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Science Writer/Editor for the Department of Neurosurgery
Science Writer/Editor for the Department of Neurosurgery
The Wnt proteins constitute a large family of extracellular signalling molecules that are found throughout the animal kingdom and are important for a wide variety of normal and pathological developmental processes. Here we describe Wnt-inhibitory factor-1 (WIF-1), a secreted protein that binds to Wnt proteins and inhibits their activities. WIF-1 is present in fish, amphibia and mammals, and is expressed during Xenopus and zebrafish development in a complex pattern that includes paraxial presomitic mesoderm, notochord, branchial arches and neural crest derivatives. We use Xenopus embryos to show that WIF-1 overexpression affects somitogenesis (the generation of trunk mesoderm segments), in agreement with its normal expression in paraxial mesoderm. In vitro, WIF-1 binds to Drosophila Wingless and Xenopus Wnt8 produced by Drosophila S2 cells. Together with earlier results obtained with the secreted Frizzled-related proteins, our results indicate that Wnt proteins interact with structurally diverse extracellular inhibitors, presumably to fine-tune the spatial and temporal patterns of Wnt activity.
View details for Web of Science ID 000079508200054
View details for PubMedID 10201374
The sexually dimorphic characteristics of the reproductive tract in Drosophila require that cells of the gonad and the genital disc be assigned sex-specific fates. We report here that DWnt-2, a secreted glycoprotein related to wingless, is a signal required for cell fate determination and morphogenesis in the developing male reproductive tract. Testes from DWnt-2 null mutant flies lack the male-specific pigment cells of the reproductive tract sheath and the muscle precursors of the sheath fail to migrate normally. However, other cell types of the testis are unaffected. DWnt-2 is expressed in somatic cells of the gonad throughout development, implicating it as a signal that can influence pigment cell fate directly. Indeed, the ectopic expression of DWnt-2 in females results in the appearance of male-specific pigment cells in otherwise morphologically normal ovaries. Thus, the presence of pigment cells is a sexually dimorphic trait that is controlled by DWnt-2 expression. DWnt-2 is also expressed in regions of the male genital disc and gonad, which we have identified as sites of contact with muscle precursor cells, suggesting that secreted DWnt-2 protein is a signal for the migration or attachment of these cells.
View details for Web of Science ID 000073299000009
View details for PubMedID 9553045
Williams syndrome (WS) is a developmental disorder with a characteristic personality and cognitive profile that is associated, in most cases, with a 2 Mb deletion of part of chromosome band 7q11.23. By applying CpG island cloning methods to cosmids from the deletion region, we have identified a new gene, called FZD3. Dosage blotting of DNA from 11 WS probands confirmed that it is located within the commonly deleted region. Sequence comparisons revealed that FZD3, encoding a 591 amino acid protein, is a novel member of a seven transmembrane domain receptor family that are mammalian homologs of the Drosophila tissue polarity gene frizzled. FZD3 is expressed predominantly in brain, testis, eye, skeletal muscle and kidney. Recently, frizzled has been identified as the receptor for the wingless (wg) protein in Drosophila. We show that Drosophila as well as human cells, when transfected with FZD3 expression constructs, bind Wg protein. In mouse, the wg homologous Wnt1 gene is involved in early development of a large domain of the central nervous system encompassing much of the midbrain and rostral metencephalon. The potential function of FZD3 in transmitting a Wnt protein signal in the human brain and other tissues suggests that heterozygous deletion of the FZD3 gene could contribute to the WS phenotype.
View details for Web of Science ID A1997WN31600016
View details for PubMedID 9147651
Receptors for Wingless and other signalling molecules of the Wnt gene family have yet to be identified. We show here that cultured Drosophila cells transfected with a novel member of the frizzled gene family in Drosophila, Dfz2, respond to added Wingless protein by elevating the level of the Armadillo protein. Moreover, Wingless binds to Drosophila or human cells expressing Dfz2. These data demonstrate that Dfz2 functions as a Wingless receptor, and they imply, in general, that Frizzled proteins are receptors for the Wnt signalling molecules.
View details for Web of Science ID A1996UX79000040
View details for PubMedID 8717036
The phenotypes caused by mutations in Wnt genes suggest that their gene products are involved in cell-to-cell communication. Wnt genes indeed encode secreted molecules, but soluble active Wnt protein has not been found. We have developed a novel cell culture assay for the Drosophila Wnt gene wingless, using a Drosophila imaginal disc cell line (cl-8; ref. 13), and measured effects on the adherens junction protein armadillo, a known genetic target of wingless. Transfection of a temperature-sensitive wingless complementary DNA into cl-8 cells increases the levels of the armadillo protein. The wingless protein does not affect the rate of synthesis of armadillo, but leads to increased stability of an otherwise rapidly decaying armadillo protein. The wingless protein in the extracellular matrix and soluble medium from donor cells also increases the levels of armadillo protein. The protein in the medium acts fast and is inhibited by an antibody to wingless protein, demonstrating that Wnt products can act as soluble extracellular signalling molecules.
