Our results indicate that proliferative homeostasis in high turnover tissues is limiting for Drosophila lifespan and highlight the importance of mechanisms that balance pro- and anti-mitotic activities. In the ISC lineage, this balance involves fine-tuning the activities of the pro-mitotic IIS and JNK signaling pathways to ensure appropriate supply of newly formed ISC daughter cells while limiting dysplasia. Accordingly, we observe moderately reduced intestinal proliferation rates in long-lived IIS mutants, as well as lifespan extension when IIS or JNK signaling are moderately reduced in the ISC lineage. This association between proliferative activity in the intestinal epithelium and lifespan is illustrated in . Strikingly, intestinal proliferation rates correlate with relative lifespan over a wide range of genotypes.
A model for the impact of regenerative capacity on lifespan.
Our results further show that the stress-protective components of the Foxo-regulated gene expression program are sufficient to maintain proliferative homeostasis, extending lifespan of the organism. Reduction of IIS activity, which extends lifespan in many organisms, is thus accompanied by the preservation of regenerative processes. While reducing IIS activity or activating Foxo in adipose tissue is sufficient to extend lifespan of flies, mice and worms 
, our results suggest that the anti-proliferative and stress-protective consequences of Foxo activation in high-turnover tissues also contribute to lifespan extension in IIS loss-of-function conditions. Interestingly, a tumor-suppressing role for Foxo in mice and C.elegans
has been reported 
, while Foxo regulates redox homeostasis in mouse HSCs 
. Reduced IIS activity thus optimizes somatic maintenance, metabolism and regenerative processes in complex metazoans, and all three physiologic consequences of IIS repression seem to contribute to achieve maximum lifespan.
It remains to be tested whether the lifespan extension commonly observed in flies exposed to dietary restriction (DR) is also associated with delayed intestinal dysplasia. Reduced IIS activity contributes to lifespan extension in DR conditions 
suggesting that reduced intestinal dysplasia might contribute to DR-induced lifespan extension.
Interestingly, the effects of JNK signaling on lifespan are more complex. JNK can extend lifespan when activated in the brain by repressing the expression of insulin-like peptides 
, thus systemically repressing IIS activity 
. Our findings reported here, however, show that JNK activation in the ISC lineage can have deleterious effects and needs to be limited to ensure longevity. Such pleiotropic consequences of JNK have also been reported in other contexts and have significant implications for the development of therapies targeting this pathway 
The importance of anti-oxidant Foxo target genes in regulating proliferative homeostasis highlights the challenging environment to which the intestinal epithelium is exposed. Apart from extraneous toxins and oxidants, the intestinal epithelium also mounts strong oxidative responses to inflammation, potentially exposing ISCs and daughter cells to high levels of oxidative stress 
. Our results show that over-expressing stress-protective proteins in the ISC lineage is sufficient to limit and optimize cellular responses to these challenges, thus preserving intestinal homeostasis longer (but not indefinitely, as a significant fraction of these animals do develop dysplasia at older ages).
Intestinal dysplasia is caused by over-proliferation of ISCs in concert with mis-differentiation of ISC progeny, and in long-lived animals both processes are prevented. Due to technical limitations of the Gal4 drivers used, however, we cannot exclude that the expression of stress-protective genes, or of IIS or JNK repressors with esgGal4 or 5961GS affects primarily the differentiation process of EBs rather than the ISC itself. While the inability of IIS mutant clones to grow, and the significant reduction in the number of pH3+ cells in IIS and JNK loss-of-function conditions and in Hsp68 and Jafrac1 over-expressing flies, demonstrates that ISC proliferation is indeed influenced by these manipulations, it is conceivable that this effect might be mediated by indirect, non-cell-autonomous limitation of ISC proliferation by EBs in these conditions. Such a feedback control of ISC division would be interesting, and further studies are needed to test this possibility.
The pattern of esgGal4 and of 5961GS expression further requires considering effects of IIS and JNK activities in other tissues on lifespan: While we can exclude the testes and salivary glands as sources of the observed effects (lifespan effects are observed in both males and females using esgGal4, expression of Jafrac1 and Hsp68 in salivary glands has no effect on lifespan, and 5961GS is not expressed in salivary glands), we cannot currently exclude a contribution of malpighian tubule stem cells. These cells also appear to respond to proliferative signals such as Hep or InR over-expression, and over-proliferate in stressed flies (GFP-labeled cells accumulate in malpighian tubules in these flies; Biteau, unpublished), but the exact mechanism of regeneration and a potential age-related dysplastic phenotype in this tissue remain unexplored. Importantly, a contribution of this somatic stem cell population to the lifespan effects reported here would further support our model of the importance of proliferative homeostasis in high-turnover tissues for Drosophila lifespan.
It is interesting that using the weaker 5961GS driver, lifespan extension in IIS and JNK loss-of-function conditions is only observed in females. This sexual dimorphism might be a consequence of a slight difference in driver activity between the sexes (no significant difference in driver activity can be observed, however), or might indicate selective sensitivity of females to intestinal dysplasia. Interestingly, intestinal turnover rates in females are higher than in males 
, indicating a potential reason for such a selective sensitivity. Accordingly, lifespan extension by esg-mediated expression of Hsp68 and Jafrac1 is also stronger in females than in males.
Based on the highly conserved regulation of regenerative processes in flies and vertebrates 
, our findings suggest that interventions that focus on maintaining regenerative capacity by improving stem and progenitor cell stress-protection hold significant promise for slowing aging in higher organisms, including humans. Interestingly, vertebrates seem to have evolved more efficient and extensive cell autonomous anti-proliferative mechanisms in stem cells than flies 
, resulting in longer-lasting maintenance of homeostasis in high-turnover tissues. The rapid decay of intestinal homeostasis in flies indicates that such control mechanisms have not been acquired in these short-lived animals, yet our data also suggest the potential for active control of proliferation rates in the intestinal epithelium by systemic insulin-like peptide levels. Interestingly, the regulation of stem cell proliferation by IIS and Foxo is conserved in mammalian systems, suggesting that similar systemic control of stem cell proliferation could be harnessed to regulate regenerative capacity and lifespan in vertebrates 
. How the maintenance of intestinal homeostasis is influenced by environmental parameters that affect systemic IIS activity is an interesting subject of further studies.