cyp6a17 Is Required for TPB in Drosophila Figure 3. cyp6a17 is expressed in the mushroom bodies. (A–E) cyp6a17 reporter expression by cyp6a17MB-Gal4.UAS-mCD8:GFP (green). (A) cyp6a17 was strongly expressed in the ab lobe (white and open triangle) and c lobe (white triangle) of the mushroom bodies. (B) The GFP expression was also detected in the ellipsoid bodies (EB, white arrowhead) and pars intercerebralis (PI, white and open arrowhead). (C) cyp6a17MB-Gal4 derived GFP was expressed in the Kenyon cells (Kc) and Calyx (Ca) of the mushroom bodies. (D) A cross section of the peduncle shows GFP staining along the outer rim that may correspond to the c lobe (red in Fig. 3E). It also shows weak circular staining in three ring structures that appear to be ab lobes. Note that there is no staining inside the rings that may correspond to the ab core. (E) Schematics of Figure (D). Red may correspond to axon fibers for c lobe; blue for a9b9; gray for ab; yellow for ab core based on their characteristic position in the peduncle. (F) Rescue of cyp6a17 mutant phenotypes by cyp6a17MB- Gal4.UAS-cyp6a17. Temperature preference profile and avoidance index (AI) of the mutant line expressing the cyp6a17 transgene (cyp6a17D173; cyp6a17MB-Gal4.UAS-cyp6a17) are shown. Black bars, AIHigh; gray bars, AILow. Two asterisks, P,0.001. doi:10.1371/journal.pone.0029800.g003 TPB (Figure 6A and Table S2). In addition, cyp6a17 RNAi knockdown by other mushroom body Gal4 lines like OK107- and c772-Gal4 also resulted in slightly weaker but significant TPB defects: OK107-Gal4 (AIHigh = 0.8060.02; AILow = 0.4060.20, p,0.001) (Figure 6B and Table S2) and c772-Gal4 (AIHigh = 0.8160.05; AILow = 0.4360.13, p,0.001) (Figure 6C & Table S2). Furthermore, cyp6a17 mutant phenotype was rescued by express- ing the wild-type cyp6a17 cDNA using the MB247-Gal4 mushroom body driver (Figure 5C and Table S2). Taken together, these data suggest that cyp6a17 expression in the mushroom bodies is necessary for normal low temperature TPB. Previous studies proposed that the PKA signaling cascade is important for TPB [8]. According to our microarray data, cyp6a17 expression is regulated by PKA in an activity-dependent manner. Thus we tested epistatic relationship between PKA and cyp6a17. It is noteworthy that TPB phenotypes induced by PKACA overex- pression were strongly suppressed by cyp6a17-RNAi (AIHigh = 0.7260.06, p = 0.08; AILow = 20.0560.12, p,0.001) (Figure 7A & Table S2). Furthermore, the transcript of cyp6a17 was significantly decreased to 0.04460.026 (p,0.001, t test) in dCrebB-17As162 mutant (Figure 7B). In rut1, which is a loss-of-function mutant for rutabaga, encoding calcium-calmodulin responsive adenylate cy- clase, cyp6a17 expression level was reduced compared with the wild-type level (0.05460.008, p,0.001, t test) (Figure 7C). On the contrary, in the dnc1/dncM14 transheterozygote, which can increase cAMP level due to a defect in Dunce phosphodiesterase, cyp6a17 expression level was slightly increased (1.4860.05, p,0.001, t test) (Figure 7D). These results suggest that cyp6a17 transcription is regulated by the cAMP-PKA signaling pathway and cyp6a17 functions downstream to PKA for TPB. Discussion Temperature preference is an important behavioral strategy for optimizing the body temperature in poikilothermal animals like Drosophila. TPB involves temperature sensing and processing of the sensory input at higher levels in the brain. Recent studies have implicated that PKA signaling in the mushroom bodies is required for TPB. However, it is largely unknown how PKA signaling pathway contributes to the behavioral response. In this study, we identified cyp6a17 as a new gene regulated by PKA signaling. Our analysis provides several pieces of evidence that cyp6a17 is required in the mushroom bodies for TPB. First, mutations in the cyp6a17 gene showed reduced TPB. Second, abnormal TPB phenotypes in cyp6a17 mutants were rescued with a wild-type cyp6a17 transgene. PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e29800