Project Summary/Abstract Cilia sparked phenomenal interest as scientists recognized them as a fundamental cellular organelle required for signaling. Untangling the specific mechanisms that regulate Sonic hedgehog (Shh) signaling within the cilium is difficult since so many mutants that disrupt ciliogenesis also affect Hh signaling. We have long focused on a small ciliary GTPase, ARL13B, that we hypothesize integrates the regulation of ciliogenesis and Hh signaling through distinct effectors and their downstream pathways. As a GTPase, single basepair mutations within the GTPase domain of ARL13B are predicted to disrupt individual effector pathways. ARL13B is highly enriched in cilia. By engineering mouse expressing only an ARL13B variant that does not localize to cilia, we genetically uncoupled the role of ARL13B in ciliogenesis from its role in signaling. We focus on mammalian neural development and through forward genetic screens identified mouse mutants in several proteins related to ARL13B. Additionally, other proteins in the ARL family of GTPases are implicated in cilia and signaling through what appear to be ARL13B related mechanisms. In the next five years, we propose using mouse mutants to define the regulatory relationships among these players in vivo and in specific cell types. These experiments will unravel ARL13B function in ciliogenesis, traffic of proteins to/within cilia, and Shh signal transduction at unprecedented resolution. Thus, our proposal will generate a molecular genetic toolkit from which the field will be poised to distinguish the regulation of cilia from that of Hh signaling. This is important to our fundamental understanding of cilia, ciliogenesis and cilia structure, as well as our basic comprehension of the Hh pathway.