Results Identification of new genes regulated by PKA in the mushroom bodies Temperature preference behavior in Drosophila depends on the level of PKA signaling in the mushroom bodies. To identify new components downstream to PKA, we carried out a genome-wide screen for genes regulated by PKA signaling in the mushroom bodies. Using the Gal4-UAS system, we increased or decreased PKA activity in the mushroom bodies by expressing dominant- negative (UAS-PKADN) or constitutively active PKA (UAS-PKACA), respectively. Expression of PKA transgenes was targeted to the mushroom bodies using the mushroom body-specific MB247-Gal4 driver [8]. PKA expression was induced for 12–16 hours in three- day-old adults by inactivating the temperature-sensitive Gal80 [24] at the restrictive temperature. We then analyzed gene- expression profiles to identify the genes showing altered expression levels in response to the high or low PKA activity. The Drosophila GeneChip (DrosGenome 2.0) was used to obtain gene expression profiles from fly heads of three different groups: (i) the control group with no PKA transgene expression, (ii) the low PKA activity group and (iii) the high PKA activity group. Transcripts that showed more than 2-fold changes from the control expression level were considered for further analysis (Figure 1A & Table S1). A total of 103 transcripts showing more than 2-fold changes were classified into three categories based on their responses to PKA activity: (i) 44 transcripts responded to PKADN but not by PKACA. Among these, 14 showed downregulation while 30 resulted in upregulation. (ii) 39 transcripts showed altered expression by PKACA (23 increases and 16 decreases). (iii) 20 transcripts showed changes by both PKADN and PKACA conditions. Among them, 8 showed decreased and 11 showed increased transcript levels by both PKADN and PKACA conditions (Figure 1B). Only one gene, cyp6a17, showed upregulation by PKACA and downregulation by PKADN. We were most interested in this gene since its expression was correlated with both higher and lower PKA activities (Figure 1C). To confirm these microarray data, we examined the PKA-dependent expression of cyp6a17 by RT-PCR and real-time-PCR. The expression level of cyp6a17 mRNA in the head was decreased to 12% of the wild-type level by inhibiting PKA activity with mushroom body-specific expression of PKADN. On the contrary, the transcript level was increased 5.6361.32 times by PKACA overexpression (Figure 1D). Hence, cyp6a17 expression in the head is regulated by PKA in an activity- dependent manner. cyp6a17 mutations affect temperature preference behavior To test whether cyp6a17 is required for normal TPB, we analyzed the effects of cyp6a17 mutations on TPB. We generated a deletion allele (cyp6a17D173) by inducing imprecise excision of the P-element P{SUPor-P}KG04448 inserted at 133 bp downstream from the translation start site (Figure 2A). The cyp6a17D173 mutation is a deletion of 1.1 kb sequence from the P-insertion site and affects only the cyp6a17 gene (Figure 2A). Consistent with the deletion mapping, the transcript for cyp6a17 was not detected in the cyp6a17D173 mutant by RT-PCR analysis, suggesting that cyp6a17D173 is a mutation (Figure 2B). Homozygous cyp6a17D173 mutant flies are viable and fertile with no visible morphological defects, suggesting that cyp6a17 is not essential for normal development and fertility. Next, we characterized the temperature preference in this deletion mutant. For TPB assays, all mutant and transgenic lines cyp6a17 Is Required for TPB in Drosophila were outcrossed to the w1118 line at least four times. cyp6a17D173 mutant flies showed reduced AI (avoidance index) for low temperature (AILow = 0.5660.06, p,0.001) (Figure 2C and Table S2). Immunocytochemcial analysis with anti-FasII antibody marker for the mushroom bodies did not reveal significant morphological defects in the brain of this mutant (Figure S1). Thus, the abnormal TPB shown in cyp6a17 loss-of-function mutant is probably not caused by morphological defects in the mushroom bodies. Specificity of the cyp6a17 gene function in TPB was also confirmed by rescue of the cyp16a17 mutant phenotypes. Abnormal TPB phenotypes of cyp6a17 mutant flies were rescued to the wild-type level by a 4.2-kb genomic DNA fragment (cyp6a17GR in Figure 2A) spanning the cyp6a17 locus (AI- High = 0.8460.04; AILow = 0.8860.01) (Figure 2A, B, & D and Table S2). These data indicate that cyp6a17 is required for normal TPB. Expression of cyp6a17 is enriched in the mushroom bodies To visualize the expression pattern of cyp6a17, we generated a cyp6a17MB-Gal4 transgenic reporter strain in which Gal4 is induced by a cyp6a17 genomic region containing the promoter. The translation start site for cyp6a17 is only 612 bp apart from the adjacent upstream gene cyp6a22. cyp6a17MB-Gal4 was created by cloning an 1,201 bp fragment upstream of the cyp6a17 translation start site into pCaSpeR vector. This fragment contains the entire 59 region of cyp6a17 including a 39 part of the adjacent cyp6a22 gene. Therefore, the 59 region of cyp6a17 includes regulatory sequences necessary for the cyp6a17 function in TPB (Figure 2A). To detect cyp6a17 expression, we crossed cyp6a17MB-Gal4 transgenic flies to a reporter line UAS-mCD8.GFP. Confocal analysis of GFP expression in the adult brain revealed a complex pattern of expression in the mushroom bodies (Figure 3A). GAL4 is expressed in the mushroom body neurons that project to a-, b- and c-lobes (Figure 3D & E), although it is not clear whether the cyp6a17MB-Gal4 is expressed in all or a subset of mushroom body neurons. In addition to the mushroom bodies, GFP was also expressed in the ellipsoid bodies (EB), pars intercerebralis (PI) and several unknown lateral neurons (Figure 3B). Abnormal TPB phenotypes in the cyp6a17D173 mutant was rescued by expressing wild-type cyp6a17 using the cyp6a17MB-Gal4 driver (AIHigh = 0.7460.03, p,0.001; AILow = 0.9060.03) (Figure 3F and Table S2). These data suggest that the cyp6a17MB-Gal4 reporter represents the pattern of endogenous cyp6a17 expression. cyp6a17 is required in the mushroom bodies for temperature preference behavior To test whether cyp6a17 is required for TPB in specific tissues, we examined the effects of reduced cyp6a17 expression by targeted knockdown using cyp6a17-specific UAS-RNAi transgenic flies. To minimize potential off-target effects on other Drosophila CYP genes, we selected a relatively small unconserved region (222 base pairs) of cyp6a17 as an RNAi target. Expression of cyp6a17-RNAi in the neuronal cells using a pan-neural Elav-Gal4 driver resulted in a significant reduction of TPB (AIHigh = 0.9060.02; AILow = 0.2260.14, p,0.001) (Figure 4A and Table S2). In contrast, cyp6a17 RNAi expression in the whole body except the head by using c290-Gal4 showed no effect on TPB (Figure 4B). These data suggest that cyp6a17 function for normal TPB is required in neurons of the brain. In the adult brain, cyp6a17MB-Gal4 expression is enriched in the mushroom bodies as well as EB and PI (Figure 3A). cyp6a17-RNAi PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e29800