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PubMedDate not supplied

Molecular characterization of pig ST8Sia IV--a critical gene for the formation of neural cell adhesion molecule and its response to sialic acid supplement in piglets.

ST8Sia IV (polysialyltransferase IV gene) encodes a key enzyme that is required for polysialic acid synthesis. Polysialic acid is a component of the neural cell adhesion molecule and is necessary for synaptic plasticity of neural cells. We characterized 5.3 kb of pig ST8Sia IV cDNA and determined its expression profile in different organs. In hippocampus, ST8Sia IV mRNA levels were increased approximately 4.5-fold in piglets with sialic acid as a milk supplement, which suggested that exogenous sialic acid is a conditionally essential nutrient for early brain development. Extensive analyses were also performed among its orthologs from human, mouse, rat, chicken, frog and zebrafish. Our results supported that the piglet is a better animal model than other nonprimate species in the studies of ST8Sia IV related metabolism and nutrition in human infants. This pig cDNA provides a basis for uncovering the roles of ST8Sia IV during piglet development and maturation.

Amino Acid Sequence↗
PubMed2006

Molecular dynamics and in vitro analysis of Connexin43: A new 14-3-3 mode-1 interacting protein.

The interaction of cellular proteins with the gap junction protein Connexin43 (Cx43) is thought to form a dynamic scaffolding complex that functions as a platform for the assembly of signaling, structural, and cytoskeletal proteins. A high stringency Scansite search of rat Cx43 identified the motif containing Ser373 (S373) as a 14-3-3 binding site. The S373 motif and the second best mode-1 motif, containing Ser244 (S244), are conserved in rat, mouse, human, chicken, and bovine, but not in Xenopus or zebrafish Cx43. Docking studies of a mouse/rat 14-3-3 homology model with the modeled phosphorylated S373 or S244 peptide ligands or their serine-to-alanine mutants, S373A or S244A, revealed that the pS373 motif facilitated a greater number of intermolecular contacts than the pS244 motif, thus supporting a stronger 14-3-3 binding interaction with the pS373 motif. The alanine substitution also reduced more than half the number of intermolecular contacts between 14-3-3 and the S373 motif, emphasizing the phosphorylation dependence of this interaction. Furthermore, the ability of the wild-type or the S244A GST-Cx43 C-terminal fusion protein, but not the S373A fusion protein, to interact with either 14-3-3 or 14-3-3zeta in GST pull-down experiments clearly demonstrated that the S373 motif mediates the direct interaction between Cx43 and 14-3-3 proteins. Blocking growth factor-induced Akt activation and presumably any Akt-mediated phosphorylation of the S373 motif in ROSE 199 cells did not prevent the down-regulation of Cx43-mediated cell-cell communication, suggesting that an Akt-mediated interaction with 14-3-3 was not involved in the disruption of Cx43 function.

14-3-3 Proteins↗
PubMed2006

Survivin increased vascular development during Xenopus ontogenesis.

Survivin is a member of the inhibitor of apoptosis proteins (IAP) family. These proteins contain one to three zinc-binding motifs termed bacculoviral IAP-binding repeats (BIRs). Survivin contains a single BIR motif. Contrary to other members that directly interact with caspases and inhibit apoptosis, Survivin is believed to have both antiapoptotic and proliferative functions. In mammals, Survivin is not detected in most adult tissues except in endothelial cells of newly formed capillaries and large blood vessels. Importantly, Survivin is highly expressed in all common human cancers. To gain a better view of Survivin expression and function during development, we used the amphibian Xenopus developmental model. We show that the genomes of X. laevis, X. tropicalis, Zebrafish, fugu pufferfish, and rainbow trout encode two different Survivin genes (Su1 and Su2), contrary to mammalian genomes, which encode a single one. In X. laevis, these two genes have a differential spatiotemporal transcription pattern. Transgenic expression of Su1 leads to an enlargement of tadpole's blood vessels with an increase in the number of endothelial cells. This effect requires a functional BIR domain and the p34/cdc2 phosphorylation site. It does not seem to rely on the antiapoptotic activity of Su1 as it is not observed in tadpoles overexpressing other antiapoptotic factors such as XIAP or BclXL. We conclude that Su1 ubiquitous gain of function leads directly or indirectly to an increase in blood vessels size via the proliferation of endothelial cells.

Amino Acid Sequence↗
PubMed2006

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.

Animals↗
PubMed2002

Some distinctive features of zebrafish myogenesis based on unexpected distributions of the muscle cytoskeletal proteins actin, myosin, desmin, alpha-actinin, troponin and titin.

