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At least 1,135 records · Page 63Linked to original sources
PubMed2002

Development and validation of an enzyme-linked immunosorbent assay to measure vitellogenin in the zebrafish (Danio rerio).

In this study, an enzyme-linked immunosorbent assay (ELISA) was developed to quantify vitellogenin (Vtg) in zebrafish (Danio rerio). Zebrafish Vtg (zf-Vtg) was purified from whole-body homogenates of estradiol-exposed zebrafish, and polyclonal antibodies against zf-Vtg were raised. Using purified zf-Vtg as a standard and anti-zf-Vtg antibodies (DR-264), a competitive ELISA method was set up and validated. The working range of the assay is from 1 to 30 ng/ml (20-80% binding), and the detection limit is 0.4 ng/ml for purified zf-Vtg. In whole-body homogenates samples, the practical detection limit is higher than that for purified Vtg (40 ng/ml) due to matrix effect. The intra- and interassay variations were 4.7% and 14%, respectively, at 50% binding (n = 36). Its usefulness to detect changes in Vtg concentration in other cyprinid fish was also tested. In addition, the assay was used to assess Vtg induction in male zebrafish exposed to 17beta-estradiol (E2). Exposure of male zebrafish to 0.1, 1, 10, and 100 microg/L of E2 for 7 d led to a Vtg induction from the lowest concentration. The results show the suitability of the developed ELISA to quantify Vtg inductions in zebrafish, the cross-reactivity of DR264 antibodies with commonly used cyprinids, and the potential of zf-Vtg induction as a sensitive biochemical endpoint that could be used to detect estrogenic properties of chemical substances.

Animals↗
PubMed1998

Zebrafish contains two pax6 genes involved in eye development.

The Pax6 genes of both vertebrates and invertebrates are expressed in the developing eye and in the central nervous system. These genes encode transcription factors with two DNA-binding domains, an N-terminal paired domain and a homeodomain separated by a flexible linker region. Ectopic eye structures are obtained upon targeted expression of Drosophila, squid, ascidian or mouse Pax6 genes in various imaginal disc primordia of Drosophila. We have previously cloned a Pax6 cDNA from zebrafish. Here we report the cloning of a novel Pax6 homolog from zebrafish denoted Pax6.2. The coding sequences of the two genes show 82% identity whereas the deduced amino acid sequences are 95% identical with complete conservation of the paired- and homeodomains. The embryonic expression patterns of Pax6.1 and Pax6.2 reveal both overlapping and discrete expression domains suggesting a division of labor between these two very similar gene products during development of brain and eye structures. Both Pax6.1 and Pax6.2 can act as transcriptional activators with Pax6.2 being more efficient than Pax6.1. Both Pax6.1 and Pax6.2 are able to induce ectopic eyes in Drosophila, while Pax2 is not, suggesting that eye induction is not a general feature of Pax family genes but a distinct characteristic of Pax6 and its direct homologs. Attempts to detect Pax6. 2 homologs in chick, mice or humans proved unsuccessful suggesting that this gene either was lost during evolution of higher vertebrates or, more likely, arose as part of a larger scale duplication of chromosome segments occurring in the zebrafish lineage.

Amino Acid Sequence↗
PubMed2006

Zebrafish foxe3: roles in ocular lens morphogenesis through interaction with pitx3.

Foxe3 is a winged helix/forkhead domain transcription factor necessary for mammalian and amphibian lens development. Human FOXE3 mutations cause anterior segment dysgenesis and cataracts. The zebrafish foxe3 cDNA was PCR amplified from 24 h post-fertilization (hpf) embryo cDNA. The zebrafish foxe3 gene consists of a single exon on chromosome 8 and encodes a 422 amino acid protein. This protein possesses 44% and 67% amino acid identity with the human FOXE3 and Xenopus FoxE4 proteins, respectively. A polyclonal antiserum was generated against a bacterial fusion protein containing the Foxe3 carboxyl terminus. The purified antiserum detects zebrafish Foxe3 on immunoblots, in embryo wholemounts, and frozen tissue sections. The zebrafish Foxe3 protein is first detected in the lens at 31hpf and is restricted to the nucleated cell population, including the epithelial and elongating fiber cells. Knockdown of Foxe3 protein using an antisense morpholino results in small lenses with multilayered epithelial cells and fiber cell dysmorphogenesis. The morphants posses normal retinas, although retinal cell proteins, including rhodopsin, are abnormally expressed in the morphant lens tissue. Functional interactions between foxe3 and pitx3 during lens development were assessed by RT-PCR and comparison of Foxe3 and Pitx3 protein expression in both foxe3 and pitx3 morphants. Immunoblots and immunohistochemistry reveal Pitx3 is expressed in the foxe3 morphant lens, while Pitx3 knockdown results in the elimination of Foxe3 expression. These data demonstrate that Foxe3 is necessary for lens development in zebrafish and that foxe3 lies genetically downstream of pitx3 in a zebrafish lens development pathway.

