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At least 703 records · Page 39Linked to original sources
PubMed2004

Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas.

Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.

Adenoviridae↗
PubMed1997

Modulation of embryonic intracellular Ca2+ signaling by Wnt-5A.

Wnt genes encode secreted proteins which are implicated in receptor-mediated cell-cell signaling events important in embryogenesis, but the second messenger systems modulated by Wnts have not been identified. We report that ectopic expression of Xwnt-5A in zebrafish embryos enhances the frequency of intracellular Ca2+ transients in the enveloping layer of the blastodisc, whereas Xwnt-8 does not. These transients are independent of extracellular Ca2+. Consistent with the observed Ca2+ transients playing a role in responses of embryos to Xwnt-5A, we report that the ligand-activated serotonin type 1C receptor, which stimulates PI cycle activity and Ca2+ signaling independent of Wnts, phenocopies embryonic responses to Xwnt-5A. These results suggest that intercellular signaling by a subset of vertebrate Wnts involves modulation of a intracellular Ca2+ signaling pathway, which may arise from phosphatidylinositol cycle activity.

Animals↗
PubMed2006

Cranio-lenticulo-sutural dysplasia is caused by a SEC23A mutation leading to abnormal endoplasmic-reticulum-to-Golgi trafficking.

Cranio-lenticulo-sutural dysplasia (CLSD) is an autosomal recessive syndrome characterized by late-closing fontanels, sutural cataracts, facial dysmorphisms and skeletal defects mapped to chromosome 14q13-q21 (ref. 1). Here we show, using a positional cloning approach, that an F382L amino acid substitution in SEC23A segregates with this syndrome. SEC23A is an essential component of the COPII-coated vesicles that transport secretory proteins from the endoplasmic reticulum to the Golgi complex. Electron microscopy and immunofluorescence show that there is gross dilatation of the endoplasmic reticulum in fibroblasts from individuals affected with CLSD. These cells also exhibit cytoplasmic mislocalization of SEC31. Cell-free vesicle budding assays show that the F382L substitution results in loss of SEC23A function. A phenotype reminiscent of CLSD is observed in zebrafish embryos injected with sec23a-blocking morpholinos. Our observations suggest that disrupted endoplasmic reticulum export of the secretory proteins required for normal morphogenesis accounts for CLSD.

Abnormalities, Multiple↗
PubMed2006

The role of heparan sulphate proteoglycans in angiogenesis.

The presence of HS (heparan sulphate) proteoglycans on the cell surface and in the extracellular environment is critical to many physiological processes including the growth of new blood vessels from pre-existing vasculature (angiogenesis). A plethora of growth factors and their receptors, extracellular matrix molecules and enzymes bind to specific sites on the HS sugar chain. For example, HS proteoglycans have profound effects on the bioactivity of the key angiogenic factor VEGF (vascular endothelial growth factor) (VEGF(165)), affecting its diffusion, half-life and interaction with its tyrosine kinase receptors. A number of HS structural features that mediate the specific binding of VEGF(165), including sulphation requirements, have been determined. In parallel, zebrafish embryos were used as a vertebrate model system to study the role in vascular development of the biosynthetic enzymes that create these specific binding sites on HS. It was discovered that knockdown of one of the HS 6-O-sulphotransferases in zebrafish with morpholino antisense oligonucleotides reduced vascular branching and corresponded to changes in the HS structure. The roles of the extracellular 6-O-sulphatase enzymes, the sulfs, in vascular development are now being investigated. Both oligosaccharides and small molecule biosynthetic enzyme inhibitors could be valuable HS-based strategies for controlling aberrant angiogenesis in diseases as diverse as cancer and heart disease.

Animals↗
PubMed2006

Differentiation of cerebellar cell identities in absence of Fgf signalling in zebrafish Otx morphants.

