Regulation of Drosophila FKH/FOXA by TOR Figure 2. FKH levels influence cell size in the larval fatbody. Pictures show confocal sections of larval fatbodies, using the fly line y w hs-flp;; Act.CD2.Gal4 UAS-GFP to drive UAS-FKH-RNAi, overexpression and control responder lines. Cells expressing the transgene are marked by the co- expression of GFP, whereas the non-fluorescent serve as wild-type controls within the same tissue sample. Larvae were reared on yeast for 64 h AED, or starved on PBS for 24 h after growing on yeast for 64 h, or treated with rapamycin for 24 h after growing on yeast for 48 h. Tissue was stained with a-GFP (green), a-CD2 (red) and DAPI (blue). (A–C, G–I) Controls show no obvious phenotype in the GFP-positive cells. (D–F) Cells expressing FKH RNAi have no growth phenotype under fed conditions, but are larger than the surrounding tissue in starved and rapamycin-treated larvae. (J–L) Cells overexpressing FKH are smaller than the surrounding tissue under fed conditions. doi:10.1371/journal.pone.0015171.g002 promising candidate growth suppressor gene downstream of TOR complex 1. It was also identified as being transcriptionally repressed by a hs-fkh transgene in late prepupae [26] and strongly induced by sugar condition (protein deprivation) in feeding larvae, but not by complete starvation [27]. Expression of an active dFOXO mutant in S2 cells leads to a moderate induction of cabut expression, while refeeding in adult flies has a weak repressing effect [28]. The CG6770 gene was first identified as a transcriptional target of starvation in Drosophila larvae, encoding a protein with vague similarity to the pancreatic P8 transcriptional regulator from mouse and human. Expression was induced by both sugar condition and complete starvation, similar to the dFOXO target gene d4E-BP [27]. In agreement with these results, CG6770 was found to be transcriptionally repressed by refeeding after starvation in adult Drosophila flies and moderately induced by expression of activated dFOXO in S2 cells [28]. It was furthermore identified as a negative regulator of cell size in a genome-wide RNAi screen [29]. We first tested whether the transcription of cabut and CG6770 is dependent on FKH levels. The driver lines used were the same as in the larval size experiments (Figure 1), that is armadillo-GAL4 for targeted RNAi and pumpless-GAL4 for ectopic expression. RNAi knockdown of FKH caused a significant decrease of cabut and CG6770 mRNA in larval extracts, while FKH overexpression induced the transcrip- tion of both genes (Figure 5A). The induction of CG6770 was several fold higher than that of cabut. We further validated the PLoS ONE | www.plosone.org 4 December 2010 | Volume 5 | Issue 12 | e15171