Application of Sonic Hedgehog to the Developing Chick Limb

  • Eva Tiecke
  • Cheryll Tickle
Part of the Methods Inmolecular Biology™ book series (MIMB, volume 397)


Here, we describe methods for applying Sonic hedgehog (Shh) to developing chick limbs. The Sonic hedgehog gene is expressed in the polarizing region, a signaling region at the posterior margin of the limb bud and application of Shh-expressing cells or Shh protein to early limb buds mimics polarizing region signaling. The polarizing region (or zone of polarizing activity) is involved in one of the best known cell-cell interactions in vertebrate embryos and is pivotal in controlling digit number and pattern. At later stages of limb development, the application of Shh protein to the regions between digit primordia can induce changes in digit morphogenesis.

Key Words

Sonic hedgehog chick embryo polarizing region limb development digit morphogenesis bead 


  1. 1.
    Tickle, C. (2004) The contribution of chicken embryology to the understanding of vertebrate limb development. Mech. Dev. 121, 1019–1029.CrossRefPubMedGoogle Scholar
  2. 2.
    Saunders, J. W. and Gasseling, M. T. (1968) Ectodermal-mesodermal in the origin of limb symmetry. In Epithelial-Mesenchymal Interactions (Fleischmeyer, R. and Billingham, R. E., eds), Williams and Wilkins, Baltimore, pp. 78–97.Google Scholar
  3. 3.
    Tickle, C., Summerbell, D., and Wolpert, L. (1975) Positional signaling and specification of digits in chick limb morphogenesis. Nature 254, 199–202.CrossRefPubMedGoogle Scholar
  4. 4.
    Smith, J. C., Tickle, C., and Wolpert, L. (1978) Attenuation of positional signaling in the chick limb by high doses of gramma-radiation. Nature 272, 612–613.CrossRefPubMedGoogle Scholar
  5. 5.
    Tickle, C. (1981) The number of polarizing region cells required to specify additional digits in the developing chick wing. Nature 289, 295–298.CrossRefPubMedGoogle Scholar
  6. 6.
    MacCabe, A. B., Gasseling, M. T. Jr., and Saunders. J. W. (1973) Spatiotemporal distribution of mechanisms that control outgrowth and anteroposterior polarization of the limb bud in the chick embryo. Mech. Ageing. Dev. 2, 1–12.CrossRefPubMedGoogle Scholar
  7. 7.
    Honig, L. S. and Summerbell, D. (1985) Maps of strength of positional signaling activity in the developing chick wing bud. J. Embryol. Exp. Morphol. 87, 163–174.PubMedGoogle Scholar
  8. 8.
    Fallon, J. F. and Crosby, G. M. (1977) Polarizing zone activity in limb buds in amniotes. In Vertebrate Limb and Somite Morphogenesis (Hinchliffe, J. R., Balls, M., and Ede, D. A., eds), Cambridge University Press, Cambridge, pp. 55–70.Google Scholar
  9. 9.
    Hornbruch, A. and Wolpert, L. (1986) Positional signaling by Hensen’s node when grafted to the chick limb bud. J. Embryol. Exp. Mophol. 94, 257–265.Google Scholar
  10. 10.
    Dollé, P., Izpisúa-Belmonte, J. C., Brown, J. M., Tickle, C., and Duboule, D. (1991) HOX-4 genes and the morphogenesis of mammalian genitalia. Genes Dev. 5, 1767–1777.CrossRefPubMedGoogle Scholar
  11. 11.
    Tickle, C., Alberts, B., Wolpert, L., and Lee, J. (1982) Local application of retinoic acid to the limb bud mimics the action of the polarizing region. Nature 296, 564–566.CrossRefPubMedGoogle Scholar
  12. 12.
    Eichele, G., Tickle, C., and Alberts, B. (1984) Microcontrolled release of biologically active compounds in chick embryos: beads of 200-microns diameter for the local release of retinoids. Anal. Biochem. 142, 542–555.CrossRefPubMedGoogle Scholar
  13. 13.
    Tickle, C., Lee, J., and Eichele, G. (1985) A quantitative analysis of the effect of all-trans-retinoic acid on the pattern of chick wing development. Dev. Biol. 109, 82–95.CrossRefPubMedGoogle Scholar
  14. 14.
    Eichele, G., Tickle, C., and Alberts, B. M. (1985) Studies on the mechanism of retinoid-induced pattern duplications in the early chick limb bud: temporal and spatial aspects. J. Cell Biol. 101, 1913–1920.CrossRefPubMedGoogle Scholar
  15. 15.
