TI - The growth-deficient phenotypes of Dlnr , chico , Dp110 and dPKB mutants are significantly suppressed by loss of dFOXO . AB - We performed genetic epistasis experiments to examine whether the growth phenotypes of DInr -signaling mutants are dependent on dFOXO function . For this purpose , we either generated double-mutant flies or investigated the double-mutant effect only in the head using the ey-Flp/FRT system . In contrast to the absence of a growth phenotype in single dFOXO mutant flies , lack of dFOXO significantly suppresses the growth-deficient phenotype observed in flies mutant for the insulin receptor SUBstrate ( IRS ) homolog chico ( Figure 4 ) . Flies mutant for chico are smaller because they have fewer and smaller cells [45] . Loss of one dFOXO copy dominantly suppresses the cell -number reduction in chico mutant flies without affecting cell size . The suppression is more pronounced when both copies of dFOXO are removed in a chico mutant background . In this situation , the chico small body-size phenotype is partially suppressed . Homozygous chico-dFOXO double-mutant flies have more , and even slightly smaller , cells than homozygous chico single mutants . It seems that removal of dFOXO accelerates the cell cycle at the expense of cell size in a chico background . We next asked whether dFOXO interacts with other components of the Drosophila insulin-signaling pathway . The ey-Flp/FRT system was used to generate heterozygous insulin-signaling mutant flies with heads homozygous for each mutation . Removal of DInr , Dp110 or dPKB leads to a characteristic 'pinhead' phenotype , which is substantially suppressed by the presence of a dFOXO loss-of-function allele on the same FRT chromosome as the insulin-signaling mutation . In all three cases , we observed a partial rather than a complete rescue of the tissue growth repression , consistent with the finding that dFOXO mutations affect only the cell -number aspect of the chico phenotype . Surprisingly , loss of dFOXO dramatically delays lethality in dPKB mutants . Complete loss of dPKB leads to larval lethality in the early third instar , but homozygous dPKB-dFOXO double mutants are able to develop into pharate adults of reduced size , most of which fail to eclose ( Figure 5l ) . The lethality associated with the complete loss of dPKB is therefore largely due to hyperactivation of dFOXO . We also observed that dFOXO interacts with the tumor suppressors dTSC1 and dPTEN . Tissue -specific removal of either gene from the head leads to a bighead phenotype ( Figure 5h , j ) . The dTSC1-/- bighead phenotype is enhanced by loss of dFOXO ( Figure 5i ) . This observation is consistent with the recently reported negative feedback loop between dS6K and dPKB . Mutant dTSC1 larvae have elevated levels of dS6K activity , which in turn downregulates dPKB activity [31] . This reduction in dPKB activity probably leads to enhanced activation of dFOXO , which in turn partially mitigates the overgrowth phenotype by slowing down proliferation . The dTSC1 phenotype can therefore be enhanced by loss of the inhibitory function of dFOXO . Unexpectedly , the dPTEN-/- bighead phenotype was slightly suppressed by dFOXO mutations ( Figure 5k ) . From the current model , it would be expected that in a dPTEN mutant dPKB activity is high and dFOXO is to a large extent inactive in the cytoplasm . Thus , removal of dFOXO function should have no effect on the dPTEN phenotype . At present , we can only speculate about possible explanations for this observation . In a parallel study , it has been shown that dFOXO can induce transcription of DInr [52] . It may be that in a dPTEN-mutant background dFOXO activates DInr expression in a negative-feedback loop . In this model , concomitant loss of dFOXO would alleviate the dPTEN overgrowth phenotype by lowering DInr levels . Another possible explanation is that dFOXO has additional functions when localized to the cytoplasm or during its nuclear export , such as interacting with other proteins . Loss of dFOXO might affect the function of interaction partners that have a role in dPTEN signaling . In summary , our epistasis analysis provides strong genetic evidence that dFOXO is required to mediate the organismal growth arrest that is elicited in insulin-signaling mutants .