cyp6a17 Is Required for TPB in Drosophila Figure 2. cyp6a17 is required for normal temperature preference behavior. (A) Exons of cyp6a17 are indicated by boxes, and coding regions are colored black. The P{SUpor-P} line has an insertion at 133 bp downstream from the translation start site. cyp6a17D173 is a mutant with a deletion of 1.1 kb, as depicted by square brackets. A genomic DNA fragments used for rescue (cyp6a17GR) is indicated by gray box. The cyp6a17-Gal4 construct contains a genomic fragment of 1.2 kb (short thick stippled box). (B) Determination of the expression level of cyp6a17 by RT-PCR. gapdh1 was used as internal control: N = 4. (C) cyp6a17D173 showed reduced TPB. (D) The abnormal TPB of cyp6a17D173 was restored by expression of the cyp6a17 genomic transgene (cyp6a17GR). Black bars, AIHigh; gray bars, AILow. One asterisk, P,0.005 and two asterisks, P,0.001. doi:10.1371/journal.pone.0029800.g002 knockdown in subsets of EB and PI neurons labeled by c232-Gal4 and dilp2-Gal4, showed little effect on TPB (Figure 4C & D and Table S2). This result is consistent with the previous report that EB and PI are not essential for TPB [8]. To exclude the possibility that cyp6a17 RNAi affects locomotor activity, we examined the motor activity of the flies by climbing behavior test. In these control assays, locomotor activity was normal at all temperature conditions tested. Next, we induced cyp6a17-RNAi in the cyp6a17-expressing cells by using cyp6a17MB-Gal4. These flies showed a reduced AI index, as seen in cyp6a17 mutants (AIHigh = 0.5760.07, p,0.001; AILow = 0.1160.07, p,0.001) (Figure 5A and Table S2). Thus, cyp6a17 function is indeed required for TPB in the cells identified by the cyp6a17 reporter. To determine whether cyp6a17 is required in the mushroom bodies, we induced cyp6a17-RNAi preferentially in the mushroom bodies using MB247-Gal4. These flies with knockdown of cyp6a17 in the mushroom bodies showed a reduced avoidance index in the TPB assay (AIHigh = 0.8960.03; AILow = 0.1460.09, p,0.001) (Figure 5B and Table S2). In the heads of these flies, cyp6a17 mRNA expression level was reduced compared with wild-type (0.5660.06, p,0.001) (Figure S2A). cyp6a17MB-Gal4 and MB247 show additional expression in surface glia [25]. To check whether cyp6a17 in surface glia is required for TPB, we induced cyp6a17-RNAi in surface glia by cyp6a17SG-Gal4 which drives strong Gal4 expression in surface glia but very weakly in the mushroom bodies (Figure S3). These flies showed normal PLoS ONE | www.plosone.org 4 December 2011 | Volume 6 | Issue 12 | e29800