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Detection of Nuclear β-catenin in Xenopus Embryos

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Wnt Signaling

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

Abstract

Immunodetection of β-catenin accumulation in the nucleus is the most direct and reliable method to determine the intensity and the spatial/temporal patterns of Wnt-dependent signaling activity. Due to the large size of the Xenopus embryo, staining must be done on sections. We present here a simple protocol to prepare cryosections and produce high-quality images of the early embryo using immunofluo-rescence. We also provide comments on various conceptual and technical issues from fixation to image collection, which may assist in optimizing immunodetection in embryos and tissues beyond the specific scope of β-catenin localization.

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References

  1. Schohl, A., Fagotto, F. (2002) β -catenin, MAPK and Smad signaling during early Xenopus development. Development 129, 37–52.

    PubMed  CAS  Google Scholar 

  2. Jung, A., Schrauder, M., Oswald, U., et al. (2001) The invasion front of human color-ectal adenocarcinomas shows co-localization of nuclear β -catenin, cyclin D1, and p16INK4A and is a region of low proliferation. Am J Pathol 159, 1613–1617.

    Article  PubMed  CAS  Google Scholar 

  3. Brabletz, T., Jung, A., Reu, S., et al. (2001) Variable beta-catenin expression in color-ectal cancers indicates tumor progression driven by the tumor environment. Proc Natl Acad Sci USA 98, 10356–10361.

    Article  PubMed  CAS  Google Scholar 

  4. Staal, F. J. T., van Noort, M., Strous, G. J., et al. (2002) Wnt signals are transmitted through N-terminally dephosphorylated β -catenin. EMBO Reports 3, 63–68.

    Article  PubMed  CAS  Google Scholar 

  5. Hendriksen, J., Fagotto, F., van der Velde, H., et al. (2005) RanBP3 enhances nuclear export of active β -catenin independently of CRM1. J Cell Biol 171, 785–797.

    Article  PubMed  CAS  Google Scholar 

  6. Henderson, B. R. (2000) Nuclear-cytoplasmic shuttling of APC regulates β -catenin subcellular localization and turnover. Nature Cell Biol 2, 653–660.

    Article  PubMed  CAS  Google Scholar 

  7. Say, Y. H., Hooper, N. M. (2007) Contamination of nuclear fractions with plasma membrane lipid rafts. Proteomics 7, 1059–1064.

    Article  PubMed  CAS  Google Scholar 

  8. Schneider, S., Steinbeisser, H., Warga, R. M., et al. (1996) β -catenin translocation into nuclei demarcates the dorsalizing centers in frog and fish embryos. Mech Dev 57, 191–198.

    Article  PubMed  CAS  Google Scholar 

  9. Schohl, A., Fagotto, F. (2003) A role for β -catenin in mesoderm induction in Xenopus. EMBO J 22, 3303–3313.

    Article  PubMed  CAS  Google Scholar 

  10. Heasman, J., Crawford, A., Goldstone, K., et al. (1994) Overexpression of cadherins and underexpression of β -catenin inhibit dorsal mesoderm induction in early Xenopus embryos. Cell 79, 791–803.

    Article  PubMed  CAS  Google Scholar 

  11. Tao, Q., Yokota, C., Puck, H., et al. (2005) Maternal Wnt11 activates the canonical Wnt signaling pathway required for axis formation in Xenopus embryos. Cell 120, 857–871.

    Article  PubMed  CAS  Google Scholar 

  12. Heasman, J. (2006) Patterning the early Xenopus embryo. Development 133, 1205–1217.

    Article  PubMed  CAS  Google Scholar 

  13. Carnac, G., Kodjabachian, L., Gurdon, J.B., et al. (1996) The homeobox gene Siamois is a target of the Wnt dorsalisation pathway and triggers organiser activity in the absence of mesoderm. Development 122, 3055–3065.

    PubMed  CAS  Google Scholar 

  14. Brannon, M., Kimelman, D. (1996) Activation of Siamois by the Wnt pathway. Dev Biol 180, 344–347.

    Article  PubMed  CAS  Google Scholar 

  15. Brannon, M., Gomperts, M., Sumoy, L., et al. (1997) A β -catenin/XTcf-3 complex binds to the siamois promoter to regulate dorsal axis specification in Xenopus. Genes Dev 11, 2359–2370.

    Article  PubMed  CAS  Google Scholar 

  16. Laurent, M.N., Blitz, I. L., Hashimoto, C., et al. (1997) The Xenopus homeobox gene twin mediates Wnt induction of goosecoid in establishment of Spemann's organizer. Development 124, 4905–4916.

    PubMed  CAS  Google Scholar 

  17. McKendry, R., Hsu, S. C., Harland, R. M., et al. (1997) LEF-1/TCF proteins mediate wnt-inducible transcription from the Xenopus nodal-related 3 promoter. Dev Biol 192, 420–431.

