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PubMed2006

Characterization of the domains of zRICH, a protein induced during optic nerve regeneration in zebrafish.

Teleost fish show a remarkable capability of nerve regeneration in their CNS, while injuries to axon fibers in the CNS of mammals result in degeneration and loss of function. Understanding this difference has biomedical consequences to humans. Both extrinsic factors from the neuronal environment and intrinsic neuronal factors seem to play a role in successful nerve regeneration. Among the intrinsic factors, a number of proteins termed axonal growth associated proteins (GAPs) are strongly induced during axon regeneration. RICH proteins are axonal GAPs that show homology to mammalian myelin marker proteins termed CNPases. Sequence analysis distinguishes three domains in these proteins. In this report, mutant versions of zebrafish RICH proteins were generated to study the roles of the domains of the protein at biochemical and cellular levels. The central CNPase homology domain was sufficient for catalytic activity. The amino terminal acidic domain causes the anomalous electrophoretic migration observed for RICH proteins. The small C-terminal domain bears an isoprenylation motif and is necessary for the interaction of zRICH with cellular membranes. At the cellular level, expression of wild-type zRICH protein in PC12 cells did not induce neurite generation. Additionally, neither the expression of wild-type zRICH nor the expression of mutant versions of the protein interfered with the levels of differentiation of PC12 cells induced by nerve growth factor, suggesting that, at least in this model of neuronal differentiation, zRICH proteins do not participate in the process of generation of neurites.

Animals↗
PubMed1993

Ultraviolet irradiation impairs epiboly in zebrafish embryos: evidence for a microtubule-dependent mechanism of epiboly.

Early morphogenesis of the teleost embryo is characterized by three orchestrated cell movements. Epiboly leads to spreading of the blastoderm over an uncleaved yolk cell while involution around the blastoderm margin and convergence movements towards the dorsal side generate the mes-endodermal inner cell sheet and the axis rudiment, respectively. Irradiation of zebrafish zygotes with ultraviolet light selectively impairs epiboly resulting in embryos with open blastopores but well-formed anterior axes. Gastrulation movements are only marginally affected by ultraviolet irradiation. Involution of marginal cells in epiboly-retarded embryos takes place prior to 50% epiboly and thus appears independent of epiboly. Expression of dorsal and anterior marker genes is unaffected by ultraviolet irradiation. The ultraviolet light effect is not restricted to the zygote stage as irradiation of later embryonic stages also impairs epiboly. The ultraviolet-sensitive targets may thus be maternally encoded components of the machinery driving epiboly. These targets appear to be microtubules: firstly, irradiated embryos show disorganized and less microtubules in the cytoplasmic layer of the yolk sphere; secondly, the ultraviolet light effect can be mimicked by the microtubule-depolymerizing agent nocodazole. We suggest that epiboly is driven, at least partially, by motors that use microtubules radiating from the yolk syncytial layer into the yolk cytoplasmic layer. Together with an observed constrictive behaviour of the blastoderm margin, we propose a two-force model of epiboly: epiboly is initiated and driven by a pulling force dependent on microtubules in the yolk cytoplasmic layer; contraction at the margin operates in addition to aid closure of the blastopore.

Animals↗
PubMed2005

Early stages of zebrafish eye formation require the coordinated activity of Wnt11, Fz5, and the Wnt/beta-catenin pathway.

During regional patterning of the anterior neural plate, a medially positioned domain of cells is specified to adopt retinal identity. These eye field cells remain coherent as they undergo morphogenetic events distinct from other prospective forebrain domains. We show that two branches of the Wnt signaling pathway coordinate cell fate determination with cell behavior during eye field formation. Wnt/beta-catenin signaling antagonizes eye specification through the activity of Wnt8b and Fz8a. In contrast, Wnt11 and Fz5 promote eye field development, at least in part, through local antagonism of Wnt/beta-catenin signaling. Additionally, Wnt11 regulates the behavior of eye field cells, promoting their cohesion. Together, these results allow us to postulate a model in which Wnt11 and Fz5 signaling promotes early eye development through the coordinated antagonism of signals that suppress retinal identity and promotion of coherence of eye field cells.

Animals↗
PubMed2003

435-bp liver regulatory sequence in the liver fatty acid binding protein (L-FABP) gene is sufficient to modulate liver regional expression in transgenic zebrafish.