Amino Acid Sequence↗
PubMed2000

Mesendoderm induction and reversal of left-right pattern by mouse Gdf1, a Vg1-related gene.

TGFbeta signals play important roles in establishing the body axes and germ layers in the vertebrate embryo. Vg1 is a TGFbeta-related gene that, due to its maternal expression and vegetal localization in Xenopus, has received close examination as a potential regulator of development in Xenopus, zebrafish, and chick. However, a mammalian Vg1 ortholog has not been identified. To isolate mammalian Vg1 we screened a mouse expression library with a Vg1-specific monoclonal antibody and identified a single cross-reactive clone encoding mouse Gdf1. Gdf1 is expressed uniformly throughout the embryonic region at 5.5-6.5 days postcoitum and later in the node, midbrain, spinal cord, paraxial mesoderm, lateral plate mesoderm, and limb bud. When expressed in Xenopus embryos, native GDF1 is not processed, similar to Vg1. In contrast, a chimeric protein containing the prodomain of Xenopus BMP2 fused to the GDF1 mature domain is efficiently processed and signals via Smad2 to induce mesendoderm and axial duplication. Finally, right-sided expression of chimeric GDF1, but not native GDF1, reverses laterality and results in right-sided Xnr1 expression and reversal of intestinal and heart looping. Therefore, GDF1 can regulate left-right patterning, consistent with the Gdf1 loss-of-function analysis in the mouse (C. T. Rankin, T. Bunton, A. M. Lawler, and S. J. Lee, 2000, Nature Genet. 24, 262-265) and a proposed role for Vg1 in Xenopus. Our results establish that Gdf1 is posttranslationally regulated, that mature GDF1 activates a Smad2-dependent signaling pathway, and that mature GDF1 is sufficient to reverse the left-right axis. Moreover, these findings demonstrate that GDF1 and Vg1 are equivalent in biochemical and functional assays, suggesting that Gdf1 provides a Vg1-like function in the mammalian embryo.

Amino Acid Sequence↗
PubMed2005

GDNF family ligand immunoreactivity in the gut of teleostean fish.

Glial-derived neurotrophic factor (GDNF), neurturin (NRTN), persephin (PSPN), and artemin (ARTN) are a group of proteins belonging to the GDNF family ligands (GFLs). GDNF, NRTN, and ARTN support the survival of central, peripheral, and autonomic neuron populations, while PSPN supports the survival of only several central neuron populations. A common receptor, RET, modulates the action of this family and a co-receptor, GFRalpha, determines RET ligand specificity. GDNF and NRTN appear to be essential for enteric nervous system (ENS) development in mammals, zebrafish, and other teleostean species. GFLs are also essential for the maintenance and plasticity of adult mammalian ENS. In this study, the distribution pattern of GFLs in the intestine of five adult fish (bass, gilt-head, scorpionfish, trout, and zebrafish) was evaluated by immunochemical and immunocytochemical analysis. The results demonstrated the presence of GDNF, NRTN, and ARTN in the gut of all species studied. They appeared to be spread in the ENS and/or endocrine cells of the intestine. These findings suggest that the presence of GFLs in fish gut is not only limited to developmental period, but could be also involved in the enteric physiology of adult species.

Animals↗
PubMed2004

Function of Cyp11a1 in animal models.

CYP11A1 encodes the P450scc enzyme that catalyzes the first and rate-limiting step of steroid biosynthesis. It is expressed in the adrenals and gonads under the control of pituitary peptide hormones. Transcription factor SF-1, which binds to the CYP11A1 promoter, plays an important role in the tissue-specific and hormonally regulated expression. Transgenic mouse experiments linking the CYP11A1 promoter to a reporter gene demonstrate the importance of the SF-1-binding site in directing balanced CYP11A1 gene expression. In addition, targeted mutagenesis of Cyp11a1 also reveals the importance of this gene in steroid secretion, gene regulation, and development of genitalia. Zebrafish cyp11a1 is expressed in the gonads and interrenal glands in the adults, similar to mammals. In addition, it is expressed in yolk syncytial layer during early embryogenesis. These two animal models complement each other for the understanding of gene regulatory pathways in human diseases related to steroid imbalance.

