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At least 919 records · Page 51Linked to original sources

PubMed2002

A defect in a novel Nek-family kinase causes cystic kidney disease in the mouse and in zebrafish.

The murine autosomal recessive juvenile cystic kidney (jck) mutation results in polycystic kidney disease. We have identified in jck mice a mutation in Nek8, a novel and highly conserved member of the Nek kinase family. In vitro expression of mutated Nek8 results in enlarged, multinucleated cells with an abnormal actin cytoskeleton. To confirm that a defect in the Nek8 gene can cause cystic disease, we performed a cross-species analysis: injection of zebrafish embryos with a morpholino anti-sense oligonucleotide corresponding to the ortholog of Nek8 resulted in the formation of pronephric cysts. These results demonstrate that comparative analysis of gene function in different model systems represents a powerful means to annotate gene function.

Amino Acid Sequence↗

PubMed2001

Overexpression of a slit homologue impairs convergent extension of the mesoderm and causes cyclopia in embryonic zebrafish.

Slit is expressed in the midline of the central nervous system both in vertebrates and invertebrates. In Drosophila, it is the midline repellent acting as a ligand for the Roundabout (Robo) protein, the repulsive receptor which is expressed on the growth cones of the commissural neurons. We have isolated cDNA fragments of the zebrafish slit2 and slit3 homologues and found that both genes start to be expressed by the midgastrula stage well before the axonogenesis begins in the nervous system, both in the axial mesoderm, and slit2 in the anterior margin of the neural plate and slit3 in the polster at the anterior end of the prechordal mesoderm. Later, expression of slit2 mRNA is detected mainly in midline structures such as the floor plate cells and the hypochord, and in the anterior margins of the neural plates in the zebrafish embryo, while slit3 expression is observed in the anterior margin of the prechordal plate, the floorplate cells in the hindbrain, and the motor neurons both in the hindbrain and the spinal cord. To study the role of Slit in early embryos, we overexpressed Slit2 in the whole embryos either by injection of its mRNA into one-cell stage embryos or by heat-shock treatment of the transgenic embryos which carries the slit2 gene under control of the heat-shock promoter. Overexpression of Slit2 in such ways impaired the convergent extension movement of the mesoderm and the rostral migration of the cells in the dorsal diencephalon and resulted in cyclopia. Our results shed light on a novel aspect of Slit function as a regulatory factor of mesodermal cell movement during gastrulation.

Animals↗

PubMed2004

The lamin CxxM motif promotes nuclear membrane growth.

We analyzed the influence of lamins on nuclear envelope growth in cultured Xenopus A6 cells by the overexpression of human lamin A, Xenopus and zebrafish lamins B2 and Drosophila lamins Dm0 and C as GFP fusion proteins. Lamins containing a CxxM motif in their primary sequence (lamins A, B2, Dm0) induced the formation of lobulated nuclei with multi-membrane-layered, highly folded nuclear membranes and intranuclear membrane assemblies, as observed by electron microscopy. Such morphological alterations were not observed with Drosophila lamin C, a lamin without this motif or with a lamin B2 mutant (B2-SxxM) where the cysteine of the CxxM motif is replaced by a serine. Drosophila lamin C mutants containing a CxxM motif behaved like B-type lamins thus confirming that this tetrapeptide is directly involved in the morphological changes we observed. Nuclear membrane proliferation could also be induced by lamin B2 in COS-7 cells and in zebrafish embryos but not by human lamin A in COS-7 cells. We speculate that the human lamin A is incompletely processed in Xenopus A6 cells and therefore behaves in this cell line like a B-type lamin. Our results indicate that the CxxM motif of B-type lamins has a dual function: it mediates lamin targeting to the inner nuclear membrane thereby promoting nuclear membrane growth.

Amino Acid Motifs↗

PubMed2006

The zebrafish (Danio rerio) embryo as a model system for identification and characterization of developmental toxins from marine and freshwater microalgae.

