Fig 1.
Expression profiles of the top 1,000 genes expressed in the PG.
Each data point represents ppm in the PG (x-axis) and relative expression ratio compared with the brain (y-axis) of each gene. Genes that are known to be involved in ecdysone biosynthesis are represented by triangles.
Table 1.
List of genes expressed >10 times in the PG than in the brain.
Table 2.
Results of RNAi driven by phm-GAL4 or 2-286-GAL4.
Fig 2.
Expression analysis of candidate genes.
(A) Quantitative real-time PCR for candidate genes in several tissues from wandering third instar larvae of D. melanogaster. Rg, ring gland; Br, brain with ventral nerve cord; Sg, salivary gland; Tr, trachea; Fb, fat body; Gu, Gut; Mt, Malpighian tubule; Ep, epidermis. The expression level of each gene was normalized using rp49 as an endogenous control. Relative expression levels were determined using the ΔΔCt method. Among tissues examined, the highest average expression level was set to 1. (B-E) In situ hybridization analysis for candidate genes in the ring gland. alas and CG33156 transcripts were detected with antisense probes but not with sense probes. B: alas antisense, C: CG33156 antisense, D: alas sense, and E: CG33156 sense.
Fig 3.
Ecdysteroids in D. melanogaster RNAi larvae.
Whole body 20E and MakA titers of RNAi and control larvae were measured by LC-MS/MS. (A) 20E and MakA titers of alas RNAi animals. (B) 20E and MakA titers of CG33156 RNAi animals. Data are from three or four biological replicates, which consisted of 10 larvae or 5 pupae for each data point. W: wandering larvae and P: prepupae. Values are mean ± S.E.M. and asterisks indicate under quantitation limit (2.5 pg/larva or 5 pg/pupa).
Fig 4.
RG morphology of D. melanogaster RNAi larvae.
RG from day 5 or day 8 larvae were visualized by mCD8::GFP under the phm-GAL4 driver. (A) Representative image of the RG from day 5 control larva (phm > mCD8::GFP). (B) and (D) Representative images of the RG from day 5 and day 8 alas RNAi larvae (phm > mCD8::GFP, alas inverted repeat), respectively. (C) and (E) Representative images of the RG from day 5 and day 8 CG33156 RNAi larvae, respectively (phm > mCD8::GFP, CG33156 inverted repeat). The morphology of the RG of RNAi larvae did not differ from that of control larvae at day 5, but the RG of RNAi larvae became larger during their extended larval period.