Animals↗
PubMed2004

A systematic genome-wide screen for mutations affecting organogenesis in Medaka, Oryzias latipes.

A large-scale mutagenesis screen was performed in Medaka to identify genes acting in diverse developmental processes. Mutations were identified in homozygous F3 progeny derived from ENU-treated founder males. In addition to the morphological inspection of live embryos, other approaches were used to detect abnormalities in organogenesis and in specific cellular processes, including germ cell migration, nerve tract formation, sensory organ differentiation and DNA repair. Among 2031 embryonic lethal mutations identified, 312 causing defects in organogenesis were selected for further analyses. From these, 126 mutations were characterized genetically and assigned to 105 genes. The similarity of the development of Medaka and zebrafish facilitated the comparison of mutant phenotypes, which indicated that many mutations in Medaka cause unique phenotypes so far unrecorded in zebrafish. Even when mutations of the two fish species cause a similar phenotype such as one-eyed-pinhead or parachute, more genes were found in Medaka than in zebrafish that produced the same phenotype when mutated. These observations suggest that many Medaka mutants represent new genes and, therefore, are important complements to the collection of zebrafish mutants that have proven so valuable for exploring genomic function in development.

Animals↗
PubMed2006

Osteocalcin and matrix Gla protein in zebrafish (Danio rerio) and Senegal sole (Solea senegalensis): comparative gene and protein expression during larval development through adulthood.

Bone Gla protein (Bgp or osteocalcin) and matrix Gla protein (Mgp) are important in calcium metabolism and skeletal development, but their precise roles at the molecular level remain poorly understood. Here, we compare the tissue distribution and accumulation of Bgp and Mgp during larval development and in adult tissues of zebrafish (Danio rerio) and throughout metamorphosis in Senegal sole (Solea senegalensis), two fish species with contrasting environmental calcium levels and degrees of skeletal reorganization at metamorphosis. Mineral deposition was investigated in parallel using a modified Alizarin red/Alcian blue protocol allowing sensitive simultaneous detection of bone and cartilage. In zebrafish, bgp and mgp mRNAs were localized in all mineralized tissues during and after calcification including bone and calcified cartilage of branchial arches. Through immunohistochemistry we demonstrated that these proteins accumulate mainly in the matrix of skeletal structures already calcified or under calcification, confirming in situ hybridization results. Interestingly, some accumulation of Bgp was also observed in kidney, possibly due to the presence of a related protein, nephrocalcin. Chromosomal localization of bgp and mgp using a zebrafish radiation hybrid panel indicated that both genes are located on the same chromosome, in contrast to mammals where they map to different chromosomes, albeit in regions showing synteny with the zebrafish location. Results in Senegal sole further indicate that, during metamorphosis, there is an increase in expression of both bgp and mgp, paralleling calcification of axial skeleton structures. In contrast with results obtained for previously studied marine fishes, in zebrafish and Senegal sole Mgp accumulates in both calcified tissues and non-mieralized vessel walls of the vascular system. These results suggest different patterns of Mgp accumulation between fish and mammals.

Animals↗
PubMed2000

The need for a simple animal model to understand sleep.

Simple animal models have allowed biologists to apply the tools of modern molecular genetics to such complex behaviors as circadian rhythms and long-term memory consolidation. The mechanisms and molecules discovered in these simple animals are evolutionarily conserved in other species, including mammals. Sleep research lacks a simple animal model because criteria based on the electroencephalogram have been met only in birds and mammals. We argue that straightforward behavioral criteria could allow the identification of a sleep-like rest state that might be useful for molecular investigations to understand the regulation and function of sleep. Candidate model systems are discussed, leading to the conclusion that several species have complementary strengths. Specifically, techniques developed for larval zebrafish can be used to visualize neural firing patterns in the living animal, and the fruit fly Drosophila melanogaster has been used successfully for molecular and genetic dissection of complex behaviors. We conclude with a hypothesis that one putative function of sleep, the optimization of neural plasticity, would also have adaptive value in simple organisms and might therefore be evolutionarily conserved.

Animals↗
PubMed2002

Distributed and concentration-invariant spatial representations of odorants by receptor neuron input to the turtle olfactory bulb.

