Regulation of Drosophila FKH/FOXA by TOR Figure 1. FKH levels influence larval body size. Pictures show 72 h old larvae (62h) which were treated at 24 h after egg deposition (AED) with 0 (A, C, E, G, I) or 50 mM (B, D, F, H, J) rapamycin for 48 h. A, B: OregonR wildtype control. C, D: y w UAS-LacZ-RNAi; arm-Gal4/+. Treating control larvae with 50 mM rapamycin leads to severe reduction of growth. E, F: w; arm-Gal4/+; UAS-FKH-RNAi/+. Untreated larvae are slightly smaller than controls, but size reduction by rapamycin feeding is less pronounced in larvae with low FKH levels. G, H: w;; ppl-Gal4/UAS-LacZ. I, J: w;; ppl-Gal4/UAS- FKH. Overexpression of FKH leads to a severe reduction in body size similar to that resulting from rapamycin feeding. At 25uC, overexpression furthermore leads to larval lethality in the 2nd instar. Rapamycin feeding of FKH-overexpressing larvae has almost no additive effect to the reduction in body size. (K) Quantitation of larval body size shows that rapamycin-treated larvae with low FKH levels are significantly larger than control larvae and that untreated larvae with high FKH levels are significantly smaller than control larvae. Significance was tested using an unpaired 2-tailed Student’s t-test. *** = p,0.001. Error bars represent standard error of the mean (SEM). doi:10.1371/journal.pone.0015171.g001 absence of nuclear signal as in the tissue from animals reared on a high-protein diet (Figure 4I, J). Significant nuclear accumulation did not occur either, but the protein was rather evenly distributed within the cells. It should be noted that under this starvation condition, dFOXO shows a strong nuclear localization (Figure 4K, L). In contrast to the condition of complete starvation, in fatbodies from rapamycin-fed (Figure 4C, D) or heterozygous TOR mutant larvae (Figure 4G, H), FKH was localized predominantly nuclear, although a prominent cytoplasmic signal remained detectable. This suggests that TOR activity is required to keep FKH protein sequestered in the cytoplasm under conditions of dietary protein abundance. Importantly, this effect of TOR signaling on FKH localization was only observed on the endogenous protein. In contrast, overexpressed FKH was found to be constitutively nuclear in the larval fatbody (Figure S2B–E) as well as in cultured S2R+ cells (Figure S2F–K) even under conditions of nutrient abundance and high insulin/TOR signaling levels. The expression of cabut and CG6770 is regulated by FKH and TOR signaling In an attempt to establish quantitative molecular readouts which may be used to monitor FKH activity under conditions of normal or impaired TOR signaling, we screened published transcriptomic datasets for Drosophila genes which are regulated by nutrient availability, rapamycin or forkhead transcription factors in several independent experiments. The gene cabut encodes a C2H2 zinc finger transcription factor which is conserved between species (the yeast ortholog is FZF1, human ortholog is KLF11), plays a role in JNK-dependent dorsal closure in the Drosophila embryo [22] and was previously identified in screens for genes involved in axon guidance and synaptogenesis [23] as well as autophagic cell death [24]. cabut caught our interest because it was found to be transcriptionally upregulated by rapamycin in S2 cells, and RNAi knockdown of the gene lead to increased cell size and proliferation [25]. It therefore is a PLoS ONE | www.plosone.org 3 December 2010 | Volume 5 | Issue 12 | e15171