The zebrafish (Danio rerio) embryo has emerged as an important model of vertebrate development. As such, this model system is finding utility in the investigation of toxic agents that inhibit, or otherwise interfere with, developmental processes (i.e. developmental toxins), including compounds that have potential relevance to both human and environmental health, as well as biomedicine. Recently, this system has been applied increasingly to the study of microbial toxins, and more specifically, as an aquatic animal model, has been employed to investigate toxins from marine and freshwater microalgae, including those classified among the so-called "harmful algal blooms" (HABs). We have developed this system for identification and characterization of toxins from cyanobacteria (i.e. "blue-green algae") isolated from the Florida Everglades and other freshwater sources in South and Central Florida. Here we review the use of this system as it has been applied generally to the investigation of toxins from marine and freshwater microalgae, and illustrate this utility as we have applied it to the detection, bioassay-guided fractionation and subsequent characterization of developmental toxins from freshwater cyanobacteria.

Animals↗

PubMed2002

A dominant-negative form of p63 is required for epidermal proliferation in zebrafish.

Epidermal stem cells play a critical role in producing the multilayered vertebrate skin. Products of the p63 gene not only mark the epidermal stem cells, but also are absolutely required for the formation of mammalian epidermis. We find that early zebrafish embryos express a dominant-negative form of p63 (DeltaNp63), which accumulates in the nucleus just as epidermal growth begins. Using antisense morpholino oligonucleotides, we show that DeltaNp63 is needed for epidermal growth and limb development and is specifically required for the proliferation of epidermal cells by inhibiting p53 activity. While the structure of fish epidermis is very different from that of higher vertebrates, our study shows that DeltaNp63 has essential and ancient role in the development of skin.

Amino Acid Sequence↗

PubMed1998

Furrow-associated microtubule arrays are required for the cohesion of zebrafish blastomeres following cytokinesis.

During the first few cleavages of the zebrafish embryo, daughter blastomeres are loosely associated immediately after furrow ingression, but then gradually cohere. Cohesion appears to be cadherin-dependent, as cadherin and beta-catenin are found at the membrane between cohering blastomeres, and blastomeres fail to cohere in calcium-free medium. Cadherin and beta-catenin are not initially found on the blastomere surface, but are deposited specifically at the furrow surface. An array of parallel microtubules is present on either side of the furrow tip during ingression, as seen by confocal microscopy of alpha-tubulin labelled embryos. Transient incubation of embryos in 1 microg/ml nocodazole at the start of furrowing, which causes a loss of the furrow array, inhibits the localization of beta-catenin to the furrow surface but does not prevent furrow ingression. During ingression, intracellular membranes are transported to the furrow, as shown by labelling with DiD or DiOC6(3). Concentration of these membranes near the furrow surface is microtubule-dependent. These findings suggest that microtubules are required for cohesion of blastomeres because they mediate trafficking of intracellular membranes to the furrow surface, where they are exocytosed and allow cohesion via cadherins.

Animals↗

PubMed1995

Expression of truncated Sek-1 receptor tyrosine kinase disrupts the segmental restriction of gene expression in the Xenopus and zebrafish hindbrain.

During development of the vertebrate hindbrain regulatory gene expression is confined to precise segmental domains. Studies of cell lineage and gene expression suggest that establishment of these domains may involve a dynamic regulation of cell identity and restriction of cell movement between segments. We have taken a dominant negative approach to interfere with the function of Sek-1, a member of the Eph-related receptor tyrosine kinase family expressed in rhombomeres r3 and r5. In Xenopus and zebrafish embryos expressing truncated Sek-1, lacking kinase sequences, expression of r3/r5 markers occurs in adjacent even-numbered rhombomeres, in domains contiguous with r3 or r5. This disruption is rescued by full-length Sek-1, indicating a requirement for the kinase domain in the segmental restriction of gene expression. These data suggest that Sek-1, perhaps with other Eph-related receptors, is required for interactions that regulate the segmental identity or movement of cells.

Amino Acid Sequence↗

PubMed1996

Labeling blastomeres with a calcium indicator: a non-invasive method of visualizing neuronal activity in zebrafish.

Injections of the calcium indicator calcium green dextran (CGD) into zebrafish embryos at the 1-4 cell stages were used to monitor the activity of neurons in larval fish. Dye was pressure injected into a single cell and the fish allowed to develop until post-hatching, when they were embedded in agar and viewed under a confocal microscope. Labeled larval cells, including identifiable neuronal classes such as Rohon-Beard cells and olfactory neurons, were clearly visible with extensive labeling of the whole fish following injections at the one cell embryonic stage, and a mosaic labeling pattern following injections at the 2 or 4 cell stages. Activity of neurons in the spinal cord, as indicated by intracellular calcium concentration changes, was observed directly by monitoring fluorescence changes of individual spinal neurons and groups of spinal neurons on a confocal microscope. Fluorescence increases of between 9 and 55% in spinal neurons were seen during escape responses produced when the fish was tapped on the tail. This technique can potentially be used to monitor the activity of any neuron or group of neurons with respect to behavior non-invasively in intact living zebrafish.

