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Gain-of-Function and Loss-of-Function Strategies in Xenopus

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Wnt Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 469))

Abstract

Xenopus embryos are particularly suited for functional experiments to investigate vertebrate embryonic development. Due to the large size of embryos and their development outside of the mother organism, they are very accessible, easy to manipulate, and allow for immediate observation of developmental phenotypes. Powerful methods have been established for both gain- and loss-of-function strategies, which build on these inherent advantages. This chapter describes injection methods used to overexpress gene products and inhibit gene expression as well as pharmacological approaches to manipulate Wnt signaling in Xenopus embryos.

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References

  1. Sokol, S., Christian, J. L., Moon, R. T., et al. (1991) Injected Wnt RNA induces a complete body axis in Xenopus embryos. Cell 67, 741–752.

    Article  PubMed  CAS  Google Scholar 

  2. Funayama, N., Fagotto, F., McCrea, P., et al. (1995) Embryonic axis induction by the armadillo repeat domain of beta-cat-enin: evidence for intracellular signaling. J Cell Biol 128, 959–968.

    Article  PubMed  CAS  Google Scholar 

  3. Itoh, K., Krupnik, V. E., & Sokol, S. Y. (1998) Axis determination in Xenopus involves biochemical interactions of axin, glycogen synthase kinase 3 and beta-catenin. Curr Biol 8, 591–594.

    Article  PubMed  CAS  Google Scholar 

  4. Moody, S. A. (1987) Fates of the blastomeres of the 32-cell-stage Xenopus embryo. Dev Biol 122, 300–319.

    Article  PubMed  CAS  Google Scholar 

  5. Moody, S. A. (1987) Fates of the blastomeres of the 16-cell stage Xenopus embryo. Dev Biol 119, 560–578.

    Article  PubMed  CAS  Google Scholar 

  6. Bauer, D. V., Huang, S., & Moody, S. A. (1994) The cleavage stage origin of Spe-mann's Organizer: analysis of the movements of blastomere clones before and during gastrulation in Xenopus. Development 120, 1179–1189.

    PubMed  CAS  Google Scholar 

  7. Urban, A. E., Zhou, X., Ungos, J. M., et al. (2006) FGF is essential for both condensation and mesenchymal-epithelial transition stages of pronephric kidney tubule development. Dev Biol 297, 103–117.

    Article  PubMed  CAS  Google Scholar 

  8. Borchers, A., David, R.,&Wedlich, D. (2001) Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification. Development 128, 3049–3060.

    PubMed  CAS  Google Scholar 

  9. Witta, S. E., Sato, S. M. (1997) XIPOU 2 is a potential regulator of Spemann's Organizer. Development 124, 1179–1189.

    PubMed  CAS  Google Scholar 

  10. Yost, C., Farr, G. H., 3rd, Pierce, S. B., et al. (1998) GBP, an inhibitor of GSK-3, is implicated in Xenopus development and oncogenesis. Cell 93, 1031–1041.

    Article  PubMed  CAS  Google Scholar 

  11. Roose, J., Molenaar, M., Peterson, J., et al. (1998) The Xenopus Wnt effector XTcf-3 interacts with Groucho-related transcriptional repressors. Nature 395, 608–612.

    Article  PubMed  CAS  Google Scholar 

  12. Wang, S., Krinks, M., Lin, K., et al. (1997) Frzb, a secreted protein expressed in the Spe-mann organizer, binds and inhibits Wnt-8. Cell 88, 757–766.

    Article  PubMed  CAS  Google Scholar 

  13. Tamai, K., Zeng, X., Liu, C., et al. (2004) A mechanism for Wnt coreceptor activation. Mol Cell 13, 149–156.

    Article  PubMed  CAS  Google Scholar 

  14. Sokol, S. Y. (1996) Analysis of Dishevelled signalling pathways during Xenopus development. Curr Biol 6, 1456–1467.

    Article  PubMed  CAS  Google Scholar 

  15. Rothbacher, U., Laurent, M. N., Deardorff, M. A., et al. (2000) Dishevelled phos-phorylation, subcellular localization and multimerization regulate its role in early embryogenesis. Embo J 19, 1010–1022.

    Article  PubMed  CAS  Google Scholar 

  16. Hoppler, S., Brown, J. D., & Moon, R. T. (1996) Expression of a dominant-negative Wnt blocks induction of MyoD in Xenopus embryos. Genes Dev 10, 2805–2817.

    Article  PubMed  CAS  Google Scholar 

  17. Christian, J. L.& Moon, R. T. (1993) Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dors-oventral pattern in the embryonic mesoderm of Xenopus. Genes Dev 7, 13–28.