We sought to characterize how odorants are represented at the level of afferent input to the olfactory bulb of the box turtle, a terrestrial reptile that, like mammals, detects airborne odorants. Using methods developed first in zebrafish, we selectively labeled olfactory receptor neurons with Calcium Green-1 dextran and imaged odorant-evoked input to glomeruli in vivo. Odorant representations were imaged at a glomerular level of resolution over a portion of the dorsal olfactory bulb and at a regional level of resolution over the entire dorsal surface. We report two new findings. First, even at low concentrations, odorants typically elicited input to a large fraction of all imaged glomeruli. Second, while the amplitude of the odorant-evoked input to glomeruli was concentration dependent, the relative pattern of input to the bulb changed only slightly over a concentration range of up to three log units. These results suggest the hypothesis that odorant representations in the turtle involve differential levels of input to many glomeruli, and that detecting relative patterns of distributed glomerular activation may be an important strategy for encoding odor quality independent of intensity.

Animals↗
PubMed2002

Zebrafish Smad7 is regulated by Smad3 and BMP signals.

Growth factors of the TGF-beta superfamily such as BMPs and Nodals are important signaling factors during all stages of animal development. Smad proteins, the cytoplasmic mediators of most TGF-beta signals in vertebrates, play central roles not only for transmission but also in controlling inductive TGF-beta signals by feedback regulation. Here, we describe cloning, expression pattern, transcriptional regulation, and functional properties of two novel zebrafish Smad proteins: the TGF-beta agonist Smad3b, and the anti-Smad Smad7. We show that zebrafish Smad3b, in contrast to the related zebrafish Smad2, can induce mesoderm independently of TGF-beta signaling. Although mammalian Smad3 was shown to inhibit expression of the organizer-specific genes goosecoid, zebrafish smad3b activates organizer genes such as goosecoid. Furthermore, we show that Smad3 and BMP signals activate smad7. Because Smad7 blocks distinct TGF-beta signals in early zebrafish development, our data provide hints for new roles of smad3 genes in the regulation and modulation of TGF-beta signals. In summary, our analyses point out differences of Smad3b and Smad2 functions in zebrafish and provide the first link of smad3 and smad7 function in context of vertebrate development.

Amino Acid Sequence↗
PubMed2005

Cloning of full-length zebrafish dcc and expression analysis during embryonic and early larval development.

Members of the DCC family play key roles in axon guidance in vertebrates as well as invertebrates. In zebrafish, only a short partial sequence of the dcc gene has been reported to date. Here, we report the cloning of full-length zebrafish dcc. Zebrafish DCC shares the typical structure of the DCC subgroup of the immunoglobulin superfamily, consisting of four immunoglobulin and six fibronectin-type III repeats in the extracellular domain, a single transmembrane domain, and an intracellular domain with three conserved motifs. As a first step toward studying the function of dcc, we analyzed its sequence and characterized its expression pattern during embryonic and larval development. dcc is expressed highly in brain and spinal cord, but distinct staining was also observed in developing pectoral fins, pancreas, intestine, and heart. Thus, dcc may play roles not only in axon guidance, but in morphogenesis and functioning of these organs as well.

Amino Acid Sequence↗
PubMed2001

GAP-43 promoter elements in transgenic zebrafish reveal a difference in signals for axon growth during CNS development and regeneration.

A pivotal event in neural development is the point at which differentiating neurons become competent to extend long axons. Initiation of axon growth is equally critical for regeneration. Yet we have a limited understanding of the signaling pathways that regulate the capacity for axon growth during either development or regeneration. Expression of a number of genes encoding growth associated proteins (GAPs) accompanies both developmental and regenerative axon growth and has led to the suggestion that the same signaling pathways regulate both modes of axon growth. We have tested this possibility by asking whether a promoter fragment from a well characterized GAP gene, GAP-43, is sufficient to activate expression in both developing and regenerating neurons. We generated stable lines of transgenic zebrafish that express green fluorescent protein (GFP) under regulation of a 1 kb fragment of the rat GAP-43 gene, a fragment that contains a number of evolutionarily conserved elements. Analysis of GFP expression in these lines confirms that the rat 1 kb region can direct growth-associated expression of the transgene in differentiating neurons that extend long axons. Furthermore, this region supports developmental down-regulation of transgene expression which, like the endogenous gene, coincides with neuronal maturation. Strikingly, these same sequences are insufficient for directing expression in regenerating neurons. This finding suggests that signaling pathways regulating axon growth during development and regeneration are not the same. While these results do not exclude the possibility that pathways involved in developmental axon growth are also active in regenerative growth, they do indicate that signaling pathway(s) controlling activation of the GAP-43 gene after CNS injury differ in at least one key component from the signals controlling essential features of developmental axon growth.

Animals↗
PubMed2006

Arteries define the position of the thyroid gland during its developmental relocalisation.

