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PubMed2006

Zebrafish G protein gamma2 is required for VEGF signaling during angiogenesis.

Vascular endothelial growth factor (VEGF) is a major mediator of pathologic angiogenesis, a process necessary for the formation of new blood vessels to support tumor growth. Historically, VEGF has been thought to signal via receptor tyrosine kinases, which are not typically considered to be G protein dependent. Here, we show that targeted knockdown of the G protein gng2 gene (Ggamma2) blocks the normal angiogenic process in developing zebrafish embryos. Moreover, loss of gng2 function inhibits the ability of VEGF to promote the angiogenic sprouting of blood vessels by attenuating VEGF induced phosphorylation of phospholipase C-gamma1 (PLCgamma1) and serine/threonine kinase (AKT). Collectively, these results demonstrate a novel interaction between Ggamma2- and VEGF-dependent pathways to regulate the angiogenic process in a whole-animal model. Blocking VEGF function using a humanized anti-VEGF antibody has emerged as a promising treatment for colorectal, non-small lung cell, and breast cancers. However, this treatment may cause considerable side effects. Our findings provide a new opportunity for cotargeting G protein- and VEGF-dependent pathways to synergistically block pathologic angiogenesis, which may lead to a safer and more efficacious therapeutic regimen to fight cancer.

Animals↗
PubMed2005

Reduced U snRNP assembly causes motor axon degeneration in an animal model for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a motoneuron disease caused by reduced levels of survival motoneuron (SMN) protein. Previous studies have assigned SMN to uridine-rich small nuclear ribonucleoprotein particle (U snRNP) assembly, splicing, transcription, and RNA localization. Here, we have used gene silencing to assess the effect of SMN protein deficiency on U snRNP metabolism in living cells and organisms. In HeLa cells, we show that reduction of SMN to levels found in SMA patients impairs U snRNP assembly. In line with this, induced silencing of SMN expression in Xenopus laevis or zebrafish arrested embryonic development. Under less severe knock-down conditions, zebrafish embryos proceeded through development yet exhibited dramatic SMA-like motor axon degeneration. The same was observed after silencing two other essential factors in the U snRNP assembly pathway, Gemin2 and pICln. Importantly, the injection of purified U snRNPs into either SMN- or Gemin2-deficient embryos of Xenopus and zebrafish prevented developmental arrest and motoneuron degeneration, respectively. These findings suggest that motoneuron degeneration in SMA patients is a direct consequence of impaired production of U snRNPs.

Animals↗
PubMed2005

Identification and characterization of a novel member of the ATP-sensitive K+ channel subunit family, Kir6.3, in zebrafish.

ATP-sensitive K+ (KATP) channels play a crucial role in coupling cellular metabolism to membrane potential. In addition to the orthologs corresponding to Kir6.1 and Kir6.2 of mammals, we have identified a novel member, designated Kir6.3 (zKir6.3), of the inward rectifier K+ channel subfamily Kir6.x in zebrafish. zKir6.3 is a protein of 432 amino acids that shares 66% identity with mammalian Kir6.2 but differs considerably from mammalian Kir6.1 and Kir6.2 in the COOH terminus, which contain an Arg-Lys-Arg (RKR) motif, an endoplasmic reticulum (ER) retention signal. Single-channel recordings of reconstituted channels show that zKir6.3 requires the sulfonylurea receptor 1 (SUR1) subunit to produce KATP channel currents with single-channel conductance of 57.5 pS. Confocal microscopic analysis shows that zebrafish Kir6.3 requires the SUR1 subunit for its trafficking to the plasma membrane. Analyses of chimeric protein between human Kir6.2 and zKir6.3 and a COOH-terminal deletion of zKir6.3 indicate that interaction between the COOH terminus of zKir6.3 and SUR1 is critical for both channel activity and trafficking to the plasma membrane. We also identified zebrafish orthologs corresponding to mammalian SUR1 (zSUR1) and SUR2 (zSUR2) by the genomic database. Both Kir6.3 and SUR1 are expressed in embryonic brain of zebrafish, as assessed by whole mount in situ hybridization. These data indicate that Kir6.3 and SUR1 form functional KATP channels at the plasma membrane in zebrafish through a mechanism independent from ER retention by the RKR motif.

