Figure 6: Hh signalling and release in mutant conditions for exosome production genes.

(a–h) Hh signalling in wing discs expressing RNAi treatments in the P compartment (b–d,f–h) and control (a,e), visualized in the A compartment using the Ptc-promotor-Trap::GFP reporter (a–d) and immunolabelling for the long-range target Ci (e–h). Detail shown at the bottom and anterior is to the left. Note in RNAi wing discs (b–d,f–h), gradients are shorter than in controls (a,e). (d,h) Box plot comparing gradient length expressed as a proportion of wing pouch length, between control discs and treatments for each reporter. (i–l) Quantification of the number of Hh-GFP (i–k) or Ihog-RFP (l) released punctate structures. Reconstruction in Z (lateral view) of wing discs expressing Hh-GFP (i) or co-expressing Hh-GFP and RNAi for AnxB11 (j) under the control of hh.Gal4 and immunostained for the transcription target Ptc (red, also a marker of the A compartment cells), note punctate structures in the A compartment, decrease after RNAi treatment. (k) Boxplot comparing the number of Hh-GFP puncta released for the control (UAS.Hh-GFP) and 10 exosome-related treatments. Note the reduction in the number of puncta. (l) Boxplot comparing the number of Ihog-RFP puncta (anterior compartment) for the control (UAS.Ihog-RFP) and seven treatments related to exosome production. Note the reduction in the number of puncta. (m–o) Endogenous externalized Hh levels decrease in mutant conditions for exosome production genes. Ex vivo staining for Hh in wild-type discs (m) or in wing disc expressing RNAi for TSG101 (n) driven by ap.Gal4 with a dorsal expression domain (marked by GFP), leaving the ventral domain in wild-type conditions as an internal control. Note the decrease in fluorescence after treatment (m,n). (o) Boxplot showing the ratio between relative intensity of the mean grey value in the dorsal (RNAi treated) versus the ventral compartment (wild type). The ratio is close to 1 in control, while the ratio for all RNAi cases is greater than 1 due to a decrease in the levels of external Hh in treated cells. Significance levels for pairwise tests (Tukey HSD or Wilcoxon, depending on Normality of data): ***P<0.001, **P<0.01, *P<0.05,+P<0.1. Scale bars,10 μm.