Although the secreted molecule Fgf8 is a key player of the isthmic organiser function, the mechanisms by which it acts remain unclear. Here, we present evidence indicating that Fgf8 is not instructive in establishing zebrafish cerebellar cell identities, although it is required for proliferation and morphogenesis of this territory. We first show that, as in mouse, lack of Otx function in zebrafish leads to transformation of the presumptive mesencephalon into an extended rhombomere 1 (r1). Expanded Fgf8 expression was proposed to be the cause of this fate transformation. However, this report demonstrates that zebrafish embryos lacking both Otx and fgf8 functions retain an extended r1 and display differentiation of at least two cerebellar cell fates. We show that this is not caused by presence of other Fgfs, which implies that in absence of Otx, Fgf function is not necessary for the differentiation of cerebellar cell types. Otx proteins are therefore potent repressors of cerebellar fates, kept out of r1 progeny by Fgf8. Because Otx transcripts are not present in presumptive r1 territory prior to fgf8 expression, Fgf8 is required to maintain, rather than induce, the posterior boundary of Otx expression. This maintenance is enough to allow cerebellar differentiation.

Animals↗
PubMed2006

Coordinated cell-shape changes control epithelial movement in zebrafish and Drosophila.

Epithelial morphogenesis depends on coordinated changes in cell shape, a process that is still poorly understood. During zebrafish epiboly and Drosophila dorsal closure, cell-shape changes at the epithelial margin are of critical importance. Here evidence is provided for a conserved mechanism of local actin and myosin 2 recruitment during theses events. It was found that during epiboly of the zebrafish embryo, the movement of the outer epithelium (enveloping layer) over the yolk cell surface involves the constriction of marginal cells. This process depends on the recruitment of actin and myosin 2 within the yolk cytoplasm along the margin of the enveloping layer. Actin and myosin 2 recruitment within the yolk cytoplasm requires the Ste20-like kinase Msn1, an orthologue of Drosophila Misshapen. Similarly, in Drosophila, actin and myosin 2 localization and cell constriction at the margin of the epidermis mediate dorsal closure and are controlled by Misshapen. Thus, this study has characterized a conserved mechanism underlying coordinated cell-shape changes during epithelial morphogenesis.

Actins↗
PubMed2005

Effects of decreased muscle activity on developing axial musculature in nicb107 mutant zebrafish (Danio rerio).

The present paper discusses the effects of decreased muscle activity (DMA) on embryonic development in the zebrafish. Wild-type zebrafish embryos become mobile around 18 h post-fertilisation, long before the axial musculature is fully differentiated. As a model for DMA, the nic(b107) mutant was used. In nic(b107) mutant embryos, muscle fibres are mechanically intact and able to contract, but neuronal signalling is defective and the fibres are not activated, rendering the embryos immobile. Despite the immobility, distinguished slow and fast muscle fibres developed at the correct location in the axial muscles, helical muscle fibre arrangements were detected and sarcomere architecture was generated. However, in nic(b107) mutant embryos the notochord is flatter and the cross-sectional body shape more rounded, also affecting muscle fibre orientation. The stacking of sarcomeres and myofibril arrangement show a less regular pattern. Finally, expression levels of several genes were changed. Together, these changes in expression indicate that muscle growth is not impeded and energy metabolism is not changed by the decrease in muscle activity but that the composition of muscle is altered. In addition, skin stiffness is affected. In conclusion, the lack of muscle fibre activity did not prevent the basal muscle components developing but influenced further organisation and differentiation of these components.

Animals↗
PubMed2005

Functional characterization of full-length TLR3, IRAK-4, and TRAF6 in zebrafish (Danio rerio).