Animals↗

PubMed1999

Pleiotropic effects of zebrafish protein-tyrosine phosphatase-1B on early embryonic development.

Protein tyrosine phosphorylation is an important mechanism of eukaryotic cell signalling which is regulated by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we report the molecular cloning of the first zebrafish protein-tyrosine phosphatase, zf-PTP-1B, the homologue of human PTP-1B. Zf-PTP-1B was catalytically active and localised to the endoplasmic reticulum, like human PTP-1B. Zf-PTP-1B was maternally expressed in zebrafish embryos, and low ubiquitous expression was detected up to day 7 of development. Microinjection of zf-PTP-1B RNA induced pleiotropic, but reproducible developmental defects. Evaluation of the live embryos at 24 h post fertilisation indicated that zf-PTP-1B induced defects in somite formation. The phenotype was dependent on protein-tyrosine phosphatase activity of zf-PTP-1B, since embryos injected with catalytically inactive zf-PTP-1B-C213S developed normally. Co-injection of wild type and inactive zf-PTP-1B led to a rescue of the zf-PTP-1B-induced phenotype, suggesting that zf-PTP-1B-C213S had dominant negative activity. The zf-PTP-1B-induced phenotype suggests that proper tyrosine phosphorylation of key proteins is essential for early development, most notably somitogenesis.

Amino Acid Sequence↗

PubMed2001

Ectopic Wnt signal determines the eyeless phenotype of zebrafish masterblind mutant.

masterblind (mbl) is a zebrafish mutation characterised by the absence or reduction in size of the telencephalon, optic vesicles and olfactory placodes. We show that inhibition of Gsk3beta in zebrafish embryos either by overexpression of dominant negative dn gsk3beta mRNA or by lithium treatment after the midblastula transition phenocopies mbl. The loss of anterior neural tissue in mbl and lithium-treated embryos is preceded by posteriorization of presumptive anterior neuroectoderm during gastrulation, which is evident from the anterior shift of marker genes Otx2 and Wnt1. Heterozygous mbl embryos showed increased sensitivity to inhibition of GSK3beta by lithium or dn Xgsk3beta that led to the loss of eyes. Overexpression of gsk3beta mRNA rescued eyes and the wild-type fgf8 expression of homozygous mbl embryos. emx1 that delineates the telencephalon is expanded and shifted ventroanteriorly in mbl embryos. In contrast to fgf8, the emx1 expression domain was not restored upon overexpression of gsk3beta mRNA. These experiments place mbl as an antagonist of the Wnt pathway in parallel or upstream of the complex consisting of Axin, APC and Gsk3beta that binds and phosphorylates beta-catenin, thereby destabilising it. mbl maps on LG 3 close to a candidate gene axin1. In mbl we detected a point mutation in the conserved minimal Gsk3beta-binding domain of axin1 leading to a leucine to glutamine substitution at position 399. Overexpression of wild-type axin1 mRNA rescued mbl completely, demonstrating that mutant axin1 is responsible for the mutant phenotype. Overexpression of mutant L399Q axin1 in wild-type embryos resulted in a dose-dependent dominant negative activity as demonstrated by the loss of telencephalon and eyes. We suggest that the function of Axin1/Mbl protein is to antagonise the Wnt signal and in doing so to establish and maintain the most anterior CNS. Our findings provide new insights into the mechanisms by which the Wnt pathway generates anteroposterior polarity of the neural plate.

Animals↗

PubMed2002

Differential expression of two somatostatin genes during zebrafish embryonic development.