ATP-Binding Cassette Transporters↗
PubMed2003

Multiple muscle cell identities induced by distinct levels and timing of hedgehog activity in the zebrafish embryo.

BACKGROUND: In the zebrafish embryo, the differentiation of distinct muscle fiber types has been shown to require the activity of members of the Hedgehog (Hh) family of secreted proteins. Evidence from other systems suggests that Hh behaves as a morphogen, inducing cell fates in a concentration-dependent manner. Exactly how Hh signaling contributes to the generation of the correct pattern of cells within the zebrafish myotome, however, has remained obscure. RESULTS: Here, we distinguish four distinct myotomal cell identities in the zebrafish embryo on the basis of their position, morphology, and gene expression patterns. Using morpholino oligonucleotides (MOs) to diminish the activities of the Hh pathway components Patched (Ptc), Fused (Fu), and Suppressor of Fused (Su(fu)), and the teratogen cyclopamine to inhibit the Hh transducer Smoothened (Smo), we show that the appropriate differentiation of each cell type depends upon the levels and range of Hh signaling within the myotome. In addition, by transiently modulating Hh activity by using cyclopamine and a heat-inducible transgene, we demonstrate that the competence of myotomal cells to respond to Hh changes with time. Finally, we show that the Gli1 and Gli2 transcription factors mediate most of the response of myotomal cells to Hh. CONCLUSIONS: Hh signaling acts in a dosage-dependent manner to specify cell fate in the zebrafish myotome. Allocation of the correct number of cells to a specific fate depends upon the range of Hh activity. The timing of exposure to Hh determines the response of cells to the signal.

Animals↗
PubMed1996

valentino: a zebrafish gene required for normal hindbrain segmentation.

Mutational analysis can serve both to identify new genes essential for patterning embryonic development and to determine their functions. Here we describe the identification and phenotypic characterization of alleles of valentino, which we recovered in a genetic screen that sought to identify mutations in the zebrafish that disrupt region-specific gene expression patterns in the embryonic brain. valentino is required for normal hindbrain segmentation and the hindbrain of valentino mutant embryos is shortened by the length of one rhombomere. We demonstrate that valentino is required cell-autonomously in the development of rhombomeres 5 and 6, and propose that valentino functions in the subdivision and expansion of a common precursor region in the presumptive hindbrain into the definitive rhombomeres 5 and 6. These results provide genetic evidence for a two-segment periodicity in the hindbrain and suggest that this periodicity arises sequentially, through the specification and later subdivision of a two-rhombomere unit, or 'protosegment'.

Animals↗
PubMed2001

Genomic organization and expression of the doublesex-related gene cluster in vertebrates and detection of putative regulatory regions for DMRT1.

Genes related to the Drosophila melanogaster doublesex and Caenorhabditis elegans mab-3 genes are conserved in human. They are identified by a DNA-binding homology motif, the DM domain, and constitute a gene family (DMRTs). Unlike the invertebrate genes, whose role in the sex-determination process is essentially understood, the function of the different vertebrate DMRT genes is not as clear. Evidence has accumulated for the involvement of DMRT1 in male sex determination and differentiation. DMRT2 (known as terra in zebrafish) seems to be a critical factor for somitogenesis. To contribute to a better understanding of the function of this important gene family, we have analyzed DMRT1, DMRT2, and DMRT3 from the genome model organism Fugu rubripes and the medakafish, a complementary model organism for genetics and functional studies. We found conservation of synteny of human chromosome 9 in F. rubripes and an identical gene cluster organization of the DMRTs in both fish. Although expression analysis and gene linkage mapping in medaka exclude a function for any of the three genes in the primary step of male sex determination, comparison of F. rubripes and human sequences uncovered three putative regulatory regions that might have a role in more downstream events of sex determination and human XY sex reversal.

Amino Acid Sequence↗
PubMed2006

Ribonucleases and angiogenins from fish.

