Confocal Analysis of Hedgehog Morphogenetic Gradient Coupled with Fluorescent In Situ Hybridization of Hedgehog Target Genes

  • Armel Gallet
  • Pascal P. Thérond
Part of the Methods Inmolecular Biology™ book series (MIMB, volume 397)


Hedgehog (Hh) family members are secreted proteins that can act at short and long range to direct cell fate decisions during developmental processes. In both Drosophila and vertebrates, the morphogenetic gradient of Hh must be tightly regulated for correct patterning. The posttranslational modification of Hh by a cholesterol adduct participates in such regulation. We have shown that cholesterol modification is necessary for the controlled long-range activity of Drosophila Hh, as observed for its vertebrate counterpart Sonic Hh. The presence of cholesterol on Hh allows the observation of large apical punctuate structures of Hh (Hh-LPSs) at a distance from the Hh source both in embryos and in imaginal discs. The Hh-LPSs apical distribution reflects the Hh gradient and is temporally regulated. Hh gradient modulation is directly related to the dynamic expression of the Hh target gene serrate (ser), shown by immunofluorescent detection of Hh coupled with fluorescent in situ hybridization of ser.

Key Words

Drosophila morphogenetic gradient Hedgehog-posttranslational modification cholesterol confocal immunofluorescence in situ hybridization serrate 


  1. 1.
    Gallet, A., Rodriguez, R., Ruel, L., and Therond, P. P. (2003) Cholesterol modification of Hedgehog is required for trafficking and movement, revealing an asymmetric cellular response to Hedgehog. Dev. Cell. 4, 191–204.CrossRefPubMedGoogle Scholar
  2. 2.
    Gallet, A. and Therond, P. P. (2005) Temporal modulation of the Hedgehog morphogen gradient by a patched-dependent targeting to lysosomal compartment. Dev. Biol. 277, 51–62.CrossRefPubMedGoogle Scholar
  3. 3.
    Bellaiche, Y., The, I., and Perrimon, N. (1998) Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1 and is needed for Hh diffusion. Nature 394, 85–88.CrossRefPubMedGoogle Scholar
  4. 4.
    Burke, R., Nellen, D., Bellotto, M., et al. (1999) Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified Hedgehog from signaling cells. Cell 99, 803–815.CrossRefPubMedGoogle Scholar
  5. 5.
    Alexandre, C., Lecourtois, M., and Vincent, J. (1999) Wingless and Hedgehog pattern Drosophila denticle belts by regulating the production of short-range signals. Development 126, 5689–5698.PubMedGoogle Scholar
  6. 6.
    Gallet, A., Ruel, L., Staccini-Lavenant, L., and Therond, P. P. (2006) Cholesterol modification is necessary for controlled planar long-range activity of Hedgehog in Drosophila epithelia. Development 133, 407–418.CrossRefPubMedGoogle Scholar
  7. 7.
    Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Lab., New York.Google Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Armel Gallet
    • 1
  • Pascal P. Thérond
    • 1
  1. 1.EMBO YIP and CNRS ATIPE, ISDBC, Centre de BiochimieUniversité de Nice Sophia AntipolisParc ValroseFrance

Personalised recommendations