Gain-of-function Shh mutants activate Smoin cisindependent of Ptch1/2 function

Sonic Hedgehog (Shh) signaling is characterized by strict non-cell autonomy; cells expressing Shh do not respond to their ligand. Here, we identify several Shh mutations that gain the ability to activate the Hedgehog (Hh) pathway in cis . This activation requires the extracellular cysteine rich domain of Smoothened, but is otherwise independent of Ptch1/2. Many of the identified mutations disrupt either a highly conserved catalytic motif found in peptidases or an a-helix domain frequently mutated in holoprosencephaly-causing SHH alleles. The expression of gain-of-function mutants often results in the accumulation of unprocessed Shh pro-peptide, a form of Shh we demonstrate is sufficient to activate the Hh response cell-autonomously. Our results demonstrate that Shh is capable of activating the Hh pathway via Smo independently of Ptch1/2, and that it harbors an intrinsic mechanism that prevents cell-autonomous activation of the pathway to favor non-cell autonomous signaling.