Hedgehog Signaling Protocols

Seijo-Barandiarán, Irene, Guerrero, Isabel, Bischoff, Marcus, Irene Seijo-Barandiarán, Isabel Guerrero, Marcus Bischoff

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.