Recently, the zebrafish, Danio rerio, has been recognized as a useful model for infectious disease and immunity. The Toll-like receptor (TLR) family is an evolutionarily conserved component of the innate immune system that responds to specific pathogen-associated molecular patterns (PAMPs) during an infection. This study reports the identification and characterization of a full-length orthologue of mammalian TLR3, and the key TLR pathway signaling molecules IRAK-4 and TRAF6 in the zebrafish. Sequence analysis of zebrafish TLR3 (zfTLR3), IRAK-4 (zfIRAK-4), and TRAF6 (zfTRAF6) revealed conserved domains shared with insect and mammalian genes. Quantitative real-time PCR showed that all three genes are expressed in a variety of adult tissues and during embryonic development. In in situ hybridization, we showed that zfTLR3, zfIRAK-4, and zfTRAF6 are present in distinct regions of the developing brain at 22hpf and that zfTRAF6 was observed in the developing medial neural tube. Overexpression of zfIRAK-4, zfTRAF6, or a mutant zfTLR3 construct was able to stimulate NF-kappaB activation in ZFL cells as measured by a cotransfected NF-kappaB-luciferase reporter plasmid. Messenger RNA expression profiles of each gene in zebrafish embryos and adults were examined by quantitative real-time PCR following infection with snakehead rhabdovirus (SHRV) or Edwardsiella tarda. Following exposure to SHRV, only zfTLR3 and zfTRAF6 mRNA transcripts were upregulated. Interestingly, exposure of fish to E. tarda resulted in an unexpected increase in mRNA expression of zfTLR3, as well as the anticipated upregulation of zfIRAK-4 and zfTRAF6 mRNA transcripts. These results demonstrate that zebrafish possess conserved TLR-signaling pathways, further emphasizing the utility of the zebrafish as a model for vertebrate immunology.

Amino Acid Sequence↗
PubMed2002

Pineal-specific expression of green fluorescent protein under the control of the serotonin-N-acetyltransferase gene regulatory regions in transgenic zebrafish.

Zebrafish serotonin-N-acetyltransferase-2 (zfAANAT-2) mRNA is exclusively expressed in the pineal gland (epiphysis) at the embryonic stage. Here, we have initiated an effort to study the mechanisms underlying tissue-specific expression of this gene. DNA constructs were prepared in which green fluorescent protein (GFP) is driven by regulatory regions of the zfAANAT-2 gene. In vivo transient expression analysis in zebrafish embryos indicated that in addition to the 5'-flanking region, a regulatory sequence in the 3'-flanking region is required for pineal-specific expression. This finding led to an effort to produce transgenic lines expressing GFP under the control of the 5' and 3' regulatory regions of the zfAANAT-2 gene. Embryos transiently expressing GFP were raised to maturity and tested for germ cell transmission of the transgene. Three transgenic lines were produced in which GFP fluorescence in the pineal was detected starting 1 to 2 days after fertilization. One line was crossed with mindbomb and floating head mutants that cause abnormal development of the pineal and an elevation or reduction of zfAANAT-2 mRNA levels, respectively. Homozygous mutant transgenic embryos exhibited similar effects on GFP expression in the pineal gland. These observations indicate that the transgenic lines described here will be useful in studying the development of the pineal gland and the mechanisms that determine pineal-specific gene expression in the zebrafish. Published 2002 Wiley-Liss, Inc.

Animals↗
PubMed2001

One-, two-, and three-color whole-mount in situ hybridization to Drosophila embryos.

This article contains detailed protocols for the localization of mRNA transcripts within whole Drosophila embryos. The procedures are based on the use of digoxigenin-, fluorescein-, and biotin-labeled antisense RNA probes for nonradioactive detection of transcripts. The labels are visualized in situ by differently colored water-insoluble precipitates using alkaline phosphatase- or beta-galactosidase-based immunoassays. First, a basic method is described that allows detection of transcript distribution(s) of one or more genes using the same color precipitate. Second, a sequential alkaline phosphatase detection method is presented that permits the visualization of two or three independent transcript patterns in multiple colors in the same embryo. Third, a shortened two-color in situ hybridization protocol is provided that employs a combination of beta-galactosidase and alkaline phosphatase colorimetric reactions for differential detection. The two-color in situ hybridization methods work equally well in Drosophila and zebrafish embryos and may therefore also be adaptable to other species.

