ins-7 influences targets of the DAF-2 insulin/IGF-1 pathway. (a) Wild-type animals expressing Pdaf-16::daf-16::gfp cultured on control RNAi bacteria exhibit diffuse DAF-16::GFP localization. (b) Subjecting these animals to ins-7 RNAi increases DAF-16::GFP nuclear localization (arrows). (c) Wild-type animals expressing Psod-3::gfp and grown on control RNAi bacteria exhibit low levels of GFP in the body and higher levels in the head region (light areas). (d) ins-7 RNAi increases Psod-3::gfp expression throughout the body.

Intestinally expressed ins-7 shortens lifespan. (a) Mean lifespan of wild-type (N2) = 17.0 ± 1.2, non-transgenic control = 16.3 ± 1.3, and Pges-1:ins-7 = 13.9 ± 0.7, P = 0.0096 (non-transgenic vs. Pges-1::ins-7). (b) Mean lifespan of Pges-1::gfp = 23.0 ± 0.6, Pges-1:ins-7 = 20.1 ± 0.6, P = 0.0007. See SI Table 1 for additional trials and statistical analysis. (c) Intestinally expressed ins-7 does not affect daf-16 expression. Quantitative RT-PCR of intestinally expressed daf-16 in wild-type and Pges-1::ins-7 backgrounds. Results are from two biological repeats; error bars represent standard deviation.

Regulation of Pins-7::gfp expression. I, intestinal cell; N, neurons. (a–g) Pins-7::gfp expression under different conditions. (a) Typical young adult, exhibiting neuronal but not intestinal gfp expression; (b) young adult expressing Pins-7::gfp in intestinal cells; (c) typical day 8 adult, exhibiting intestinal as well as neuronal gfp expression (d) typical Pins-7::gfp; daf-2(e1370) mutant, with no intestinal expression (see h); (e) typical Pins-7::gfp; daf-16(mu86) mutant, with pronounced intestinal expression; (f) typical Pins-7::gfp animal subjected to daf-18/PTEN RNAi; and (g) typical starved wild-type animal, with sharply reduced ins-7::gfp expression; (h) percentage of wild-type (n = 100) and mutant Pins-7::gfp worms (n = 84 for daf-16, n = 102 for daf-2) expressing GFP fluorescence in intestinal cells. Standard error of the proportion (SEP). A Z test for two proportions shows that daf-2 and daf-16 mutants are each significantly different from the vector control at the 99% confidence level (*).

Inhibiting DAF-16 regulation of ins-7 decreases the ability of intestinal DAF-16 to affect DAF-16 activity in other tissues. All animals carry Psod-3::gfp. (a) Schematic illustration of the animal's head. (b–d) The upper edge of the animal's head is indicated by the dashed yellow line, and arrowheads point to muscle nuclei. (b) Control wild-type animals express Psod-3::gfp in the pharynx but not in surrounding head tissue. (c) Increasing intestinal DAF-16 levels by using Pges-1::daf-16 increases Psod-3::gfp expression throughout the head. [This increased Psod-3::gfp expression requires increased DAF-16 activity in nonintestinal tissues, because it is not seen in a daf-16(−) background (14).] (d) Constitutive expression of ins-7 from the ges-1 intestinal promoter (Pges-1::ins-7) reduces the ability of intestinal Pges-1::daf-16 to stimulate Psod-3::gfp expression in the head. (e) Semiquantification for b–d (n ≥ 109). (f and g) Pges-1::daf-16 animals with (*) and without Pges-1::ins-7. Animals carrying Pges-1::ins-7 have red intestinal fluorescence from the coinjected Pges-1::rfp construct (see Materials and Methods and SI Fig. 6).