The current myofibrillogenesis model is based mostly on in vitro cell cultures and on avian and mammalian embryos in situ. We followed the expression of actin, myosin, desmin, alpha-actinin, titin, and troponin using immunofluorescence microscopy of zebrafish (Danio rerio) embryos. We could see young mononucleated myoblasts with sharp striations. The striations were positive for all the sarcomeric proteins. Desmin distribution during muscle maturation changes from dispersed aggregates to a perinuclear concentration to striated afterwards. We could not observe desmin-positive, myofibrillar-proteins-negative cells, and we could not find any non-striated distribution of sarcomeric proteins, such as stress fiber-like structures. Some steps, like fusion before striation, seem to be different in the zebrafish when compared with the previously described myogenesis sequences.

Actinin↗
PubMed2006

Behavioral phenotyping in zebrafish: comparison of three behavioral quantification methods.

The zebrafish has been popular in developmental biology and genetics, but its brain function has rarely been studied. High-throughput screening of mutation or drug-induced changes in brain function requires simple and automatable behavioral tests. This article compares three behavioral quantification methods in four simple behavioral paradigms that test a range of characteristics of adult zebrafish, including novelty-induced responses, social behavior, aggression, and predator-model-induced responses. Two quantification methods, manual recording and computerized videotracking of location and activity, yielded very similar results, suggesting that automated videotracking reliably measures activity parameters and will allow high-throughput screening. However, observation-based event recording of posture patterns was found generally not to correlate with videotracking measures, suggesting that further refinement of automated behavior quantification may be considered.

Aggression↗
PubMed2001

Developmental analysis of ceruloplasmin gene and liver formation in zebrafish.

Formation of the liver in zebrafish has been analyzed during normal embryogenesis using ceruloplasmin (Cp) as a specific marker. The asymmetric expression of Cp has been detected in dorsal endoderm at 16 hpf and later in the early hepatic cells in the yolk sac. The liver primordium can be detected after 32 hpf. In oep-/- mutant, which lacks dorsal endoderm, the liver fails to form. In the notochordless flh-/- mutant, the asymmetry of the liver has been lost. Therefore the notochord, dorsal endoderm and endoderm of the yolk sac play a role in liver formation in zebrafish.

Amino Acid Sequence↗
PubMed1999

Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation.

Oscillations of cytosolic free calcium levels have been shown to influence gene regulation and cell differentiation in a variety of model systems. Intercellular calcium waves thus present a plausible mechanism for coordinating cellular processes during embryogenesis. Herein we report use of aequorin and a photon imaging microscope to directly observe a rhythmic series of intercellular calcium waves that circumnavigate zebrafish embryos over a 10-h period during gastrulation and axial segmentation. These waves first appeared at about 65% epiboly and continued to arise every 5-10 min up to at least the 16-somite stage. The waves originated from loci of high calcium activity bordering the blastoderm margin. Several initiating loci were active early in the wave series, whereas later a dorsal marginal midline locus predominated. On completion of epiboly, the dorsal locus was incorporated into the developing tail bud and continued to generate calcium waves. The locations and timing at which calcium dynamics are most active appear to correspond closely to embryonic cellular and syncytial sites of known morphogenetic importance. The observations suggest that a panembryonic calcium signaling system operating in a clock-like fashion might play a role during vertebrate axial patterning.

Aequorin↗
PubMed2003

Movement through Slits: cellular migration via the Slit family.

First isolated in the fly and now characterised in vertebrates, the Slit proteins have emerged as pivotal components controlling the guidance of axonal growth cones and the directional migration of neuronal precursors. As well as extensive expression during development of the central nervous system (CNS), the Slit proteins exhibit a striking array of expression sites in non-neuronal tissues, including the urogenital system, limb primordia and developing eye. Zebrafish Slit has been shown to mediate mesodermal migration during gastrulation, while Drosophila slit guides the migration of mesodermal cells during myogenesis. This suggests that the actions of these secreted molecules are not simply confined to the sphere of CNS development, but rather act in a more general fashion during development and throughout the lifetime of an organism. This review focuses on the non-neuronal activities of Slit proteins, highlighting a common role for the Slit family in cellular migration.

Animals↗
PubMed2004

Genetic interactions underlying otic placode induction and formation.