We have identified the cDNAs of two new zebrafish preprosomatostatins, PPSS1 and PPSS3, in addition to the previously cloned PPSS2 (Argenton et al., 1999). PPSS1 is the orthologue of mammalian PPSSs, with a conserved C-terminal SS-14 sequence, PPSS2 is a divergent SS precursor and PPSS3 is a cortistatin-like prohormone. Using whole-mount in situ hybridisation, we have analysed the expression of PPSS1 and PPSS2 in zebrafish embryos up to 5 days post fertilisation. PPSS1 was expressed in the developing pancreas and central nervous system (CNS), whereas PPSS2 expression was exclusively pancreatic. In the CNS, PPSS1 was detected in several areas, in particular in the vagal motor nucleus and in cells that pioneer the tract of the postoptic commissure. PPSS1 was also expressed transiently in the telencephalon and spinal motor neurons. In all areas but the telencephalon PPSS1 was coexpressed with islet-1.

Amino Acid Sequence↗

PubMed1999

beta-thymosin is required for axonal tract formation in developing zebrafish brain.

beta-Thymosins are polypeptides that bind monomeric actin and thereby function as actin buffers in many cells. We show that during zebrafish development, &bgr;-thymosin expression is tightly correlated with neuronal growth and differentiation. It is transiently expressed in a subset of axon-extending neurons, essentially primary neurons that extend long axons, glia and muscle. Non-neuronal expression in the brain is restricted to a subset of glia surrounding newly forming axonal tracts. Skeletal muscle cells in somites, jaw and fin express beta-thymosin during differentiation, coinciding with the time of innervation. Injection of beta-thymosin antisense RNA into zebrafish embryos results in brain defects and impairment of the development of beta-thymosin-associated axon tracts. Furthermore, irregularities in somite formation can be seen in a subset of embryos. Compared to wild-type, antisense-injected embryos show slightly weaker and more diffuse engrailed staining at the midbrain-hindbrain boundary and a strong reduction of Isl-1 labeling in Rohon Beard and trigeminal neurons. The decreased expression is not based on a loss of neurons indicating that beta-thymosin may be involved in the maintenance of the expression of molecules necessary for neuronal differentiation. Taken together, our results strongly indicate that beta-thymosin is an important regulator of development.

Amino Acid Sequence↗

PubMed1992

Retinoic acid-induced duplication of the zebrafish retina.

Exogenous treatment of zebrafish embryos with retinoic acid induces a duplication of the retinas during development. These effects occur only when retinoic acid is applied within a 2-hr period prior to and during the initial formation of the optic primordia, and they are concentration-dependent. Light microscopic examination reveals that the second retina derives from cells in the ventral region of the developing eyecup that normally become pigment epithelial cells. Two distinct ganglion cell fields are usually observed in eyes with duplicated retinas. Bundles of axons from each ganglion cell field join as they leave the eye and innervate the contralateral tectum.

Animals↗

PubMed2006

Shuffling of cis-regulatory elements is a pervasive feature of the vertebrate lineage.

BACKGROUND: All vertebrates share a remarkable degree of similarity in their development as well as in the basic functions of their cells. Despite this, attempts at unearthing genome-wide regulatory elements conserved throughout the vertebrate lineage using BLAST-like approaches have thus far detected noncoding conservation in only a few hundred genes, mostly associated with regulation of transcription and development. RESULTS: We used a unique combination of tools to obtain regional global-local alignments of orthologous loci. This approach takes into account shuffling of regulatory regions that are likely to occur over evolutionary distances greater than those separating mammalian genomes. This approach revealed one order of magnitude more vertebrate conserved elements than was previously reported in over 2,000 genes, including a high number of genes found in the membrane and extracellular regions. Our analysis revealed that 72% of the elements identified have undergone shuffling. We tested the ability of the elements identified to enhance transcription in zebrafish embryos and compared their activity with a set of control fragments. We found that more than 80% of the elements tested were able to enhance transcription significantly, prevalently in a tissue-restricted manner corresponding to the expression domain of the neighboring gene. CONCLUSION: Our work elucidates the importance of shuffling in the detection of cis-regulatory elements. It also elucidates how similarities across the vertebrate lineage, which go well beyond development, can be explained not only within the realm of coding genes but also in that of the sequences that ultimately govern their expression.

Algorithms↗

PubMed2005

FoxA3 and goosecoid promote anterior neural fate through inhibition of Wnt8a activity before the onset of gastrulation.

