Expression and characterization of nonmammalian selenoprotein P in the zebrafish, Danio rerio

BACKGROUND: Selenoprotein P is a protein of considerable intrigue, due to its unusual composition and requirements for its biosynthesis. Whereas most selenoproteins contain a single selenocysteine residue, the human, bovine and rodent selenoprotein P genes encode proteins containing 10-12 selenocysteines. Selenoprotein P genes have, to date, only been reported in mammals, and the function of the protein remains elusive. RESULTS: Herein, we report the identification and characterization of nonmammalian selenoprotein P in the zebrafish Danio rerio. Sequencing of the cDNA revealed the presence of 17 selenocysteine codons, the highest number reported in any protein. Two histidine-rich regions present in the mammalian selenoprotein P sequences are conserved in the zebrafish protein, and two SECIS elements are present in the 3' untranslated region. Whole-mount in situ hybridization of zebrafish embryos revealed high levels of expression of selenoprotein P mRNA in fertilized eggs and in the yolk sac of developing embryos. Transient transfection of the cDNA in mammalian cells resulted in efficient expression of the full-length secreted selenoprotein. A single N-glycosylation site is predicted, and shown to be utilized. CONCLUSIONS: Discovery of selenoprotein P in the zebrafish opens a previously unavailable avenue for genetic investigation of the functions of this unusual protein.     

Artemin-like immunoreactivity in the zebrafish, Danio rerio

Artemin is a member of the glial cell line-derived neurotrophic factor (GDNF) family. It is a neurotrophic factor that supports neurite migration and outgrowth and survival of the sympathetic and sensory nervous system. Artemin has been studied in human and murine tissues, but no study has been devoted to nonmammalian species. Zebrafish is a teleost fish belonging to the family Ciprinidae, which is becoming an important model species for genetic and developmental studies. Thus, the aim of the present investigation was to evaluate, by immunochemical and immunocytochemical analyses, the tissue distribution pattern of artemin in zebrafish. Different isoforms of artemin with corresponding different molecular weights were detected in the brain, muscle, testis, ovary, kidney, gut, and gills of zebrafish by Western blot analysis. Immunocytochemical analysis showed artemin-like immunoreactivity in different cell types: in glial cells and rare neurons of the central nervous system, taste buds, retina, neuromasts of the lateral line, dorsal root ganglia, sympathetic ganglia, gill epithelium, tubular kidney epithelium, gut epithelium and ganglia, pancreas, thyroid, hypothalamus, testis, and ovary. These results indicate a wide distribution of artemin-like immunoreactivity in adult zebrafish, related to the presence of different forms of artemin. These findings might suggest a complex maturation pattern of artemin, whose forms could also exert different roles in zebrafish tissues.     

New kappa opioid receptor from zebrafish Danio rerio

A cDNA that encodes a kappa opioid receptor like from zebrafish (ZFOR3) has been cloned and characterized. The encoded protein is 377 residues long and presents 70% identity with the mammalian kappa receptors, although less homology is found in the amino- and carboxyl-terminus as well as in the extracellular loops. In situ hybridization studies have revealed that ZFOR3 mRNA is highly expressed in particular brain areas that coincide with the expression of the kappa opioid receptor in other species. When ZFOR3 is stably expressed in HEK293 cells, [(3)H]-diprenorphine binds with high affinity (K(D)=1.05+/-0.26 nM), being this value on the same range as those reported for mammalian kappa opioid receptors. On the other hand, the selective agonist for mammalian kappa receptors U69,593 does not bind to ZFOR3. [(3)H]-diprenorphine binding is readily displaced by the peptidic ligand dynorphin A and by the non-endogenous compounds bremazocine, naloxone and morphine, although with different affinities. Our results demonstrate that ZFOR3 is a unique model to study the kappa opioid receptor functionality.     

Isolation and characterization of the laure olfactory behavioral mutant in the zebrafish, Danio rerio.

To initiate a genetic analysis of olfactory development and function in the zebrafish, Danio rerio, we developed a behavioral genetic screen for mutations affecting the olfactory sensory system. First, we characterized olfactory responses of wild-type zebrafish to various odors. We found that 3-day-old juvenile zebrafish reacted to the amino acid L-cysteine with an aversive behavioral response. We isolated one mutant, laure (lre), which showed no aversive behavioral response to L-cysteine at 3 days of development, and carried out a preliminary characterization of this mutant's defects. We found that lre mutant fish were also defective in their response to L-serine and L-alanine, but not to taurocholic acid, as young adults. In addition, lre mutant fish had significantly fewer primary olfactory sensory neurons than normal, and the axons of these neurons did not form the characteristic axon termination pattern in the developing olfactory bulb. Nevertheless, the olfactory epithelium of lre mutant fish showed normal or near normal electrophysiological responses to several odorants. Our data suggest that the behavioral defects observed in the lre mutant result from the disruption of the developing olfactory sensory neurons and their axonal connections within the olfactory bulb. The isolation of the lre mutant shows that our behavior-based screen represents a viable approach for carrying out a genetic dissection of olfactory behaviors in this vertebrate model system.     

