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Using 32-Cell Stage Xenopus Embryos to Probe PCP Signaling

  • Hyun-Shik LeeEmail author
  • Sergei Y. Sokol
  • Sally A. Moody
  • Ira O. Daar
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 839)

Abstract

Use of loss-of function (via antisense Morpholino oligonucleotides (MOs)) or over-expression of proteins in epithelial cells during early embryogenesis of Xenopus embryos, can be a powerful tool to understand how signaling molecules can affect developmental events. The techniques described here are useful for examining the roles of proteins in cell–cell adhesion, and planar cell polarity (PCP) signaling in cell movement. We describe how to target specific regions within the embryos by injecting an RNA encoding a tracer molecule along with RNA encoding your protein of interest or an antisense MO to knock-down a particular protein within a specific blastomere of the embryo. Effects on cell–cell adhesion, cell movement, and endogenous or exogenous protein localization can be assessed at later stages in specific targeted tissues using fluorescent microscopy and immunolocalization.

Key words

Planar cell polarity Xenopus Immunofluorescence Blastomeres Cell movement 

References

  1. 1.
    Simons, M., Mlodzik, M. (2008) Planar cell polarity signaling: from fly development to human disease. Annu Rev Genet 42, 517–540.PubMedCrossRefGoogle Scholar
  2. 2.
    Vladar, E.K., Antic, D., Axelrod, J.D. (2009) Planar cell polarity signaling: the developing cell’s compass. Cold Spring Harb Perspect Biol 1, a002964.PubMedCrossRefGoogle Scholar
  3. 3.
    Lee, M., Vasioukhin, V. (2008) Cell polarity and cancer--cell and tissue polarity as a non-canonical tumor suppressor. J Cell Sci 121, 1141–1150.PubMedCrossRefGoogle Scholar
  4. 4.
    Guo, P., Weinbaum, A.M., and Weinstein, S. (2003) A dual-pathway ultrastructural model for the tight junction of rat proximal tubule epithelium. Am J Physiol Renal Physiol 285, 241–257.Google Scholar
  5. 5.
    Handler, J.S. (1989) Overview of epithelial polarity. Annu Rev Physiol 51, 729–740.PubMedCrossRefGoogle Scholar
  6. 6.
    Rodriguez-Boulan, E., Powell, S.K. (1992) Polarity of epithelial and neuronal cells. Annu Rev Cell Biol 8, 395–427.PubMedCrossRefGoogle Scholar
  7. 7.
    Fleming, T.P., McConnell, J., Johnson, M.H., Stevenson, B.R. (1989) Development of tight junctions de novo in the mouse early embryo: control of assembly of the tight junction-specific protein, ZO-1. J Cell Biol 108, 1407–1418.PubMedCrossRefGoogle Scholar
  8. 8.
    Eckert, J.J., Fleming, T.P. (2008) Tight junction biogenesis during early development. Biochim Biophys Acta 1778, 717–728.PubMedCrossRefGoogle Scholar
  9. 9.
    Moody, S.A. (1987) Fates of the blastomeres of the 32-cell-stage Xenopus embryo. Dev Biol 122, 300–319.PubMedCrossRefGoogle Scholar
  10. 10.
    Lee, H.S., Nishanian, T.G., Mood, K., Bong, Y.S., Daar, I.O. (2008) EphrinB1 controls cell-cell junctions through the Par polarity complex. Nat Cell Biol 8, 979–986.CrossRefGoogle Scholar
  11. 11.
    Moore, K.B., Mood, K., Daar, I.O., Moody, S.A. (2004) Morphogenetic movements underlying eye field formation require interactions between the FGF and ephrinB1 signaling pathways. Dev Cell 6, 55–67.PubMedCrossRefGoogle Scholar
  12. 12.
    Lee, H.S., Bong, Y.S., Moore, K.B., Soria, K., Moody, S.A., Daar, I.O. (2006) Dishevelled mediates ephrinB1 signalling in the eye field through the planar cell polarity pathway. Nat Cell Biol 8, 55–63.PubMedCrossRefGoogle Scholar
  13. 13.
    Lee, H.S., Mood, K., Battu, G., Ji, Y.J., Singh, A., Daar, I.O. (2009) Fibroblast growth factor receptor-induced phosphorylation of ephrinB1 modulates its interaction with Dishevelled. Mol Biol Cell 20, 124–33.PubMedCrossRefGoogle Scholar
  14. 14.
    Dale, L. and Slack, J.M.W. (1987) Fate map for the 32-cell stage of Xenopus laevis. Development 99, 527–551.PubMedGoogle Scholar
  15. 15.
    Klein, S.L. (1987) The first cleavage furrow demarcates the dorsal-ventral axis in Xenopus embryos. Dev Biol 120, 299–304.PubMedCrossRefGoogle Scholar
  16. 16.
    Masho, R. (1990) Close correlation between the first cleavage plane and the body axis in early Xenopus embryos. Dev Growth Diff 32, 57–64.CrossRefGoogle Scholar
  17. 17.
    Nieuwkoop, P.D. and Faber, J. (1967) Normal Table of Xenopus laevis, 2nd Ed. North-Holland, Amsterdam.Google Scholar
  18. 18.
    Bauer, D.V., Huang, S. and Moody, S.A. (1994). The cleavage stage origin of Spemann’s Organizer: Analysis of the movements of blastomere clones before and during gastrulation in Xenopus. Development 120, 1179–1189.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Hyun-Shik Lee
    • 1
    Email author
  • Sergei Y. Sokol
    • 2
  • Sally A. Moody
    • 3
  • Ira O. Daar
    • 4
  1. 1.School of Life Sciences, College of Natural SciencesKyungpook National UniversityDaeguSouth Korea
  2. 2.Department of Developmental and Regenerative BiologyMount Sinai School of MedicineNew YorkUSA
  3. 3.Department of Anatomy and Regenerative BiologyThe George Washington University Medical CenterWashingtonUSA
  4. 4.Laboratory of Cell and Developmental SignalingNational Cancer Institute-FrederickFrederickUSA

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