Figure 1: Hh and key signalling components are present in EVs and cytonemes.

(a) Schematic representation of a cross-section of the wing disc showing the expression of Hh in both epithelia; disc proper (DP) and peripodial membrane. (b) Transverse section of a hh.Gal4>UAS.Hh-GFP wing disc which has been ex vivo stained with anti-GFP antibody for 1 h at low temperature to avoid Hh-GFP internalization. Note the co-localization of Hh-GFP and externalized GFP (red) mainly at the basolateral part of the disc epithelium (bottom of image). (c) Confocal sections of a similar hh.Gal4>UAS.Hh-GFP wing disc ex vivo stained with anti-GFP antibody. Note that the co-localization of green and red (externalized Hh) is increased in more basolateral sections. The internalized Hh-GFP at the A compartment is in green (white arrow heads). Note also that in the most basal section Hh puncta appear to be aligned in a ‘beads on a string’ arrangement, suggesting they associate to cytonemes (see also ref. 6). (d) Ex vivo staining using an anti-GFP antibody in a UAS.Hh-GFP/UAS.Ihog-RFP; hh.Gal4/tubGal80^ts wing disc after 24 h at the restrictive temperature. Note the co-localization between Hh and Ihog (arrow heads) in the A compartment and also the presence of externalized Hh at the cellular extensions marked by the Ihog protein. (e) Dlp expression in a tubGal80^ts; UAS.Ihoh-YFP/hh.Gal4 wing disc for 24 h. (e) Insets in e showing the co-localization of puncta of Ihog and Dlp (arrow heads) in the A compartment. (f) Ectopic expression of UAS-Ihog-YFP and UAS-Disp using the hh.Gal4; tubGal80^ts system for 24 h. (f) Insets in f show the co-localization of puncta of both proteins (arrow heads) in the A compartment. (g) Ex vivo staining using an anti-Disp antibody in a UAS-Disp/UAS-iHo-CFP; hh.Gal4/tubGal80^ts wing disc after 24 h at the restrictive temperature. Note the co-localization of Disp, Dlp and Ihog in the A compartment cells (arrow heads) (insets g). Scale bars,10 μm.