Alkaline Phosphatase↗
PubMed1999

In vivo recording from identifiable neurons of the locomotor network in the developing zebrafish.

The zebrafish is a popular model for developmental studies due to its accessibility by cellular, molecular and genetic approaches. As a complement to these other methods, we have devised an exposed hindbrain/spinal cord preparation in the curarized zebrafish embryo and larva that permits intracellular labeling and patch clamp recording from individually identified sensory neurons, motoneurons and interneurons in vivo. Regular bursts of synaptic potentials and action potentials were observed under whole-cell current clamp in embryonic motoneurons and in some identified interneurons. Larval neurons showed prolonged depolarizations with synaptically driven bursts of action potentials. Frequent spontaneous synaptic potentials were observed and synaptic currents were effectively space clamped. It is thus feasible to study in vivo the properties of identifiable neurons of the developing locomotor network in the zebrafish, including their synaptic activity, firing patterns and interconnections.

Action Potentials↗
PubMed2001

Sequence and expression pattern of ziro7, a novel, divergent zebrafish iroquois homeobox gene.

We have isolated the zebrafish ziro7 gene, a novel, divergent member of the Iroquois family. ziro7 is expressed at early epiboly stages in the dorsal half of the zebrafish embryo, with a higher level in the dorso-lateral margin. From mid-gastrulation stages onward, ziro7 is expressed in a large transversal stripe in the future neural plate, which subsequently divides into thinner stripes located in the diencephalon, midbrain and hindbrain.

Amino Acid Sequence↗
PubMed2005

MicroRNA expression in zebrafish embryonic development.

MicroRNAs (miRNAs) are small noncoding RNAs, about 21 nucleotides in length, that can regulate gene expression by base-pairing to partially complementary mRNAs. Regulation by miRNAs can play essential roles in embryonic development. We determined the temporal and spatial expression patterns of 115 conserved vertebrate miRNAs in zebrafish embryos by microarrays and by in situ hybridizations, using locked-nucleic acid-modified oligonucleotide probes. Most miRNAs were expressed in a highly tissue-specific manner during segmentation and later stages, but not early in development, which suggests that their role is not in tissue fate establishment but in differentiation or maintenance of tissue identity.

Animals↗
PubMed2005

Zebrafish vps33b, an ortholog of the gene responsible for human arthrogryposis-renal dysfunction-cholestasis syndrome, regulates biliary development downstream of the onecut transcription factor hnf6.

Arthrogryposis-renal dysfunction-cholestasis syndrome (ARC) is a rare cause of cholestasis in infants. Causative mutations in VPS33B, a gene that encodes a Class C vacuolar sorting protein, have recently been reported in individuals with ARC. We have identified a zebrafish vps33b-ortholog that is expressed in developing liver and intestine. Knockdown of vps33b causes bile duct paucity and impairs intestinal lipid absorption, thus phenocopying digestive defects characteristic of ARC. By contrast, neither motor axon nor kidney epithelial defects typically seen in ARC could be identified in vps33b-deficient larvae. Biliary defects in vps33b-deficient zebrafish larvae closely resemble the bile duct paucity associated with knockdown of the onecut transcription factor hnf6. Consistent with this, reduced vps33b expression was evident in hnf6-deficient larvae and in larvae with mutation of vhnf1, a downstream target of hnf6. Zebrafish vhnf1, but not hnf6, increases vps33b expression in zebrafish embryos and in mammalian liver cells. Electrophoretic mobility shift assays suggest that this regulation occurs through direct binding of vHnf1 to the vps33b promoter. These findings identify vps33b as a novel downstream target gene of the hnf6/vhnf1 pathway that regulates bile duct development in zebrafish. Furthermore, they show that tissue-specific roles for genes that regulate trafficking of intracellular proteins have been modified during vertebrate evolution.

