TCER-1 Links Reproductive Signals to Longevity Figure 4. tcer-1 is required for the up-regulation of DAF-16/FOXO-target genes in glp-1 mutants. (A–X) Expression of DAF-16/FOXO- target genes in wild-type worms (left column), glp-1 mutants (middle column), and tcer-1; glp-1 mutants (right column). Animals were observed on day 2 of adulthood using transcriptional GFP reporters for individual genes. Bar graph representation of the data for each gene is shown in the far- right column. In each experiment, worms were classified into those with High (red bars), Medium (orange bars), and Low (green bars) GFP levels. ‘n’ signifies the total number of worms examined in two or three trials. Error bars represent the standard error of the mean. The elevated expression observed in glp-1 mutants was reduced in the case of gpd-2 (A–D), dod-8 (E–H), nnt-1 (I–L), K07B1.4 (M–P) and T21D12.9 (Q–T). Expression of sod-3 was elevated further (U–X). In (A–C), (M–O), and (Q–S), images were observed at 2006magnification. The insets in these panels show intestinal cells of the same strains imaged at 4006. In (E–G), (I–K), and (U–W), images of intestinal cells were observed at 4006. For more information about the expression patterns of these genes, and other DAF-16/FOXO-targets, see Table S3. doi:10.1371/journal.pgen.1000639.g004 TCER-1::GFP levels in daf-2(e1370) mutants. We found that daf-2 mutants did not have elevated TCER-1::GFP levels. In contrast, TCER-1 levels were significantly reduced (Figure 6H, compare with Figure 3C). TCER-1 levels were also reduced in long-lived eat- 2(ad1116) mutants (Figure S5), and we found that tcer-1 overex- pression further extended the lifespan of eat-2(ad1116) mutants (Figure S5; Table S2C). These data suggested that the specificity of TCER-1 for the reproductive pathway might be achieved, at least in part, by the regulation of its level in the animal. kri-1 controls the intestinal expression of tcer-1 How is TCER-1 up-regulation controlled by the reproductive system? To begin to address this question, we asked whether daf-16 itself, or genes that are known to influence DAF-16/FOXO nuclear localization in response to germline loss [13], were involved. We found that the daf-16(mu86) mutation did not alter TCER-1::GFP levels in wild-type or in germline-deficient animals (Figure 7A). Likewise, neither daf-9 nor daf-12 mutations, both of which partially inhibit DAF-16/FOXO nuclear localiza- PLoS Genetics | www.plosgenetics.org 6 September 2009 | Volume 5 | Issue 9 | e1000639