(A, B) Dynamic behaviour of cytonemes emanating from the A compartment abdominal histoblasts, monitored by the actin cytoskeleton marker GMA-GFP alone (the actin-binding domain of moesin fused to GFP) (A), or co-expressed with Ihog-RFP (B), after 24 hr of expression using the ptc.Gal4 driver. Hh-receiving cells produce highly dynamic filopodia visualized by expressing GMA-GFP (A), which are more stabilized when co-expressing Ihog-RFP (B). (C–E) Violin plots represent filopodia maximum extent (C), lifetime (D), and elongation velocity (Ve) and retraction velocity (Vr) measurements (E). Statistical analysis was done to compare the expression, for 24 hr, of GMA-GFP alone or co-expressed with Ihog-RFP in Hh-receiving cells (ptc.Gal4) or Hh-producing cells (hh.Gal4). Coloured diamonds indicate the mean of the data and black lines the median. *p<0.05, **p<0.01, ***p<10−3, ****p<10−4. Scale bars represent 10 μm. (F) Interfering with A cytonemes extension (by transient co-expression of UAS.ihog-RFP together with UAS.scar-RNAi, UAS.dia-RNAi, UAS.cpa RNAi or UAS.cher-RNAi for 30 hr using the ptc.Gal4 driver) affects the spreading of the Hh gradient in the wing disc, monitored by Ptc and Ci expressions (by transiently expressing the UAS-RNAi lines and not UAS.ihog-RFP for 30 hr). Graph representing the average of 5 discs in 3 independent experiments (error bars represent SDs). (G) Two Ihog-RFP expressing clones induced in the wing pouch, one in the A compartment and another in the P compartment, in lateral, basal and transverse (Z-stack) sections. Note that very distant cells contact at the basal region through cytonemes oriented towards the A/P compartment border (arrow). (H) A ptc.Gal4, tub.Gal80ts>UAS.ihog-RFP wing disc after 24 hr at the restrictive temperature and stained with α-Hh antibody. Note that A compartment cytonemes expressing Ihog-RFP capture Hh produced by the P compartment cells (arrows). The data shown were consistent in at least three independent experiments with an average of 5–10 discs in each experiment. Bars, 10 µm.