SummaryHedgehog (Hh) signalling is important in development, stem cell biology and disease. In a variety of tissues, Hh acts as a morphogen to regulate growth and cell fate specification. Several hypotheses have been proposed to explain morphogen movement, one of which is transport via filopodia-like protrusions called cytonemes. Here, we analyse the mechanism underlying Hh movement in the wing disc and the abdominal epidermis of Drosophila. We show that, in both epithelia, cells generate cytonemes in regions of Hh signalling. These protrusions are actin-based and span several cell diameters. Various Hh signalling components localise to cytonemes, as well as to punctate structures that move along cytonemes and are probably exovesicles. Using in vivo imaging, we show that cytonemes are dynamic structures and that Hh gradient establishment correlates with cytoneme formation in space and time. Indeed, mutant conditions that affect cytoneme formation reduce both cytoneme length and Hh gradient length. Our results suggest that cytoneme-mediated Hh transport is the mechanistic basis for Hh gradient formation.

The Hedgehog signalling pathway is crucial for development, adult stem cell maintenance, cell migration and axon guidance in a wide range of organisms. During development, the Hh morphogen directs tissue patterning according to a concentration gradient. Lipid modifications on Hh are needed to achieve graded distribution, leading to debate about how Hh is transported to target cells despite being membrane-tethered. Cytonemes in the region of Hh signalling have been shown to be essential for gradient formation, but the carrier of the morphogen is yet to be defined. Here we show that Hh and its co-receptor Ihog are in exovesicles transported via cytonemes. These exovesicles present protein markers and other features of exosomes. Moreover, the cell machinery for exosome formation is necessary for normal Hh secretion and graded signalling. We propose Hh transport via exosomes along cytonemes as a significant mechanism for the restricted distribution of a lipid-modified morphogen.

Hedgehog can signal both at a short and long-range, and acts as a morphogen during development in various systems. We studied the mechanisms of Hh release and spread using the Drosophila wing imaginal disc as a model system for polarized epithelium. We analyzed the cooperative role of the glypican Dally, the extracellular factor Shifted (Shf, also known as DmWif), and the Immunoglobulin-like (Ig-like) and Fibronectin III (FNNIII) domain-containing transmembrane proteins, Interference hedgehog (Ihog) and its related protein Brother of Ihog (Boi), in the stability, release and spread of Hh. We show that Dally and Boi are required to prevent apical dispersion of Hh; they also aid Hh recycling for its release along the basolateral part of the epithelium to form a long-range gradient. Shf/DmWif on the other hand facilitates Hh movement restrained by Ihog, Boi and Dally, establishing equilibrium between membrane attachment and release of Hh. Furthermore, this protein complex is part of thin filopodia-like structures or cytonemes, suggesting that the interaction between Dally, Ihog, Boi and Shf/DmWif is required for cytoneme-mediated Hh distribution during gradient formation.