in either eat-2 which lead to reduced food intake by impaired pharyngeal pumping [8], or in pep-2 mutants which display compromised intestinal uptake of dietary peptides [9]. PHA-4 was recently identified as the forkhead transcription factor which is necessary to increase lifespan under multiple conditions of dietary restriction, such as lowering the concentration of bacteria fed to the worms in culture or eat-2 mutations, but not under conditions of lowered insulin signaling [10], making it a candidate for a transcriptional effector downstream of TOR signaling. This working hypothesis was confirmed by the finding that PHA4 is required for the lifespan extension elicited by reduced LET-363/ TOR or RSKS-1/S6 kinase levels, both of which are independent of DAF-16 function [11]. These observations prompted us to investigate a possible link between TOR signaling and the transcription factor Fork head (FKH), which among the 18 Drosophila forkhead proteins is the only one belonging to the FoxA subfamily [12]. Here we present evidence that in addition to its established role in embryonic development and regulation of salivary gland cell death in the larva, FKH controls cell and organismal size, that its subcellular localization is regulated by TOR signaling and that it is necessary for the expression of rapamycin- and starvation-responsive genes as well as for rapamycin-induced inhibition of growth. For the first time in Drosophila, we describe an interaction between TOR and a FoxA protein, which is in agreement with the observations made in C. elegans. In addition, our findings yield novel insights about the regulation of growth by FKH, and how FKH and dFOXO are partially redundant in the regulation of d4E-BP expression. Results Alteration of FKH levels elicits organism and cell size phenotypes which are sensitive to nutrient conditions and TOR signaling As in other organisms, TOR signaling in Drosophila is involved in the nutrient-dependent regulation of cellular and organismal growth [13,14]. We therefore first asked whether an alteration of FKH levels in vivo would have an effect on growth. As homozygous fkh mutants are embryonic lethal [15], we applied the GAL4/UAS system to either knock down FKH in larval tissues by RNA interference or to raise FKH levels by ectopic overexpression, each under different nutrient conditions. As shown in Figure 1, overexpression of FKH in the larval fatbody induced by the pumpless driver causes a severe reduction in body size (Figure 1I and K), similar to the one resulting from TOR inhibition induced by rearing the larvae on rapamycin-containing food (Figure 1B). Rapamycin does not significantly further reduce the size of FKH- overexpressing animals (Figure 1J). Moderate ubiquitous RNAi knockdown of FKH with the armadillo driver leads to a slight decrease in larval size under fed conditions (Figure 1E). Rapamy- cin feeding causes a reduction in larval size via inhibition of TOR signaling. However, this phenotype is less strong in animals with repressed FKH levels (Figure 1F and K), suggesting that FKH is required for rapamycin-induced growth inhibition. Expression of a negative control inverted repeat directed against lacZ had no significant impact on larval size under both conditions (Figure 1C, D and K), ruling out unspecific effects of double-stranded RNA expression. Likewise, ectopic expression of b-galactosidase in UAS-lacZ control animals had no effect on larval size (Figure 1G, H and K). Quantitative realtime PCR was performed to measure the efficiency of fkh knockdown by RNAi in this experimental setup, and fkh transcript levels were found to be reduced by 60% compared to wildtype larvae (Figure S1). Regulation of Drosophila FKH/FOXA by TOR In a second set of experiments, we analyzed the effect of FKH overexpression or knockdown on cell size. The ‘FLP-out’ system [16] was used to overexpress or silence FKH in cell clones in the larval fatbody. The effects on cell size were similar to those on organismal size and are displayed in Figure 2, the quantitative and statistical analysis of cell size phenotypes is shown in Figure 3. For each genotype and condition, we compared the size of transgene- expressing cells which are marked by the co-expression of nuclear green fluorescent protein (GFP) to the size of the non-fluorescent wild-type cells within the same tissue sample. Cells overexpressing FKH are significantly smaller than wild-type cells in fed animals (Figure 2J and 3B) but not in starved larvae (Figure 2K and 3B). In rapamycin-fed larvae, clonal FKH expression slightly reduced cell size, but to a much lesser extent compared to yeast-fed animals (Figure 2L and 3B). This indicates that cells are most susceptibe to growth inhibition by ectopic FKH expression under conditions of dietary protein abundance and active TOR signaling. Conversely, RNAi-induced silencing of FKH expression in cell clones had no significant effect on cell size in fatbodies of fed larvae (Figure 2D and 3A), but under conditions of starvation or rapamycin feeding, FKH knockdown elicited increased cell growth compared to control cells within the same tissue (Figure 2E, F and 3A). The observation that cells with low FKH levels are larger than the neighboring wild-type cells in tissues subjected to starvation or TOR inhibition supports the model that FKH is required for the inhibition of growth in response to these conditions. As in the larval body size experiments described above, fat body cell size was unaffected by clonal expression of the negative control constructs UAS-lacZ-RNAi (Figure 2D–F and 3A) or UAS-lacZ (Figure 2J–L and 3B). In this experimental setup, the FKH phenotypes observed correspond qualitatively to those of negatively regulating TOR signaling components. As shown in Figure S4, co-expression of TSC1 and TSC2 [17], negative upstream regulators of TOR, strongly reduces cell size in feeding larvae, while the size reduction is much less pronounced in starved animals. Conversely, expression of the small GTPase Rheb, which activates TOR signaling, leads to a mild cell size increase under conditions of nutrient abundance and to a stronger one under starvation, as has been reported previously [18,19]. Subcellular localization of FKH depends on nutrient and TOR signaling levels In the next step, we sought to investigate whether nutrient or TOR signaling levels have an influence on the subcellular localization of FKH protein. To monitor localization of the endogenous protein in tissues, we generated an affinity-purified polyclonal antibody against FKH. To ascertain the specificity of the new tool, western blot and immunohistochemical control experiments were performed which showed that the antibody could be used to detect endogenous as well as transgenically encoded FKH protein in larval extracts with very little unspecific background (Figure S2A). Furthermore, immunostainings visual- ized endogenous and overexpressed epitope-tagged FKH protein, and it could be observed that the main nuclear signal recognized by the antibody is indeed FKH (Figure S2B–E). Based on these control experiments, we then analyzed endogenous FKH localization in the larval fatbody under conditions of different nutrient and TOR signaling levels. When larvae were fed yeast paste, which contains a higher fraction of dietary proteins and amino acids compared to sugar-cornmeal-based fly food [20,21], FKH was found to be localized in the cytoplasm of fatbody cells and almost completely excluded from the nuclei (Figure 4A, B and 4E, F). In larvae starved on PBS, nuclear exclusion was not observed, and the contours of the nuclei were not visible due to the PLoS ONE | www.plosone.org 2 December 2010 | Volume 5 | Issue 12 | e15171