    Article  PubMed  CAS  Google Scholar 

  18. Yang, J., Tan, C., Darken, R. S., et al. (2002) Beta-catenin/Tcf-regulated transcription prior to the midblastula transition. Development 129, 5743–5752.

    Article  PubMed  CAS  Google Scholar 

  19. Hsieh, J. C., Rattner, A., Smallwood, P. M., et al. (1999) Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein. Proc Natl Acad Sci USA 96, 3546–3551.

    Article  PubMed  CAS  Google Scholar 

  20. Heasman, J., Kofron, M., & Wylie, C. (2000) Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach. Dev Biol 222, 124–134.

    Article  PubMed  CAS  Google Scholar 

  21. Klein, P. S. & Melton, D. A. (1996) A molecular mechanism for the effect of lithium on development. Proc Natl Acad Sci USA 93, 8455–9.

    Article  PubMed  CAS  Google Scholar 

  22. Phiel, C. J. & Klein, P. S. (2001) Molecular targets of lithium action. Annu Rev Pharmacol Toxicol 41, 789–813.

    Article  PubMed  CAS  Google Scholar 

  23. Meijer, L., Skaltsounis, A. L., Magiatis, P., et al. (2003) GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol 10, 1255–1266.

    Article  PubMed  CAS  Google Scholar 

  24. Sato, N., Meijer, L., Skaltsounis, L., et al. (2004) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 10, 55–63.

    Article  PubMed  CAS  Google Scholar 

  25. Liu, J., Wu, X., Mitchell, B., et al. (2005) A small-molecule agonist of the Wnt signaling pathway. Angew Chem Int Ed Engl 44, 1987–1990.

    Article  PubMed  CAS  Google Scholar 

  26. Gao, Z. H., Seeling, J. M., Hill, V., et al. (2002) Casein kinase I phosphorylates and destabilizes the beta-catenin degradation complex. Proc Natl Acad Sci USA 99, 1182–1187.

    Article  PubMed  CAS  Google Scholar 

  27. Lee, E., Salic, A., & Kirschner, M. W. (2001) Physiological regulation of [beta]-catenin stability by Tcf3 and CK1epsilon. J Cell Biol 154, 983–993.

    Article  PubMed  CAS  Google Scholar 

  28. Braun, M. M., Etheridge, A., Bernard, A., et al. (2003) Wnt signaling is required at distinct stages of development for the induction of the posterior forebrain. Development 130, 5579–5587.

    Article  PubMed  CAS  Google Scholar 

  29. Maurus, D., Heligon, C., Burger-Schwar-zler, A., et al. (2005) Noncanonical Wnt-4 signaling and EAF2 are required for eye development in Xenopus laevis. Embo J 24, 1181–1191.

    Article  PubMed  CAS  Google Scholar 

  30. Pandur, P., Lasche, M., Eisenberg, L. M., et al. (2002) Wnt-11 activation of a non-canonical Wnt signalling pathway is required for cardiogenesis. Nature 418, 636–641.

    Article  PubMed  CAS  Google Scholar 

  31. Hamilton, F. S., Wheeler, G. N., & Hoppler, S. (2001) Difference in XTcf-3 depen-dency accounts for change in response to beta-cat-enin-mediated Wnt signalling in Xenopus blastula. Development 128, 2063–2073.

    PubMed  CAS  Google Scholar 

  32. Yost, C., Torres, M., Miller, J. R., et al. (1996) The axis-inducing activity, stability, and subcellular distribution of beta-cat-enin is regulated in Xenopus embryos by glycogen synthase kinase 3. Genes Dev 10, 1443–1454.

    Article  PubMed  CAS  Google Scholar 

  33. Tada, M. & Smith, J. C. (2000) Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway. Development 127, 2227–2238.

    PubMed  CAS  Google Scholar 

  34. Tamai, K., Semenov, M., Kato, Y., et al. (2000) LDL-receptor-related proteins in Wnt signal transduction. Nature 407, 530–535.

    Article  PubMed  CAS  Google Scholar 

  35. Molenaar, M., van de Wetering, M., Oost-erwegel, M., et al. (1996) XTcf-3 transcription factor mediates beta-catenin-induced axis formation in Xenopus embryos. Cell 86, 391–399.

    Article  PubMed  CAS  Google Scholar 

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Lavery, D.L., Hoppler, S. (2008). Gain-of-Function and Loss-of-Function Strategies in Xenopus . In: Vincan, E. (eds) Wnt Signaling. Methods in Molecular Biology, vol 469. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-469-25

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  • DOI: https://doi.org/10.1007/978-1-60327-469-25

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-468-5

  • Online ISBN: 978-1-60327-469-2

  • eBook Packages: Springer Protocols

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