Hml-apo flies show a defective leg phenotype, which can be rescued by blocking apoptosis, pharmacological inhibition of NOS and antibiotic treatment.
(A) Control adults (HFP/+) where legs were normal. (B-C’) Both Hid- and Grim-expressing adults showed defective legs ranging from shortened leg segments (mild phenotype, B and C) to the complete absence of a leg (strong phenotype, B’ and C’). In both cases, phenotypes were most pronounced for the 3rd leg pair. Arrows indicate the defective leg phenotype (B, B’, C and C’). (D) Inhibiting apoptosis by co-expressing UAS-grim28.2 with UAS-p35 rescued the defective leg phenotype. (E-G’’’) shows isolated legs including (E) normal control adult legs (E, HFP/+) and defective legs in both Hid and Grim lines (F, G, and G’) in non-treated conditions. (E”) Feeding L-NAME (a pharmacological inhibitor of NOS) to 3rd instar larvae, rescued the leg defects in both Hid and Grim adult flies (F” and G’’’) while feeding D-NAME (inactive isomer of NAME) did not (F’ and G”). (H) Quantification of the defective leg phenotype in apoptotic adults, and after rescue with antibiotic treatment and upon co-expressing UAS-grim28.2 with UAS-p35. Defective legs were found in both Hid and Grim lines. Hid lines showed a stronger phenotype (such as a complete absence of legs, bracket 1) and a higher frequency of defective legs than Grim adults. Using a different food source (DIM) rescued the defective leg phenotype in Hid- but not Grim-expressing flies (bracket 2) and the same was true for antibiotics treatment (bracket 3). Blocking apoptosis using UAS-p35 also rescued the defective leg phenotype (right part). (I) Quantification of defective leg penetrance after treatment (D-NAME and L-NAME) compared to non-treated flies. Data presented are means ± SD; t test: * p<0.05; **p<0.01 (n = 81, 120 and 62 for controls, 189, 114 and 62 for Hid-expressing flies and 206, 120 and 72 for Grim-expressing flies).