Liver fatty acid binding protein (L-FABP) is a small protein that is thought to play an important role in the intracellular binding and trafficking of long chain fatty acids in the liver. Expression of the gene encoding the zebrafish liver fatty acid binding protein is regulated by a 435-bp distal region (-1944 to -1510) of the L-FABP promoter. The 435-bp sequence is sufficient for gene activation in the liver primordia (or bud) and continues to be active in the adult liver when positioned adjacent to the SV40 basal promoter and linked directly to green fluorescent protein. The 435-bp sequence region has two distinct liver regulatory elements, A (-1944 to -1623) and B (-1622 to -1510), and contains multiple putative consensus binding sites. The element A sequence includes two consensus HFH and one HNF-1alpha site and the element B sequence includes one consensus HNF-3beta site. Deletion of an internal 435-bp fragment (-1944 to -1510) including the A and B elements totally ablated the liver-specific activity of the zebrafish L-FABP gene promoter. Deletion of either of the two elements reduces the liver activity. Mutation of the HNF-1alpha site or either of the two HFH sites in the A element or the HNF-3beta site in the B element significantly altered specificity in the liver primordia of transient expression embryos. The importance of the HNF-1alpha consensus binding site in the A element and the HNF-3beta consensus binding site in the B element within the 435-bp distal region of the L-FABP promoter region suggests that combinatorial interactions between multiple regulatory factors are responsible for the gene expression of L-FABP in the liver.

Animals↗
PubMed2005

Characterization of zebrafish PSD-95 gene family members.

The PSD-95 family of membrane- associated guanylate kinases (MAGUKs) are thought to act as molecular scaffolds that regulate the assembly and function of the multiprotein signaling complex found at the postsynaptic density of excitatory synapses. Genetic analysis of PSD-95 family members in the mammalian nervous system has so far been difficult, but the zebrafish is emerging as an ideal vertebrate system for studying the role of particular genes in the developing and mature nervous system. Here we describe the cloning of the zebrafish orthologs of PSD-95, PSD-93, and two isoforms of SAP-97. Using in situ hybridization analysis we show that these zebrafish MAGUKs have overlapping but distinct patterns of expression in the developing nervous system and craniofacial skeleton. Using a pan-MAGUK antibody we show that MAGUK proteins localize to neurons within the developing hindbrain, cerebellum, visual and olfactory systems, and to skin epithelial cells. In the olfactory and visual systems MAGUK proteins are expressed strongly in synaptic regions, and the onset of expression in these areas coincides with periods of synapse formation. These data are consistent with the idea that PSD-95 family members are involved in synapse assembly and function, and provide a platform for future functional studies in vivo in a highly tractable model organism.

Animals↗
PubMed2005

Current perspectives in intronic micro RNAs (miRNAs).

MicroRNAs (miRNAs), small single-stranded regulatory RNAs capable of interfering with intracellular messenger RNAs (mRNAs) that contain either complete or partial complementarity, are useful for the design of new therapies against cancer polymorphism and viral mutation. Numerous miRNAs have been reported to induce RNA interference (RNAi), a post-transcriptional gene silencing mechanism. Intronic miRNAs, derived from introns by RNA splicing and Dicer processing, can interfere with intracellular mRNAs to silence that gene expression. The intronic miRNAs differ uniquely from previously described intergenic miRNAs in the requirement of type II RNA polymerases (Pol-II) and spliceosomal components for its biogenesis. Several kinds of intronic miRNAs have been identified in Caenorhabditis elegans, mouse and human cells; however, neither their function nor application has been reported. To this day, the computer searching program for miRNA seldom include the intronic portion of protein-coding RNAs. The functional significance of artificially generated intronic miRNAs has been successfully ascertained in several biological systems such as zebrafishes, chicken embryos and adult mice, indicating the evolutionary preservation of this gene regulation system in vivo. Multiple miRNAs can be generated from the same cluster of introns; however, non-homologous miRNAs may have different targets and functions while homologous miRNA may be derived from different intronic clusters. Taken together, the model of intronic miRNA-mediated transgenic animals provides a tool to investigate the mechanism of miRNA-associated diseases in vivo and will shed light on miRNA-related therapies.

Animals↗
PubMed1999

Dishevelled: at the crossroads of divergent intracellular signaling pathways.