    Article  PubMed  CAS  Google Scholar 

  18. Christian, J. L., Moon, R. T. (1993) Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dor-soventral pattern in the embryonic meso-derm of Xenopus. Genes Dev 7, 13–28.

    Article  PubMed  CAS  Google Scholar 

  19. 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 

  20. Yasuo, H., Lemaire, P. (2001) Role of Goo-secoid, Xnot and Wnt antagonists in the maintenance of the notochord genetic programme in Xenopus gastrulae. Development 128, 3783–3793.

    PubMed  CAS  Google Scholar 

  21. Shi, D. L., Bourdelas, A., Umbhauer, M., et al. (2002) Zygotic Wnt/ β -catenin signaling preferentially regulates the expression of Myf5 gene in the mesoderm of Xenopus. Dev Biol 245, 124–135.

    Article  PubMed  CAS  Google Scholar 

  22. Reintsch, W. E., Habring-Mueller, A., Wang, R. W., et al. (2005) β -catenin controls cell sorting at the notochord-somite boundary independently of cadherin-medi-ated adhesion. J. Cell Biol. 170, 675–686.

    Article  PubMed  CAS  Google Scholar 

  23. Wolda, S. L., Moody, C. J., Moon, R. T. (1993) Overlapping expression of Xwnt-3A and Xwnt-1 in neural tissue of Xenopus lae-vis embryos. Dev Biol 155, 46–57.

    Article  PubMed  CAS  Google Scholar 

  24. Saint-Jeannet, J. P., He, X., Varmus, H. E., et al. (1997) Regulation of dorsal fate in the neuraxis by Wnt-1 and Wnt-3a. Proc Natl Acad Sci USA 94, 13713–13718.

    Article  PubMed  CAS  Google Scholar 

  25. Kazanskaya, O., Glinka, A., Niehrs, C. (2000) The role of Xenopus dickkopf1 in prechordal plate specification and neural patterning. Development 127, 4981–4992.

    PubMed  CAS  Google Scholar 

  26. Logan, C. Y., Miller, J. R., Ferkowicz, M. J., et al. (1999) Nuclear β -catenin is required to specify vegetal cell fates in the sea urchin embryo. Development 126, 345–357.

    PubMed  CAS  Google Scholar 

  27. Imai, K., Takada, N., Satoh, N., et al. (2000) β -catenin mediates the specification of endo-derm cells in ascidian embryos. Development 127, 3009–3020.

    PubMed  CAS  Google Scholar 

  28. Mohamed, O. A., Clarke, H. J., Dufort, D. (2004) β -catenin signaling marks the prospective site of primitive streak formation in the mouse embryo. Dev Dyn 231, 416–424.

    Article  PubMed  CAS  Google Scholar 

  29. Roeser, T., Kessel, M. (1999) Nuclear β -catenin and the development of bilateral symmetry in normal and LiCl-exposed chick embryos. Development 126, 2955–2965.

    PubMed  CAS  Google Scholar 

  30. Momoi, A., Yoda, H., Steinbeisser, H., et al. (2003) Analysis of Wnt8 for neural posteri-orizing factor by identifying Frizzled 8c and Frizzled 9 as functional receptors for Wnt8. Mech Dev 120, 477–489.

    Article  PubMed  CAS  Google Scholar 

  31. Peifer, M., Wieschaus, E. (1990) The segment polarity gene armadillo encodes a functionally modular protein that is the Drosophila homolog of human plakoglobin. Cell 63, 1167–1176.

    Article  PubMed  CAS  Google Scholar 

  32. Peifer, M., Sweeton, D., Casey, M., et al. (1994) wingless signal and Zeste-white 3 kinase trigger opposing changes in the intra-cellular distribution of armadillo. Development 120, 369–380.

    PubMed  CAS  Google Scholar 

  33. Tolwinski, N. S., Wieschaus, E. (2001) Armadillo nuclear import is regulated by cytoplasmic anchor Axin and nuclear anchor dTCF/Pan. Development 128, 2107–2117.

    PubMed  CAS  Google Scholar 

  34. Wiechens, N., Fagotto, F. (2001) Crm1-and Ran-independent nuclear export of beta-catenin. Curr Biol 11, 18–27.

    Article  PubMed  CAS  Google Scholar 

  35. Tolwinski, N. S., Wehrli, M., Rives, A., et al. (2003) Wg/Wnt signal can be transmitted through arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity. Dev Cell 4, 407–418.

    Article  PubMed  CAS  Google Scholar 

  36. Krieghoff, E., Behrens, J., Mayr, B. (2006) Nucleo-cytoplasmic distribution of Beta-catenin is regulated by retention.J Cell Sci 119, 1453–1463.