For the first time fish RNases have been isolated and characterized. Their functional and structural properties indicate that they belong to the RNase A superfamily (or tetrapod RNase superfamily), now more appropriately described as the vertebrate RNase superfamily. Our findings suggest why previously repeated efforts to isolate RNases from fish tissues have met with no success; fish RNases have a very low ribonucleolytic activity, and their genes have a low sequence identity with those of mammalian RNases. The investigated RNases are from the bony fish Danio rerio (or zebrafish). Their cDNAs have been cloned and expressed, and the three recombinant proteins have been purified to homogeneity. Their characterization has revealed that they have indeed a very low RNA-degrading activity, when compared with that of RNase A, the superfamily prototype, but comparable with that of mammalian angiogenins; that two of them have angiogenic activity that is inhibited by the cytosolic RNase inhibitor. These data and a phylogenetic analysis indicate that angiogenic fish RNases are the earliest diverging members of the vertebrate superfamily, suggesting that ribonucleases with angiogenic activity were the ancestors of all ribonucleases in the superfamily. They later evolved into both mammalian angiogenins and, through a successful phylogenesis, RNases endowed with digestive features or with diverse bioactivities.

Amino Acid Sequence↗
PubMed2004

The development of semicircular canals in the inner ear: role of FGFs in sensory cristae.

In the vertebrate inner ear, the ability to detect angular head movements lies in the three semicircular canals and their sensory tissues, the cristae. The molecular mechanisms underlying the formation of the three canals are largely unknown. Malformations of this vestibular apparatus found in zebrafish and mice usually involve both canals and cristae. Although there are examples of mutants with only defective canals, few mutants have normal canals without some prior sensory tissue specification, suggesting that the sensory tissues, cristae, might induce the formation of their non-sensory components, the semicircular canals. We fate-mapped the vertical canal pouch in chicken that gives rise to the anterior and posterior canals, using a fluorescent, lipophilic dye (DiI), and identified a canal genesis zone adjacent to each prospective crista that corresponds to the Bone morphogenetic protein 2 (Bmp2)-positive domain in the canal pouch. Using retroviruses or beads to increase Fibroblast Growth Factors (FGFs) for gain-of-function and beads soaked with the FGF inhibitor SU5402 for loss-of-function experiments, we show that FGFs in the crista promote canal development by upregulating Bmp2. We postulate that FGFs in the cristae induce a canal genesis zone by inducing/upregulating Bmp2 expression. Ectopic FGF treatments convert some of the cells in the canal pouch from the prospective common crus to a canal-like fate. Thus, we provide the first molecular evidence whereby sensory organs direct the development of the associated non-sensory components, the semicircular canals, in vertebrate inner ears.

Animals↗
PubMed2001

Expression pattern of two zebrafish genes, cxcr4a and cxcr4b.

We cloned and mapped two novel zebrafish genes, cxcr4a and cxcr4b, which are closely related to mammalian CXCR4. Expression analysis by reverse transcription-polymerase chain reaction and in situ hybridization demonstrated that these two genes are expressed in most cell lineages known to express Cxcr4 in mammals. These genes are co-expressed in lateral mesoderm and posterior midbrain. The transcripts of cxcr4a were detected in interneurons and endoderm, whereas cxcr4b was specifically expressed in sensory neurons, motoneurons and cerebellum. In the lateral mesoderm, cxcr4b transcripts appeared earlier than those of cxcr4a. Thus, the function of mammalian CXCR4 could be split between the two zebrafish genes. These genes probably derived from the genome duplication event, which occurred during the evolution of teleosts. Similar pairs of Cxcr4 may exist in other species, where genome duplication has occurred.

Amino Acid Sequence↗
PubMed2006

Gene order data from a model amphibian (Ambystoma): new perspectives on vertebrate genome structure and evolution.

