Identification of a Frizzled-like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases.
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In Drosophila, members of the Frizzled family of tissue-polarity genes encode proteins that appear to function as cell-surface receptors for Wnts. The Frizzled genes belong to the seven transmembrane class of receptors (7TMR) and have on their extracellular region a cysteine-rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in the hedgehog signalling pathway). We have identified, using BLAST, sequence similarity between the cysteine-rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development. These include the muscle-specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and ROR1 and ROR2. At present, the ligands for these developmental tyrosine kinases are unknown. Our results suggest that Wnt-like ligands may bind to these developmental tyrosine kinases (+info)
Differential expression assay of chromosome arm 8p genes identifies Frizzled-related (FRP1/FRZB) and Fibroblast Growth Factor Receptor 1 (FGFR1) as candidate breast cancer genes.
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Deletions and amplifications are frequent alterations of the short arm of chromosome 8 associated with various types of cancers, including breast cancers. This indicates the likely presence of tumor suppressor genes and oncogenes. In the present study, we have used the expressed sequence tag (EST) map of 8p11-21 to assemble a set of available cDNAs representing genes from this region. DNA arrays were prepared for expression analysis and search for genes potentially involved in breast cancer. Underexpresion in tumoral breast cells (versus normal breast) was observed for 15 transcripts. Among these, the Frizzled-related gene FRP1/FRZB, was turned off in 78% of breast carcinomas, suggesting that the lack of its product may be associated with malignant transformation. Overexpression in tumoral breast cells was observed for 13 genes. The FGFR1 gene, that encodes a tyrosine kinase receptor for members of the fibroblast growth factor family, was identified as a good candidate for one amplification unit. Taken together, our results demonstrate that such a strategy can rapidly identify genes with an altered pattern of expression and provide candidate genes for malignancies. (+info)
Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein.
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Biochemical studies of Wnt signaling have been hampered by difficulties in obtaining large quantities of soluble, biologically active Wnt proteins. In this paper, we report the production in Drosophila S2 cells of biologically active Xenopus Wnt8 (XWnt8). Epitope- or alkaline phosphatase-tagged XWnt8 proteins are secreted by concentrated S2 cells in a form that is suitable for quantitative biochemical experiments with yields of 5 and 0.5 mg per liter, respectively. Conditions also are described for the production in 293 cells of an IgG fusion of the cysteine-rich domain (CRD) of mouse Frizzled 8 with a yield of 20 mg/liter. We demonstrate the use of these proteins for studying the interactions between soluble XWnt8 and various Frizzled proteins, membrane anchored or secreted CRDs, and a set of insertion mutants in the CRD of Drosophila Frizzled 2. In a solid phase binding assay, the affinity of the XWnt8-alkaline phosphatase fusion for the purified mouse Frizzled 8-CRD-IgG fusion is approximately 9 nM. (+info)
Notch and wingless regulate expression of cuticle patterning genes.
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The cell surface receptor Notch is required during Drosophila embryogenesis for production of epidermal precursor cells. The secreted factor Wingless is required for specifying different types of cells during differentiation of tissues from these epidermal precursor cells. The results reported here show that the full-length Notch and a form of Notch truncated in the amino terminus associate with Wingless in S2 cells and in embryos. In S2 cells, Wingless and the two different forms of Notch regulate expression of Dfrizzled 2, a receptor of Wg; hairy, a negative regulator of achaete expression; shaggy, a negative regulator of engrailed expression; and patched, a negative regulator of wingless expression. Analyses of expression of the same genes in mutant N embryos indicate that the pattern of gene regulations observed in vitro reflects regulations in vivo. These results suggest that the strong genetic interactions observed between Notch and wingless genes during development of Drosophila is at least partly due to regulation of expression of cuticle patterning genes by Wingless and the two forms of Notch. (+info)
Establishment of the dorsal-ventral axis in Xenopus embryos coincides with the dorsal enrichment of dishevelled that is dependent on cortical rotation.