Formation of the nervous system initially requires the acquisition of neural identity, which is achieved through the inhibition of epidermalizing factors. A regional patterning then takes place within the neural plate through the activity of caudalizing factors. These two processes are tightly regulated early in development by the dorsal organizer. Here, we show that, in zebrafish embryos, two transcription factors, FoxA3 and Goosecoid, coexpressed at the dorsal blastula margin, are required for the definition of anterior neural fate. Their inactivation results in deletions of anterior head structures associated with an increase of Wnt8 activity at the dorsal blastula margin. These phenotypes can be fully rescued by overexpression of Wnt inhibitors or by inactivation of wnt8a. Altogether, foxA3 and goosecoid cooperate to promote formation of anterior neural tissue by protecting, as early as blastula stage, presumptive anterior neural cells from an irreversible caudalization by the posteriorizing factor Wnt8a.

Amino Acid Sequence↗

PubMed2006

BMP signaling restricts hemato-vascular development from lateral mesoderm during somitogenesis.

The bone morphogenetic protein (BMP) signaling pathway is essential during gastrulation for the generation of ventral mesoderm, which makes it a challenge to define functions for this pathway at later stages of development. We have established an approach to disrupt BMP signaling specifically in lateral mesoderm during somitogenesis, by targeting a dominant-negative BMP receptor to Lmo2+ cells in developing zebrafish embryos. This results in expansion of hematopoietic and endothelial cells, while restricting the expression domain of the pronephric marker pax2.1. Expression of a constitutively active receptor and transplantation experiments were used to confirm that BMP signaling in lateral mesoderm restricts subsequent hemato-vascular development. The results show that the BMP signaling pathway continues to function after cells are committed to a lateral mesoderm fate, and influences subsequent lineage decisions by restricting hemato-vascular fate in favor of pronephric development.

Animals↗

PubMed2004

Tuberculous granuloma formation is enhanced by a mycobacterium virulence determinant.

Granulomas are organized host immune structures composed of tightly interposed macrophages and other cells that form in response to a variety of persistent stimuli, both infectious and noninfectious. The tuberculous granuloma is essential for host containment of mycobacterial infection, although it does not always eradicate it. Therefore, it is considered a host-beneficial, if incompletely efficacious, immune response. The Mycobacterium RD1 locus encodes a specialized secretion system that promotes mycobacterial virulence by an unknown mechanism. Using transparent zebrafish embryos to monitor the infection process in real time, we found that RD1-deficient bacteria fail to elicit efficient granuloma formation despite their ability to grow inside of infected macrophages. We showed that macrophages infected with virulent mycobacteria produce an RD1-dependent signal that directs macrophages to aggregate into granulomas. This Mycobacterium-induced macrophage aggregation in turn is tightly linked to intercellular bacterial dissemination and increased bacterial numbers. Thus, mycobacteria co-opt host granulomas for their virulence.

Animals↗

PubMed2006

Distinct delta and jagged genes control sequential segregation of pancreatic cell types from precursor pools in zebrafish.

The different cell types of the vertebrate pancreas arise asynchronously during organogenesis. Beta-cells producing insulin, alpha-cells producing glucagon, and exocrine cells secreting digestive enzymes differentiate sequentially from a common primordium. Notch signaling has been shown to be a major mechanism controlling these cell-fate choices. So far, the pleiotropy of Delta and Jagged/Serrate genes has hindered the evaluation of the roles of specific Notch ligands, as the phenotypes of knock-out mice are lethal before complete pancreas differentiation. Analyses of gene expression and experimental manipulations of zebrafish embryos allowed us to determine individual contributions of Notch ligands to pancreas development. We have found that temporally distinct phases of both endocrine and exocrine cell type specification are controlled by different delta and jagged genes. Specifically, deltaA knock-down embryos lack alpha cells, similarly to mib (Delta ubiquitin ligase) mutants and embryos treated with DAPT, a gamma secretase inhibitor able to block Notch signaling. Conversely, jagged1b morphants develop an excess of alpha-cells. Moreover, the pancreas of jagged2 knock-down embryos has a decreased ratio of exocrine-to-endocrine compartments. Finally, overexpression of Notch1a-intracellular-domain in the whole pancreas primordium or specifically in beta-cells helped us to refine a model of pancreas differentiation in which cells exit the precursor state at defined stages to form the pancreatic cell lineages, and, by a feedback mediated by different Notch ligands, limit the number of other cells that can leave the precursor state.

Animals↗