Originally discovered in the fruit fly named Drosophila melanogaster, the pathway known as Hedgehog (Hh) signaling has since been shown to play essential roles in homeostasis and tissue development that are conserved across species ranging from planaria to mammals. Importantly, Hh signaling, which is dysregulated drives familial and sporadic cancers, such as medulloblastoma, basal cell carcinoma (BCC), and rhabdomyosarcoma. Several synthetic small-molecule inhibitors of the Hh pathway have been developed, leading the FDA approval of vismodegib and sonidegib for metastatic or locally advanced BCC and medulloblastoma. Therefore, further studies of the Hh pathway are vital for improving our knowledge of basic mechanisms of animal development, and it will provide unexplored avenues for novel therapeutics.

Recent biophysical and cell biological breakthroughs considerably advanced our understanding of the unique molecular mechanisms underlying the mammalian Hh signaling pathway. These include the requirement of the microtubule-based organelle called the primary cilium for Hh signaling and dynamic localization of many Hh pathway components in this organelle, sterol regulation of Hh signaling, and structures of Hh pathway components obtained by X-ray crystallography and cryo-electron microscopy. The objective of this Methods Collection is to serve as a resource for analyzing Hh signal transduction through such cell biological, biochemical, and biophysical methodologies. These methods should be helpful in developing assays to screen for novel genes or small molecules that can modulate Hh pathway activity with the potential to aid in the development of new tools to fight human disease.