    Riddle, R. D., Johnson, R. L., Laufer, E., and Tabin, C. (1993) Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75, 1401–1416.CrossRefPubMedGoogle Scholar
  16. 16.
    Lopez-Martinez, A., Chang, D. T., Chiang, C., et al. (1995) Limb-pattern activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage. Curr. Biol. 5, 791–796.CrossRefPubMedGoogle Scholar
  17. 17.
    Yang, Y., Drossopoulou, G., Chuang, P. T., et al. (1997) Relationship between dose, distance and time in Sonic Hedgehog-mediated regulation of anteroposterior polarity in the chick limb. Development 124, 4393–4404.PubMedGoogle Scholar
  18. 18.
    Zeng, X., Goetz, J. A., Suber, L. M., Scott, W. J. Jr., Schreiner, C. M., and Robbins, D. J. (2001) A freely diffusible form of Sonic hedgehog mediates long-range signalling. Nature 411, 716–720.CrossRefPubMedGoogle Scholar
  19. 19.
    Gritli-Linde, A., Lewis, P., McMahon, A. P., and Linde, A. (2001) The whereabouts of a morphogen: direct evidence for short-and graded long-range activity of Hedgehog signaling peptides. Dev. Biol. 236, 364–386.CrossRefPubMedGoogle Scholar
  20. 20.
    Zuniga, A., Haramis, A. P., McMahon, A. P., and Zeller, R. (1999) Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds. Nature 401, 598–602.CrossRefPubMedGoogle Scholar
  21. 21.
    Marigo, V., Scott, M. P., Johnson, R. L., Goodrich, L. V., and Tabin, C. J. (1996) Conservation in Hedgehog signaling: an induction of a chicken patched homolog by Sonic hedgehog in the developing limb. Development 122, 1225–1233.PubMedGoogle Scholar
  22. 22.
    Lewis, K. E., Drossopoulou, G., Paton, I. R., et al. (1999) Expression of ptc and gli genes in talpid3 suggests bifurcation in Shh pathway. Development 126, 2397–2407.PubMedGoogle Scholar
  23. 23.
    Drossopoulou, G., Lewis, K. E., Sanz-Ezquerro, J. J., et al. (2000) A model for anteroposterior patterning of the vertebrate limb based on sequential long-and short-range Shh signaling and Bmp signaling. Development 127, 1337–1348.PubMedGoogle Scholar
  24. 24.
    Sanz-Ezquerro, J. J. and Tickle, C. (2000) Autoregulation of Shh expression and Shh induction of cell death suggest a mechanism for modulating polarising activity during chick limb development. Development 127, 4811–4823.PubMedGoogle Scholar
  25. 25.
    Dahn, R. D. and Fallon, J. F. (2000) Interdigital regulation of digit identity and homeotic transformation by modulated BMP signaling. Science 289, 438–441.CrossRefPubMedGoogle Scholar
  26. 26.
    Sanz-Ezquerro, J. J. and Tickle, C. (2003) Fgf signaling controls the number of phalanges and tip formation in developing digits. Curr. Biol. 13, 1830–1836.CrossRefPubMedGoogle Scholar
  27. 27.
    Ros, M. A., Simandl, B. K., Clark, A. W., and Fallon, J. (2000) Methods for manipulating the chick limb bud to study gene expression, tissue interactions and patterning. In Developmental Biology Protocols III (Tuan, R. S. and Lo, C. W. eds), Humana, Totowa, NJ.Google Scholar
  28. 28.
    Hamburger, V. and Hamilton, H. L. (1951) A series of normal stages in the development of the chick embryo. J. Morph. 88, 49–92.CrossRefGoogle Scholar
  29. 29.
    Duprez, D., Fournier-Thibault, C., and Le Douarin, N. (1998) Sonic Hedgehog induces proliferation of committed skeletal muscle cells in the chick limb. Development 125, 495–505.PubMedGoogle Scholar
  30. 30.
    Duprez, D. M., Kostakopoulou, K., Francis-West, P. H., Tickle, C., and Brickell, P. M. (1996) Activation of Fgf-4 and HoxD gene expression by BMP-2 expressing cells in the developing chick limb. Development 122, 1821–1828.PubMedGoogle Scholar
  31. 31.
    de la Pompa, J. L. and Zeller, R. (1993) Ectopic expression of genes during chicken limb pattern formation using replication defective retroviral vectors. Mech. Dev. 43, 187–198.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Eva Tiecke
    • 1
  • Cheryll Tickle
    • 1
  1. 1.Division of Cell and Developmental Biology, School of Life SciencesUniversity of DundeeDundeeUK

Personalised recommendations