[26]. We found that cyp6a17 mRNA is expressed in the adult body including hindgut, but about 30% more expression is detected in the head (Figure S2B). Using a reporter construct regulated by a 59 region of the cyp6a17 gene, we showed that cyp6a17 expression is highly enriched in the mushroom bodies, especially ab and c lobes. Three observations strongly support that this reporter reflects the pattern of cyp6a17 gene expression. Firstly, cyp6a17 RNAi driven by mushroom body-specific Gal4 lines causes TPB defects. Secondly, cyp6a17 RNAi using cyp6a17MB-Gal4 also results in similar TPB phenotypes. Thirdly, cyp6a17 expression induced by cyp6a17MB-Gal4 rescues cyp6a17 mutant phenotypes. These data indicate that the mushroom body regions labeled by cyp6a17 reporter are the sites where cyp6a17 function is required for TPB. To our knowledge, cyp6a17 is the first gene of this family shown to be expressed in the mushroom bodies with specific function in TPB. However, it is possible that other cell types in addition to the mushroom bodies might also be involved in the TPB phenotype. Cytochrome P450 proteins are known to participate in the synthesis of steroid hormone, thereby regulating homeostasis and thermal responses [27,28]. Because cyp6a17 is also expressed in PI and EB regions that have been implicated in neuroendocrine regulation [29], cyp6a17 may play a role in the neuroendocrine system. However, it has been shown that the PI and EB are not required for TPB [8]. Furthermore, our data show that the cyp6a17 mutant deficits in TPB are rescued by cyp6a17 expression in the mushroom bodies but not in subsets of PI and EB neurons. Thus, it appears that the cyp6a17 function for TPB is required mainly in the mushroom bodies rather than the PI and EB cells. There are 83 CYP family genes in Drosophila, excluding 7 pseudogenes. Of these, only several CYP genes have been characterized for their expression patterns and functions. Some CYP proteins function together in a protein complex system for catalyzing oxidative reactions [30]. However, many CYP genes show tissue-specific expression profiles, suggesting that CYP genes may play distinct functions in diverse tissues. This possibility is supported by our finding that cyp6a17 has a specific function in the mushroom bodies as a necessary component for controlling the temperature preference behavior. In addition to cyp6a17, other CYP genes might also play important roles in TPB. cAMP-PKA signaling in the mushroom bodies is important for learning and memory in Drosophila [31], but downstream events are not well understood. Our study identified a cytochrome P450 gene as a downstream factor for PKA signaling in TPB. Given the potential relationship between temperature and stress related genes [32–34], this work could provide clues to understanding the relationship between stress response and temperature preference behavior. Although we focused on characterizing the function of cyp6a17 in this study, our genome-wide screen identified many other genes that showed strong response to either PKACA or PKADN (Table S1). Further analysis of these genes might identify additional genetic factors involved in PKA-dependent TPB and their relationship with cyp6a17. Until now, there is no report to our knowledge that Drosophila CYP is involved in temperature preference behavior. It is an intriguing question how PKA-dependent regulation of cyp6a17 is functionally related to TPB. Since steroid hormone can modulate several kinds of behavior, neuronal homeostasis could be an important factor for TPB. Consistent with this idea, it has been shown that Drosophila dystroglycan plays a role in modulating energy homeostasis and the thermal responses [35]. Cyp6a17 is known to have monooxygenase activity [13]. Hence, it is plausible that cyp6a17 might be involved in the synthesis of steroid hormones, thereby modulating temperature preference behavior. cyp6a17 may also participate in the regulation of ATP level to modulate cyp6a17 Is Required for TPB in Drosophila energy homeostasis in neurons. Detailed analysis of these possibilities in the future will help provide mechanistic insights into the function of cyp6a17 for TPB. Materials and Methods Drosophila strains and transgenic lines Fly stocks were raised on standard cornmeal food at 25uC and 40–50% relative humidity. w1118 and Canton-S were used as wild- type strains. UAS-PKADN and PKACA were provided by D. Kalderon; other lines were provided as follows: MB247, c772 (T. Zars), c309 (L. C. Griffith), MB247; tub-GAL80ts (G. Roman), P{SUpor-P}KG04448, c232, c290 and dilp2-Gal4 (Bloomington stock center). All mutant and transgenic alleles used in this study were outcrossed to the w1118 line at least four times. Microarray analysis Total RNA was extracted from adult heads. After extracting total RNA, microarrays were generated by Digital Genomics Inc. (Seoul, Korea). Modified cDNA from total RNA was hybridized to GeneChipH Drosophila Genome 2.0 Array. Image acquisition and data extraction from microarray experiments were analyzed by Affymetrix GeneChipH Scanner 3000 7G and Affymetrix GCOS1.4 software, respectively. Data were normalized by MAS5 and RMA (Robust Multi-array Average), and differentially expressed genes were selected by comparing control and transgenic lines. The functional category of selected genes was classified using the DAVID2007 (http://david.abcc.ncifcrf.gov/ home.jsp) program. Temperature preference behavior assay and climbing assay TPB assays were performed as described previously [8,36]. 1–3 day old adult files of mixed gender were collected without anesthetization in vials containing fresh food during morning hours. After 3 days under a 12 hr light/dark incubation cycle, the collected flies were placed in the test device. A temperature gradient from 15 to 45uC with a slope of 0.75uC/cm was produced in an aluminum block (42 length624 width67 cm heights). Electronic thermal sensors were embedded in the block every 3 cm, and the gradient was established using cold and hot circulating water chambers at each end. A glass plate with five separate lanes was placed 2.5 mm above the block, creating suitable space for flies to walk. The glass plate was coated with quinine sulfate powder, an aversive stimulus for Drosophila, to prevent flies from escaping the temperature gradient by resting on the walls or roof of the lane. 1% yeast paste was applied on the block for flies to stay on the block. Adult flies were placed between the aluminum block and the glass plate, allowed to move for 25 min in the dark, and photographed with a digital camera with 2–5 msec exposure at the end of each assay. Avoidance indices against low temperature (AILow) and high temperature (AIHigh) were calculated from the following formulae: AILow = NInt2NLow/ NInt+NLow and AIHigh = NInt2NHigh/NInt+NHigh. NLow, NHigh and NInt are the numbers of flies in the region below 23.4uC, above 26.7uC, and between 23.4 and 26.7uC, respectively. A population of mixed sexes was tested for TPB. The number of flies was recorded by using digital photographs and processed with Microsoft Excel, and one-way ANOVA followed by Dunnett’s post test was performed. All data points in the figures are represented as the mean and the standard error of the mean (s.e.m.), unless mentioned otherwise. For climbing test to check the locomotor activity, groups of ten flies were transferred into vials and incubated for 1 hr at 25uC for PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e29800