ARL13B regulates Sonic Hedgehog signaling from outside primary cilia

ARL13B is a regulatory GTPase highly enriched in cilia. Complete loss ofArl13bdisrupts cilia architecture, protein trafficking and Sonic hedgehog signaling. To determine whether ARL13B is required within cilia, we knocked in a cilia-excluded variant of ARL13B (V358A) and showed it retains all known biochemical function. We found that ARL13B^V358Aprotein was expressed but could not be detected in cilia, even when retrograde ciliary transport was blocked. We showedArl13b^V358A/V358Amice are viable and fertile with normal Shh signal transduction. However, in contrast to wild type cilia,Arl13b^V358A/V358Acells displayed short cilia and lacked ciliary ARL3 and INPP5E. These data indicate that ARL13B’s role within cilia can be uncoupled from its function outside of cilia. Furthermore, these data imply that the cilia defects upon complete absence of ARL13B do not underlie the alterations in Shh transduction, which is unexpected given the requirement of cilia for Shh transduction.