Figure S2.

Cellular Analysis of the Reduced Tracheal Coverage Caused by Nutrient Restriction or Reduced Insulin Signaling, Related to Figures 1 and 2

(A to D) Staining with an anti-DSRF antibody that labels the tracheal terminal cell nuclei indicates that these cells are present in 3^rd-instar larvae exposed to a mild nutrient restriction (B, not significantly different from the tracheal terminal cell number of well-fed larvae (A). See also quantifications to the right of (A) and (B). This rules out tracheal terminal cell death as the reason for the reduced intestinal tracheal coverage in nutrient-restricted larvae. Similarly, no differences in tracheal terminal cell number are apparent upon reduction of insulin signaling in DSRF>InR-RNAi larvae of the same stage (D, not significantly different from the tracheal terminal cell number of GAL4 (C) or UAS controls (data not shown). See also quantifications to the right of C and D. In all panels, DAPI is used in blue to highlight the gut tissue, and asterisks are used above the tracheal terminal nuclei to highlight them. All panels show the mid-hindgut but the same results were obtained in other intestinal regions (data not shown). (E to P) Quantifications of the total length of tracheal arbours (E to J) and the ratio between this length and the gut area covered by the tracheal arbours (K to P) in the mid-hindgut of well-fed (8% yeast) versus nutrient-restricted (2% yeast) 1^st-, 2^nd- and 3^rd-instar OreR larvae (E to G and K to M) or DSRF>InR-RNAi larvae versus GAL4 and UAS controls (H to J and N to P). Tracheal coverage (as quantified by the length/area ratio in K to M for each larval instar) is mildly reduced in 2^nd-instar larvae that have been nutrient restricted (L, p = 0.02), becoming strongly reduced by the 3^rd-instar stage (M, p < 0.0001). However, the total length of these tracheal arbours increases with each larval instar (E to G), although not at the same rate as in well-fed larvae (G, p < 0.0001). n = 14–20/set. This suggests that the reduced tracheal coverage results from slower growth of these tracheal arbours. The same is true for the DSRF>InR-RNAi larvae (H to J and N to P). For tracheal length: in I, p = 0.02 versus UAS control but not significant versus GAL4 control, and in J, p < 0.0001 versus either GAL4 or UAS controls. For tracheal coverage: in O, p < 0.001 versus UAS control but not significant versus GAL4 control, and in P, p < 0.0001 versus either GAL4 or UAS controls. n = 13-18/set. (Q and R) Morphology of the tracheal terminal cell arbours of well-fed (Q, 8% yeast) or nutrient-restricted (R, 2% yeast) 3^rd-instar larvae expressing the membrane-tagged reporter cd8-GFP from DSRF-GAL4. As the panels and quantification to the right of the panels show, tracheal coverage is reduced in nutrient-restricted 3^rd-instar larvae (p < 0.0001, n = 26-29/set). This further confirms that the phenotype observed with DIC optics (an imaging technique best suited to the analysis of gas-filled tracheae), results, at least partly, from reduced branching rather than gas filling. (S and T) The same is true for DSRF > cd8-GFP, PI3K-DN (p < 0.0001 versus DSRF>cd8-GFP control, n = 22-25/set).