA into specific sites within the genome. The specificity and efficiency of this system also make it ideal for applications in which precise genome modifications are required (e.g., stock improvement). Future work should establish whether alternative recombination systems (e.g., phiC31 integrase) can improve the utility of this system. After the fish system is fully established, it would be interesting to explore its application to genome engineering in other organisms.

Animals↗

PubMed1999

Zebrafish YAC, BAC, and PAC genomic libraries.

Numerous positional cloning projects directed at isolating genes responsible for the myriads of observed developmental defects in the zebrafish are anticipated in the very near future. In this chapter, we have reviewed the YAC, BAC, and PAC large-insert genomic resources available to the zebrafish community. We have discussed how these resources are screened and used in a positional cloning scheme and have pointed out frequently formidable logistical considerations in the approach. Despite being extremely tedious, positional cloning projects in the zebrafish will be comparatively easier to accomplish than in human and mouse, because of unique biological advantages of the zebrafish system. Moreover, the ease and speed at which genes are identified and cloned should rapidly increase as more mapping reagents and information become available, thereby paving the way for meaningful biological studies.

Animals↗

PubMed2003

Characterization of rainbow trout and zebrafish eukaryotic initiation factor 2alpha and its response to endoplasmic reticulum stress and IPNV infection.

The cDNAs of rainbow trout and zebrafish eIF2alpha have been isolated and found to encode proteins of similar molecular weight and isoelectric point to the alpha-subunit of the human translational initiation factor, eIF2. The rainbow trout (36.0kDa) and zebrafish (36.2kDa) eIF2alphas share 93 and 91% identity to the human protein, respectively, and are recognized by antibodies raised to the human form. In mammals, the phosphorylation of the alpha-subunit of eIF2 plays a key role in the regulation of protein synthesis in response to a range of cellular stresses. Regions corresponding to the human phosphorylation and kinase-docking sites are identical in the proteins of both fish species, as are residues that interact with the eIF2 recycling factor, eIF2B. Moreover, both recombinant rainbow trout and zebrafish eIF2alphas can be phosphorylated in vitro by the mammalian heme-sensitive eIF2alpha-kinase, HRI/HCR, as well as the interferon-inducible, dsRNA sensitive kinase, PKR. Phosphorylation of rainbow trout and zebrafish eIF2alpha can also occur in vivo. RTG-2 and ZFL cells subjected to endoplasmic reticulum (ER) stress by treatment with the Ca(2+)-ionophore A23187 showed increased levels of eIF2alpha phosphorylation, suggesting similarity between the ER stress response in fish and other higher eukaryotes. Furthermore, RTG-2 cells responded to treatment with poly(I).poly(C) or to infection by infectious pancreatic necrosis virus, IPNV, by increasing eIF2alpha phosphorylation. These data imply that RTG-2 cells express the interferon-induced eIF2alpha-kinase, PKR and suggests that the interferon/eIF2alpha/PKR response to virus infection may be a conserved vertebrate characteristic. Overall these data are consistent with the premise that fish are able to regulate protein synthesis in response to cellular stresses through phosphorylation of eIF2alpha.

Amino Acid Sequence↗

PubMed2003

Identification of a putative calcium-binding protein as a dioxin-responsive gene in zebrafish and rainbow trout.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) is a widespread environmental contaminant that causes multiple effects in vertebrates. TCDD elicits its toxicity through aryl hydrocarbon receptor (AhR)-mediated modulation of gene regulation, increasing intracellular free calcium, and inducing calcium-mediated apoptosis in cell culture. Two TCDD-responsive cDNAs, which encode putative calcium-binding proteins, have been isolated from zebrafish and rainbow trout. The zebrafish and rainbow trout sequences are 88% similar to each other at the amino acid level and are orthologs of the human S100A4 calcium-binding protein. In zebrafish liver cell culture, treatment with TCDD increases S100A4a mRNA abundance. In juvenile rainbow trout, S100A4 mRNA was constitutively expressed in the heart, kidney, intestine, and spleen, but not in the liver. Exposure to TCDD significantly increased rainbow trout S100A4 mRNA abundance in the rainbow trout kidney. Taken together, these findings demonstrate in zebrafish and rainbow trout that dioxin increases expression of this EF-hand calcium-binding protein gene in a tissue-dependent fashion. However, demonstration that the encoded S100A4 proteins actually bind calcium and play a role in dioxin toxicity will require further study.

Amino Acid Sequence↗

PubMed1999

Cloning and characterization of the zebrafish (Danio rerio) aryl hydrocarbon receptor.

