Advertisement

Rho GTPases pp 321-337 | Cite as

Using Zebrafish for Studying Rho GTPases Signaling In Vivo

  • Shizhen Zhu
  • Boon Chuan Low
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 827)

Abstract

Rho small GTPases play pivotal roles in a variety of dynamic cellular processes including cytoskeleton rearrangement, cell migration, cell proliferation, cell survival, and gene regulation. However, their functions in vivo are much less understood. Recently, the zebrafish, Danio rerio has emerged as a powerful model organism for developmental and genetic studies. Zebrafish embryos have many unique characteristics, such as optical transparency, external fertilization and development, and amenability for various molecular manipulations including morpholino oligo-mediated gene knockdown, mRNA or DNA overexpression-induced gain of function or rescue, in situ hybridization (ISH) with riboprobes for gene expression, western blot for protein analysis, small-molecule inhibition on signaling pathways, and bioimaging for tracking of molecular events. Taking many of such advantages, we have demonstrated the role of rhoA small GTPase in the control of gastrulation cell movements and cell survival during early zebrafish embryogenesis, linking RhoA functions to at least the noncanonical Wnt, Mek/Erk, and Bcl2 signaling nodes in vivo. Here, we describe the use of such techniques, including gene knockdown by morpholino oligo, functional rescue by mRNA overexpression, microinjection, ISH, western blot analysis and pharmacological inhibition of signaling pathways by small molecule inhibitors, with special considerations on their merits, potential drawbacks, and adaptation which could pave the way to our better understanding of the roles of various classes of small GTPases in regulating cell dynamics and development in vivo.

Key words

Rho small GTPase Zebrafish Mopholino oligos mRNA rescue Microinjection In situ hybridization Western blot Small molecule inhibitor 

Notes

Acknowledgments

This work was supported in part by the Mechanobiology Institute, National University of Singapore, co-funded by the National Research Foundation and the Ministry of Education, Singapore, and also from a grant from the Biomedical Research Council of Singapore, and a fellowship from the Friends for Life.

