Fig. 4: Excess TOR activity leads to hypoxia in a tissue-size-dependent manner.
a-a’ Fluorescence from SyHREns is altered by modulation of Rheb levels. Overexpression of Rheb in the P compartment (with hh-GAL4) enhances the SyHREns signal. Note that fluorescence intensity in the A compartment of Rheb-overexpressing discs is higher than in the A compartment of control discs lacking the UAS-Rheb transgene (\(\varnothing\)), suggesting a non-autonomous effect on SyHREns activity. Knockdown of Rheb reduces SyHREns signal also both autonomously and non-autonomously. Representative images are shown in (a) (all discs imaged and processed under identical conditions, allowing intensities to be compared) and quantification of wing pouch fluorescence intensity is shown in (a’). For images shown here and in subsequent figures, the hh-GAL4 expression domain (P compartment) is recognised by the absence of Ci staining and indicated with a double-headed arrow. The A-P boundary was outlined manually (based on Ci staining) with a dotted white line. Scale bar = 100 μm. n ≥ 13 discs for each genotype, except for data of RhebRNAi where n = 11. After assessing normality of the data, statistical significance was assessed by a two-sided unpaired Wilcoxon signed-rank test. b-b’ Representative images of discs stained with anti-V5 show that Rheb overexpression in the P compartment (with hh-GAL4) stimulates FlHypox significantly more than FlHypoxP850A. For analysis of the fluorescence from the ubi-mCherry module from the same samples, see Supplementary Fig. 5a, b. Fluorescence in the P compartment was normalised to that in the A compartment by calculating the P/A ratio plotted in b’. Scale bar = 100 μm. n ≥ 14 for each condition, except for data of FlHypox in \(\varnothing\) where n = 12. Depending on the normality of the data, statistical significance was assessed by either a two-sided unpaired t test or a two-sided unpaired Wilcoxon signed-rank test. c-c’ Fluorescence from SyHREns increases upon Rheb overexpression in the P compartment. The fluorescence P/A ratio plotted in c’ shows that this effect is more pronounced in late L3 discs than in mid-L3. Scale bar = 100 μm. n ≥ 10 for each time point. After confirming normality of the data, statistical significance was assessed by a two-sided unpaired t test.
