Oligomerization and Endocytosis of Hedgehog is Necessary for its Efficient Exovesicular Secretion

Abstract

Hedgehog (Hh) is a secreted morphogen, involved in both short and long range signalling necessary for tissue patterning during development. It is unclear how this dually lipidated protein is transported over a long range in the aqueous milieu of interstitial spaces. We had previously shown that the long range signalling of Hh requires its oligomerization. Here we showthat Hh is secreted in the form of exovesicles. Fluorescence correlation spectroscopy (FCS) and electron microscopy based measurements confirmed Hh secretion on vesicles with a heterogeneous size distribution and a distinct biochemical composition. These are derived by the endocytic delivery of cell surface Hh to multi vesicular bodies (MVBs) via an endosomal sorting complex required for transport (ECSRT)-dependent process. Perturbations of ESCRT proteins have a selective effect on long-range Hh signalling in Drosophila wing imaginal discs. Importantly oligomerization-defective Hh is inefficiently incorporated into exovesicles and shows a narrower distribution of sizes. The defects in exovesicular secretion of this mutant directly correlate to its poor endocytic delivery to MVBs. These results provide evidence that nanoscale organization of Hh regulates the secretion of Hh on ESCRT-derived exovesicles, which in turn act as a vehicle for long range signalling.