Primary cilia mediate vertebrate Hedgehog signaling through the regulation of Smoothened and Gli2
- Author(s): Santos, Nicole
- Advisor(s): Reiter, Jeremy F
- et al.
During development, the Hedgehog (Hh) signaling pathway controls growth, cell fate decisions and morphogenesis. Hh signaling has long been an intense area of research, as defects in this pathway are associated with a number of congenital defects and cancers. Within the past few years, studies have illuminated that vertebrate Hh signaling requires primary cilia, microtubule-based organelles that project from the surface of most cells. To elucidate how cilia regulate Hh signaling, we chose to focus on the two central activators of the pathway, the seven transmembrane protein, Smoothened, and its downstream effector, Gli2.
Vertebrate Smo (vSmo) translocation to the primary cilium is a necessary and regulated step in the Hh pathway. Cilia, however, are dispensable in Drosophila Hh signaling and dSmo requires other mechanisms for activation. To explore how Smo activation and the requirement for the primary cilium have evolutionarily diverged, we examined vSmo phosphorylation and generated a panel of dSmo-vSmo chimeric proteins. We demonstrate that vSmo may be phosphorylated by CKIä/å, proteins which localize to the base of the primary cilium. Although phosphorylation by Grk2 and interaction with âarrestin2 have been implicated in vSmo ciliary localization and activation, we show that neither are necessary. Through our chimeric analyses, we observe that the cytoplasmic tail, which contains a previously described ciliary localization motif, is necessary, but not sufficient, for ciliary translocation.
Gli2 is the primary transcriptional activator of the Hh pathway and has been shown to be enriched at the primary cilium, particularly upon pathway activation. However, the molecular mechanisms that regulate this translocation remain largely unexplored. To investigate Gli2 subcellular dynamics, we used recombinant gene technology to specifically modify the Gli2 locus in mouse embryonic stem cells. We generated a knock-in mouse model expressing Gli2-GFP and find that this allele functions as a hypomorph in vivo. We demonstrate how specific domains and post-translational modifications regulate Gli2 nuclear enrichment, binding partners and function. We identify a central region of Gli2 that is required, but not sufficient for Gli2 ciliary localization. Together, these studies provide insight into how Gli2 may be functioning at the subcellular level, particularly at the primary cilium.