Our osm-9p::gfp expression and ascr#3 avoidance behavior results both suggested that DAF-3 SMAD and DAF-5 SNO/SKI are acting as a negative regulators of osm-9 in PD ADL neurons (see Figure 3). However, mutation of part of the potential DAF-3 binding site (Mutation 2) resulted in a decrease in osm-9 expression in CON adults. We observed significantly increased GFP expression in ADL neurons of CON adults carrying the Mutation 2 construct in the daf-3(mgDf90), daf-5(e1386), and daf-3(mgDf90); daf-5(e1386) mutant strains compared to wild-type. We were unable to obtain PD adults in these mutant strains to examine GFP levels. DAF-3 has been shown to negatively regulate the genes daf-7, daf-8, and myosin gene myo-2 by binding to their upstream regulatory sequences (Thatcher et al., 1999; Park et al., 2010). Thus, DAF-3 and DAF-5 may be inhibiting activity of an unknown protein that promotes osm-9 expression when they cannot bind to the PD motif. n ≥ 20 animals over at least 3 biologically independent trials. osm-9p::gfp data in wild-type, daf-3(mgDf90), and daf-5(e1386) and Mutation 2 reproduced from Figures 2C and 3A for reference. Data for strains carrying Mutation 2 are averages of values for two independent extrachromosomal lines. * Significantly different from wild-type, & significantly different from Mutation 2, One-way ANOVA, LSD posthoc correction, p<0.05. Error bars represent S.E.M. (Figure 3—figure supplement 1—source data 1).