When PDF signaling was disrupted,
the expression of both RC and RE remained rhythmic ( Figures 2A and 2B) and, as with the control flies, GSK-3 inhibitor review maintained a fixed phase relationship to that of Clk. Similar to expression patterns previously described for the clock genes in response to disruptions of the PDF pathway, both RC and RE showed a phase delay and a phase advance in Pdf01 and Pdfr5304 mutant flies, respectively, relative to wild-type controls under free-running conditions ( Figures 2A and 2B and Tables S1 and S2). Moreover, the profile of desat1 transcript expression of the Pdfr5304; +; Pdf01 double mutant displayed a relationship ( Figures 2A–2C and Table R428 solubility dmso S1) identical to that previously described for the clock genes (compare to Figure 1). To confirm the role of PDF signaling in influencing the free-running period of the oenocyte clock, we generated a clock-controlled luciferase reporter derived from the regulatory sequence of the desat1-RE promoter. The RE promoter targets transgene expression specifically to the adult male oenocytes and reproductive organs ( Billeter et al., 2009). With the desat1-luciferase reporter (desat1-luc), it was possible to
continuously monitor the molecular rhythm of the oenocyte clock in living flies over many days in constant conditions. In wild-type control flies, desat1-luc expression was significantly rhythmic with an estimated periodicity of approximately 25 hr ( Figures 3A and 3B, top row), reproducing the circadian expression of the endogenous desat1-RE transcript. When placed in the mutant genetic background of either Pdf01 or Pdfr5304, the desat1-luc reporter ran with a long period of >28 hr ( Figures 3A and 3B, bottom row). Importantly, the introduction of a single transgenic copy of the wild-type Pdf gene (Pdfresc) rescued the long period phenotype of Pdf01, restoring the period to near wild-type length ( Figure 3C). Thus, Pdf and Pdfr maintain the period of the oenocyte clock and desat1 expression. The
level of desat1 expression in the oenocytes directly correlates with the amount of the sex pheromones 7-T, 5-T, and 7-P expressed on the cuticular surface of male D. melanogaster ( Krupp et al., 2008). GBA3 Therefore, we predicted that the effects on the circadian expression pattern of desat1 in response to disruptions in PDF signaling would produce corollary changes in sex pheromone expression. We compared the sex pheromone expression profiles of wild-type controls to that of Pdf01 and Pdfr5304 mutant flies, during the subjective day and night on DD6. Canton-S control flies expressed 7-T, 5-T, and 7-P at all times of the day with significantly higher levels occurring during the subjective night ( Figure 4A), a time roughly corresponding with the observed peak in desat1 expression.