Genetic Analysis of the Vertebrate Hedgehog-Signaling Pathway Using Muscle Cell Fate Specification in the Zebrafish Embryo

  • Sudipto Roy
Part of the Methods Inmolecular Biology™ book series (MIMB, volume 397)


Over the recent years, a large number of embryological studies with the zebrafish have provided substantial evidence of its usefulness for the investigation of the genetic and cellular basis of vertebrate development. With regard to the Hedgehog (Hh) pathway, forward as well as reverse genetic approaches in this organism have not only validated the roles of evolutionarily conserved players of the signaling cascade, but have also contributed to the isolation of several novel components that had remained unidentified through screens in other animal models. Here, the author describes a whole mount antibody labeling method that allows the detection of three unique muscle cell fates in the zebrafish embryo, which are induced by distinct levels and timing of Hh-signaling activity. This technique provides a rapid and convenient assay that can be utilized for the evaluation of effects of loss- or gain-of-function of any gene on the levels of Hh pathway activation during embryogenesis.

Key Words

Hedgehog zebrafish slow muscle muscle pioneer fast muscle antibody labeling 


  1. 1.
    Schauerte, H. E., van Eeden, F. J., Fricke, C., Odenthal, J., Strahle, U., and Haffter, P. (1998) Sonic hedgehog is not required for the induction of the medial floor plate cells in the zebrafish. Development 125, 2983–2993.PubMedGoogle Scholar
  2. 2.
    Koudijs, M. J., den Broeder, M. J., Keijser, A., et al. (2005) The zebrafish mutants dre, uki, and lep encode negative regulators of the Hedgehog signaling pathway. PLos Genet. 1(2), e19.CrossRefPubMedGoogle Scholar
  3. 3.
    Nakano, Y., Kim, H. R., Kawakami, A., Roy, S., Schier, A. F., and Ingham, P. W. (2004) Inactivation of dispatched 1 by the chameleon mutation disrupts Hedgehog signaling in the zebrafish embryo. Dev. Biol. 269, 381–392.CrossRefPubMedGoogle Scholar
  4. 4.
    Barresi, M. J., Stickney, H. L., and Devoto, S. H. (2000) The zebrafish slow-muscle-omitted gene product is required for Hedgehog signal transduction and the development of slow muscle identity. Development 127, 2189–2199.PubMedGoogle Scholar
  5. 5.
    Varga, Z. M., Amores, A., Lewis, K. E., et al. (2001) Zebrafish Smoothened functions in ventral neural tube specification and axon tract formation. Development 128, 3497–3509.PubMedGoogle Scholar
  6. 6.
    Chen, W., Burgess, S., and Hopkins, N. (2001) Analysis of the zebrafish Smoothened mutant reveals conserved and divergent functions of Hedgehog activity. Development 128, 2385–2396.PubMedGoogle Scholar
  7. 7.
    Karlstrom, R. O., Tyurina, O. V., Kawakami, A., et al. (2003) Genetic analysis of zebrafish gli1 and gli2 reveals divergent requirements for gli genes in vertebrate development. Development 130, 1549–1564.CrossRefPubMedGoogle Scholar
  8. 8.
    Karlstrom, R. O., Talbot, W. S., and Schier, A. F. (1999) Comparative synteny cloning of zebrafish you-too: mutations in the Hedgehog target gli2 affect ventral forebrain patterning. Genes Dev. 13, 388–393.CrossRefPubMedGoogle Scholar
  9. 9.
    Wolff, C., Roy, S., Lewis, K. E., et al. (2004) Iguana encodes a novel zinc-finger protein with coiled-coil domains essential for Hedghog signal transduction in the zebrafish embryo. Genes Dev. 18, 1565–1576.CrossRefPubMedGoogle Scholar
