Abstract
In this chapter, we explain different strategies to analyze the extracellular Hedgehog (Hh) morphogen distribution and Hh intracellular trafficking by immunohistochemistry techniques. For this purpose, it has been very useful to have a transgenic fly line that expresses a Hh-green fluorescent protein (GFP) fusion protein. These flies can be used to study the way Hh spreads through the anterior compartment where it signals, and analyze in detail how Hh is internalized by its receptor Patched. In addition, this Hh-GFP fusion made without lipid modifications (cholesterol or palmitic acid) can be used to investigate the function of these lipids on Hh in terms of spreading, internalization, and signaling abilities.
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References
Torroja, C., Gorfinkiel, N., and Guerrero, I. (2005) Mechanisms of Hedgehog gradient formation and interpretation. J. Neurobiol. 64(4), 334–356.
Takei, Y., Ozawa, Y., Sato, M., Watanabe, A., and Tabata, T. (2004) Three Drosophila EXT genes shape morphogen gradients through synthesis of heparan sulfate proteoglycans. Development 131, 73–82.
Torroja, C., Gorfinkiel, N., and Guerrero, I. (2004) Patched controls the Hedgehog gradient by endocytosis in a dynamin-dependent manner, but this internalization does not play a major role in signal transduction. Development 131, 2395–2408.
Callejo, A., Torroja, C., Quijada, L., and Guerrero, I. (2006) Hedgehog lipid modifications are required for Hedgehog stabilization in the extracellular matrix. Development 133, 471–483.
Calleja, M., Moreno, E., Pelaz, S., and Morata, G. (1996) Visualization of gene expression in living adult Drosophila. Science 274, 252–255.
de Celis, J. F. and Bray, S. (1997) Feed-back mechanisms affecting boundary in the Drosophila wing. Development 124, 3241–3251.
Pignoni, F. and Zipursky, S. L. (1997) Induction of Drosophila eye development by decapentaplegic. Development 124, 271–278.
Struhl, G. and Basler, K. (1993) Organizing activity of wingless protein in Drosophila. Cell 72, 527–540.
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.
Pepinsky, R. B., Zeng, C., Wen, D., et al. (1998) Identification of a palmitic acid-modified form of human Sonic hedgehog. J. Biol. Chem. 273, 14,037–14,045.
Brand, A. H. and Perrimon, N. (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415.
Ashburner, M. (1989) Drosophila. A Laboratory Handbook, Cold Spring Harbor Laboratory Press, New York.
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Quijada, L., Guerrero, I. (2007). Detecting Tagged Hedgehog with Intracellular and Extracellular Immunocytochemistry for Functional Analysis. In: Horabin, J.I. (eds) Hedgehog Signaling Protocols. Methods Inmolecular Biology™, vol 397. Humana Press. https://doi.org/10.1007/978-1-59745-516-9_8
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DOI: https://doi.org/10.1007/978-1-59745-516-9_8
Publisher Name: Humana Press
Print ISBN: 978-1-58829-692-4
Online ISBN: 978-1-59745-516-9
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