1 (4.1 kb) and 1.6 kb 59 upstream promoter sequence were amplified using the following primers: Forward- 59 GCA AGT ATT TGA GCA CTA CTG TCA AGG GC 39, Reverse- 59 AGT CGA CCT GCA GGC ATG CAA GCT TTG CTT TCT GCG ATC CCG CTC 39. The gfp construct (1.9 kb) was amplified from the vector pPD95.75 using the following primers: Forward- 59 AGC TTG CAT GCC TGC AGG TCG ACT 39, Reverse- 59 AAG GGC CCG TAC GGC CGA CTA GTA GG 39. The complete gfp fusion construct (7.6 kb) was amplified by pooling the products of the above two PCRs and using the following primers: Forward: 59 GCC GGT CAT GCT CTT CTT CAA C 39, Reverse- 59 GGA AAC AGT TAT GTT TGG TAT ATT GGG AAT GTA TTC TG 39. The co-injection marker Podr-1::rfp was amplified from the plasmid pCF155. Transgenic strains To generate the TCER-1::GFP expressing worms, Ptcer-1::tcer- 1::gfp was injected as described earlier [63] at 50 ng/ml or 20 ng/ ml, along with the co-injection marker Podr-1::rfp (75 ng/ml), into N2 worms (see Table S6). As a control, Podr-1::rfp alone was injected into N2 worms (75 ng/ml). The resulting transgenic control strains (CF2144, CF2145) had mean lifespans that were the same as N2. Lifespan analysis Lifespan assays in general were conducted as previously described [64]. For glp-1 mutant lifespan assays, eggs were incubated at 20uC for 2–6 hrs, transferred to 25uC to eliminate germ cells, then shifted back to 20uC on day 1 of adulthood for the rest of their lifespan. All other lifespan assays were performed at 20uC. In all cases, the L4 stage was counted as day 0 of adulthood. In all experiments with TCER-1::GFP, worms were examined for Podr-1::rfp co-injection marker under a Leica MZ16F stereomi- croscope (Wetzlar, Germany) on day 0 and isolated for lifespan experiments. The non-transgene carrying siblings were used as negative controls in the same experiment. glp-1 mutant strains used in lifespan assays were completely sterile. Fertile strains were transferred every other day to fresh plates until progeny production ceased. Animals that crawled off the plate, exploded, bagged, or became contaminated were censored. Stata 10.0 and 8.2 (Stata Corporation, Texas, USA) and (for some lifespans) Statview 5.0.1 (SAS) softwares were used to calculate mean life spans and perform statistical analyses. P values were determined using log-rank (Mantel-Cox) statistics. RNAi experiments We screened a C. elegans chromosome II RNAi library to identify RNAi clones that suppressed the extended lifespan of glp-1 mutants. Details of a similar screen were described earlier [13]. All RNAi experiments were performed as described previously [65,66]. In general, RNAi clones were inoculated overnight at 37uC in LB medium containing 10 mg/ml tetracycline and 100 mg/ml carbenicillin, and seeded onto NG-carbenicilin plates supplemented with 0.1 M IPTG. For all lifespan experiments, and the RNAi screen, worms were exposed to RNAi clones from hatching. All RNAi clones were confirmed by sequencing (M13- forward primer) and upon start of every experiment, by PCR (T7 primers). Clones were obtained from the libraries described previously [65,66]. pAD12 (empty vector) was used as the negative control, and pAD43 (daf-16 RNAi) as the positive control for the screen and for individual lifespans [43]. The tcer-1 RNAi clone also targets a partial duplication of tcer-1, the gene ZK1127.6, whose predicted protein product contains only FF domains. Transcrip- tional and full-length translational GFP-tagged reporters for TCER-1 Links Reproductive Signals to Longevity ZK1127.6 showed no basal expression or induction on germline ablation suggesting that it is a non-functional duplication of tcer-1 (ZK1127.9). Laser ablations Laser ablations of germ-cell precursors (Z2, Z3) were performed using a Zeiss Axiophot with a laser attachment (Photonics Instruments, USA) as described earlier [67]. Briefly, eggs were transferred to fresh plates and left at 20uC for 1–3 hrs. Hatched L1 larvae were mounted on slides with 2% Agarose pads containing 1.5 mM Sodium Azide anesthetic. As intact controls, worms were subjected to all the steps of this process except for ablation. Ablated worms (and controls) were removed from the slide and grown at 20uC until required for GFP assays or lifespan analysis. GFP assays Eggs of worms carrying the Pdaf-16::daf-16::gfp reporter construct and the different DAF-16/FOXO-target gene reporters (in wild-type, glp-1 mutant and tcer-1; glp-1 mutant backgrounds) were incubated at 20uC for 2–6 hrs, transferred to 25uC to eliminate germ cells, then shifted back to 20uC on day 1 of adulthood. GFP assays were conducted on day 2 of adulthood, using a Leica MZ16F (Wetzlar, Germany) stereomicroscope with standard fluorescence filter sets. All assays were performed blind after initial familiarization of GFP levels in control plates by the experimenter. Microscopy All fluorescence images were captured using a Retiga EXi Fast1394 CCD digital camera (QImaging, Burnaby, BC, Canada) attached to a Zeiss Axioplan 2 compound microscope (Zeiss Corporation, Jena, Germany). Openlab 4.0.2 software (Improvi- sion, Coventry, U.K.) was used for image acquisition. GFP assays were conducted on a Leica MZ16F (Wetzlar, Germany) stereomicroscope with fluorescence filter sets or (in the case of Pnnt-1::gfp) the Zeiss Axioplan 2 compound microscope mentioned above. Preliminary image processing was performed using Photo- shop 10 (Adobe Creative Suite 3, USA). Q-PCRs Total RNA was isolated from synchronized populations of approximately 15,000 day 1 daf-2 and tcer-1; daf-2 mutants. For comparing, tcer-1(2) and tcer-1(2); tcer-1 OE strains, worms were picked manually for RNA isolation. Eggs were transferred to fresh plates. Day 0 (L4) transgenic animals carrying tcer-1(2); tcer-1 OE were isolated using a Leica MZ16F stereomicroscope with standard fluorescence filters. 2,000–5,000 worms were picked manually for RNA isolation for each biological replicate. A similar number of tcer- 1 mutants were also collected manually. Both strains were allowed to grow for 24 hrs and on day 1 of adulthood used for RNA isolation. Total RNA was extracted using TRIzol reagent (Invitro- gen) and purified using Qiagen RNAeasy Mini Kit. cDNA was generated using Protoscript First Strand cDNA Synthesis Kit (New England Biolabs). SybrGreen real-time Q-PCR reactions were performed on an Applied Biosystems 7300 Real Time PCR System. The primers used in this study are listed in Table S7. Supporting Information Figure S1 Effect of tcer-1 RNAi on TCER-1::GFP expression. Found at: doi:10.1371/journal.pgen.1000639.s001 (1.37 MB TIF) Figure S2 Effect of tcer-1 overexpression on the lifespan of germline-precursor (Z2, Z3) ablated worms. PLoS Genetics | www.plosgenetics.org 11 September 2009 | Volume 5 | Issue 9 | e1000639