The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice.

During development, extracellular cues guiding cell fate determination are provided by morphogens. One mechanism by which morphogens are proposed to traverse extracellular space is by traveling along specialized filopodia called cytonemes. These cellular highways extend between signal-producing and -receiving cells to enable direct morphogen delivery. Although genetic studies support cytoneme involvement in morphogen transport, mechanistic insight into how they are regulated is limited owing to technical challenges associated with performing cell biological analysis of the delicate filopodial structures. Here, we introduce a fixation method whereby cultured cell cytonemes can be preserved for imaging studies, allowing investigation of cytoneme regulation using standard cell biological techniques. Using this method, we examined Hedgehog-containing cytonemes and identified a role for the Hedgehog deployment protein Dispatched in cytoneme stabilization. We demonstrate that Hedgehog and Dispatched colocalize in cytonemes, and that cholesterol-modified Hedgehog acts through Dispatched to increase cytoneme occurrence. Live imaging suggests that this occurs through Dispatched-mediated slowing of cytoneme retraction rates. Dispatched-induced cytoneme modulation was recapitulated in wing imaginal discs of transgenic Drosophila, providing evidence that cultured cell cytoneme analysis is predictive of in vivo functionality.

Hedgehog ligands activate an evolutionarily conserved signaling pathway that provides instructional cues during tissue morphogenesis, and when corrupted, contributes to developmental disorders and cancer. The transmembrane protein Dispatched is an essential component of the machinery that deploys Hedgehog family ligands from producing cells, and is absolutely required for signaling to long-range targets. Despite this crucial role, regulatory mechanisms controlling Dispatched activity remain largely undefined. Herein, we reveal vertebrate Dispatched is activated by proprotein convertase-mediated cleavage at a conserved processing site in its first extracellular loop. Dispatched processing occurs at the cell surface to instruct its membrane re-localization in polarized epithelial cells. Cleavage site mutation alters Dispatched membrane trafficking and reduces ligand release, leading to compromised pathway activity in vivo. As such, convertase-mediated cleavage is required for Dispatched maturation and functional competency in Hedgehog ligand-producing cells.

The use of ASA may be associated with an increased risk of developing a SCH during the first trimester. The increased frequencies of SCH in pregnancies of patients attending a fertility clinic compared to women from a general obstetrical practice was highly correlated with the use of ASA.