Biochemical and genetic studies indicate

Biochemical and genetic studies indicate that the endosomal sorting complex required for transport (ESCRT) machinery is crucial for Hh exovesicle packaging and morphogen gradient function 39., 43., 44.. In vertebrates, ESCRT proteins promote the release of Shh exovesicles to maintain progenitor cell pools during VX-770 development [44]. In flies, knockdown of ESCRT or exovesicle proteins reduced long-range Hh signaling activity, consistent with an evolutionarily conserved role for ESCRT in Hhs release 39., 43.. Imaging of Drosophila wing imaginal discs revealed puncta containing both Disp and Hh on the surface of extracellular exovesicles (Figure 3A, Key Figure) [39]. These exovesicles purified from cultured Drosophila cells were competent to activate Hh reporter gene expression when provided exogenously, indicating that vesicular Hh is operative for signaling [39]. Although the effects of Disp loss on Hh exovesicle localization were not tested, its presence with ligand inside the structures argues for its involvement in this release mechanism.
Disp Transports Hhs on Cytonemes How Disp directs Hh family ligands to establish their morphogen gradients and initiate long-range responses remains a topic of significant debate. Early models proposed that Hh family morphogen gradients formed by free diffusion and that Disp might function to package Hh molecules into multimers that could shield its lipid modifications from the aqueous extracellular environment (Figure 3B) 19., 46.. Chaperone-assisted diffusion and cell-to-cell movement, in which Hh sequentially shuttles along neighboring cell membranes via heparan sulfate proteoglycans (HSPGs), have also been proposed as possible distribution mechanisms (Figure 3C,D) 42., 47., 48., 49., 50.. In such cases, the contribution of Disp would be likely to be limited to transferring Hhs from producing cell membranes to extracellular chaperones or adjacent cell HSPGs (Figure 3C,D). More recently, evidence has accumulated in support of a model in which Hhs-producing cells actively distribute morphogen along specialized filopodia called cytonemes (Figure 3E,F). These actin-based cellular extensions contain Disp and the Hh coreceptors Cdon/Ihog and Boc/Boi and provide a conduit on which Hhs can be transported far from their site of synthesis 28., 33., 39., 51., 52., 53.. Disp- and Hh-containing exovesicles are found along cytonemes that reach across basal sections of Drosophila wing imaginal discs (Figure 3E,F) [39]. In addition, cytonemes containing the Hh receptor Ptch have been documented to extend from signal-receiving cells in both fly and vertebrate systems. These Ptch-containing extensions connect with signal-producing cell cytonemes to receive Hhs across cytoneme tips through what might function as a ‘morphogenetic synapse’ (Figure 3F) 49., 53., 54., 55.. Recent studies performed using both cultured cells and Drosophila imaginal discs support an active role for Disp in controlling cytoneme dynamics. Disp overexpression increased cytoneme occurrence rates and its knockdown reduced occurrence 28., 33.. How Disp promotes cytoneme occurrence is not yet clear. However, Disp-expressing cells showed slower cytoneme retraction rates than nonexpressing cells, raising the possibility that Disp might influence actin dynamics or filopodial tip adhesion molecules to increase cytoneme durations [28]. Disruption of actin nucleation in Drosophila by knockdown of the formin protein Diaphanous shortened cytonemes and disrupted long-range signaling, indicating that targeting of the actin cytoskeleton could be a feasible mechanism to control cytoneme activity 52., 54., 55.. Determination of the intermediaries facilitating communication between Disp and actin regulators is likely to be an important step toward understanding cytoneme contribution to Hh morphogen gradient formation.
Concluding Remarks Detailed examination of Disp function in cytonemes will be crucial in understanding how it actively contributes to and reinforces Hhs morphogen gradients. Determination of Disp structure will be necessary to understand its molecular mechanisms of action during gradient establishment. Structural studies are likely to provide insights into the function of the Disp SSD and to reveal how Furin cleavage impacts Disp activity, and may define the basis of Disp collaboration with Scube2. Structural studies could also provide information about how deleterious HPE disease mutations compromise Disp activity (Box 1).