TI - Overexpressed dFOXO is responsive to insulin signaling and nutrient levels , inducing organ -size reduction and cell death . AB - To assess whether dFOXO has a key function in insulin signaling like that of DAF-16 in C.elegans , we tested whether overexpression of wild-type or mutant forms of hFOXO3a and dFOXO could antagonize insulin signaling . Elimination of the three PKB consensus phosphorylaTION sites in mammalian FOXO3a prevents its PHOSphorylation , subsequent binding to 14-3-3 proteins and sequestration in the cytoplasm [12] . This leads to constitutive nuclear localization of the mutant FOXO3a and transcriptional activation of its target genes . Assuming that blocking the PKB signal would have the same activating effect on dFOXO , we overexpressed wild-type and triple PKB-PHOSphorylation-mutant variants of both dFOXO and human FOXO3a . Furthermore , we identified an EP transposable element insertion in the second dFOXO intron , which permits the GAL4 -induced overexpression of endogenous dFOXO ( Figure 1d ) . We used the GMR-Gal4 construct to drive UAS -dependent expression in postmitotic cells in the eye imaginal disc [38] . While expression of wild-type hFOXO3a in the developing eye did not result in a visible phenotype ( Figure 2b ) , hFOXO3a-TM expression caused pupal lethality . Few escaper flies eclosed and displayed a strong necrotic eye phenotype ( Figure 2c ) . A block of cell differentiation and necrosis was also observed when hFOXO3a-TM was expressed in cell clones in the developing eye ( Figure 2d ) . Assuming that the lack of a phenotype observed upon UAS-hFOXO3a expression is due to hFOXO3a inactivation by endogenous DInr signaling in the eye disc , we performed the same experiment in a background of reduced insulin signaling . Indeed , in the presence of a dominant-negative ( DN ) form of Dp110 ( encoding the PI 3-kinase catalytic subunit ) [39] , hFOXO3a expression induced a necrotic phenotype similar to the one observed with the hyperactive PHOSphorylation mutant ( Figure 2f ) . To confirm that hFOXO3a is responsive to Drosophila insulin signaling and rule out artificial coexpression effects , we expressed hFOXO3a in flies mutant for either dPKB ( Figure 2g ) or Dp110 ( not shown ) , and observed similar phenotypes to those seen upon coexpression of Dp110DN . Drosophila FOXO has qualitatively similar , but stronger effects . Expressing the wild-type form of dFOXO causes a weak eye-size reduction and disruption of the ommatidial pattern even in a wild-type background ( Figure 2h , i ) , and the phenotype is strongly affected by Dp110DN as well ( Figure 2j ) . The UAS-dFOXO-TM transgene appears to cause lethality even in the absence of a Gal4 driver , as we did not obtain viable transgenic lines with this construct . Furthermore , we examined the effects of nutrient deprivation on FOXO-expressing tissues . If nutrient availability is limited , FOXO should be more active in response to lowered insulin signaling . Indeed , we observed that the overexpression phenotypes of both hFOXO3a and dFOXO are enhanced under conditions of starvation . Drosophila larvae that are starved until 70 h after egg laying ( AEL ) die within a few days . But if the onset of nutrient deprivation occurs after they have surpassed the metabolic '70 h change' [40,41] , they survive and develop into small adult flies . We therefore subjected larvae expressing hFOXO3a or dFOXO ( under GMR control ) to either protein starvation ( sugar as the only energy source ) or complete starvation , starting 80-90 h AEL , and analyzed the effect on the adult's eyes . Both phenotypes ( Figure 2k , n ) were progressively exacerbated by protein starvation ( Figure 2l , o ) and complete starvation ( Figure 2m ) , the latter condition being accompanied by early adult or larval lethality , in the case of hFOXO3a or dFOXO , respectively . The resulting phenotypes are due to the FOXO transgenes , as wild-type control flies that have been starved during development display only a body-size reduction while maintaining normal proportions and normal eye structure . The dFOXO overexpression phenotype ( Figure 2i , j ) does not appear to be caused by the activation of any of the known cell -death pathways . Expression of the caspase inhibitors p35 or DIAP1 , or of p21 , an inhibitor of p53 -induced apoptosis [42] , and loss of eiger , which encodes the Drosophila homolog of tumor necrosis factor ( TNF ) [43] , did not suppress the eye phenotype ( data not shown ) . In agreement with our results , it was observed in a parallel study that the GMR-dFOXO overexpression phenotype is insensitive to caspase inhibitors , and is not accompanied by increased acridine-orange-detectable apoptosis in the imaginal disc [44] . It therefore remains unclear whether high levels of nuclear dFOXO induce a specific caspase -independent cell -death program or whether nuclear accumulation of overexpressed dFOXO leads to secondary necrosis in a rather nonspecific fashion . Furthermore , the necrotic eye phenotype does not reflect the phenotype observed following a complete block in insulin signaling . Loss-of-function mutations in insulin-signaling components reduce cell size and cell number but do not increase cell death in larval tissues [45,46] . In summary , our overexpression experiments are consistent with a model in which , under normal conditions , excess FOXO transcription factor is PHOSphorylated by dPKB and kept inactive in the cytoplasm . Under conditions of reduced insulin-signaling activity or nutrient deprivation , dFOXO or hFOXO3a protein translocates to the nucleus and induces growth arrest and necrosis .