The formation of the otic placode is a complex process requiring multiple inductive signals. In zebrafish, fgf3 and fgf8, dlx3b and dlx4b, and foxi1 have been identified as the earliest-acting genes in this process. fgf3 and fgf8 are required as inductive signals, whereas dlx3b, dlx4b, and foxi1 appear to act directly within otic primordia. We have investigated potential interactions among these genes. Depletion of either dlx3b and dlx4b or foxi1 leads to a delay of pax2a expression in the otic primordia and reduction of the otic vesicle. Depletion of both foxi1 and dlx3b results in a complete ablation of otic placode formation. A strong synergistic interaction is also observed among foxi1, fgf3, and fgf8, and a weaker interaction among dlx3b, fgf3, and fgf8. Misexpression of foxi1 can induce expression of pax8, an early marker for the otic primordia, in embryos treated with an inhibitor of fibroblast growth factor (FGF) signaling. Conversely, morpholino knockdown of foxi1 blocks ectopic pax8 expression and otic vesicle formation induced by misexpression of fgf3 and/or fgf8. The observed genetic interactions suggest a model in which foxi1 and dlx3b/dlx4b act in independent pathways together with distinct phases of FGF signaling to promote otic placode induction and development.

Animals↗
PubMed1998

Zebrafish hox clusters and vertebrate genome evolution.

HOX genes specify cell fate in the anterior-posterior axis of animal embryos. Invertebrate chordates have one HOX cluster, but mammals have four, suggesting that cluster duplication facilitated the evolution of vertebrate body plans. This report shows that zebrafish have seven hox clusters. Phylogenetic analysis and genetic mapping suggest a chromosome doubling event, probably by whole genome duplication, after the divergence of ray-finned and lobe-finned fishes but before the teleost radiation. Thus, teleosts, the most species-rich group of vertebrates, appear to have more copies of these developmental regulatory genes than do mammals, despite less complexity in the anterior-posterior axis.

Animals↗
PubMed2004

Primordial germ cell migration.

Mutational and antisense screens in Drosophila and zebrafish, and transcriptional profiling and time-lapse analysis in the mouse, have contributed greatly to our understanding of PGC development. In all three systems, the behavior of PGCs is controlled by growth factors which signal through G-protein coupled receptors and/or tyrosine kinase receptors. Additionally, regulated cell-cell and cell-substrate adhesion is important for PGC motility. Finally, localized growth factors may control PGC survival and consequently PGC position. Chemotaxis, regulated adhesion and cell survival are important for multiple migration processes which occur during development and disease. PGC migration shares these features.

Animals↗
PubMed2005

EphA4 is required for cell adhesion and rhombomere-boundary formation in the zebrafish.

The formation of boundaries between or within tissues is a fundamental aspect of animal development. In the developing vertebrate hindbrain, boundaries separate molecularly and neuroanatomically distinct segments called rhombomeres. Transplantation studies have suggested that rhombomere boundaries form by the local sorting out of cells with different segmental identities. This sorting-out process has been shown to involve repulsive interactions between cells expressing an Eph receptor tyrosine kinase, EphA4, and cells expressing its ephrinB ligands. Although a model for rhombomere-boundary formation based on repulsive Eph-ephrin signaling is well established in the literature, the predictions of this model have not been tested in loss-of-function experiments. Here, we eliminate EphA4 and ephrinB2a proteins in zebrafish with antisense morpholinos (MO) and find that rhombomere boundaries are disrupted in EphA4MO embryos, consistent with a requirement for Eph-ephrin signaling in boundary formation. However, in mosaic embryos, we observe that EphA4MO cells and EphA4-expressing cells sort from one another, an observation that is not predicted by the Eph-ephrin repulsion model but instead suggests that EphA4 promotes cell adhesion within the rhombomeres in which it is expressed. Differential cell adhesion is known to be an effective mechanism for cell sorting. We therefore propose that the well-known EphA4-dependent repulsion between rhombomeres operates in parallel with the EphA4-dependent adhesion within rhombomeres described here to drive the cell sorting that underlies rhombomere-boundary formation.

Animals↗
PubMed2002

Zebrafish mutagenesis yields eye morphological mutants with retinal and lens defects.

A chemical mutagenesis to identify zebrafish eye morphological mutants was performed by screening F(3) larvae at 5 and 7 days post-fertilization (dpf) for changes in eye or pupil size. Based on histological analysis, four different phenotypic classes were obtained. The two Class I and three Class II mutants are all characterized by small eyes and exhibit defects in early retinal development or unregulated cell death, respectively. The single Class III mutant has reduced ocular pigmentation. The three Class IV mutants display defects in the ocular lens, including one mutant line with normal sized eyes and pupils that develops lens opacity at 7 dpf.

Animals↗
PubMed2002

Morpholino phosphorodiamidate oligonucleotides in zebrafish: a recipe for functional genomics?

