Regulation of Drosophila FKH/FOXA by TOR Figure 4. Subcellular localization of FKH is modulated by nutrient availability and TOR signaling. Confocal sections of larval fatbody are shown. For each section, one panel shows only the signal derived from the FKH or dFOXO antibody in green, and a second panel an additional nuclear counterstain with DAPI pseudo-colored in red. (A, B) FKH is excluded from the nucleus in fatbodies of wildtype 2nd instar larvae fed on yeast. (C, D) FKH accumulates in the nucleus in fatbodies of wildtype larvae treated with 50 mM rapamycin. (E, F) Closeup of A, B. (G, H) FKH is nuclear in heterozygous TOR mutants (y w; dTORDP/+). (I, J) Nuclear exclusion of FKH is not observed in larvae subjected to starvation on PBS. (K, L) In contrast to FKH, dFOXO shows a clear nuclear localization in wildtype larvae starved on PBS. Scale bar is 20 mm. doi:10.1371/journal.pone.0015171.g004 as well as CG6770 expression in larvae (Figure 5B). Transcription of both genes was likewise elevated in heterozygous and homozygous TOR mutant larvae (Figure 6B). The highest levels of CG6770 expression, which apparently is more sensitive to TOR signaling levels than cabut expression, were measured in rapamy- cin-fed larvae which also expressed transgenic FKH (Figure 5C), although the difference in CG6770 transcript levels to the FKH- expressing larvae without rapamycin is not significant. FKH is required for the response to rapamycin and lowered TOR levels Based on the observations described above, we addressed whether FKH plays a functional role of physiological relevance downstream of TOR. Several lines of evidence indicate that FKH is required for the induction of cabut and CG6770 as well as for growth inhibition under conditions of low TOR signaling. First, target gene mRNA levels in FKH-RNAi larvae fed with rapamycin were lower than in wild-type or lacZ-RNAi control animals subjected to the same treatment (Figure 6A), indicating that FKH function is required for full expression of both genes upon TOR inhibition. However, both genes still appear to be induced by rapamycin in FKH-RNAi larvae, because FKH knockdown also reduces basal cabut and CG6770 transcript levels in the absence of rapamycin. Second, larvae with reduced FKH levels were less susceptible to growth inhibition by rapamycin (Figure 1B and F). Likewise, individual cells in which FKH expression was silenced had a growth advantage over the neighboring wildtype cells under conditions of starvation or TOR inhibition (Figures 2 and 3). Third, the augmented transcription of CG6770 and cabut in heterozygous TOR mutant larvae was reversed to wild-type levels in double mutant animals carrying a single copy of the fkh1 or the fkh6 loss-of-function allele [15] in addition to the TOR mutation (Figure 6B). Fourth, presence of the fkh1 allele significantly increased the body weight of adult flies heterozygous for a TOR mutation (Figure 6C). Taken together, these results demonstrate that FKH function is required in vivo for the inhibition of organismal and cellular growth and the induction of target gene transcription under conditions of low TOR pathway activity. Impact of FKH and TOR on the expression of the dFOXO target d4E-BP Finally, we investigated the convergence of transcriptional regulation downstream of the insulin and TOR signaling modules. More specifically, we measured the impact of FKH and TOR signaling on the expression of the dFOXO target gene d4E-BP/ Thor. The O subfamily forkhead transcription factor dFOXO is negatively regulated by insulin signaling and activates transcription PLoS ONE | www.plosone.org 6 December 2010 | Volume 5 | Issue 12 | e15171