Extended Data Fig. 6: Internalized Dpp is recycled and spreads in the tissue: Dpp^Timer and recycling Rab proteins.

a, Functionality of Dpp^Timer. Left, control disc, expressing sfGFP-mKate2-Dpp under the control of the GAL4/UAS expression system (Dpp^Timer). Centre, dpp mutant disc, the wing imaginal disc is outlined with the white dashed line. Right, dpp mutant disc expressing Dpp^Timer. Note that the mutant phenotype seen in the central image is rescued. b, Scatter plot of sfGFP and mKate2 pixel intensities and linear fit to obtain the calibration factor F (see Supplementary Information section 2.6.3). n = 23 beads. c, Confocal images of the Dpp^Timer gradient in the wing disc (sfGFP, top and mKate2, bottom). d, Relative concentration profiles of mature sfGFP and mKate2 plotted against the distance from the Dpp source (see Supplementary Information section 2.6.3), corresponding to the intensity profiles measured from the images in c. These intensity profiles represent the relative amounts of sfGFP and mature mKate2 molecules. e, Adjusted fluorescence intensity profiles for sfGFP (g^*(x)) and mature mKate2 (r^*(x)) which are proportional to the respective concentration profiles. X-axis represents the distance from the source. Red dashed line is positioned at the anterior-posterior boundary. Note that both in the source and in the region of the target closer to the source, there are less mature mKate2 molecules, confirming that Dpp molecules are younger closer to the source. f, Plotted relative age (A(x)) of Dpp molecules as a function of position calculated from the calibrated profiles in e. Note that as molecules move away from the source they become older on average: A(x) increases to plateau at values close to 1. n, number of biologically independent samples. Shaded areas, s.e.m (e, f). g–j, Effect of pH on the Timer. g, Control of the bafilomycin treatment. Confocal images of a ROI in discs incubated with a LysoSensor^TM probe for 30 min before (top) and after (bottom) incubation in control Clone 8 medium (right) or bafilomycin solution (left). h, Effect of pH on sfGFP and mKate2 in the Dpp^Timer. Confocal images of sfGFP (left) and mKate2 (right) of Dpp^Timer before (top) and after (bottom) neutralization of pH to 7 following bafilomycin treatment for 30 min. i, Fluorescence signal decrease of sfGFP and mKate2 owing to acidic pH in intracellular compartments. Percentage decrease of fluorescence from pH 7 (discs after bafilomycin treatment) to the acidic environment in intracellular compartments (discs before bafilomycin treatment). Note that the decrease is very similar for both fluorophores. j, Normalized fluorescence intensity of sfGFP (blue) and mKate2 (orange) in purified Timer molecules in solutions at different pH. Data normalized to the intensity at pH 7.4. The number of biologically independent samples for this analysis: n_pH5.86 = 8; n_pH6.4 = 7; n_pH7.4 = 7; n_pH7.9 = 5. Data represented as mean values ± s.e.m. Note, that the difference between the normalized intensity of sfGFP and mKate2 at the different pH value is not significant (p-value>0.05; two-tailed two sample t-test). k, Confocal images of eGFP-Dpp^LOP in control condition (top) and after RNAi through expression of dsRNA for the recycling Rab proteins, Rab11 (middle) and Rab4 (bottom) in posterior target cells. l, Spatial fluorescence profiles of eGFP-Dpp^LOP corresponding to control (top), Rab11RNAi (middle) and Rab4RNAi (bottom) conditions in k. m, Decay length λ of eGFP-Dpp^LOP gradient versus posterior compartment length l for control (n = 157), pent² discs (n = 63) and Rab4RNAi (n = 39). Dots, binned data; bars, s.e.m. Control and pent² data as in Fig. 2a, Extended Data Fig. 7f. n, Average eGFP-Dpp^LOP decay length in control and Rab11RNAi conditions. Difference between the two conditions is significant as determined by a two-tailed, two sample t-test with unequal variances, p-value = 0.0034. o, Recycling rate in control and Rab4RNAi conditions, determined by the nanobody uptake assay. Number of curves for each condition is n = 4. Difference between the two conditions is significant; two-tailed, two sample t-test with unequal variances, p-value < 0.0001. Rab4RNAi expression was driven by means of the thermosensitive Gal4Gal80^ts system (29 °C). p–r, Scaling of eGFP-Dpp^LOP. p, Dpp gradient profiles of discs from 40 to 160 µm posterior length. Each individual profile was fitted to an exponential function with an offset (see Supplementary Information section 2.1.2) and the offset returned from the fit was subtracted. q, Normalized Dpp gradient profiles. Each profile was normalized to the amplitude C₀ of its exponential fit in the ordinates (C(r)/C₀) and to the posterior length l of the corresponding wing disc in the abscissas (r=x/l). Shaded area, s.e.m. Black line, average normalized profile. r, Density plot of q: Colour-code corresponds to the fraction of the number of gradients passing through a certain r, C(r)/C₀ bin. Scale bars, 100 µm (a) and 10 µm (c, g, h, k).

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