The aryl hydrocarbon receptor (AhR) mediates the toxicity of 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds in vertebrates. To further establish zebrafish as a vertebrate model to study the molecular mechanism of TCDD toxicity, we have isolated and characterized the cDNA encoding the zebrafish aryl hydrocarbon receptor (zfAhR2). Analysis of the deduced protein sequence revealed the 1027 amino acid protein is approximately 200 amino acids longer than previously isolated receptors. zfAhR2 is homologous to previously cloned PAS proteins within the basic helix-loop-helix and PAS domains. The C-terminal domain of zfAhR2 diverges from the mammalian AhR at position 420, and does not contain a Q-rich domain. zfAhR2 mRNA is first detected by Northern blot analysis at 24 h post fertilization, and expression increases throughout early development. Treatment of zebrafish embryos and zebrafish liver cells with graded doses of TCDD results in a dose-dependent increase in zfAhR2 mRNA. The time course for zfAhR2 and cytochrome P4501A mRNA induction by TCDD are similar. In vitro produced zfAhR2 protein dimerizes with the rainbow trout aryl hydrocarbon receptor nuclear translocator (rtARNTb) and binds dioxin response elements derived from the rainbow trout CYP1A gene. Finally, transient coexpression of zfAhR2 and rtARNTb in COS-7 cells results in a TCDD dose-related increase in transcription driven by the rainbow trout CYP1A promoter and enhancer.

Amino Acid Sequence↗

PubMed1999

Effects of lithium on pigmentation in the embryonic zebrafish (Brachydanio rerio).

Pigment cell precursors of the embryonic zebrafish give rise to melanophores, xanthophores and/or iridophores. Cell signaling mechanisms related to the development of pigmentation remain obscure. In order to examine the mechanisms involved in pigment cell signaling, we treated zebrafish embryos with various activators and inhibitors of signaling pathways. Among those chemicals tested, LiCl and LiCl/forskolin had a stimulatory effect on pigmentation, most notable in the melanophore population. We propose that the inositol phosphate (IP) pathway, is involved in pigment pattern formation in zebrafish through its involvement in the: (1) differentiation/proliferation of melanophores; (2) dispersion of melanosomes; and/or (3) synthesis/deposition of melanin. To discern at what level pigmentation was being effected we: (1) counted the number of melanophores in control and experimental animals 5 days after treatment; (2) measured tyrosinase activity and melanin content; and (3) employed immunoblotting techniques with anti-tyrosine-related protein-2 and anti-melanocyte-specific gene-1 as melanophore-specific markers. Although gross pigmentation increased dramatically in LiCl- and LiCl/forskolin treated embryos, the effect on pigmentation was not due to an increase in the proliferation of melanophores, but was possibly through an increase in melanin synthesis and/or deposition. Collectively, results from these studies suggest the involvement of an IP-signaling pathway in the stimulation of pigmentation in embryonic zebrafish through the synthesis/deposition of melanin within the neural crest-derived melanophores.

Animals↗

PubMed1999

Neuropeptide Y receptor subtype with unique properties cloned in the zebrafish: the zYa receptor.

Neuropeptide Y (NPY) belongs to a family of structurally related neuroendocrine peptides for which five different G-protein-coupled receptor subtypes have been cloned in mammals. To identify additional subtypes we have performed PCR with degenerate primers in different species. We describe here the cloning and pharmacological profile of a unique NPY receptor subtype in the zebrafish that has tentatively been called the zYa receptor. It has 46-50% amino acid identity to the mammalian Y1, Y4 and y6 receptors and the previously cloned zebrafish receptors zYb and zYc, and only about 27% to Y2 and Y5. The zYa receptor binds NPY and PYY from mammals as well as zebrafish with high affinities and has a K(d) of 28 pM for porcine (125)I-PYY. It has a unique binding profile displaying some features in common with each of the mammalian Y1, Y2 and Y5 receptors. In a microphysiometer assay the receptor responds with extracellular acidification. Chromosomal mapping in the zebrafish genome of zYa, zYb and zYc receptor genes indicates a possible orthologous relationship between zYc and mammalian y6, but identifies no obvious mammalian ortholog for zYa (zYb is a recent copy of zYc in the fish lineage). These results imply that previous studies of NPY in fishes, which have striven to interpret the effects within the framework of mammalian Y1, Y2, and Y5 receptors, need to be reevaluated. Thus, the sequence comparisons, pharmacological properties, and chromosomal localization suggest that the zYa receptor is a novel NPY receptor subtype which is likely to be present also in mammals.

Amino Acid Sequence↗

PubMed1999

Exo-rhodopsin: a novel rhodopsin expressed in the zebrafish pineal gland.

The zebrafish, a useful animal model for genetic studies, has a photosensitive pineal gland, which has an endogenous circadian pacemaker entrained to environmental light-dark cycles [G.M. Cahill, Brain Res. 708 (1996) 177-181]. Although pinopsin has been found in the pineal glands of birds and reptiles, the molecular identity responsible for fish pineal photosensitivity remains unclear. This study reports identification of a novel opsin gene expressed in the zebrafish pineal gland. The deduced amino acid sequence is similar to, but not identical (74% identity) with that of canonical rhodopsin in the zebrafish retina. This novel rhodopsin is expressed in the majority of pineal cells but not in retinal cells, and hence named exo-rhodopsin after extra-ocular rhodopsin. This study first shows that two different rhodopsin genes are expressed in an individual animal each within a unique location. A phylogenetic analysis indicated that the exo-rhodopsin gene was produced by a duplication of the rhodopsin gene at an early stage in the ray-finned fish lineage. As expected, the exo-rhodopsin gene was found in the medakafish and European eel genomes, suggesting strongly that exo-rhodopsin is a pineal opsin common to teleosts. Identification of exo-rhodopsin in the zebrafish provides an opportunity for studying the role of pineal photoreceptive molecules by using genetic approaches.