View details for Web of Science ID A1994NB98500048
View details for PubMedID 8127369
We have characterized the molecular nature of mutations in wingless (wg), a segment polarity gene acting during various stages of Drosophila development. Embryo-lethal alleles have undergone mutations in the protein-encoding domain of the gene, including deletions and point mutations of conserved residues. In a temperature sensitive mutation, a conserved cysteine residue is replaced by a serine. In embryo-viable alleles, the wg transcriptional unit is not affected. Immunostaining of mutant embryos shows that the embryo-lethal alleles produce either no wg antigen or a form of the protein that is retained within cells. Interestingly, embryos mutant for the segment polarity gene porcupine show a similar retention of the wg antigen. We have also transfected wild type wg alleles into Drosophila tissue culture cells, which then display wg protein on the cell surface and in the extracellular matrix. In similar experiments with mutant alleles, the proteins are retained in intracellular compartments and appear not to be secreted. These data provide further evidence that wg acts as a secreted factor and suggest that porcupine provides an accessory function for wg protein secretion or transport.
View details for Web of Science ID A1993MM12000042
View details for PubMedID 8262072
Antibodies to phosphotyrosine were used in immunoprecipitation experiments to determine if post-translational modification of the platelet-derived growth factor (PDGF) receptor was required for the acquisition of ligand-induced tyrosine kinase activity. In intact Balb/c 3T3 fibroblasts, only the fully processed 180-kDa receptor was activated (tyrosine-phosphorylated) by PDGF. In a cell-free assay, however, the tyrosine-phosphorylated forms of the 160- and 145-kDa PDGF receptor precursors were also detected. These activated precursors were immunoprecipitated after brief (5-15 min) metabolic labeling periods. Thus the receptor could bind PDGF and induce tyrosine kinase activity shortly after translation. Unlike the mature form of the receptor, the 160-kDa receptor precursor was resistant to digestion with endo-alpha-N-acetylgalactosaminidase and thus did not contain O-linked oligosaccharides. Since this receptor precursor was activated by PDGF in the cell-free assay, the addition of O-linked sugars must not be necessary for ligand binding activity. Incubation of cells with tunicamycin completely inhibited N-linked glycosylation of the PDGF receptor. Nevertheless, PDGF still induced tyrosine phosphorylation of the 130-kDa aglycoreceptor in lysates of tunicamycin-treated cells. Thus, the addition of N-linked oligosaccharides was also not required for receptor activation. These findings show that the PDGF receptor acquired the ability to be activated by ligand cotranslationally or immediately after translation and that the addition of N- or O-linked oligosaccharides was not required for ligand binding and tyrosine kinase activities.
View details for Web of Science ID A1989U866500008
View details for PubMedID 2542300
The effect of ligand binding on platelet-derived growth factor (PDGF) receptor conformation was examined using peptide antibodies directed against specific receptor domains. Antiserum 83, which was directed to the receptor's carboxyl terminus (residues 934-951), preferentially immunoprecipitated the ligand-activated form of the PDGF receptor from 35S-labeled BALB/c 3T3 cells. By contrast, two antisera directed against other receptor sequences precipitated unactivated and activated receptors equally well. Denatured receptors were recognized equally by all antisera, even 83. Thus, ligand activation caused a change in PDGF receptor conformation that enhanced accessibility of the antibody to the carboxyl terminus. The activated receptor conformation was induced by three different forms of PDGF (AA and BB homodimers and AB heterodimers) and was reversed by suramin, a polyanionic compound that dissociates PDGF from the receptor. The inhibitory effect of suramin on receptor conformation was abolished by the phosphatase inhibitor, sodium orthovanadate, suggesting that receptor phosphorylation mediated the conformational change. In a cell-free assay, the change in receptor conformation was induced by PDGF only in the presence of ATP and was inhibited by adenyl-5'-yl imidodiphosphate, a nonhydrolyzable analog of ATP. The functional significance of receptor conformation was examined in Chinese hamster ovary fibroblasts transfected with wild-type or mutated forms of the PDGF receptor. When receptor tyrosine kinase activity was abolished by a mutation of the ATP binding site the receptor no longer underwent PDGF-induced conformational change and did not mediate PDGF-induced mitogenesis even though 125I-PDGF binding was normal. These findings show that ligand binding elicits a phosphorylation-dependent change in PDGF receptor conformation that may be important for receptor function.
View details for Web of Science ID A1988Q091700004
View details for PubMedID 2843499