Extended Data Fig. 8: Gradient scaling by recycling: HSPGs.

a, b, Scaling analysis for control and dally mutants. Left, decay length λ of eGFP-Dpp (a) and PMad gradients (b) plotted as a function of posterior compartment length l. Raw and binned data (bars, s.e.m.) are shown together with a linear regression to the raw data. Right, bar plots showing the slopes ϕ of corresponding linear regressions for control experimental conditions (blue) compared to dally mutant experimental conditions (red). Number of biologically independent samples: n = 93 (control) and n = 39 (dally^gem) (a); n = 43 (control) and n = 36 (dally^gem) (b). ****p-value < 0.00001; two-tailed two sample t-test with unequal variances. Bars, confidence intervals at 95%. c, Sets of parameter values satisfying the constraints given by all the experimental assays represented in (k_on, k_off), (k_on, D₀) and (k, k_off) planes in the four experimental conditions: eGFP-Dpp discs of 144 µm and 80 µm average posterior length, pent² (average length, 130 µm) mutant and dally^gem mutant discs (average length, 174 µm). d, Stacked bar chart showing the relative contribution of the different modules to λ² (described in Fig. 1e,f) in the four experimental conditions compared to the theoretical values of parameters in the extracellular diffusion (ExD) and transcytosis regimes of transport (Tr). e, GBP-Alexa555 signal intensity as a function of time in discs expressing eGFP-Dpp^Gal4 in control discs (left), dally^gem mutant discs (middle) and control discs following treatment with PI-PLC for 1h (right). Lines, fits to the phenomenological equation describing the internalized signal intensity dynamics C_T(t). f, Values of k_N, k_r and k₀ estimated by the nanobody uptake assay in control discs, dally^gem mutant discs and PI-PLC treated discs expressing eGFP-Dpp^Gal4. g, Internalized GBP-Alexa555 fluorescence as a function of time in discs expressing eGFP-Dpp^CRISPR (control), discs expressing eGFP-Dpp^CRISPR and sflRNAi (sflRNAi) and control discs (no GFP-Dpp). Number of biologically independent samples: n = 3 for each condition. Data represented as the average curve. Shaded area, s.e.m. h, i, Confocal images of eGFP-Dpp^CRISPR (left) and internalized GBP-Alexa555 (right) after 85 min of incubation with the nanobody in control discs (h) and discs expressing sflRNAi in the posterior compartment (i). Posterior compartment, to the right from the GFP-Dpp source boundary. j, Decay length of the eGFP-Dpp^CRISPR gradient λ as a function of the posterior compartment width l. Red line, linear regression to the raw data. bars, s.e.m. eGFP-Dpp^CRISPR was visualized by means of a nanobody uptake assay (Methods). Number of biologically independent samples n = 38. k, Slope ϕ of the linear regressions for scaling plots corresponding to eGFP-Dpp^LOP (LOP) and eGFP-Dpp^CRISPR (CRISPR). Bars, confidence intervals of the fitted slope. l, Confocal images of photoconverted GBP-Dendra2* in eGFP-Dpp^CRISPR-expressing discs at different times after photoconversion (post-conversion). Before photoconversion, discs were incubated in GBP-Dendra2* solution for 45 min and extracellular GBP-Dendra2 was removed by an acid wash, so that only internalized GBP-Dendra2 is remaining. PhotoconvOgradient outside of the photoconverted region. m, The values of k_N, k_r and k₀ estimated by the nanobody uptake parameters for large discs expressing eGFP-Dpp^CRISPR versus eGFP-Dpp^LOP. Bars, confidence intervals of the fits. Number of biologically independent samples n = 10 (eGFP-Dpp^CRISPR) and n = 13 (eGFP-Dpp^LOP). Scale bar, 10 µm (h, l).

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