Hsieh, J. C., Rattner, A., Smallwood, P. M. & Nathans, J. Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein. Proc. Natl. Acad. Sci. USA 96, 3546-3551

Johns Hopkins University, Baltimore, Maryland, United States
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 04/1999; 96(7):3546-51. DOI: 10.1073/pnas.96.7.3546
Source: PubMed

ABSTRACT 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.

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Available from: Amir Rattner, Jun 27, 2015
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    • "Wnt signals have been suggested to be important for the generation of RPE cells (Fujimura et al., 2009; Steinfeld et al., 2013), but whether Wnt activity plays any role in the specification of neural retina cells has not been determined. We therefore first tested whether Wnt activity is required for the specification of neural retina cells, by culturing stage 10 OVL explants and stage 13 OV explants together with a soluble Frizzled receptor (Frizzled-conditioned medium) to inhibit Wnt activity (Gunhaga et al., 2003; Hsieh et al., 1999). Stage 10 OVL explants cultured alone or in the presence of Frizzled generated Rax2 + and Vsx2 + neural retina cells, whereas no FoxG1 + cells were detected (supplementary material Fig. S8A,B). "
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    ABSTRACT: The eye has served as a classical model to study cell specification and tissue induction for over a century. Nevertheless, the molecular mechanisms that regulate the induction and maintenance of eye-field cells, and the specification of neural retina cells are poorly understood. Moreover, within the developing anterior forebrain, how prospective eye and telencephalic cells are differentially specified is not well defined. In the present study, we have analyzed these issues by manipulating signaling pathways in intact chick embryo and explant assays. Our results provide evidence that at blastula stages, BMP signals inhibit the acquisition of eye-field character, but from neural tube/optic vesicle stages, BMP signals from the lens are crucial for the maintenance of eye-field character, inhibition of dorsal telencephalic cell identity and specification of neural retina cells. Subsequently, our results provide evidence that a Rax2-positive eye-field state is not sufficient for the progress to a neural retina identity, but requires BMP signals. In addition, our results argue against any essential role of Wnt or FGF signals during the specification of neural retina cells, but provide evidence that Wnt signals together with BMP activity are sufficient to induce cells of retinal pigment epithelial character. We conclude that BMP activity emanating from the lens ectoderm maintains eye-field identity, inhibits telencephalic character and induces neural retina cells. Our findings link the requirement of the lens ectoderm for neural retina specification with the molecular mechanism by which cells in the forebrain become specified as neural retina by BMP activity. © 2015. Published by The Company of Biologists Ltd.
    Development 05/2015; 142(10):1850-9. DOI:10.1242/dev.123653 · 6.46 Impact Factor
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    • "The Wnt ligand binds immediately to its receptor gene, Frizzled, and this binding site is divided into the canonical and non-canonical pathways (1,2). Among them, the canonical pathway is dependent on β-catenin and affects cancer cell adhesion. "
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    ABSTRACT: Previously, we examined various apoptosis pathways in the AGS gastric cancer cell line using Capsosiphon fulvescens glycoprotein (Cf-GP). In this study, we focused on the downregulation of the Wnt-1 signaling pathway and cell cycle arrest. Upregulation of the Wnt signaling pathway has been observed in various cancer cells. The Wnt signal ligand acts in both canonical and non-canonical pathways. Among them, Wnt-1 was dependent on the canonical pathway. Here, we show inhibition of Wnt-1 signaling, β-catenin and transcription factors in AGS cells via Cf-GP. First, we examined the Frizzled receptor and Wnt-1 signal-related proteins including Axin, LRP, β-catenin, APC and GSK-3β. In addition, the expression levels of transcription factors Tcf/LEF were determined by western blot analysis and RT-PCR. Based on the data, we confirmed downregulation of the Wnt-1 signaling pathway by Cf-GP. Also, we determined the expression levels of cell cycle-related proteins cyclin D and c-myc, and looked for cell cycle arrest by cell cycle test analysis. We found that AGS cells arrested in the G0/G1 phase by Cf-GP. These results provide a mechanism of AGS cell inhibition through the downregulation of Wnt-1 signaling by Cf-GP.
    International Journal of Oncology 08/2013; 43(5). DOI:10.3892/ijo.2013.2079 · 3.03 Impact Factor
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    • "Fzd and LRP are transmembrane proteins. The extracellular domain of Fzd contains a cysteine-rich domain, involved in Wnt binding [84] followed by a seven-pass transmembrane region. The extracellular domain of LRP is composed by EGF-like repeats involved in the interaction with Fzd receptor [85]. "
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    ABSTRACT: Hematopoietic stem cells (HSC), which reside in the marrow of adult mammals and sustain hematopoiesis for the lifetime of the organism, are specified and generated during embryonic development. We are just beginning to understand how HSC develop from more primitive cells and the complexity of the signaling pathways involved. In this work, we review the role of two crucial pathways, Notch and Wnt, in the specification and development of HSC and their nascent microenvironment, the arterial vessels.
    Blood Cells Molecules and Diseases 08/2013; 51(4). DOI:10.1016/j.bcmd.2013.07.005 · 2.65 Impact Factor
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