During the development of multicellular organisms the formation of complex patterns relies on specific cell-cell signaling events. For tissues to become spatially organized and cells to become committed to specialized fates it is absolutely crucial for proper development that the underlying signaling systems receive and route information correctly. Recently, a wealth of genetic and biochemical experimental data has been collected about prevalent evolutionary conserved signaling families, such as the Wnts, Dpp/BMPs, and Hedgehogs, in flies, worms, and vertebrates. Paradoxically, members of a particular signaling family often have receptors with similar biochemical binding properties, though they activate different intracellular pathways in vivo and can be phenotypically distinguished. How are their specific biological responses then generated? With respect to signaling specificity in Wnt pathways, Dishevelled is an intriguing protein; in Drosophila melanogaster it is required in two distinct signaling pathways, that share Frizzled receptors of similar structure, but have distinct intracellular signaling routes. Recent results suggest that Dishevelled is a multifunctional protein at the crossroads of divergent Wnt/Fz pathways. Dishevelled appears to be a key factor in Wnt signaling to read' signals coming from the plasma membrane and route them into the correct intracellular pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Glycogen synthase kinase-3 and Axin function in a beta-catenin-independent pathway that regulates neurite outgrowth in neuroblastoma cells.

We have sought to determine the roles of beta-catenin and the Wnt signaling pathway in neurite outgrowth using a model cell system, the Neuro-2a neuroblastoma cell line. Activation of the Wnt signaling pathway disrupts a multiprotein complex that includes beta-catenin, Axin, and glycogen synthase kinase-3 (GSK-3), which would otherwise promote the phosphorylation and degradation of beta-catenin. Stabilized beta-catenin accumulates in the cytosol and in the nucleus; in the nucleus it binds to TCF family transcription factors, forming a bipartite transcriptional activator of Wnt target genes. These events can be mimicked by lithium (Li(+)), which inhibits GSK-3 activity. Both Li(+) and the GSK-3 inhibitor SB415286 induced neurite outgrowth of Neuro-2a cells. Li(+)-induced neurite outgrowth did not require beta-catenin-/TCF-dependent transcription, and increasing levels of beta-catenin either by transfection or using Wnt-3A was not sufficient to induce neurite outgrowth. Interestingly, Axin, which is also a substrate for GSK-3, was destabilized by Li(+) and ectopic expression of Axin inhibited Li(+)-induced neurite outgrowth. Deletion analysis of Axin indicated that this inhibition required the GSK-3 binding site, but not the beta-catenin binding site. Our results suggest that a signaling pathway involving Axin and GSK-3, but not beta-catenin, regulates Li(+)-induced neurite outgrowth in Neuro-2a cells.

Animals↗
PubMed2000

The establishment of Spemann's organizer and patterning of the vertebrate embryo.

Molecular studies have begun to unravel the sequential cell-cell signalling events that establish the dorsal-ventral, or 'back-to-belly', axis of vertebrate animals. In Xenopus and zebrafish, these events start with the movement of membrane vesicles associated with dorsal determinants. This mediates the induction of mesoderm by generating gradients of growth factors. Dorsal mesoderm then becomes a signalling centre, the Spemann's organizer, which secretes several antagonists of growth-factor signalling. Recent studies have led to new models for the regulation of cell-cell signalling during development, which may also apply to the homeostasis of adult tissues.

Amino Acid Sequence↗
PubMed2006

Planar cell polarity signalling couples cell division and morphogenesis during neurulation.

Environmental and genetic aberrations lead to neural tube closure defects (NTDs) in 1 out of every 1,000 births. Mouse and frog models for these birth defects have indicated that Van Gogh-like 2 (Vangl2, also known as Strabismus) and other components of planar cell polarity (PCP) signalling might control neurulation by promoting the convergence of neural progenitors to the midline. Here we show a novel role for PCP signalling during neurulation in zebrafish. We demonstrate that non-canonical Wnt/PCP signalling polarizes neural progenitors along the anteroposterior axis. This polarity is transiently lost during cell division in the neural keel but is re-established as daughter cells reintegrate into the neuroepithelium. Loss of zebrafish Vangl2 (in trilobite mutants) abolishes the polarization of neural keel cells, disrupts re-intercalation of daughter cells into the neuroepithelium, and results in ectopic neural progenitor accumulations and NTDs. Remarkably, blocking cell division leads to rescue of trilobite neural tube morphogenesis despite persistent defects in convergence and extension. These results reveal a function for PCP signalling in coupling cell division and morphogenesis at neurulation and indicate a previously unrecognized mechanism that might underlie NTDs.

Animals↗
PubMed2004

Disruption of dimerization and substrate phosphorylation inhibit factor inhibiting hypoxia-inducible factor (FIH) activity.

