To increase PDFR signaling, we overexpressed a membrane-tethered PDF peptide (t-PDF). Expression of this peptide C59 wnt research buy in PDF-negative neurons is known to result in phenotypes reminiscent to those of flies with high PDF levels (Choi et al.,

2009). Strikingly, we could rescue rhythmicity in DD in 60% of flies with one of the t-PDF transgenic line (40% in the other; Figure 3E; Table 1). Importantly, a scrambled version of the t-PDF (t-SCRB) was totally unable to do so. LD behavior was not rescued with t-PDF, however. Thus, hyperactivation of PDFR can partially suppress the phenotypes associated with downregulation of GW182. This result, combined with all the results presented above, clearly demonstrates that GW182 is an essential element of the PDFR pathway. GW182 plays a central role in miRNA-mediated translation silencing. It does so by interacting with AGO1, which binds directly to miRNAs (Eulalio et al., 2009a). Unfortunately, we could not determine directly whether AGO1 is important for GW182′s circadian function. AGO1 null mutants are lethal, one of the two AGO1 RNAi line showed no phenotype, and the other RNAi line

is, as mentioned above almost completely lethal when combined with TD2 (or even in the absence of DCR2). A few unhealthy escapers were obtained. Not surprisingly, they were arrhythmic both in DD and LD, with very low activity levels (data not shown). To determine whether GW182 works with AGO1 to regulate circadian behavior, we used a rescue strategy. We generated two transgenes resistant to the gw182 shRNA by mutagenizing extensively the binding http://www.selleckchem.com/products/LY294002.html site for this shRNA without affecting the amino acid sequence of the GW182 protein ( Figure 4A). The first transgene encodes a wild-type GW182 (GW), while the other encodes a mutant protein (GWAA) in which the 12 N-terminal glycine-tryptophane (GW) motifs critical for AGO1 binding were changed to alanines (AA) ( Eulalio et al., 2009b). We then coexpressed the shRNA and the resistant constructs with the TD2 combination. As expected, rhythmicity was restored in DD with the wild-type gw182 transgene (although frequently with a long period

phenotype; see below), and under a LD cycle, both the morning peak and the evening Sodium butyrate were entirely normal in phase and amplitude ( Figures 4B and 4C; Tables 1 and S2). This definitely establishes that all the phenotypes we observed with the gw182 dsRNAs are caused by GW182 downregulation. Importantly, the GWAA mutant had a very limited ability to rescue the GW182 downregulation phenotype. None of the five mutant lines we obtained could rescue behavior under LD ( Figures 4B and 4C; data not shown). Three of the five lines did not rescue behavior in DD ( Tables 1 and S2). One line showed very weak rescue in DD. The strongest rescue was observed with GWAA line #7, with about 50% of flies being rhythmic in constant conditions. Amplitude of these rhythms was weaker than in control flies.