UC Berkeley

The Hedgehog (Hh) signaling pathway is critical for patterning during embryonic development in most animals. The three key components in mammals are the ligand Sonic Hedgehog (Shh), its receptors Patched1 and -2 (Ptch1/2), and the pathway activator Smoothened (Smo). In the absence of Shh, Smo is inhibited by a small antagonist that is thought to be transported across the cell membrane by Ptch1/2. In the genetic and functional analyses of single pathway components, three-dimensional aggregates of cells with varying genotypes pose a valuable proxy for the in vitro study of signaling events. I describe a protocol for the generation and analysis of such in vitro organoids where specific roles (like producing and responding cell) can be assigned to a fraction of the cells based on their genotype. Release of the ligand Shh from producing cells and the inhibition of Smo by Ptch1/2 in responding cells in the absence of Shh are central aspects of this thesis. I demonstrate a function for the zinc coordination center of Shh in the release of the protein from the cell into the extracellular matrix. Furthermore, I present the known Smo inhibitor 7-Dehydrocholesterol (7DHC) as a putative cargo for Ptch1/2. Ptch1/2 are not only required for 7DHC to be inhibitory but also exacerbate the Smo inhibitory effect of 7DHC.