Basal cytonemes at the A/P compartment border. (A) Left: line drawing of a wing disc viewed from the columnar epithelium basal side. The green line indicates the A/P border; boxes mark the hinge (upper) and pouch (lower). Right: transverse sections showing the concavity of the pouch in early and late L3 discs. (B,C) Frontal (B) and sagittal (C) optical projections of the pouch region of an unfixed early L3 disc expressing BAC-encoded Ptc:mCherry. Ptc:mCherry puncta were present in the A compartment, mostly near the compartment border; Ptc:mCherry puncta were also present in the P compartment. (D,E) Quantification of Ptc:mCherry puncta along anteroposterior (D, n=5 discs) and apicobasal (E, n=5 discs) axes in the P compartment (standard box plot and whisker diagrams based on minimum, first quartile, median, third quartile and maximum values; ‘whiskers’ show minima and maxima). (F-F″) Frontal sections (F,F′) and sagittal projection (F″) of the hinge region, showing membrane-marked A cells (ptc-Gal4 UAS-CD8:GFP) and marked P cells (hh-nlsDsRed). A cell cytonemes (dashed line) project along the basal surface. (G,G′,H,H′) Hinge region, with marked membranes of A cells (ptc-Gal4 UAS-CD8:mCherry; G,G′) and BAC-encoded Ptc:mCherry (H,H′). Arrows indicate motile cytonemes (G,G′); arrowheads indicate motile Ptc:mCherry-containing puncta (H,H′). (I) Kymograph describing the movement of 12 Ptc:mCherry-containing puncta. (J-J″) Motile puncta with fluorescent BAC-expressed Ptc:mCherry and Hh:GFP in A compartment, and A compartment marked cytonemes (ptc-Gal4 UAS-CD4:IFP2.0). Data set: 922 cytonemes in a 50×50 µm area of the ventral hinge region (n=25 discs); 133 cytonemes had 386 Ptc:mCherry puncta, 114 of which also had Hh:GFP; 789 cytonemes had neither Ptc:mCherry nor Hh:GFP puncta. Scale bars: 10 µm in B; 5 µm in F, G, H and J′′.

Cytoneme transport is required for Hh dispersion and signaling. (A-G) Sagittal (A-C) images of L3 wing pouch, BAC-expressed Ptc:mCherry and Hh:GFP. The spread of Hh:GFP into A cells and Ptc levels was reduced by dia RNAi (ptc-Gal4 UAS-dia RNAi) (B,D,E) and SCAR RNAi (ptc-Gal4 UAS-SCAR RNAi) (C,F,G) in the ptc domain. (H-N) A compartment cytonemes marked with CD8:mCherry or CD8:GFP were shorter and less abundant under conditions of dia (I), SCAR (J) and Nrg (K) depletion in the ptc domain (ptc-Gal4), and under conditions of Syb depletion (L) in the P compartment (hh-Gal4, UAS-Syb RNAi dpp-LexA lexO-mCherryCAAX). (M,N) Standard box plot and whisker diagrams based on minimum, first quartile, median, third quartile and maximum values; ‘whiskers’ show minima and maxima. P-values (t-test), n=8 for all genotypes: control and dia RNAi-treated length (P=5.8E-08) and number comparisons (P=0.00025); control and SCAR RNAi-treated length (P=8.87E-08) and number comparisons (P=0.00042). SCAR RNAi- and dia RNAi-treated length (P=0.32) and number comparisons (P=0.50); control and Syb RNAi-treated length (P=8.92E-17) and number comparisons (P=0.0018); control and Nrg RNAi-treated length (P=1.28E-11) and number comparisons (P=0.00054). (O-U) α-Ptc staining at the wing pouch A/P compartment border for control (O), dia-depleted (P), SCAR-depleted (R), Nrg-depleted (T) and Syb-depleted (U) discs. Hh:CD2 reverses the effects of dia RNAi and SCAR RNAi on Ptc (Q,S). Scale bars: 10 µm in A; 5 µm in H.

Cytonemes extend direct contacts across the A/P compartment border. (A-A‴) Optical sections at basal (A) and more apical elevations of unfixed disc (A′-A‴) with the P compartment marked with mCherry (hh-Gal4 UAS-CD8:mCherry), membrane-tethered GFP¹¹ in the dpp domain (dpp-LexA lexO-CD4:GFP¹¹), and membrane-tethered GFP^1-10 in the P compartment (hh-Gal4 UAS-CD4:GFP^1-10). GRASP marked the juxtaposition of A and P compartments at basolateral elevations, and broader domain and cellular projections (asterisk) at the basal surface. Arrowheads indicate GRASP displaced from the border in more apical sections (A,A′,A‴). (B) Sagittal image of disc fixed and stained with α-Dlg antibody and with GRASP fluorescence (dpp-LexA lexO-CD4:GFP¹¹ hh-Gal4 UAS-CD4:GFP^1-10) that extends basolaterally and along the basal surface (arrow). (C) GRASP fluorescence of an unfixed disc with the A compartment marked with mCherry (dpp-LexA lexO-CD4:GFP¹¹ lexO-mCherryCAAX hh-Gal4 UAS-CD4:GFP^1-10). (D,E) GRASP fluorescence of fixed discs with the A/P compartment border identified by α-Ptc (blue) staining and marked with dashed lines. Arrowheads in E indicate cytonemes lacking GRASP fluorescence. (D,D′) Genotype: dpp-LexA lexO-CD4:GFP¹¹ lexO-mCherryCAAX hh-Gal4 UAS-CD4:GFP^1-10; (E,E′) genotype: dpp-LexA lexO-CD4:GFP¹¹ hh-Gal4 UAS-CD4:GFP^1-10 UAS-CD8:mCherry. (F) GRASP fluorescence with membrane-tethered GFP¹¹ in A compartment cells marked with mCherry and Syb-tethered GFP^1-10 in P compartment cells (dpp-LexA lexO-CD4:GFP¹¹ lexO-mCherryCAAX hh-Gal4 UAS-Syb:GFP^1-10). Scale bars: 5 µm in A, D and E; 10 µm in C.

Hh dispersion in the A and P compartments. (A-B′) BAC-encoded Hh:GFP and Ptc:mCherry at frontal (A-A″) and sagittal (B-B′) planes. White dashed lines surround a clone lacking Hh:GFP BAC; red dashed lines indicate the A/P compartment border. GFP fluorescence is reduced in the clone except at the basal surface. (B″) GFP fluorescence intensity in regions bounded by the yellow dashed lines in B′ at the indicated planes. (C,D) Frontal images showing Hh:GFP expressed by the transgene (hh-Gal4 tub-Gal80^ts UAS-Hh:GFP) following a shift to 29°C. Red dashed lines indicate the A/P compartment border. The subcellular localization of Hh:GFP in the P compartment was mostly apical at 8 h, and more basal later. The localization of Hh:GFP in A compartment cells was mostly basal at 12 h and spread more apical later. (D) Graphical representation of Hh:GFP fluorescence at the indicated planes.