During vertebrate development, the thyroid gland undergoes a unique relocalisation from its site of induction to a distant species-specific position in the cervical mesenchyme. We have analysed thyroid morphogenesis in wild-type and mutant zebrafish and mice, and find that localisation of growing thyroid tissue along the anteroposterior axis in zebrafish is linked to the development of the ventral aorta. In grafting experiments, ectopic vascular cells influence the localisation of thyroid tissue cell non-autonomously, showing that vessels provide guidance cues in zebrafish thyroid morphogenesis. In mouse thyroid development, the midline primordium bifurcates and two lobes relocalise cranially along the bilateral pair of carotid arteries. In hedgehog-deficient mice, thyroid tissue always develops along the ectopically and asymmetrically positioned carotid arteries, suggesting that, in mice (as in zebrafish), co-developing major arteries define the position of the thyroid. The similarity between zebrafish and mouse mutant phenotypes further indicates that thyroid relocalisation involves two morphogenetic phases, and that variation in the second phase accounts for species-specific differences in thyroid morphology. Moreover, the involvement of vessels in thyroid relocalisation sheds new light on the interpretation of congenital thyroid defects in humans.

Animals↗
PubMed2006

Olfactory imprinting is correlated with changes in gene expression in the olfactory epithelia of the zebrafish.

Odors experienced as juveniles can have significant effects on the behavior of mature organisms. A dramatic example of this occurs in salmon, where the odors experienced by developing fish determine the river to which they return as adults. Further examples of olfactory memories are found in many animals including vertebrates and invertebrates. Yet, the cellular and molecular bases underlying the formation of olfactory memory are poorly understood. We have devised a series of experiments to determine whether zebrafish can form olfactory memories much like those observed in salmonids. Here we show for the first time that zebrafish form and retain olfactory memories of an artificial odorant, phenylethyl alcohol (PEA), experienced as juveniles. Furthermore, we demonstrate that exposure to PEA results in changes in gene expression within the olfactory sensory system. These changes are evident by in situ hybridization in the olfactory epithelium of the developing zebrafish. Strikingly, our analysis by in situ hybridization demonstrates that the transcription factor, otx2, is up regulated in the olfactory sensory epithelia in response to PEA. This increase is evident at 2-3 days postfertilization and is maintained in the adult animals. We propose that the changes in otx2 gene expression are manifest as an increase in the number of neuronal precursors in the cells olfactory epithelium of the odor-exposed fish. Thus, our results reveal a role for the environment in controlling gene expression in the developing peripheral nervous system.

Animals↗
PubMed2001

A dual role for the zebrafish unplugged gene in motor axon pathfinding and pharyngeal development.

On their way toward their synaptic targets, motor growth cones encounter multiple choice points, where they are confronted with trajectory choices. We have previously shown that the zebrafish unplugged gene acts as a somite-derived cue controlling pathway choice of primary motor axons. Here, we demonstrate that this trajectory choice is not exclusively controlled by a single unplugged-dependent process, but depends on the coordinated function of additional cues. We also show that secondary motor neurons, most similar to those in birds and mammals, depend on the unplugged gene to navigate a choice point, suggesting that primary and secondary motor neurons share common mechanisms controlling axonal path selection. Moreover, we show that the unplugged gene plays an additional role guiding secondary motor axons through a single segmental nerve. Finally, we report that unplugged larvae display a striking pharyngeal arch defect, consistent with a dual function of the unplugged gene in axonal guidance and cell motility.

Alleles↗
PubMed2003

Extraretinal and retinal hedgehog signaling sequentially regulate retinal differentiation in zebrafish.

Hedgehog (Hh) signaling is required for eye development in vertebrates; known roles in the zebrafish include regulation of eye morphogenesis and ganglion cell and photoreceptor differentiation. We employed a temporally selective Hh signaling knockdown strategy, by using antisense morpholino oligonucleotides or the teratogenic alkaloid cyclopamine, in order to dissect the separate roles of Hh signaling arising from specific sources. We also examined the eye phenotype of zebrafish slow muscle-omitted (smu) mutants, which lack a functional smoothened gene, encoding a component of the Hh signal transduction pathway. We find that Hh signaling from extraretinal sources is required for the initiation of retinal differentiation, but this involvement may be independent of the effects of Hh signaling on optic stalk development. We also find that Hh signals from ganglion cells participate in propagating expression of ath5, and we suggest that the effects of Hh signals from the retinal pigmented epithelium on photoreceptor differentiation may be mediated by the transcription factor rx1.

Animals↗