Figure 4.

Autophagy inhibition delays midgut removal that is suppressed by inhibition of PI3K pathway. Morphology of midguts at 0 h RPF (left) and +4 h RPF (middle) showing (a) control (NP1-GAL4/+) compared with (b) PTEN expression (NP1-GAL4/+ UAS-PTEN/+) and (c) TSC1/2 expression (NP1-GAL4/+ UAS-TSC1, UAS-TSC2/+) showing decreased size with contracted gastric caeca at 0 h RPF and smaller midgut at +4 h RPF. Histological analysis of paraffin sections at +12 h RPF (right) shows significant midgut contraction in (b) PTEN expression and (c) TSC1/2 expression compared with control (a) (NP1-GAL4/+). (d) Inhibition of autophagy severely delays midgut degradation. Morphology of midguts at 0 h RPF (left) and +4 h RPF (middle) show a significant delay in midgut histolysis in (d) Atg1^IR (NP1-GAL4/+ UAS-Atg1^IR/+) compared with (a) control as seen by the presence of less-contracted gastric caeca at +4 h RPF (arrows). Histological analysis of paraffin sections at +12 h RPF (right) shows an enlarged midgut in (d) Atg1^IR compared with (a) control. The combined expression of (e) Atg1^IR PTEN and (f) Atg1^IR TSC1/2 suppressed the delay in midgut histolysis with greater contraction of gastric caeca at +4 h RPF (arrows) compared with (d) Atg1^IR alone. The delayed midgut histolysis in (d) Atg1^IR is reduced by co-expression of (e) PTEN or (f) TSC1/2 at +12 h RPF. Scale bars in (a) represent 200 μm in all panels. (g) Mosaic clones of PTEN (hsFLP; mCherry-Atg8a/+ Act>CD2>GAL4, UAS-nlsGFP/UAS-PTEN) marked by GFP (green) and DNA (blue) have smaller cell size with increased autophagy compared with the neighbouring cells. Scale bar in (g) is 50 μm. (h) Quantification of gastric caeca size (average pixels±S.D.) at 0 h RPF from control, PTEN, TSC1/2, Atg1^IR, Atg1^IR PTEN and Atg1^IR TSC1/2 (N=10, 12, 9, 8, 5 and 12, respectively). *P<0.001 (compared with the control). (i) Quantification of gastric caeca size (average pixels±S.D.) at +4 h RPF measured from control, PTEN, TSC1/2, Atg1^IR, Atg1^IR PTEN and Atg1^IR TSC1/2 (N=8, 8, 9, 9, 10 and 9, respectively). *P<0.001 (compared with the control). (j) Caspase activity was measured from −4 h RPF larval lysates on DEVD-AMC, represented as pmol DEVD/min. Data are mean from three independent experiments, with error bars representing S.E.M. *P<0.05 (compared with control)