HIF (hypoxia-inducible factor) is an alphabeta transcription factor that modulates the hypoxic response in many animals. The cellular abundance and activity of HIF-alpha are regulated by its post-translational hydroxylation. The hydroxylation of HIF is catalysed by PHD (prolyl hydroxylase domain) enzymes and FIH (factorinhibiting HIF), all of which are 2-oxoglutarate- and Fe(II)-dependent dioxygenases. FIH hydroxylates a conserved asparagine residue in HIF-alpha (Asn-803), which blocks the binding of HIF to the transcriptional co-activator p300, preventing transcription of hypoxia-regulated genes under normoxic conditions. In the present paper, we report studies on possible mechanisms for the regulation of FIH activity. Recently solved crystal structures of FIH indicate that it is homodimeric. Site-directed mutants of FIH at residues Leu-340 and Ile-344, designed to disrupt dimerization, were generated in order to examine the importance of the dimeric state in determining FIH activity. A single point mutant, L340R (Leu-340-->Arg), was shown to be predominantly monomeric and to have lost catalytic activity as measured by assays monitoring 2-oxoglutarate turnover and asparagine hydroxylation. In contrast, the I344R (Ile-344-->Arg) mutant was predominantly dimeric and catalytically active. The results imply that the homodimeric form of FIH is required for productive substrate binding. The structural data also revealed a hydrophobic interaction formed between FIH and a conserved leucine residue (Leu-795) on the HIF substrate, which is close to the dimer interface. A recent report has revealed that phosphorylation of Thr-796, which is adjacent to Leu-795, enhances the transcriptional response in hypoxia. Consistent with this, we show that phosphorylation of Thr-796 prevents the hydroxylation of Asn-803 by FIH.

Amino Acid Sequence↗
PubMed1999

Membrane-anchored plakoglobins have multiple mechanisms of action in Wnt signaling.

In Wnt signaling, beta-catenin and plakoglobin transduce signals to the nucleus through interactions with TCF-type transcription factors. However, when plakoglobin is artificially engineered to restrict it to the cytoplasm by fusion with the transmembrane domain of connexin (cnxPg), it efficiently induces a Wnt-like axis duplication phenotype in Xenopus. In Xenopus embryos, maternal XTCF3 normally represses ventral expression of the dorsalizing gene Siamois. Two models have been proposed to explain the Wnt-like activity of cnxPg: 1) that cnxPg inhibits the machinery involved in the turnover of cytosolic beta-catenin, which then accumulates and inhibits maternal XTCF3, and 2) that cnxPg directly acts to inhibit XTCF3 activity. To distinguish between these models, we created a series of N-terminal deletion mutations of cnxPg and examined their ability to induce an ectopic axis in Xenopus, activate a TCF-responsive reporter (OT), stabilize beta-catenin, and colocalize with components of the Wnt signaling pathway. cnxPg does not colocalize with the Wnt pathway component Dishevelled, but it does lead to the redistribution of APC and Axin, two proteins involved in the regulation of beta-catenin turnover. Expression of cnxPg increases levels of cytosolic beta-catenin; however, this effect does not completely explain its signaling activity. Although cnxPg and Wnt-1 stabilize beta-catenin to similar extents, cnxPg activates OT to 10- to 20-fold higher levels than Wnt-1. Moreover, although LEF1 and TCF4 synergize with beta-catenin and plakoglobin to activate OT, both suppress the signaling activity of cnxPg. In contrast, XTCF3 suppresses the signaling activity of both beta-catenin and cnxPg. Both exogenous XLEF1 and XTCF3 are sequestered in the cytoplasm of Xenopus cells by cnxPg. Based on these data, we conclude that, in addition to its effects on beta-catenin, cnxPg interacts with other components of the Wnt pathway, perhaps TCFs, and that these interactions contribute to its signaling activity.

Animals↗
PubMed2004

Developmental roles of pufferfish Hox clusters and genome evolution in ray-fin fish.