Animals↗
PubMed2006

FGF signaling mediates regeneration of the differentiating cerebellum through repatterning of the anterior hindbrain and reinitiation of neuronal migration.

To address the regenerative capability of the differentiating hindbrain, we ablated the cerebellum in wild-type and transgenic zebrafish embryos. These larvae showed no obvious locomotive malfunction several days after the ablation. Expression analysis and in vivo time-lapse recording in GFP (green fluorescent protein)-transgenic embryos indicate that cerebellar neuronal cells can regenerate from the remaining anterior hindbrain. The onset of regeneration is accompanied by repatterning within the anterior hindbrain. Inhibition of FGF signaling immediately after cerebellar ablation results in the lack of regenerating cerebellar neuronal cells and the absence of cerebellar structures several days later. Moreover, impaired FGF signaling inhibits the repatterning of the anterior hindbrain and the reexpression of rhombic lip marker genes soon after cerebellar ablation. This demonstrates that the hindbrain is highly plastic in recapitulating early embryonic differentiation mechanisms during regeneration. Moreover, the regenerating system offers a means to uncouple cerebellar differentiation from complex morphogenetic tissue rearrangements.

Animals↗
PubMed2002

Regulation by protein kinase A switching of axonal pathfinding of zebrafish olfactory sensory neurons through the olfactory placode-olfactory bulb boundary.

Cumulative evidence suggests that neural network formation requires an ingenious regulation of the attractive and repulsive responses of growing axons to guidance cues. We examined the role of intracellular protein kinase A (PKA) signaling in the axonal pathfinding of olfactory sensory neurons in transparent zebrafish embryos. Microinjection of an olfactory marker protein gene promoter-driven double-cassette vector directed the expression of both the dominant form of PKA and green fluorescent protein fused with the microtubule-associated protein tau in the same olfactory neurons. The dominant-negative form of PKA enhanced the turning of olfactory neuron axons in the olfactory placode, whereas the disturbance effect of the constitutively active form on the axonal pathfinding was prominent in the olfactory bulb. Consistently, forskolin treatment severely inhibited the axonal extension in the olfactory bulb, but not in the olfactory placode. These results suggest that the switching of PKA signaling in developing olfactory sensory neurons is important for axonal pathfinding through the boundary between the olfactory placode and the olfactory bulb in vivo. We thus propose that the regulation of PKA signaling plays a key role in the long-distance axonal pathfinding through intermediate guideposts.

Amino Acid Sequence↗
PubMed1996

A new tinman-related gene, nkx2.7, anticipates the expression of nkx2.5 and nkx2.3 in zebrafish heart and pharyngeal endoderm.

The Drosophila homeobox gene tinman and its vertebrate homologs Nkx-2.5 and Nkx-2.3 are critical determinants of cardiac development. We report here the identification of a new tinman-related gene, nkx2.7, as well as orthologs of Nkx-2.5 and Nkx-2.3 in the zebrafish. Analysis of their expression in the developing zebrafish embryo reveals that nkx2.7 transcripts are the first to appear in cardiac mesodermal and pharyngeal endodermal precursors of the anterior hypoblast, anticipating both temporally and spatially the later expression of nkx2.5 and nkx2.3 in these lineages. The preeminence of nkx2.7 in these embryonic lineages is consistent with a key role in cell fate determination, perhaps in part through the induction of nkx2.5 and nkx2.3. The findings provide the first molecular clues as to the spatial organization of endodermal and cardiac mesodermal precursors in the zebrafish hypoblast immediately following gastrulation. They suggest a coordinate role for these three tinman-related genes in the development of the heart and pharyngeal arches, and reinforce the paradigm of gene duplication and subspecialization between Drosophila and vertebrate species. The results provide a framework in which to analyze potential changes in tinman-related gene expression during abnormal zebrafish development.

Amino Acid Sequence↗