Death scoring and timed sample collections

hdc mutant alleles were balanced with fluorescent TM3-cherry chromosome to select against the presence of fluorescence the homozygous mutants during embryonic and early larval stages. For death scoring during development, flies were allowed to lay eggs on agar plates supplied with fresh yeast paste for 2–3 h and 15–30 embryos were collected and split into plates containing standard fly food. The embryos were scored for genotype and monitored throughout development at 25°C in time intervals of 1–5 h during the daytime, while death events were scored till pupal stages. For pupariation timing, larvae were synchronized by allowing flies to lay eggs on agar plates supplied with fresh yeast paste for 2–3 h, and 20–30 freshly eclosed L1 per genotype were incubated at 25°C until they reach the pupal stage. Pupariation time was scored every 3–4 h during the daytime. The data obtained were ordered by time and cumulative percentages of pupariation and were analyzed in GraphPad Prism 8.4.3 Software.