    Article  PubMed  CAS  Google Scholar 

  37. Fagotto, F., Funayama, N., Gluck, U., et al. (1996) Binding to cadherins antagonizes the signaling activity of beta-catenin during axis formation in Xenopus. J Cell Biol 132, 1105–1114.

    Article  PubMed  CAS  Google Scholar 

  38. Rosin-Abersfeld, R., Townsley, F., Bienz, M. (2000) The APC tumor suppressor has a nuclear export function. Nature 406, 1009–1012.

    Article  Google Scholar 

  39. Neufeld, K. L., Zhang, F., Cullen, B. R., et al. (2000) APC-mediated downregula-tion of beta-catenin activity involves nuclear sequestration and nuclear export. EMBO Reports 1, 519–523.

    PubMed  CAS  Google Scholar 

  40. Wiechens, N., Heinle, K., Englmeier, L., et al. (2004) Nucleo-cytoplasmic Shuttling of Axin, a Negative Regulator of the Wnt- β -Catenin Pathway. J Biol Chem 279, 5263–5267.

    Article  PubMed  CAS  Google Scholar 

  41. Cong, F., Varmus, H. (2004) Nuclear-cyto-plasmic shuttling of Axin regulates subcellu-lar localization of β -catenin. Proc Natl Acad Sci USA 101, 2882–2887.

    Article  PubMed  CAS  Google Scholar 

  42. Larabell, C. A., Torres, M., Rowning, B. A., et al. (1997) Establishment of the dorso-ventral axis in Xenopus embryos is presaged by early asymmetries in β -catenin that are modulated by the Wnt signaling pathway. J Cell Biol 136, 1123–1136.

    Article  PubMed  CAS  Google Scholar 

  43. Rowning, B. A., Wells, J., Wu, M., et al. (1997) Microtubule-mediated transport of organelles and localization of β -catenin to the future dorsal side of Xenopus eggs. Proc Natl Acad Sci USA 94, 1224–1229.

    Article  PubMed  CAS  Google Scholar 

  44. Faure, S., Lee, M. A., Keller, T., et al. (2000) Endogenous patterns of TGF β superfamily signaling during early Xenopus development. Development 127, 2917–2931.

    PubMed  CAS  Google Scholar 

  45. Lemaire, P., Gurdon, J. B. (1994) A role for cytoplasmic determinants in mesoderm patterning: cell-autonomous activation of the goosecoid and Xwnt-8 genes along the dorsoventral axis of early Xenopus embryos. Development 120, 1191–1199.

    PubMed  CAS  Google Scholar 

  46. Hausen, P., Dreyer, C. (1981) The use of polyacrylamide as an embedding medium for immunohistochemical studies of embryonic tissues. Stain Technol 56, 287–293.

    PubMed  CAS  Google Scholar 

  47. Fagotto, F. (1999) The Wnt pathway in Xenopus development in (Guan, J. -L., ed.) Signaling through Cell Adhesion, pp. 303–356, Boca Raton, CRC Press.

    Google Scholar 

  48. Fagotto, F., Gumbiner, B. M. (1994) β -catenin localization during Xenopus embry-ogenesis: accumulation at tissue and somite boundaries. Development 120, 3667–3679.

    PubMed  CAS  Google Scholar 

  49. Dent, J. A., Cary, R. B., Bachant, J. B., et al. (1992) Host cell factors controlling vimen-tin organization in the Xenopus oocyte. J Cell Biol 119, 855–866.

    Article  PubMed  CAS  Google Scholar 

  50. Torpey, N. P., Heasman, J., Wylie, C. C. (1992) Distinct distribution of vimentin and cytokeratin in Xenopus oocytes and early embryos. J Cell Sci 101, 151–160.

    PubMed  CAS  Google Scholar 

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Acknowledgments

The work of FF laboratory is supported by funds NCIC #17162 and NSERC # 261679. We would like to acknowledge the invaluable contribution of Mrs. Anne Schohl to the establishment of the original protocol for β-catenin nuclear localization.

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Authors and Affiliations

  1. Department of Biology, McGill University, Montreal, Quebec, Canada

    François Fagotto & Carolyn M. Brown

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  1. François Fagotto
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  2. Carolyn M. Brown
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Editors and Affiliations

  1. University of Melbourne, VIC 3010, Parkville, Victoria, Australia

    Elizabeth Vincan

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© 2008 Springer Science+Business Media, LLC

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Fagotto, F., Brown, C.M. (2008). Detection of Nuclear β-catenin in Xenopus Embryos. In: Vincan, E. (eds) Wnt Signaling. Methods in Molecular Biology, vol 469. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-469-2_23

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  • DOI: https://doi.org/10.1007/978-1-60327-469-2_23

  • 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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