ire-1 and xbp-1 contribute to the longevity of daf-2(−) animals. (A) Reduction of ire-1 activity by the null mutation ok799 shortened the lifespan of daf-2(e1370) mutants more than it shortened the lifespan of wild type. (B) The xbp-1(zc12) mutation shortened the lifespan of daf-2(e1370) mutants more than it shortened wild-type lifespan. (C) The xbp-1(zc12) mutation did not further shorten the lifespan of ire-1(−); daf-2(−) double mutants, suggesting that these genes function in a common pathway. [Compare survival curves of ire-1(−); daf-2(−) and ire-1(−); daf-2(−) xbp-1(zc12) mutants.] Note that xbp-1(zc12) mutation shortened the lifespan of daf-2(e1370) mutants, but not as much as did an ire-1(ok799) deletion. [Compare survival curves of ire-1(−); daf-2(−) and daf-2(−) xbp-1(zc12) mutants.] As xbp-1(zc12) is likely to eliminate xbp-1 function, this finding implies that ire-1 contributes to the longevity of daf-2 mutants in part through an xbp-1–independent pathway. For additional lifespan data, see Table S1. Also, see Fig. S1 for data showing that other ER stress–response genes, namely abu-11, pek-1, and atf-6, are not involved in this pathway.

 The ire-1/xbp-1 pathway is set at a lower level in daf-2(−) mutants. (A) Bar graph representing the mean ratio of spliced/unspliced xbp-1 transcripts, multiplied by a factor of 100, in three independent biological experiments. Control lane (yellow bar) presents the xbp-1 transcripts ratio in wild-type animals treated with tunicamycin. The rest of the bars present steady-state xbp-1 transcripts ratio in day-1 wild-type, ire-1, daf-2, and daf-16; daf-2 animals. These animals have not been treated with tunicamycin (blue bars). Error bars represent SEM. Asterisks mark Student t test values of P < 0.01. See Fig. S2 for data showing a representative RT-PCR experiment. (B) Representative fluorescence micrographs (100-fold magnification) of day-3 adults harboring an integrated Phsp-4::gfp transgene in a wild-type or daf-2(e1370) background. daf-2 mutants expressed lower levels of the Phsp-4::gfp transgene than did wild type. wild type, n = 9, relative mean fluorescence intensity (m) = 1 ± 0.14; daf-2(e1370), n = 10, m = 0.34 ± 0.07, P = 0.0012. (C) daf-2(e1370) mutants expressed less Phsp-4::gfp transgene than did wild type even upon heat shock. Phsp-4::gfp expression at 20 °C or upon 3 h of heat shock at 30 °C. Arrows point at anterior and posterior ends of the animals.

 daf-2(−) mutants are resistant to ER stress. Eggs from wild-type or daf-2(e1370) animals containing xbp-1(zc12), ire-1(ok799), pek-1(ok275), or atf-6(ok551) mutations were grown in the presence of 0, 2, or 5 μg/mL tunicamycin (corresponding to the blue, red, and off-white bars in the graph). Percentage of eggs that developed into mature adults was scored. Student t test values were calculated between daf-2(+) and daf-2(−) backgrounds. Each experiment was repeated independently with similar effects. Each strain was scored on three independent plates. Error bars represent SEM for repeat plates within the experiment. Asterisks mark Student t test values of P < 0.01.

 dox-1 promotes longevity, but not ER stress resistance, in daf-2 mutants. (A) Reduction of dox-1 activity by RNAi shortened the lifespan of daf-2(mu150) mutants more than it shortened the lifespan of daf-2(+) animals. Animals were in a fer-15(b26); fem-1(hc17) background to prevent reproduction. Similar results were seen in daf-2(e1370) mutants (Table S1). (B) Reduction of dox-1 did not increase the set-point of the ire-1/xbp-1 branch of the UPR in daf-2 mutants. Bar graph showing the mean intensity of day-2 adults expressing a Phsp-4::gfp transgene. daf-2(e1370), n = 13, relative mean fluorescence intensity (m) = 21.9 ± 2.2; dox-1(RNAi); daf-2(e1370), n = 17, m = 23.3 ± 2.3, P = 0.677. See Fig. S4 for representative micrographs and wild-type data. (C) dox-1 expression was up-regulated in daf-2 mutants. Bar graph showing the fraction of animals expressing high levels of Pdox-1::gfp. Day-1 adults of each strain were classified as expressing high or low GFP levels (Fig. S4). The fraction of animals expressing high levels of Pdox-1::gfp was 1.35 fold higher in transgenic daf-2(e1370) mutants than in wild-type animals (P = 0.037), but was not significantly increased in daf-2(e1370) xbp-1(tm2457) double mutants. The fraction of animals expressing high levels of Pdox-1::gfp was 1.52 fold higher in transgenic daf-2(e1368) mutants than in wild-type animals (P = 0.007), but was not increased in daf-2(e1368) xbp-1(tm2457) double mutants. The fraction of animals expressing high levels of Pdox-1::gfp was higher by 1.8 fold in transgenic daf-2(e1370) mutants than in wild-type animals (P = 0.026), but was not significantly increased in daf-16(mu86); daf-2(e1370) double mutants. Error bars represent SEM of at least two independent trials. Asterisks mark Student t test values of P < 0.05.

 daf-16 contributes to the ER stress resistance in daf-2 mutants. (A) Representative fluorescence micrographs of day-1 adults harboring an integrated Phsp-4::gfp transgene in a daf-2(e1370) or daf-16(mu86); daf-2(e1370) background. daf-16 deletion (and inactivation of daf-16 by RNAi) increased the level of Phsp-4::gfp in daf-2(−) mutants. daf-2(e1370), n = 20, relative mean fluorescence intensity (m) = 1 ± 0.06; daf-16(mu86); daf-2(e1370), n = 28, m = 2.70 ± 0.2, P < 0.0001. Error bars represent SEM. Arrows point at anterior and posterior ends of the animals. (B) Tunicamycin resistance developmental assay. Eggs from wild-type, daf-2(e1370), and daf-16(mu86); daf-2(e1370) animals were grown in the presence of 0 or 5 μg/mL tunicamycin (corresponding to the blue and off-white bars in the graph). Percentage of eggs that developed into mature adults was scored. Student t test values were calculated relative to daf-16(−); daf-2(−). Asterisks mark Student t test values of P < 0.05. This experiment was repeated independently with similar effects. (C) Model for how XBP-1 extends lifespan, increases ER stress resistance, and also lowers XBP-1 levels when insulin/IGF-1 signaling is reduced. In daf-2 mutants, multiple transcription factors, such as DAF-16/FOXO, HSF-1, ELT-3, and SKN-1 are activated. These can act in a combinatorial fashion with XBP-1 to turn on new longevity genes and new ER stress response genes (both in purple), which, unlike the “normal” ER stress–protective genes (in red), cannot be induced by XBP-1 alone. This combinatorial control results in a wider array of longevity and ER stress–protective genes, both of which contribute to the longevity of daf-2 mutants. In addition, as part of a negative feedback loop, the wider array of ER stress protective genes expressed in daf-2 mutants by xbp-1 can better reduce the level of unfolded proteins in the ER and improve ER homeostasis. Improved ER homeostasis would, in turn, dictate a lower basal activity level of the ire-1/xbp-1 pathway. In this model, the enhanced ER stress resistance of daf-2 mutants is completely xbp-1–dependent, and yet, because the ER stress response is more efficient, the set point of XBP-1 and its normal downstream targets is lower than in wild type.