Figure 5

DAF-16 and PHA-4 function in parallel during dauer recovery. (A-E) Transcriptional regulation of pha-4 expression. (A) pha-4 gene structure (adapted from [49]). (B) The potential DAF-16 binding site in the second intron of pha-4 and flanking sequences are conserved in the corresponding intron of C. briggsae. The potential DAF-16 binding site is shown in red, capital letters. (C) A daf-2(e1370) L4 larva carrying a pha-4::gfp reporter with the potential DAF-16 binding site shows gfp expression in head neurons. (D) A daf-2(e1370) L4 larva carrying a pha-4::gfp reporter with the potential DAF-16 binding site mutated shows diminished gfp expression in head neurons. Two transgenic lines were made from each construct. 20 animals were examined from each transgenic line. Over 50% of animals showed representative expression patterns as shown in this panel. Pictures were taken using the same exposure time. (E) Increased pha-4 transcription upon starvation. Semi-quantitative RT-PCR experiments were performed to assess pha-4 transcript levels in well-fed N2 L3 larvae with low DAF-16 activity (lane 1); starved N2 L3 larvae with high DAF-16 activity (lane 2); well-fed daf-16(mgDf47) L3 larvae and starved daf-16(mgDf47) L3 larvae with no DAF-16 activity (lanes 3 and 4). RT-PCR experiments were performed three times with consistent results using three independent RNA preparations. (F) The daf-16 mutation and pha-4 RNAi have additive effects on dauer recovery. A daf-16(mgDf47); daf-7(e1372) double mutant treated with pha-4 RNAi showed more severe dauer recovery defects compared to either the daf-16(mgDf47); daf-7(e1372) mutant control or the daf-7(e1372) mutant treated with pha-4 RNAi. Percentages of dauer recovery, numbers of animals scored, and p values for t-tests are shown in Table 1. The entire experiment was performed twice with triplicates for each treatment.