Figure 1. Reduction of tcer-1 function suppresses the lifespan extension produced by germline ablation. (A) Effect of tcer-1 RNAi on the lifespan of germline-defective mutants. glp-1 (germline- defective) mutants grown on bacteria containing control empty vector (m = 27.061.1, n = 112/114), daf-16 RNAi bacteria (m = 15.260.2, n = 110/113, P vs. control,0.0001), and tcer-1 RNAi bacteria (m = 18.160.4, n = 96/114, P vs. control,0.0001). Wild-type (N2) worms grown on bacteria containing control empty vector shown for comparison (m = 19.560.3, n = 81/99, P vs. glp-1(2),0.0001). (B) Effect of tcer-1 RNAi on the lifespan of wild-type (N2) worms. N2 worms grown on bacteria containing control empty vector (m = 19.960.6, n = 49/72), daf-16 RNAi bacteria (m = 16.760.5, n = 62/72, P vs. control,0.0001), and tcer-1 RNAi bacteria (m = 18.760.5, n = 66/72, P vs. control = 0.2, P vs. daf-16 RNAi = 0.002). (C) Effect of the tcer-1(tm1452) mutation on the lifespan of glp-1 mutants and wild-type worms. glp-1(2): m = 32.960.3, n = 233/258; tcer-1(2); glp-1(2): m = 21.860.5, n = 77/115, P vs. glp- 1(2),0.0001; wild type (N2): m = 18.960.5, n = 60/121; tcer-1(2): m = 19.060.4, n = 96/110, P vs. N2 = 0.84. Additional repeats of these experiments are shown in Table S1. doi:10.1371/journal.pgen.1000639.g001 relatively resistant to RNAi in C. elegans, this finding suggests that intestinal TCER-1 function, like intestinal DAF-16/FOXO, is likely to be particularly important for promoting longevity. TCER-1 Links Reproductive Signals to Longevity tcer-1 overexpression extends the lifespan of wild-type, fertile worms The finding that loss of the germline elevated TCER-1 levels raised the possibility that TCER-1 might play a rate-limiting step in this longevity pathway. To test this, we asked whether overexpressing tcer-1 in wild-type animals might bypass the requirement for germline loss and extend the lifespan of fertile animals. We found that tcer-1 overexpression produced a modest but consistent increase in the lifespan of wild-type worms (average mean lifespan extension ,15%; Figure 3D and 3E; Table S2A and S2B). We also found that, in keeping with its role in the reproductive pathway, tcer-1 overexpression did not produce any further increase in the extended lifespans of worms whose germ cells had been eliminated either by the glp-1 mutation (Figure 3F; Table S2C) or by laser ablation (Figure S2; Table S2C). tcer-1 is required for expression of DAF-16/FOXO-target genes in response to germline removal To understand why tcer-1 was required for loss of the germ cells to extend lifespan, we asked whether tcer-1 might impact DAF-16/ FOXO function. DAF-16/FOXO undergoes nuclear accumula- tion primarily in intestinal cells following germline removal and we first explored the possibility that tcer-1 was required for this step. However, we found that in tcer-1(tm1452) worms lacking a germline, DAF-16/FOXO accumulated normally in intestinal nuclei (Figure S3). Curiously, we noticed a small increase in the levels of DAF-16::GFP, largely in the cytoplasm of intestinal cells, in these animals (Figure S3; see Discussion). Next, we examined the effect of tcer-1(tm1452) on DAF-16/ FOXO’s transcriptional output following germline loss. Many putative (direct or indirect) DAF-16/FOXO target genes have been identified [12,22–25], but only a few, sod-3, dod-8, gpd-2 and nnt-1, are known to be up-regulated by germline removal [3]. To increase the repertoire of such genes, we obtained available transgenic worm strains expressing GFP-tagged transcriptional reporters for additional putative DAF-16/FOXO targets [38] (see Table S3). Germline precursors of each of these strains were laser ablated, and the animals were examined as adults for changes in expression. We found several genes whose expression was sharply elevated upon germline removal, including K07B1.4, T21D12.9, F52H3.5, aat-1 and pssy-1 (see Table S3). We then asked whether the up-regulation of these new genes, as well as the known daf-16-dependent germline-regulated genes, required tcer-1. We found that this was the case for six of the nine genes we examined. The up-regulation of gpd-2, dod-8, nnt-1, K07B1.4, and T21D12.9 was strongly attenuated in tcer-1; glp-1 mutants (Figure 4A–4T) and pssy-1 expression was moderately reduced (data not shown). In contrast, aat-1 expression remained unaltered in two trials and showed a small increase in expression in one (data not shown). These findings suggest that loss of TCER-1 may eliminate at least a portion of DAF-16/FOXO’s transcrip- tional response to germline depletion. Unexpectedly, sod-3 and F52H3.5 levels were further increased upon tcer-1 inactivation (Figure 4U–4X and Figure S4, respectively; see Discussion). tcer-1 overexpression up-regulates DAF-16/FOXO-target genes As tcer-1 reduction of function reduced the expression of many DAF-16/FOXO-target genes, we asked if TCER-1 overexpression would increase the expression of the same DAF-16/FOXO-target genes that were downregulated in tcer-1(2); glp-1(2) mutants. Using quantitative RT-PCR (Q-PCR), we found that several such genes (dod-8, gpd-2, nnt-1, K07B1.4 and pssy-1) showed a modest but PLoS Genetics | www.plosgenetics.org 3 September 2009 | Volume 5 | Issue 9 | e1000639