Figure 2. Decay is required for caspase activity but not midgut degradation.

(A) Expression of effector caspases drice, decay, dcp-1 and damm during midgut morphogenesis from third instar larvae, L3 (-8 hr RPF), late third instar larvae, late L3 (-4 hr to -1 hr RPF), and white prepupal, WPP (0 hr RPF) by qPCR. (B) Verification of decay knockdown in the midgut from late third instar larvae (-4 to -1 hr RPF) by qPCR. NP1-GAL4 UAS-decay-IR (NP1 decay) results in greatly reduced levels of decay compared to wild type (WT) and UAS-decay-IR alone (+/decay). In (A) and (B) the transcript levels were normalized against the internal control gene rp49 and represented as relative expression. (C) Compared to the high levels of caspase activity detected in wild type (WT) and dronc^I24/I29 drice^Δ1 midgut, NP1-GAL4 UAS-decay-IR (decay), NP1-GAL4 UAS-decay-IR; dronc drice (decay dronc drice) and NP1-GAL4 UAS-p35 (p35) midgut have greatly reduced activity. The combined decay knockdown and p35 expression (NP1/+; UAS-decay-IR/UAS-p35) had no detectable activity. Caspase activity was measured from -4 to -1 hr RPF midgut lysates on DEVD-AMC and represented as pmol DEVD/min. In (A-C) the data are mean from three experiments, with error bars representing SEM. Histolysis of (D) wild type (WT); (E) NP1-GAL4/UAS-decay-IR (decay-IR); (F) NP1-GAL4/UAS-decay-IR; dronc drice (decay-IR dronc drice); (G) NP1-GAL4/+;UAS-p35/+ (p35); and (H) NP1-GAL4;UAS-decay-IR/UAS-p35 (decay-IR p35). Morphology of midgut at +4 hr RPF (left) showing (D) WT; (E) decay-IR; (F) decay-IR; dronc drice; (G) p35; and (H) decay-IR p35 undergo histolysis at similar rates, with contraction of gastric caeca (arrows). Histological analysis of paraffin sections at +12 hr RPF (right) shows that larval midgut contraction is similar in (D) WT; (E) decay-IR; (F) decay-IR; dronc drice; (G) p35; and (H) decay-IR p35.