Taste bud development in the zebrafish, Danio rerio.

Taste buds are chemosensory endorgans consisting of modified epithelial cells. Fish and other vertebrates use their taste bud cells to sample potential food, either selecting or rejecting substances according to their edibility. The adult gustatory system in fish has been studied thoroughly, including regeneration experiments. Taste buds occur in the epithelia of the lips, the mouth cavity, the oropharyngeal cavity, and also in the skin of the barbels, the head, and sometimes even all over the body surface. Despite its importance for feeding, little is known about the ontogeny of the fish taste system. We examined the development of taste buds in the zebrafish on the light microscopical and the scanning and transmission electron microscopical levels. Taste buds develop later than the olfactory organ and the solitary chemosensory cells, two other chemosensory systems in aquatic vertebrates. The first few taste bud primordia are visible within the epithelia of lips and gill arches 3 to 4 days after fertilization, and the first few taste buds with open receptor areas appear on the lips and simultaneously on the gill arches 4-5 days after fertilization, which coincides with the onset of feeding. Taste buds in the mouth cavity, on the head, and on the barbels are formed later in development. As seen in other fish, zebrafish taste buds contain elongate dark and light cells, termed according to their electron density. Dark cells with a cell apex of many short microvilli appear first, followed by the light cells with one large microvillus. In addition, the zebrafish has a third fusiform cell type, which appears last. This cell type is low in electron density and has a brush-like apical ending with several small microvilli. This cell type has not been described previously. Furthermore, in zebrafish, the ontogenetic processes of taste bud formation differ from regenerative processes described in the literature.     

Developmental plasticity of ventilatory control in zebrafish, Danio rerio

To determine whether development of ventilatory control in zebrafish (Danio rerio) exhibits plasticity, embryos were exposed to hypoxia, hyperoxia or hypercapnia for the first 7 days post-fertilization. Their acute reflex breathing responses to ventilatory stimuli (hypoxia, hypercapnia and external cyanide) were assessed when they had reached maturity (3 months or older). Zebrafish reared under hyperoxic conditions exhibited significantly higher breathing frequencies at rest (283+/-27min(-1) versus 212+/-16min(-1) in control fish); breathing frequency was unaffected in adult fish subjected to hyperoxia for 7 days. The respiratory responses of fish reared in hyperoxic water to acute hypoxia, hypercapnia or external cyanide were blunted (hypoxia, cyanide) or eliminated (hypercapnia). Adult fish exposed for 7 days to hyperoxia showed no change in acute responses to these stimuli. The respiratory responses to acute hypoxia, hypercapnia or external cyanide of fish reared under hypoxic or hypercapnic conditions were similar to those in fish reared under normal conditions. A subset of all fish examined exhibited episodic breathing; an analysis of breathing patterns demonstrated that fish reared under hypercapnic conditions had an increased tendency to display episodic breathing. The results of this study reveal that there is flexibility in the design and functioning of the embryonic or larval respiratory system in zebrafish.     

Use of the zebrafish (Danio rerio) to define hematopoiesis

Hematopoiesis in the vertebrate is characterized by the induction of ventral mesoderm to form hematopoietic stem cells and the eventual differentiation of these progenitors to form the peripheral blood lineages. Several genes have been implicated in the differentiation and development of hematopoietic and vascular progenitor cells, yet our understanding of the discrete steps involved in the induction of these cells from the ventral mesoderm is still incomplete. One method of delineating these processes is based on the use of lower vertebrates. The zebrafish (Danio rerio) is an especially robust vertebrate system for both isolating and characterizing genes involved in these processes. Hematopoietic mutants have been generated with defects in many of the steps of both the primitive and definitive hematopoietic programs. Cloning of the genes that underlie these mutations should yield valuable details of hematopoiesis and may have therapeutic implications for bone marrow transplantation and stem cell gene therapy.     

Maternal transfer and protective role of antibodies in zebrafish Danio rerio

Maternal transfer of antibodies from mother to eggs has been reported in various species of fishes, and these antibodies have been proposed to play immune roles in developing embryos and larvae. However, firm evidence supporting this remains lacking. In this study, we clearly demonstrated that immunization of female zebrafish with the hapten-carrier complex, trinitrophenylated bovine serum albumin (TNP-BSA), induced a significant increase in anti-TNP antibody production in the mothers, which in turn induced a marked increase in anti-TNP antibody level in their eggs. Microinjection of anti-zebrafish IgM antibody into early embryos (to neutralize endogenous antibody action) resulted in a remarkable decrease in the resistance of recipient embryos to pathogenic Aeromonas hydrophila, whereas injection of BSA or anti-β-actin monoclonal antibody into the same stage embryos had little effect on their resistance to the pathogen. Moreover, the content of endogenous antibodies in eggs was clearly correlated with their antibacterial activity against A. hydrophila. This is the first report showing that maternally transferred antibodies in fish can protect early embyros/larvae from the attack of pathogens like A. hydrophila.     

