Fig 2. Ecdysone production is dependent on proper snail levels in the PG.

(A) Developmental arrest caused by PG>snail-RNAi was partially rescued by ecdysone feeding. Bar graphs show the percentage of larvae that survived to the indicated stages. Fifty L2 larvae of each genotype were used as a starting population. Error bars represent standard deviation. *p < 0.05 (one-way ANOVA). (B) Left panel: example vials from experiments shown in (A). Larvae on the vial wall indicate that wandering behavior had occurred. Right panel: control and snail-RNAi PP reared on 20E supplemented food. (C) Ecdysteroid titers in whole-body larvae were measured at various time points during early L3 stage. Error bars represent standard deviation. *p < 0.5, ***p < 0.001 (two-way ANOVA). (D) Survival rates of animals conditionally expressing snail in the PG. Gal80^ts; phm22>snail-cDNA embryos/larvae were initially reared at 18°C and then shifted to the restrictive temperate (29°C) at the indicated time points to induce the expression of the snail-cDNA. Bar graphs show the percentage of animals that reach indicated developmental stages. Error bars represent standard deviation. (E) qPCR analysis showing the expression levels of six ecdysone biosynthetic genes in snail-RNAi RGs. RGs were dissected at 24 hr after L2 to L3 molt. (F) qPCR results showing the expression of the six ecdysone biosynthetic genes in hs>snail-cDNA RGs. The expression of each gene in snail overexpression was normalized to the expression in the control (hs-Gal4> y^[1]w^[67c23]). (A-C, E) Control/ctrl.: UAS-Dicer2; phm22-Gal4>w¹¹¹⁸. snail-RNAi: UAS-Dicer2; phm22-Gal4>UAS-snail-RNAi. (E and F) Error bar represents the 95% confidence interval. *p < 0.05, **p < 0.01, ***p < 0.001 (Student t test). Underlying data for this figure can be found in S1 Data. 20E, 20-hydroxyecdysone; AED, after egg deposition; dib, disembodied; Gal80^ts, Gal80 temperature-sensitive allele; L1, first instar; L2, second instar; L3, third instar; nvd, neverland; n.s., not significant; PP, pupae; PG, prothoracic gland; phm, phantom; PP, pupae; qPCR, quantitative PCR; RG, ring gland; RNAi, RNA interference; sad, shadow; SM, agar-cornmeal-based standard medium; spok, spookier; sro, shroud.