Morpholino phosphorodiamidate anti-sense oligonucleotides (MPOs) have recently emerged as a tool for gene-specific knockdown. MPOs have a great potential for both therapeutic applications and functional genomics. In particular, zebrafish are well suited for gene function studies using MPOs owing to their rapid external development, transparent embryos and the ease of delivery of the intervening MPOs. This paper describes principles of MPO action and the application of MPOs for gene function studies and therapeutics. In the field of functional genomics, the MPO strategy has been most successfully used to study gene function in zebrafish. Over 35 mutations have been successfully phenocopied and over 30 novel gene functions have been analysed in zebrafish using MPOs during the last two years. The essential controls that are required to avoid misinterpretation of experimental data when using MPOs for gene function analysis will also be described.

Amides↗
PubMedDate not supplied

Strain-dependent effects of developmental ethanol exposure in zebrafish.

Developmental ethanol exposure from maternal consumption of alcoholic beverages and many other consumer products has been linked to developmental abnormalities in humans and animal models. The sensitivity of an individual to ethanol-induced perturbation of developmental processes is strongly influenced by genetic factors. In this study, we show that there are strain- and dose-dependent differences in sensitivity to developmental ethanol exposure in zebrafish (Danio rerio), suggesting that genetic variation within regulatory factors, influencing critical developmental pathways, is responsible for these differences. Embryos/larvae from genetically distinct strains of zebrafish [Ekkwill (EK), AB, and Tuebingen (TU)] were treated with different concentrations of ethanol. Embryo/larval survival, neurocranial and craniofacial skeletal development, and CNS cell death were analyzed. EK was the most resistant strain to the embryolethal effects of ethanol exposure but had the greatest increase in ethanol-induced cell death. AB survival was affected moderately, as were the neurocranial and craniofacial skeletal structures and ethanol-induced cell death. TU had the lowest survival rate but was the most resistant to alterations in neurocranial and craniofacial skeletal elements. No single strain is the most sensitive or the most resistant to any of the phenotypes examined, suggesting that alcohol influences each of these pathways independently. Further analysis of the molecular and biochemical pathways underlying the strain-dependent differences reported herein could lead to a significant advancement in our mechanistic understanding of the teratogenic effects of ethanol in humans.

Alcohol-Induced Disorders, Nervous System↗
PubMed2003

PCR-based cloning of an intronless zebrafish no tail gene.

The zebrafish no tail gene (ntl) is indispensable for the formation of the notochord and tail structure. Here, we show the presence of an intronless ntl gene in zebrafish, which we designated cryptail (ctl). ctl could not be found in any zebrafish genomic resources examined and was only just cloned by a PCR-based approach that relied on its lack of introns and homology to ntl. The amplifiable region of ctl was confined to the transcribed region of ntl. ctl thus appeared to have been generated by reverse transcription of ntl mRNA, like a processed pseudogene. ctl was very polymorphic even in individual fish, but had no missense mutations. This may suggest that ctl has a physiological function in zebrafish.

Animals↗
PubMed1999

Formation of cone mosaic of zebrafish retina.

In the zebrafish retina, four types of cone photoreceptor cells (or cones) with different sensitive frequencies are arranged in a regular pattern, named "cone mosaic". A pair of small cones, one sensitive to red and the other sensitive to green, is in close contact and forms a "double cone". In addition, there are two kinds of single cones, sensitive to blue and to UV, respectively. We study characteristics of cell-differentiation rules that realize stable formation of cone mosaic. Assumptions are: undifferentiated cells are arranged in a regular square lattice, and they are one of the three types (B, U, and D cells). A D cell has two parts (G and R-parts) and takes one of the four directions. The cells change their cell type and orientation following a continuous-time Markovian chain. The state transtion occurs faster if it increases the stabilities of the focal cell, in which the stability is the sum of affinities with neighboring cells. After the transient period, the system may reach a stable pattern (pre-pattern). The pattern becomes fixed later when the cells are fully differentiated in which B cells, U cells, and D cells become blue-sensitive, UV-sensitive, and double cones, respectively. We search for the combinations of affinities between cell states that can generate the same cone mosaic patterns as in zerbrafish retina. Successful transition rules give (1) zero or small affinity with the pairs of cell states that are absent in the zebrafish cone mosaic (lambda(UR), lambda(BG)and the contact of two cells of the same type); (2) a large affinity between a part of D cells and a non-D cell (lambda(UG)and lambda(BR)); and (3) a positive affinity of an intermediate magnitude between two non-D cells (lambda(BU)) and between two parts of D cells (lambda(GR)). The latter should be of a magnitude of about 60-90% of the former. The time needed to form a regular pattern increases with the lattice size if all the cells start pre-pattern formation simultaneously. However, the convergence time is shortened considerably if the pre-pattern formation occurs only in a narrow band of morphogenetic cell layer that sweeps from one end of the lattice to the other.

Animals↗