daf-16/FOXO promotes the activity of ligand-bound DAF-12/NHR to coordinate dauer recovery and post-dauer seam cell fate

The mechanisms by which developmental pathways are modulated to accommodate developmental arrest are poorly understood. In adverse environments, dauer diapause can interrupt developmental progression after the second larval molt. During continuous (non-dauer) development, a heterochronic molecular timer consisting primarily of microRNAs and their targets controls the progression of stage-specific cell fates in lateral hypodermal seam cells. In unfavorable conditions, the DAF-16/FOXO transcription factor and the DAF-12 nuclear hormone receptor in its ligand-free state promote dauer formation and oppose the expression of let-7 family microRNAs, thus pausing developmental progression during dauer. Surprisingly, we found that daf-16(0) post-dauer adults showed reiterative heterochronic defects including missing or gapped adult alae, lack of seam-cell fusion, and reduced expression of the adult-specific col-19p::gfp marker. Consistent with these adult cell fate defects, let-7- family microRNA expression was reduced in daf-16(0) post-dauer larvae. Addition of the DAF-12 ligand, dafachronic acid, suppressed the reiterative phenotypes in daf-16(0) post-dauer animals. Notably, addition of dafachronic acid to daf-16 mutants rescued levels of let-7 family transcriptional reporters but did not affect levels of these reporters in daf-16(+) control strains. Dafachronic acid is synthesized from cholesterol which is normally sequestered in the intestinal lumen during dauer. We found that a fluorescent cholesterol analog was not maintained in daf-16 mutant larvae during dauer recovery. Consistent with this finding, timed auxin-mediated depletion of DAF-16 indicated that daf-16 is required before dauer formation, rather than after dauer, to prevent reiterative seam cell fates in post-dauer larvae and adults. Furthermore, depletion of DAF-16 from the intestine recapitulated defects in adult cell fate. Taken together, we propose a model whereby daf-16 acts prior to dauer formation to enable dafachronic acid synthesis by retaining cholesterol during dauer recovery. Ligand-bound DAF-12 then promotes dauer recovery and expression of let-7 family microRNAs, thereby promoting adult cell fate. Thus, daf-16 and daf-12 coordinate dauer exit with the resumption of a developmental pathway.