A subtracted cDNA library from the zebrafish (Danio rerio) embryonic inner ear

A database was built that consists of 4694 sequence contigs of approximately 18,000 reads of cDNAs isolated from the microdissected otocysts of zebrafish embryos at 20-30 hour postfertilization, following subtraction with a pool of liver cDNAs from adult fish. These sequences were compared with those of public databanks. Significant similarity were recorded and organized in a relational database at http://www.genoscope.cns.fr/zie. A first group of 2067 sequences correspond to 1428 known zebrafish genes or ESTs present in the Danio rerio section of UniGene. A second group of 302 sequences encode putative proteins that showed significant similarity (50%-100%) with 302 nonzebrafish proteins in the nr databank, a public databank containing an exhaustive nonredundant collection of protein sequences from different species (ftp://ftp.ncbi.nlm.nih.gov/blast/db/nr). The remaining 2325 (49.5%) sequence contigs or singletons showed no significant similarity with sequences available in public databanks. Several genes known to be expressed in the developing inner ear were represented in the present database, in particular genes involved in hair cell differentiation or innervation The occurrence of these genes validates the outcome of this study as the first collection of ESTs preferentially expressed in the zebrafish inner ear during the period of hair cell differentiation and neuroblast delamination from the otic vesicle epithelium. Novel zebrafish genes also involved in these processes are thus likely to be represented among the sequences obtained herein, for which no homology was found in the D. rerio section of UniGene. [The sequence data from this study have been submitted to EMBL under accession nos. AL714032-AL731531].     

Rodlet cells and the sensory systems in zebrafish (Danio rerio)

This preliminary work was designed to study, using routine procedures for light and transmission electron microscopy, the presence of rodlet cells (RCs) in or near the sensory systems of 12 adult specimens (4.0 ± 1.2 cm, LT ± SD) of zebrafish, Danio rerio Hamilton, 1822. Rodlet cells, characterized by a distinctive cell cortex (range, 0.4–1.5 μm in thickness) and conspicuous inclusions named “rodlets,” have a round to ovoid nucleus with irregular outline. Mature RCs are 11.5 ± 1.2 μm (mean ± SD) long and 7.8 ± 1.1 μm (mean ± SD) wide. These cells are more numerous near neuromasts enclosed by an epithelial roof and/or ossified canal wall. In contrast, very few RCs were noticed near superficial neuromasts. Based on the presence of RCs around the two cranial neuromasts of each fish, a variable number from 1 to 15 rodlet cells was found (10.4 ± 3.6, mean ± SD). The RCs were located 1.5 μm (minimal) to 73.3 μm (maximal) from the neuromast (27.9 ± 17.2, mean ± SD). Moreover, RCs were found in olfactory epithelium and in proximity to some taste buds. Interestingly, RCs were absent in the inner ear, eye, and brain. Anat Rec, 2007. © 2007 Wiley‐Liss, Inc.     

The development of eye movements in the zebrafish (Danio rerio)

We investigated the development of oculomotor activity in zebrafish embryos and larvae of ages 48-96 hrs postfertilization (hpf). The optokinetic response (OKR: smooth tracking movements evoked by a rotating striped drum) improved steadily after its onset at 73 hpf, and by 96 hpf had a achieved a gain (eye velocity/drum velocity) of 0.9, comparable to adult performance. Reset movements (the fast phase of optokinetic nystagmus) developed over 75–81 hpf. The vestibuloocular reflex (VOR: compensatory eye movements evoked by passive rotation of the head) developed over 74–81 hpf, and the associated reset movements, over 76–81 hpf. The VOR was qualitatively normal in dark-reared fish, which excludes an essential role for visual experience in its early development. Spontaneous saccadic movements (the fast shift of eye position) appeared between 81 and 96 hpf, and at 96 hpf had maximum velocities that were comparable to adults. These results are compared to, and found to be incompatible with, two earlier ideas of motor development: behavioral “differentiation” and “encephalization.” © 1997 John Wiley & Sons, Inc. Dev Psychobiol 31: 267–276, 1997     

Isolation,characterization, and efficacy of bacteriophages isolated against Citrobacter spp. an in vivo approach in a zebrafish model (Danio rerio)