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Examination of the subcellular localization of Dishevelled (Dsh) in fertilized Xenopus eggs revealed that Dsh is associated with vesicle-like organelles that are enriched on the prospective dorsal side of the embryo after cortical rotation. Dorsal enrichment of Dsh is blocked by UV irradiation of the vegetal pole, a treatment that inhibits development of dorsal cell fates, linking accumulation of Dsh and specification of dorsal cell fates. Investigation of the dynamics of Dsh localization using Dsh tagged with green fluorescent protein (Dsh-GFP) demonstrated that Dsh-GFP associates with small vesicle-like organelles that are directionally transported along the parallel array of microtubules towards the prospective dorsal side of the embryo during cortical rotation. Perturbing the assembly of the microtubule array with D(2)O, a treatment that promotes the random assembly of the array and the dorsalization of embryos, randomizes translocation of Dsh-GFP. Conversely, UV irradiation of the vegetal pole abolishes movement of Dsh-GFP. Finally, we demonstrate that overexpression of Dsh can stabilize beta-catenin in Xenopus. These data suggest that the directional translocation of Dsh along microtubules during cortical rotation and its subsequent enrichment on the prospective dorsal side of the embryo play a role in locally activating a maternal Wnt pathway responsible for establishing dorsal cell fates in Xenopus. (+info)
Two novel Xenopus frizzled genes expressed in developing heart and brain.
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A family of genes related to the Drosophila wingless receptor frizzled have been found in vertebrates. We have cloned full length cDNAs of two novel frizzled genes from embryonic Xenopus tissue. We are calling them Xfz7 and Xfz9 (for Xenopus frizzled) because their deduced peptide sequences show extensive similarity to other vertebrate frizzled molecules. Xfz7 is closely related to human, chick and mouse frz-7 and Xfz9 is most related to human FZD9 and mouse fzd9. Xfz7 is expressed in a broad, complex and dynamic pattern beginning at gastrulation. At later stages Xfz7 expression is found in neural crest, neural tube, eye, pronephric duct and the heart. Xfz9 expression in contrast is more restricted to the neuroectoderm and, at later stages of development, to the dorsal regions of the mid- and hindbrain. (+info)
Frizzled and Dfrizzled-2 function as redundant receptors for Wingless during Drosophila embryonic development.
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In cell culture assays, Frizzled and Dfrizzled2, two members of the Frizzled family of integral membrane proteins, are able to bind Wingless and transduce the Wingless signal. To address the role of these proteins in the intact organism and to explore the question of specificity of ligand-receptor interactions in vivo, we have conducted a genetic analysis of frizzled and Dfrizzled2 in the embryo. These experiments utilize a small gamma-ray-induced deficiency that uncovers Dfrizzled2. Mutants lacking maternal frizzled and zygotic frizzled and Dfrizzled2 exhibit defects in the embryonic epidermis, CNS, heart and midgut that are indistinguishable from those observed in wingless mutants. Epidermal patterning defects in the frizzled, Dfrizzled2 double-mutant embryos can be rescued by ectopic expression of either gene. In frizzled, Dfrizzled2 mutant embryos, ectopic production of Wingless does not detectably alter the epidermal patterning defect, but ectopic production of an activated form of Armadillo produces a naked cuticle phenotype indistinguishable from that produced by ectopic production of activated Armadillo in wild-type embryos. These experiments indicate that frizzled and Dfrizzled2 function downstream of wingless and upstream of armadillo, consistent with their proposed roles as Wingless receptors. The lack of an effect on epidermal patterning of ectopic Wingless in a frizzled, Dfrizzled2 double mutant argues against the existence of additional Wingless receptors in the embryo or a model in which Frizzled and Dfrizzled2 act simply to present the ligand to its bona fide receptor. These data lead to the conclusion that Frizzled and Dfrizzled2 function as redundant Wingless receptors in multiple embryonic tissues and that this role is accurately reflected in tissue culture experiments. The redundancy of Frizzled and Dfrizzled2 explains why Wingless receptors were not identified in earlier genetic screens for mutants defective in embryonic patterning. (+info)
The Drosophila STE20-like kinase misshapen is required downstream of the Frizzled receptor in planar polarity signaling.
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The Drosophila misshapen (msn) gene is a member of the STE20 kinase family. We show that msn acts in the Frizzled (Fz) mediated epithelial planar polarity (EPP) signaling pathway in eyes and wings. Both msn loss- and gain-of-function result in defective ommatidial polarity and wing hair formation. Genetic and biochemical analyses indicate that msn acts downstream of fz and dishevelled (dsh) in the planar polarity pathway, and thus implicates an STE20-like kinase in Fz/Dsh-mediated signaling. This demonstrates that seven-pass transmembrane receptors can signal via members of the STE20 kinase family in higher eukaryotes. We also show that Msn acts in EPP signaling through the JNK (Jun-N-terminal kinase) module as it does in dorsal closure. Although at the level of Fz/Dsh there is no apparent redundancy in this pathway, the downstream effector JNK/MAPK (mitogen-activated protein kinase) module is redundant in planar polarity generation. To address the nature of this redundancy, we provide evidence for an involvement of the related MAP kinases of the p38 subfamily in planar polarity signaling downstream of Msn. (+info)