Figure 2. Inactivation of DAF-2::degron by the auxin-inducible degradation (AID) upregulated downstream reporters and caused dauer entry at any temperature.

(A) A schematic illustration of the DAF-2 signaling pathway. DAF-2 phosphorylates the transcription factors DAF-16 and SKN-1 through a cascade of kinases and sequesters them to the cytosol. Lower DAF-2 levels or activity leads to de-phosphorylation and translocation of DAF-16 and SKN-1 to the nucleus and expression of target genes like sod-3 and gst-4, respectively. (B) One hour exposure to auxin led to nuclear translocation of DAF-16::GFP in a concentration-dependent manner in DAF-2::degron; daf-16::gfp animals, but not in daf-16::gfp at the L4 stage. n > 94 from two (for daf-16::gfp) or three (for DAF-2::degron; daf-16::gfp) independent experiments. **: p < 0.01, ****: p < 0.0001. (C) Exposure to 1 mM auxin activates the reporter sod-3::GFP in DAF-2::degron; sod-3::gfp but not in animals that carry sod-3::gfp alone. L4 animals were exposed to 1 mM auxin or DMSO for 24 hr and GFP levels were scored (i.e., at day 1 of adulthood). n > 140 from two (for sod-3::gfp) or four (for DAF-2::degron; sod-3::gfp) independent experiments. ****: p < 0.0001. (D) Exposure to 1 mM auxin activates the reporter gst-4::GFP in DAF-2::degron; gst-4::gfp but not in animals that carry gst-4::gfp alone. L4 animals were exposed to 1 mM auxin or DMSO for 24 hr and GFP levels were scored (i.e., at day 1 of adulthood). n > 111 from two (for gst-4::gfp) or four (for DAF-2::degron; gst-4::gfp) independent experiments. ****: p < 0.0001. (E) Auxin treatment of DAF-2::degron affected development. Synchronized L1 put at low concentrations (1–50 μM auxin) shows reduced growth speed, and their offspring enters dauer. At high concentrations (100 and 1000 μM), the L1 animals arrest and enter the dauer stage after a few days. (F) Representative pictures of growth impairment caused by auxin-mediated degradation of DAF-2 in DAF-2::degron animals at different concentrations. Bar = 1 mm. (G) Dauer entry of DAF-2::degron animals at 1 mM auxin was temperature-independent, but the time needed for dauer entry is temporally scaled. To distinguish dauer animals from pre-dauer animals, they were treated for 15 min with 1% SDS. Only dauer animals survived SDS treatment. (H) Microscope pictures of DAF-2::degron animals after dauer entry (right column) and control animals (left column) kept for the same time at 15°C, 20°C, and 25°C. Control animals were on their second day of adulthood when the auxin-treated counterparts entered the dauer stage. Bar = 1 mm. For (B–D), see Source data 1 for raw data and statistics.

Figure 2—figure supplement 1. Inactivation of DAF-2::degron by the auxin-inducible degradation (AID) upregulated downstream reporters and caused dauer entry at any temperature.

(A) The nuclear translocation of DAF-16::GFP in DAF-2::degron; daf-16::gfp animals shown after 30 min or 1 hr of 1 mM auxin treatment. Bar = 100 µm. (B) Five-hour exposure to auxin led to further nuclear translocation of DAF-16::GFP in a concentration-dependent manner in DAF-2::degron; daf-16::gfp, but not in daf-16::gfp. n > 94 from two (for daf-16::gfp) or three (for DAF-2::degron; daf-16::gfp) independent experiments. ****: p < 0.0001. (C) Dauer alae of daf-2(e1370) and DAF-2::degron animals. Arrows indicate dauer alae-specific prongs. Bar = 100 μm. (D) Percentages of animals carrying red fluorescent beads in the intestine. n > 100 animals from three independent experiments. Error bar represents s.d. ****: p < 0.0001. (E) Images of control and dauer pharynxes. The pharynxes of DAF-2::degron animals on auxin were indistinguishable from other daf-2 mutant dauers. Black arrows indicate wild-type pharynx, and white arrows indicate dauer-typical pharynx constriction. Bar = 50 μm. For (A, D), see Source data 1 for raw data and statistics.

Figure 2—figure supplement 2. Depletion of DAF-2::degron at mid-L1 promoted daf-16-dependent dauer entry.

(A) Determination of the decision time point between dauer entry or continuation of normal development of DAF-2::degron animals at 25°C. The animals were synchronized for 2 days in M9 at 20°C, then put onto NGM OP50 plates at 20°C. Next, DAF-2::degron animals were put on plates supplemented with auxin after the indicated time on the x-axis and shifted to 25°C. The ratio of dauers and adult animals was determined 2 days later. Each time point was repeated four to seven times with n = 976–2022. The red line shows the average, black dots indicate single measurements. (B) Determination of the decision time point between dauer entry or continuation of normal development of DAF-2::degron animals at 25°C. The animals were synchronized for 2 days in M9 at 20°C, then put onto NGM OP50 plates at 20°C. Next, DAF-2::degron animals were put on plates supplemented with auxin after the indicated time on the x-axis and shifted to 20°C. The ratio of dauers and adult animals was determined 3 days later. Each time point was repeated four to seven times with n = 735–1791. The red line shows the average, black dots indicate single measurements. Note that dauers at 20°C tend to crawl off the plate, the graph is only an approximation, and there are likely more dauers on the plate. (C) Gonadal cell count of L1 animals after 24–25 hr on OP50. The dotted red line divides a younger population that enters dauer and an older population that continues development. The percentages indicate two arbitrarily divided groups with few and many gonadal cells to match the observation in panel A that approximately 50% of the animals continue development at 24–25 hr. The experiment was repeated five times with n = 289. (D) Simultaneous knockdown of degron-tagged DAF-16 in DAF-2::degron; daf-16::mNG::degron led to fertile adults. Bar = 2 μm. (E) 10 mM auxin causes embryonic lethality. The same amount of egg suspension was put on plates, and the number of offspring was counted several days later. This experiment was repeated four times for DAF-2::degron and two times for wild type. Error bar represents s.d. *: p < 0.05. For (A–C, E), see Source data 1 for raw data and statistics.