  10. 10.
    Sekimizu, K., Nishioka, N., Sasaki, H., Takeda, H., Karlstrom, R. O., and Kawakami, A. (2004) The zebrafish iguana locus encodes Dzip1, a novel zinc-finger protein required for proper regulation of Hedgehog signaling. Development 131, 2521–2532.CrossRefPubMedGoogle Scholar
  11. 11.
    Kawakami, A., Nojima, Y., Toyoda, A., et al. (2005) The zebrafish secreted matrix protein You/Scube2 is implicated in long range regulation of Hedgehog signaling. Curr. Biol. 15, 480–488.CrossRefPubMedGoogle Scholar
  12. 12.
    Woods, I. G. and Talbot, W. S. (2005) The you gene encodes an EGF-CUB protein essential for Hedgehog signaling in zebrafish. PLoS Biol. 3, e66.CrossRefPubMedGoogle Scholar
  13. 13.
    Wolff, C., Roy, S., and Ingham, P. W. (2003) Multiple muscle cell identities induced by distinct levels and timing of Hedgehog activity in the zebrafish embryo. Curr. Biol. 13, 1169–1181.CrossRefPubMedGoogle Scholar
  14. 14.
    Tay, S. Y., Ingham, P. W., and Roy, S. (2005) A homologue of the Drosophila kinesin-like protein Costal2 regulates Hedgehog signal transduction in the vertebrate embryo. Development 132, 625–634.CrossRefPubMedGoogle Scholar
  15. 15.
    Wilbanks, A. M., Fralish, G. B., Kirby, M. L., Barak, L. S., Li, Y. X., and Caron, M. G. (2004) β-arrestin 2 regulates zebrafish development through the Hedgehog signaling pathway. Science 306, 2264–2267.CrossRefPubMedGoogle Scholar
  16. 16.
    Devoto, S. H., Melancon, E., Eisen, J. S., and Westerfield, M. (1996) Identification of separate slow and fast muscle precursor cells in vivo, prior to somite formation. Development 122, 3371–3380.PubMedGoogle Scholar
  17. 17.
    Lewis, K. E., Currie, P. D., Roy, S., Schauerte, H., Haffter, P., and Ingham, P. W. (1999) Control of muscle cell-type specification in the zebrafish embryo by Hedgehog signaling. Dev. Biol. 216, 469–480.CrossRefPubMedGoogle Scholar
  18. 18.
    Hammerschmidt, M., Bitgood, M. J., and McMahon, A. P. (1996) Protein kinase A is a common negative regulator of Hedgehog signaling in the vertebrate embryo. Genes Dev. 10, 647–658.CrossRefPubMedGoogle Scholar
  19. 19.
    Roy, S., Wolff, C., and Ingham, P. W. (2001) The u-boot mutation identifies a Hedgehog-regulated myogenic switch for fiber-type diversification in the zebrafish embryo. Genes Dev. 15, 1563–1576.CrossRefPubMedGoogle Scholar
  20. 20.
    Hatta, K., Bremiller, R., Westerfield, M., and Kimmel, C. B. (1991) Diversity of expression of Engrailed-like antigens in zebrafish. Development 112, 821–832.PubMedGoogle Scholar
  21. 21.
    Westerfield, M. (ed.) (2000) The Zebrafish Book, University of Oregon Press, Oregon.Google Scholar
  22. 22.
    Nusslein-Volhard, C. and Dahm, R. (ed.) (2002) Zebrafish: A Practical Approach, Oxford University Press, Oxford.Google Scholar
  23. 23.
    Detrich, H. W. 3rd, Zon, L. I., and Westerfield, M. (ed.) (2004) Zebrafish: Cellular and Developmental Biology, Elsevier Academic Press, San Diego.Google Scholar
  24. 24.
    Detrich, H. W. 3rd, Zon, L. I., and Westerfield, M. (ed.) (2004) Zebrafish: Genetics, Genomics and Informatics, Elsevier Academic Press, San Diego.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Sudipto Roy
    • 1
  1. 1.Institute of Molecular and Cell BiologyProteosSingapore

Personalised recommendations