Abstract
The Hedgehog (Hh) signaling pathway is a regulator of patterning, cell migration and axon guidance during development as well as of homeostatic events in adult organs. It is highly conserved from Drosophila to humans. In many contexts during development, Hh appears to function as a morphogen; it spreads from producing cells to trigger concentration dependent responses in target cells, leading to their specification. During production, Hh undergoes two lipid modifications resulting in a highly hydrophobic molecule. The processes that create lipid-modified Hh for release from producing cells and that move it to target cells in a graded manner are complex. While most of the work done trying to explain Hh gradient formation is based on immunohistochemical studies in steady state, in vivo imaging in intact organisms is the finest technique to study gradient formation in real time. Both the wing imaginal disc epithelium and the adult abdominal epidermis of Drosophila are well suited for in vivo imaging. They allow us to observe the behavior of cells and fluorescently labeled proteins, without interfering with development. Here, we describe in vivo imaging methods for these two epithelia, which allowed us to study Hh transport along specialized cytoplasmic protrusions called cytonemes.
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Acknowledgements
We are very grateful to Carmen Ibáñez for technical assistance. We also thank the confocal microscopy facility of the CBMSO for skillful technical assistance. Work was supported by grant BFU2011-25987 from the Spanish MINECO and by an institutional grant to the CBMSO from the Fundación Areces to I.G. I.S. was financially supported by an FPI fellowship of the Spanish MINECO.
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Video 1
In vivo imaging of cytonemes in the abdominal epidermis. Cytonemes are visualized by Ihog-CFP expression in all P compartment cells. The histoblasts (hb) replace the larval epithelial cells (LEC) during morphogenesis. Along the compartment border, cytonemes point anteriorly (arrows). Movie begins at approx. 35 h after puparium formation. 150 s time interval between frames. Segment A2 shown. Anterior is to the left. (MOV 9107 kb)
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Seijo-Barandiarán, I., Guerrero, I., Bischoff, M. (2015). In Vivo Imaging of Hedgehog Transport in Drosophila Epithelia. In: Riobo, N. (eds) Hedgehog Signaling Protocols. Methods in Molecular Biology, vol 1322. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2772-2_2
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DOI: https://doi.org/10.1007/978-1-4939-2772-2_2
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