TCER-1 Links Reproductive Signals to Longevity Figure 2. A TCER-1::GFP fusion protein is widely expressed and rescues the shortened lifespan of tcer-1; glp-1 mutants. (A–C) tcer-1 expression in adult somatic tissues. TCER-1::GFP is expressed in nuclei of adult neurons (‘n’, head neurons shown by arrow in (A)), hypodermis (‘h’, (B)), muscles (‘m’, arrow in (C)), and intestinal cells (‘i’, arrowhead in (C)). The asterisk in (A) indicates expression of the co-injection marker, Podr- 1::RFP, in olfactory neurons. (D) Rescue of the shortened lifespan of tcer-1(2); glp-1(2) mutant by the TCER-1::GFP fusion protein (tcer-1 OE). glp- 1(2): m = 31.160.7, n = 204/241; tcer-1(2); glp-1(2): m = 23.460.5, n = 81/119, P vs. glp-1(2),0.0001; tcer-1(2); glp-1(2); tcer-1 OE: m = 32.460.3, n = 111/116, P vs. glp-1(2) = 0.5, P vs. tcer-1(2); glp-1(2),0.0001; tcer-1(2): m = 19.160.2, n = 50/64. See Table S1. C for additional trials of this experiment. doi:10.1371/journal.pgen.1000639.g002 statistically significant increase in expression when tcer-1 was overexpressed (Figure 5A). In addition, the lifespan extension obtained by tcer-1 overexpression required the presence of wild- type daf-16 (Figure 5B; Table S4). Together these findings suggest that tcer-1 overexpression may extend the lifespan of wild-type, fertile animals by stimulating expression of DAF16/FOXO-target genes that are induced in response to germline loss. tcer-1 is not required for DAF-16/FOXO to extend lifespan in insulin/IGF-1 pathway mutants Since daf-16 is also required for the doubling of lifespan produced by reducing insulin/IGF-1 signaling [16,39], we examined the role of tcer-1 in this longevity pathway too. We found that neither tcer-1(tm1452) nor tcer-1 RNAi suppressed the extended lifespans of the insulin/IGF-1-receptor mutants daf- 2(e1370) or daf-2(e1368) (Figure 6A and 6B; Table S5). Therefore, TCER-1 is not part of the insulin/IGF-1 pathway. Consistent with this, we found that TCER-1 was not required in daf-2(e1370) mutants for the expression of any of the DAF-16/FOXO targets that were tcer-1-dependent in the reproductive pathway. The tcer- 1(tm1452) mutation either had no effect, or in some cases produced a small increase in expression (Figure 6C and 6D). Removing the germ-cell precursors of daf-2 mutants further doubles their already long lifespan [1,4]. Similarly, we found that overexpressing tcer-1 in daf-2(e1370) mutants extended their long lifespan by another 6–12% (Figure 6E; Table S2C). In contrast, we did not observe a further extension of lifespan when tcer-1 was overexpressed in germline-depleted worms (Figure 3F, Figure S2; Table S2C). These observations support the model that tcer-1 functions in the germline longevity pathway but not the insulin/ IGF-1 longevity pathway. Not all longevity pathways in C. elegans are daf-16 dependent. For example, the longevity response to caloric restriction caused by the feeding-defective mutant eat-2(ad1116) [40] is daf-16 independent, as is the longevity response to inhibition of mitochondrial respiration caused by isp-1(qm150) [41–43]. We found that tcer-1 was not required for either of these mutations to extend lifespan (Figure 6F and 6G; Table S5). These findings further reinforced the interpretation that TCER-1 is not a general longevity factor but instead extends lifespan specifically in response to reproductive signals. The ability of TCER-1 overexpression to activate DAF-16/ FOXO-dependent gene expression and extend lifespan suggested that the up-regulation of TCER-1 is a key switch-point by which loss of the germ cells triggers a longevity response. This up-regulation could also be part of the mechanism by which TCER-1 activity is specifically directed towards this pathway and not towards the insulin/IGF-1 pathway. To test this possibility, we examined PLoS Genetics | www.plosgenetics.org 4 September 2009 | Volume 5 | Issue 9 | e1000639