BACKGROUND: Because amphibians arise from a branch of the vertebrate evolutionary tree that is juxtaposed between fishes and amniotes, they provide important comparative perspective for reconstructing character changes that have occurred during vertebrate evolution. Here, we report the first comparative study of vertebrate genome structure that includes a representative amphibian. We used 491 transcribed sequences from a salamander (Ambystoma) genetic map and whole genome assemblies for human, mouse, rat, dog, chicken, zebrafish, and the freshwater pufferfish Tetraodon nigroviridis to compare gene orders and rearrangement rates. RESULTS: Ambystoma has experienced a rate of genome rearrangement that is substantially lower than mammalian species but similar to that of chicken and fish. Overall, we found greater conservation of genome structure between Ambystoma and tetrapod vertebrates, nevertheless, 57% of Ambystoma-fish orthologs are found in conserved syntenies of four or more genes. Comparisons between Ambystoma and amniotes reveal extensive conservation of segmental homology for 57% of the presumptive Ambystoma-amniote orthologs. CONCLUSION: Our analyses suggest relatively constant interchromosomal rearrangement rates from the euteleost ancestor to the origin of mammals and illustrate the utility of amphibian mapping data in establishing ancestral amniote and tetrapod gene orders. Comparisons between Ambystoma and amniotes reveal some of the key events that have structured the human genome since diversification of the ancestral amniote lineage.

Ambystoma↗
PubMed2005

Identification of residues in human neuroglobin crucial for Guanine nucleotide dissociation inhibitor activity.

Neuroglobin (Ngb) is a recently discovered vertebrate heme protein that is expressed in the brain and can reversibly bind oxygen. We previously demonstrated that ferric human Ngb binds to the alpha-subunits of heterotrimeric G proteins (Galpha) and acts as a guanine nucleotide dissociation inhibitor (GDI) for Galpha. Here we have investigated the interaction between Ngb and Galpha in more detail. We report that zebrafish Ngb, which shares about 50% amino acid sequence identity with human Ngb, does not have a GDI activity for Galpha. By carrying out exon swapping between zebrafish and human Ngb and site-directed mutagenesis, we have identified several residues that are crucial for the GDI activity of human Ngb.

Amino Acid Sequence↗
PubMed1999

Maternal mRNA localization of zebrafish DAZ-like gene.

Members of the DAZ gene family encode RNA-binding proteins and have been shown to play a pivotal role in gametogenesis. In Xenopus, a DAZ-like gene encodes an RNA component of the germ plasm. We have identified a zebrafish DAZ homologue, zDazl. zDazl mRNA was expressed in gonads of both sexes. In ovary, it was localized in the cortex of oocytes. At the onset of embryogenesis, maternal zDazl mRNA was detected at the vegetal pole. It migrated toward blastomeres through cytoplasmic streams as early embryogenesis proceeded. This is the first report showing maternal mRNA localization at the vegetal pole in fish and the existence of mRNA streams in the yolk cytoplasm.

Animals↗
PubMed2004

A new group of tyrosine recombinase-encoding retrotransposons.

A wide variety of novel tyrosine recombinase (YR)-encoding retrotransposons were identified using data emerging from the various eukaryotic genome sequencing projects. Although many of these elements are clearly members of the previously described DIRS group of YR retrotransposons, a substantial number, including elements from a variety of fungi and animals, belong to a distinct and previously unrecognized group. We refer to these latter elements as the Ngaro group after a representative from zebrafish. Like the members of the DIRS group, Ngaro elements encode proteins bearing reverse transcriptase (RT) and ribonuclease H (RH) domains similar to those of long terminal repeat (LTR) retrotransposons. Phylogenetic analyses based on alignments of RT/RH and YR domains, however, indicate that Ngaro and DIRS are anciently diverged groups. Differences in coding capacity also support the distinction between the two groups. For instance, we found that DIRS elements all encode a protein domain which is similar in sequence to the DNA methyltransferases of certain bacteriophages, whereas this domain is absent from all Ngaro elements. Together, the Ngaro and DIRS groups of YR retrotransposons contain elements with an astonishing diversity in structures, with variations in the nature of the associated repeat sequences and in the arrangement and complement of coding regions. In addition they contain elements with some surprising features, such as spliceosomal introns and long overlapping open reading frames.

Amino Acid Sequence↗
PubMed2002

Secreted Frizzled-related proteins: searching for relationships and patterns.

