HSF- 1 also acts downstream of DAF- 7 to regulate development. A, Inactivation of HSF-1 enhances, while its hyperactivation decreases, dauer development in daf-7(−) null mutant genetic backgrounds. Bars represent S.E.M. For each single mutants vs. double mutants or RNAi combination, p<0.001, except for the daf-7(m62) mutant background, where p<0.01 (Student’s t-test). For each genotype, at least 150 animals were tested. B, The structure of daf-9 gene encoding two isoforms. Boxes represent exons, connecting lines indicates introns. A conserved HSF-1 binding site (the red arrow) can be found in the second intron of the longer daf-9 transcript. C, This regulatory element is conserved in the daf-9 genomic environment of other Caenorhabditis species. Highly conserved nucleotides are in red. D, HSF-1 upregulates daf-9. Fluorescent images showing daf-9::gfp expression in a wild-type, a daf-11(m47) single mutant and a daf-11(m47); hsf-1(sy441) double mutant L2 larva at 20°C. HSF-1 deficiency suppresses hyperactivation of daf-9 in animals defective for DAF-11. Fluorescent images were captured with the same exposure time. 91% of the daf-11(−); hsf-1(−) double mutant animals displayed weak (wild-type levels) daf-9 expression (N=166). p<0.0001; Student’s t-test. Enhanced expression of a daf-9::gfp reporter at 25°C, as compared with that obtained at 20°C. HSF-1 is required for higher temperature-induced ectopic expression of daf-9. E, Our epistasis model showing regulatory interactions among HSF-1, DAF-7 and DAF-9. HSF-1 both promotes (through repressing daf-7) and inhibits (through upregulating daf-9) dauer formation. Thus, it acts both upstream and downstream of DAF-7 to modulate development.