Amino Acid Sequence↗

PubMed2002

Development of the locomotor network in zebrafish.

The zebrafish is a leading model for studies of vertebrate development and genetics. Its embryonic motor behaviors are easy to assess (e.g. for mutagenic screens), the embryos develop rapidly (hatching as larvae at 2 days) and are transparent, permitting calcium imaging and patch clamp recording in vivo. We review primarily the recent advances in understanding the cellular basis for the development of motor activities in the developing zebrafish. The motor activities are generated largely in the spinal cord and hindbrain. In the embryo these segmented structures possess a relatively small number of repeating sets of identifiable neurons. Many types of neurons as well as the two types of muscle cells have been classified based on their morphologies. Some of the molecular signals for cellular differentiation have been identified recently and mutations affecting cell development have been isolated. Embryonic motor behaviors appear in sequence and consist of an early period of transient spontaneous coiling contractions, followed by the emergence of twitching responses to touch, and later by the ability to swim. Coiling contractions are generated by an electrically coupled network of a subset of spinal neurons whereas a chemical (glutamatergic and glycinergic) synaptic drive underlies touch responses and swimming. Swimming becomes sustained in larvae once the neuromodulatory serotonergic system develops. These results indicate many similarities between developing zebrafish and other vertebrates in the properties of the synaptic drive underlying locomotion. Therefore, the zebrafish is a useful preparation for gaining new insights into the development of the neural control of vertebrate locomotion. As the types of neurons, transmitters, receptors and channels used in the locomotor network are being defined, this opens the possibility of combining cellular neurophysiology with forward and reverse molecular genetics to understand the principles of locomotor network assembly and function.

Animals↗

PubMed2000

Immunocytochemical localization of neuronal and inducible nitric oxide synthase in the retina of zebrafish, Brachydanio rerio.

Several previous studies have revealed the distribution of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in the retina of various species. However, nothing has been reported on the nNOS and iNOS expression in zebrafish retina, even though it has been used as an effective model for developmental studies. In this study on nNOS and iNOS immunoreactivity (IR) in the zebrafish retina, iNOS-IR signals were detected in the ganglion cell layer (GC), the inner plexiform layer (IP), the outer plexiform layer (OP) and the photoreceptor layer (PR). nNOS-IR signals were also intensely detected in the GC, IP, OP and PR. These signals were very similar to the iNOS-IR signals, except for some minor differences in relative intensities. This could be explained by the speculation that the synthesis of nitric oxide by iNOS may represent a compensatory mechanism in the absence of nNOS. We described for the first time the distribution of nNOS and iNOS-IR cells in zebrafish retina. This could provide the basis for further study on the nNOS and iNOS properties of zebrafish retina.

Animals↗

PubMed2000

Muscle regulatory factor gene: zebrafish (Danio rerio) myogenin cDNA.

Myogenin is one of the basic helix-loop-helix proteins that regulate muscle-specific gene expression. Using reverse transciption-polymerase chain reaction (RT-PCR), 5'- and 3'-rapid amplification of cDNA ends (RACE), zebrafish myogenin cDNA was cloned from mRNA of embryos at 10-96 h post-fertilization. The cDNA, at 1384 base pairs (bp), contained a 771-bp open reading frame with 113- and 500-bp flanking regions at the 5'- and 3'-ends, respectively. The deduced amino acid sequences of zebrafish myogenin encoded a 256-amino-acid polypeptide. In a comparison with myogenin of carp, trout, Xenopus, chicken and human, zebrafish myogenin shared 90.9, 77.6, 70.3, 62.9 and 51.5% amino acid identity, respectively. The basic helix-loop-helix domains in myogenin are all conserved. The molecular phylogenic tree demonstrated that myogenin of zebrafish is more closely related to that of fish than to the myogenin of other vertebrates.

Amino Acid Sequence↗

PubMed2000

Cloning and characterization of cDNA encoding zebrafish Danio rerio NM23-B gene.

A full-length zebrafish NM23-B cDNA was cloned and sequenced. The zebrafish NM23-B cDNA consists of 624bp with an open reading frame of 153 amino acids. NM23-B mRNA of approximately 0.7kb is present in adult zebrafish tissues. Zebrafish NM23-B his-tagged protein (17kDa) was produced in E. coli and characterized by binding and UV-cross-linking to a single-stranded telomeric repeat (TTAGGG)(6). This is the first report to show that fish have a NM23-H2 homologue that is similar to that in humans.

Amino Acid Sequence↗