The pufferfish skeleton lacks ribs and pelvic fins, and has fused bones in the cranium and jaw. It has been hypothesized that this secondarily simplified pufferfish morphology is due to reduced complexity of the pufferfish Hox complexes. To test this hypothesis, we determined the genomic structure of Hox clusters in the Southern pufferfish Spheroides nephelus and interrogated genomic databases for the Japanese pufferfish Takifugu rubripes (fugu). Both species have at least seven Hox clusters, including two copies of Hoxb and Hoxd clusters, a single Hoxc cluster, and at least two Hoxa clusters, with a portion of a third Hoxa cluster in fugu. Results support genome duplication before divergence of zebrafish and pufferfish lineages, followed by loss of a Hoxc cluster in the pufferfish lineage and loss of a Hoxd cluster in the zebrafish lineage. Comparative analysis shows that duplicate genes continued to be lost for hundreds of millions of years, contrary to predictions for the permanent preservation of gene duplicates. Gene expression analysis in fugu embryos by in situ hybridization revealed evolutionary change in gene expression as predicted by the duplication-degeneration-complementation model. These experiments rule out the hypothesis that the simplified pufferfish body plan is due to reduction in Hox cluster complexity, and support the notion that genome duplication contributed to the radiation of teleosts into half of all vertebrate species by increasing developmental diversification of duplicate genes in daughter lineages.

Animals↗
PubMed1999

[The head inducer Cerberus in a multivalent extracellular inhibitor].

Cerberus encodes for a secreted protein which when overexpressed ventrally in a Xenopus embryo induces head differentiation without trunk (Bouwmeester et al., 1996). We have recently shown that Cerberus can bind BMP-4 (Bone Morphogenetic Protein-4), Xnr-1 (Xenopus Nodal-related 1) and Xwnt-8 in the extracellular space (Piccolo et al., 1999). We present here studies showing that Cerberus does not have a receptor nor a dedicated transduction pathway but rather acts as an extracellular inhibitor. Our results suggest that the action of Cerberus in head induction can be explained by an inhibitory activity upstream of the Nodal-related and BMP-4 receptors. In addition, using dominant negative receptor mutants which block both the Xnr-1 and BMP-4 transduction pathways, we show that this double inhibition is sufficient for head induction in ventral mesoderm explants.

Animals↗
PubMed2003

Functional and structural analyses of cryptochrome. Vertebrate CRY regions responsible for interaction with the CLOCK:BMAL1 heterodimer and its nuclear localization.

Mouse mCRY1 and zebrafish zCRY1a and zCRY3 belong to the DNA photolyase/Cryptochrome family. mCRY1 and zCRY1a repress CLOCK:BMAL1-mediated transcription, whereas zCRY3 does not. Reciprocal chimeras between zCRY1a and zCRY3 were generated to determine the zCRY1a regions responsible for nuclear translocation, interaction with the CLOCK:BMAL1 heterodimer, and repression of CLOCK:BMAL1-mediated transcription. Three regions, RD-2a-(126-196), RD-1-(197-263), and RD-2b-(264-293), were identified. Proteins in this family consist of an N-terminal alpha/beta domain and a C-terminal helical domain connected by an interdomain loop. RD-2a is within this loop, RD-1 is at the N-terminal 50 amino acids, and RD-2b at the following 31 amino acid residues of the helical domain. Either RD-2a or RD-1 is required for interaction with the CLOCK: BMAL1 heterodimer, and either RD-1 or RD-2b is required for the nuclear translocation of CRY. Both of these functions are prerequisites for the transcriptional repressor activity. The functional nuclear localizing signal in the RD-2b region also was identified. The sequence is well conserved among repressor-type CRYs, including mCRY1. Mutations in the nuclear localizing signal of mCRY1 reduce the extent of its nuclear localization. These findings show that both nuclear localization and interaction with the CLOCK:BMAL heterodimer are essential for transcriptional repression by CRY.

Amino Acid Sequence↗
PubMed2002

Weakness of a giant: mutations of the sarcomeric protein titin.

In 2002, three reports described for the first time mutations in the sarcomeric protein titin associated with dilated cardiomyopathy in humans. Despite different locations (Z-line region, Z-I transitional zone, N2B region, half A band region) all mutations resulted in heart failure. In addition, an N2B mutation was found in zebrafish embryos with ventricular dilatation and cardiac insufficiency. It is concluded that titin mutations have significant functional consequences and need to be studied intensively in the future.

Cardiomyopathy, Dilated↗
PubMed2004

Morphogens, their identification and regulation.

During the course of development, cells of many tissues differentiate according to the positional information that is set by the concentration gradients of morphogens. Morphogens are signaling molecules that emanate from a restricted region of a tissue and spread away from their source to form a concentration gradient. As the fate of each cell in the field depends on the concentration of the morphogen signal, the gradient prefigures the pattern of development. In this article, we describe how morphogens and their functions have been identified and analyzed, focusing on model systems that have been extensively studied.

Animals↗