Characterizing oxysterol trafficking networks
A small set of regio- and stereochemically defined oxysterols activate Hh signaling by binding to Smo and directing its trafficking to the primary cilium. However, the mechanisms that deliver oxysterols to Smo have yet to be defined. We will synthesize oxysterol probes based on active metabolite structures to achieve proteome-wide identification of specific oxysterol-binding proteins in live, Hh-pathway signaling cells. Biochemical and functional interrogation of novel oxysterol interactors that influence Hh pathway activity will define the networks that link Hh signaling to sterol trafficking.
Identifying endogenous sterol regulators
A wealth of biochemical evidence indicates that cholesterol metabolites influence Hh signaling by directly regulating the functions of Ptch and Smo. A key step in understanding these regulatory mechanisms is to identify bone fide endogenous molecules that can fulfill their proposed roles. Using CRISPR-based interference and activation of sterol biosynthesis genes we will identify metabolic elements that alter Hh pathway state. Analysis of sterol profiles in cells overexpressing or lacking genetic regulators of Gli activity will pinpoint endogenous molecules for assessment in Hh signaling assays.
Defining the Hh morphogen gradient
The morphogenetic potential of the Hh ligands relies critically on the appendage of a cholesterol molecule through a unique autoprocessing mechanism. We will fashion bump-and-hole pairs comprising mutant Hh proteins and synthetic sterol analogs to enable visualization and control of Hh cholesteroylation in vivo. These bump-and-hole Hh ligands will be employed for super resolution imaging of Hh internalization in cells and expressed in transgenic zebrafish for light-induced stimulation of Hh autoprocessing at specific stages of embryonic development.