Citrobacter infections are becoming an increasingly significant health problem in aquaculture in South-Eastern countries. The objective of this study was to isolate and evaluate the potential of lytic bacteriophages against Citrobacter infections. TEM analysis revealed that the isolated phages Citrophage MRM19 and Citrophage MRM57 were identified to be Siphovirus and Podovirus family of the order Caudovirales. The phage life-cycle studies showed that Citrophage MRM19 had an adsorption time of 18 ± 1 min and a latency period of 25 ± 3 min with burst size of 110 ± 20 phages/infected cell and Citrophage MRM57 had an adsorption time of 15 ± 1 min and a latency period of 25 ± 2 min with burst size of 50 ± 5 phages/infected cell. In vitro studies indicated that the bacterial load was reduced by 5 and 7 log units within 12 h by Citrophage MRM19 and Citrophage MRM57. The in vivo efficacy of the phages was studied using zebrafish (Danio rerio) as a model organism in low-scale tanks. The study unveiled that the use of phages increased the survival up to 17%, 23%, and 26% in the case of Citrophage MRM19, Citrophage MRM57, and phage cocktail treatment, respectively. Our study indicated that bacteriophages are suitable biocontrol agents against Citrobacter spp. especially in aquaculture industry.     

斑马鱼3D解剖互动系统的制作

目的制作斑马鱼三维(3D)解剖互动系统软件。方法采用光学显微镜和微计算机断层扫描技术(Micro-CT)研究斑马鱼的组织构造,并收集其形态结构图像。使用Photoshop对获得的连续切片(HE染色)的图片进行校正,使用Autodesk 3ds Max 2012 64-bit-English软件进行三维模型建立。结果获得了斑马鱼骨骼的3D成像和连续切片图像(雄鱼144张,雌鱼179张),实现了斑马鱼重要器官的3D重构,建立了斑马鱼3D解剖互动系统(V 1.0),获得了计算机软件著作权登记证书(证书号:软著登字第0989966号)。结论斑马鱼3D解剖互动系统软件制作的完成为研究斑马鱼立体形态学奠定了基础。     

Epistatic regulation of behavioural and morphological traits in the zebrafish (Danio rerio)

There is currently a lack of studies examining epistasis in general, and specifically for behavioural traits of evolutionary significance. The advent of more efficient analytical methods for exploring epistasis in QTL studies removes the computational restraint on this type of analysis and suggests that performing further analyses of existing datasets may reveal a more complete picture of the genetic architecture of the traits. Here we report the results from an epistatic QTL analysis of an F2 cross between a wild population and a standard laboratory strain of zebrafish. This further analysis was performed using a simultaneous search to identify epistatically interacting QTL affecting behavioural and morphological traits and uncovered several novel epistatic interactions that reached either genome-wide or suggestive significance levels as determined by a randomisation testing approach. These results provide novel insight into the genetic architecture of the regulation of behavioural as well as morphological phenotypes and call for more studies of epistasis for this group of traits.Edited by Jeanne Wehner     

Gene expression patterns underlying proximal-distal skeletal segmentation in late-stage zebrafish, Danio rerio.

Timing and pattern of expression of ten candidate segmentation genes or gene pairs were reviewed or examined in developing median fins of late-stage zebrafish, Danio rerio. We found a general correspondence in timing and pattern of expression between zebrafish fin radial segmentation and tetrapod joint development, suggesting that molecular mechanisms underlying radial segmentation have been conserved over 400 million years of evolution. Gene co-expression during segmentation (5.5-6.5 mm SL) is similar between tetrapods and zebrafish: bmp2b, bmp4, chordin, and gdf5 in interradial mesenchyme and ZS; bapx1, col2a1, noggin3, and sox9a in chondrocytes. Surprisingly, wnt9a is not expressed in the developing median fins, though wnt9b is detected. In contrast to all other candidate segmentation genes we examined, bapx1 is not expressed in the caudal fin, which does not segment. Together, these data suggest a scenario of gene interactions underlying radial segmentation based on the patterns and timing of candidate gene expression.     

Neurotrophin receptors in taste buds of adult zebrafish (Danio rerio)

TrkB plays crucial roles in the development and maintenance of taste buds in mammals. In this study we investigated the presence and cell localization of Trks (TrkA, TrkB and TrkC) in taste buds of the zebrafish (Danio rerio) using Western blot and immunohistochemistry. Proteins of 140 and 145 kDa, identified as full-length TrkA and TrkB, were detected. Conversely, the anti-TrkC antibody recognized a protein lower than expected (100 kDa). In agreement with these results the sensory cells of taste buds, displayed TrkA- and TrkB-like, but not TrkC-like, immunoreactivity. TrkA and TrkB co-existed in the same taste buds, but remains to be clarified whether or not they are co-expressed in the same cells. Present results demonstrate that as for mammals neurotrophins might play a role in sensory cells of the teleostean taste buds.