Germline removal causes increased expression of NHR-49 under regulation of DAF-16 and TCER-1.

A–E: Elevation of NHR-49::GFP in germline-less animals by DAF-16 and TCER-1. NHR-49::GFP fluorescence observed in wild type (N) worms (A) (n = 219) and glp-1 (B) (n = 383), daf-16;glp-1 (C) (n = 175) and tcer-1;glp-1 (D) (n = 267) mutants. The increased GFP in glp-1 is visible in intestinal nuclei (compare A and B) and is abolished in daf-16;glp-1 animals (C). tcer-1;glp-1 mutants exhibit high expression in some gut cells but no GFP in others (D). The bar graph in E shows the quantification of these data obtained from day 2 young adults of each strain classified into those with high (green), medium (peach), and low (gray) GFP. In the tcer-1;glp-1 bar, the high GFP class is shown as spotted green to indicate that these worms showed high but mosaic intestinal expression. F: Selective effect of daf-16 and tcer-1 RNAi on NHR-49::GFP in glp-1 mutants. NHR-49::GFP fluorescence in wild-type animals (N2, solid bars) and in glp-1 background (striped bars) observed in day 2 adults. Worms were grown on bacteria containing empty control vector (EV) or those expressing dsRNA targeting daf-16, tcer-1 or nhr-49. GFP classification is the same as in E. daf-16 or tcer-1 RNAi treatments suppress the increased GFP seen in glp-1 mutants, but not in wild-type worms (both strains were tested simultaneously). In the N2 background, n = 175, 113, 136 and 64, respectively for EV, daf-16, tcer-1 or nhr-49 RNAi, respectively. In the glp-1 background, n = 206, 146, 202 and 81, respectively for EV, daf-16, tcer-1 or nhr-49 RNAi, respectively. In E and F, ‘n’ signifies the total number of worms examined in three-to-five independent trials. G, H: The control of nhr-49 mRNA levels by DAF-16 and TCER-1 in fertile vs. germline-less adults. Q-PCR analysis used to compare the mRNA levels of nhr-49 between wild type (N2), glp-1, daf-16;glp-1 and tcer-1;glp-1 day 2 adults grown under similar conditions (G) as well as day 2 adults of N2, nhr-49, daf-16 and tcer-1 single mutants (H). Strains are represented on the X-axis and relative expression levels are on the Y-axis. The asterisks represent the statistical significance of the differences in expression in an unpaired, two-tailed t-test with P values 0.05 (*) and 0.005 (**). Error bars in E–H represent the standard error of the mean. In G, the difference between glp-1 and tcer-1;glp-1 was statistically significant in four of seven biological replicates (each with three technical replicates), but did not achieve significance when data from all the trials were combined.