References

  1. 1.
    Pyati U.J., Look A.T., and Hammerschmidt, M. (2007) Zebrafish as a powerful vertebrate model system for in vivo studies of cell death. Semin Cancer Biol 17, 154–165PubMedCrossRefGoogle Scholar
  2. 2.
    Lieschke G.J, and Currie, P.D. (2007) Animal models of human disease: zebrafish swim into view. Nat Rev Genet 8, 353–367PubMedCrossRefGoogle Scholar
  3. 3.
    Chakraborty, C., Hsu, C.H., Wen, Z.H., Lin, C.S., and Agoramoorthy, G. (2009) Zebrafish: a complete animal model for in vivo drug discovery and development. Curr Drug Metab 10, 116–124PubMedCrossRefGoogle Scholar
  4. 4.
    Peal, D.S., Peterson, R.T., and Milan, D. (2010) Small molecule screening in zebrafish. J Cardiovasc Transl Res 3, 454–460PubMedCrossRefGoogle Scholar
  5. 5.
    Lam, S.H., Wu, Y.L., Vega, V.B., Miller, L.D., Spitsbergen, J., Tong, Y., Zhan, H., Govindarajan, K.R., Lee, S., Mathavan, S., Murthy, K.R., Buhler, D.R., Liu, E.T., and Gong, Z. (2006) Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression. Nat Biotechnol 24, 73–75PubMedCrossRefGoogle Scholar
  6. 6.
    Storer, N.Y., and Zon L.I. (2010) Zebrafish models of p53 functions. Cold Spring Harb Perspect Biol 2, a001123PubMedCrossRefGoogle Scholar
  7. 7.
    Kari, G., Rodeck, U., and Dicker, A.P. (2007) Zebrafish: an emerging model system for human disease and drug discovery. Clin Pharmacol Ther 82, 70–80PubMedCrossRefGoogle Scholar
  8. 8.
    Veldman, M.B., and Lin, S. (2008) Zebrafish as a developmental model organism for pediatric research. Pediatr Res 64, 470–476PubMedCrossRefGoogle Scholar
  9. 9.
    Zhu, S., Liu, L., Korzh, V., Gong, Z., and Low, B.C. (2006) RhoA acts downstream of Wnt5 and Wnt11 to regulate convergence and extension movements by involving effectors Rho kinase and Diaphanous: use of zebrafish as an in vivo model for GTPase signaling. Cell signal 18, 359–372PubMedCrossRefGoogle Scholar
  10. 10.
    Bakkers, J., Kramer, C., Pothof, J., Quaedvlieg, N.E., Spaink, H.P., and Hammerschmidt, M. (2004) Has2 is required upstream of Rac1 to govern dorsal migration of lateral cells during zebrafish gastrulation. Development 131, 525–537PubMedCrossRefGoogle Scholar
  11. 11.
    Matthews, H.K., Marchant, L., Carmona-Fontaine, C., Kuriyama, S., Larraín, J., Holt, M.R., Parsons, M., and Mayor, R. (2008) Directional migration of neural crest cells in vivo is regulated by Syndecan-4/Rac1 and non-canonical Wnt signaling/RhoA. Development 135, 1771–1780PubMedCrossRefGoogle Scholar
  12. 12.
    Kardash, E., Reichman-Fried, M., Maitre, J.L., Boldajipour, B., Papusheva, E., Messerschmidt, E.M., Heisenberg, C.P., and Raz, E. (2010) A role for Rho GTPases and cell-cell adhesion in single-cell motility in vivo. Nat Cell Biol 12, 47–53PubMedCrossRefGoogle Scholar
  13. 13.
    Smolen, G.A., Schott, B.J., Stewart, R.A., Diederichs, S., Muir, B., Provencher, H.L., Look, A.T., Sgroi, D.C., Peterson, R.T., Haber, D.A. (2007) A Rap GTPase interactor, RADIL, mediates migration of neural crest precursors. Genes Dev 21, 2131–2136PubMedCrossRefGoogle Scholar
  14. 14.
    Tsai, I.C., Amack, J.D., Gao, Z.H., Band, V., Yost. H.J., and Virshup, D.M. (2007) A Wnt-CKIvarepsilon-Rap1 pathway regulates gastrulation by modulating SIPA1L1, a Rap GTPase activating protein. Dev Cell 12, 335–347PubMedCrossRefGoogle Scholar
  15. 15.
    Palamidessi, A., Frittoli, E., Garre, M., Faretta, M., Mione, M., Testa, I., Diaspro, A., Lanzetti, L., Scita, G., and Di Fiore, P.P. (2008) Endocytic trafficking of Rac is required for the spatial restriction of signaling in cell migration. Cell 134, 135–147PubMedCrossRefGoogle Scholar
  16. 16.
    Zhu, S., Korzh, V., Gong, Z., and Low, B.C. (2008) RhoA prevents apoptosis during zebrafish embryogenesis through activation of Mek/Erk pathway. Oncogene 27, 1580–1589PubMedCrossRefGoogle Scholar
  17. 17.
    Nasevicius, A., and Ekker, S.C. (2000) Effective targeted gene ‘knockdown’ in zebrafish. Nat Genet 26, 216–220PubMedCrossRefGoogle Scholar
  18. 18.
    Bill, B.R., Petzold, A.M., Clark, K.J., Schimmenti, L.A., and Ekker, S.C. (2009) A primer for morpholino use in zebrafish. Zebrafish 6, 69–77PubMedCrossRefGoogle Scholar
  19. 19.
    Robu, M.E., Larson, J.D., Nasevicius, A., Beiraghi, S., Brenner, C., Farber, S.A., and Ekker, S.C. (2007) p53 activation by knockdown technologies. PLoS Genet 3, e78PubMedCrossRefGoogle Scholar
  20. 20.
    Hyatt, T.M., and Ekker, S.. (1999) Vectors and techniques for ectopic gene expression in zebrafish. Methods Cell Biol 59, 117–126PubMedCrossRefGoogle Scholar
  21. 21.
    Xu, Q. (1999) Microinjection into zebrafish embryos. Methods Mol Biol 127, 125–132PubMedCrossRefGoogle Scholar
  22. 22.
    Stuart, G.W., McMurray, J.V., and Westerfield, M. (1988) Replication, integration and stable germ-line transmission of foreign sequences injected into early zebrafish embryos. Development 103, 403–412PubMedGoogle Scholar
  23. 23.
    Rosen, J.N., Sweeney, M..F, and Mably, J.D. (2009) Microinjection of zebrafish embryos to analyze gene function. J Vis Exp 25, pii: 1115. doi:  10.3791/1115
  24. 24.
    Yuan, S., and Sun, Z. (2009) Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos. J Vis Exp 27, pii: 1113. doi:  10.3791/1113
  25. 25.
    Wang, W., Liu, X., Gelinas, D., Ciruna, B., and Sun, Y. (2007) A fully automated robotic system for microinjection of zebrafish embryos. PLoS One 2, e862PubMedCrossRefGoogle Scholar
  26. 26.
    Holder, N., and Xu, Q. (1999) Microinjection of DNA, RNA, and protein into the fertilized zebrafish egg for analysis of gene function. Methods Mol Biol 97, 487–490PubMedGoogle Scholar
  27. 27.
    Broadbent, J., and Read, E.M. (1999) Wholemount in situ hybridization of Xenopus and zebrafish embryos. Methods Mol Biol 127, 57–67PubMedCrossRefGoogle Scholar
  28. 28.
    Brend, T., and Holley, S.A. (2009) Zebrafish whole mount high-resolution double fluorescent in situ hybridization. J Vis Exp 25, pii: 1229. doi:  10.3791/1229
  29. 29.
    Paffett-Lugassy, N.N., and Zon, L.I. (2005) Analysis of hematopoietic development in the zebrafish. Methods Mol Med 105, 171–198PubMedGoogle Scholar
  30. 30.
    Link, V., Shevchenko, A., and Heisenberg, C.P. (2006) Proteomics of early zebrafish embryos. BMC Dev Biol 6, 1PubMedCrossRefGoogle Scholar
  31. 31.
    Guo, S. (2009) Using zebrafish to assess the impact of drugs on neural development and function. Expert Opin Drug Discov 4, 715–726PubMedCrossRefGoogle Scholar
  32. 32.
    Westerfield, M. (2000) The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). Univ. of Oregon Press, Eugene.Google Scholar
  33. 33.
    Kimmel, C.B., Ballard, W.W., Kimmel, S.R. Ullmann, B., and Schilling, T.F. (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203, 253–310PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of Pediatric OncologyDana-Farber Cancer Institute, Harvard Medical SchoolBostonMA
  2. 2.Cell Signaling and Developmental Biology Laboratory, Department of Biological Sciences, Mechanobiology InstituteNational University of SingaporeSingaporeSingapore

Personalised recommendations