Secreted Frizzled-related proteins (SFRPs) are modulators of the intermeshing pathways in which signals are transduced by Wnt ligands through Frizzled (Fz) membrane receptors. The Wnt networks influence biological processes ranging from developmental cell fate, cell polarity and adhesion to tumorigenesis and apoptosis. In the five or six years since their discovery, the SFRPs have emerged as dynamically expressed proteins able to bind both Wnts and Fz, with distinctive structural properties in which cysteine-rich domains from Fz- and from netrin-like proteins are juxtaposed. The abundant expression of SFRP genes in the early embryo, altered expression patterns in disease states, and potential significance in the evolution of the vertebrate body plan, make these intriguing molecules relevant to investigations in diverse fields of biology and biomedical sciences.

Animals↗
PubMed2007

Theoretical study of interactions between muscle aldolase and F-actin: insight into different species.

Interactions of the glycolytic enzyme, fructose-1,6-bisphosphate aldolase (aldolase), with F-actin may be one mechanism for the colocalization of glycolytic enzymes. Examination of these interactions in different animal species tests this hypothesis by observing whether binding sites are conserved across species. Brownian dynamics (BD) simulations provide descriptions of such protein-protein interactions with the muscle isoforms of zebra fish and human aldolase. The results are compared with previous results obtained for rabbit muscle and yeast. The aldolase binding groove previously determined in rabbit muscle is conserved in both the human and fish muscle isoforms. The nonspecific radial free energies of interaction are similar with fish being slightly weaker than human and rabbit: human, -2.27 +/- 0.05 kcal/mol; rabbit, -2.0 +/- 0.04 kcal/mol; and fish, -1.5 +/- 0.03 kcal/mol. BD results show a large Boltzmann population of complexes formed around the A/D and B/C grooves of aldolase with the most feasible binding mode comprising two aldolase subunits to subdomain I region of the actin subunits. These results show that the location of the important residues and binding site for fish and human aldolase is very similar to that in rabbit and that in different animals the binding site is conserved. This suggests that the binding interaction between aldolase and F-actin is general in animal muscles and is rendered possible and energetically favorable through the conservation of this binding site.

Actins↗
PubMed2002

Role of glycogen synthase kinase-3 in cancer: regulation by Wnts and other signaling pathways.

Although glycogen synthase kinase-3 (GSK-3) is but one of more than a thousand distinct serine/threonine kinases present in the mammalian genome, this enzyme has attracted attention for its role in a diverse range of cellular processes and its positioning at a nexus of several signaling pathways that are important in cancer and other human diseases. The association of GSK-3 with widely different functions, from glycogen metabolism to fruit fly segmentation and slime mold differentiation, was initially perplexing. However, as the context of the biological processes involving this enzyme has been clarified, unifying themes have emerged that begin to explain its pleiotropic nature. Unlike most protein kinases involved in signaling, GSK-3 is active in unstimulated, resting cells. Its activity is inactivated during cellular responses and its substrates therefore tend to be dephosphorylated. As more of these targets have been identified and the effects of their modification by GSK-3 determined, most have been found to be functionally inhibited by GSK-3. Hence, this kinase appears to act as a general repressor, keeping its targets switched off or inaccessible under resting conditions. The rarity of this form of regulation is perhaps related to the diversity of its targets. Over the past decade, the importance of GSK-3 has been established by three significant properties: its remarkable evolutionary conservation, allowing analysis in genetically tractable organisms; its involvement in the Wnt/wingless signaling pathway; and its inhibition by agonists of the prosurvival phosphatidylinositol 3' kinase (P13'K) pathway. This review covers recent advances in understanding the physiological roles of this enzyme, particularly in the context of cancer.

Animals↗
PubMed2003

Wnt signaling in the thymus.

Wnt proteins are secreted signaling molecules that regulate cell-to-cell interactions during embryogenesis in many different tissues and species. Wnt signaling is required for normal thymocyte development, most dramatically at the pro-T-cell stage, although recent reports also indicate a role for Wnt proteins in later stages of thymocyte differentiation. The Wnt cascade induces the interaction of the normally cytoplasmic cofactor beta-catenin with the nuclear Tcf and Lef transcription factors. Active Wnt signaling is an absolute requirement for T-cell development, as demonstrated by the complete block in thymocyte development observed in the absence of Tcf1 and Lef1, or in the presence of extracellular Wnt inhibitors.

Animals↗