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Characterization of Drosophila Melanogaster Cytochrome P450 Genes

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Characterization of Drosophila Melanogaster Cytochrome P450 Genes Characterization of Drosophila melanogaster cytochrome P450 genes Henry Chung, Tamar Sztal, Shivani Pasricha, Mohan Sridhar, Philip Batterham, and Phillip J. Daborn1 Centre for Environmental Stress and Adaptation Research, Department of Genetics, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, 3010, Australia Edited by May R. Berenbaum, University of Illinois, Urbana, IL, and approved February 4, 2009 (received for review November 28, 2008) Cytochrome P450s form a large and diverse family of heme- structure of sensory bristles (23), and Cyp4g1, an oenocyte- containing proteins capable of carrying out many different enzy- specific P450 required for correct triacylglycerol composition matic reactions. In both mammals and plants, some P450s are (24). P450s are also involved in behavioral phenotypes in D. known to carry out reactions essential for processes such as melanogaster, with Cyp6a20 associated with aggressive behavior hormone synthesis, while other P450s are involved in the detoxi- in males (25, 26) and Cyp4d21 necessary for efficient male fication of environmental compounds. In general, functions of mating (27). In the cockroach Diploptera punctata, Cyp15a1 is insect P450s are less well understood. We characterized Drosophila involved in the biosynthesis of juvenile hormone III (28). melanogaster P450 expression patterns in embryos and 2 stages of It has been suggested that the large complement of P450s in third instar larvae. We identified numerous P450s expressed in the insect genomes (most insects possess around 100 different fat body, Malpighian (renal) tubules, and in distinct regions of the P450s) is necessary to protect the insect from the diverse array midgut, consistent with hypothesized roles in detoxification pro- of harmful compounds in its environment (29). Insect P450s cesses, and other P450s expressed in organs such as the gonads, capable of metabolizing allelochemicals have been identified corpora allata, oenocytes, hindgut, and brain. Combining expres- (30, 31). For example, CYP6B1v1 and CYP6B3v1 from the black sion pattern data with an RNA interference lethality screen of swallowtail butterfly, Papilio polyxenes, can metabolize the individual P450s, we identify candidate P450s essential for devel- furanocoumarins it is exposed to in its diet (32, 33). Numerous opmental processes and distinguish them from P450s with poten- individual P450s have also been implicated in insecticide resis- tial functions in detoxification. tance in different insect populations (34–38). Approaches, such as sex-specific expression of P450s, induc- detoxification genes ͉ in situ hybridization ͉ insecticide resistance ͉ tion capacity of P450s by various exogenous compounds, and multigene family ͉ RNAi transcriptional profiling have been useful in characterizing P450 functions (39–42). In an effort to distinguish P450s with essential ytochrome P450s form a diverse and important gene super- endogenous functions from those involved in detoxification, we Cfamily present in virtually all organisms. Genome sequenc- characterized expression patterns of D. melanogaster P450s by in ing projects have identified a large number of P450 sequences in situ hybridization and performed a P450 RNA interference eukaryotes (1–5). Originally identified as monooxygenases, P450 (RNAi) knockdown screen. enzymes are now known to catalyze an extremely diverse range Results of chemical reactions important both in developmental processes and in the detoxification of foreign compounds (6–8). The sequenced, isogenic D. melanogaster strain y; cn bw sp (43) P450s that perform essential functions in mammals and plants was used in this study, as the complement of P450s in this strain have been identified. For example, in humans, CYP17A1 and has been annotated (4). In y; cn bw sp, most of the 86 P450 genes CYP19A1 catalyze steps in the production of androgens and were experimentally distinguishable from paralogs, except for estrogens (9). CYP26B1 metabolizes retinoic acid and is involved the recently described duplication of Cyp12d1 (Cyp12d1-d and in the fate of germ cells in the testes of mice (10). CYP4F2 is Cyp12d1-p), which only differ by 3 nucleotides in the coding involved in postabsorptive elimination of gamma-tocopherol region (www.flybase.org). We classified both Cyp12d1-d and forms of vitamin E (11). Mutations in human P450s also lead to Cyp12d1-p as Cyp12d1, bringing the number of P450s analyzed various diseases (12, 13). Plant P450s are essential for catalyzing to 85. Using RT-PCR with P450 specific primers, 70 P450s were steps in the synthesis of many compounds, including phenylpro- amplified from cDNA isolated from embryos and 81 P450s were panoids, lipids, phytohormones, and carotenoids (14, 15). Roles amplified from cDNA isolated from third instar larvae, with 69 P450s amplified from both embryos and larvae. Cyp6t3, for P450s in the detoxification of xenobiotics, for example EVOLUTION Cyp308a1, and Cyp313a2 were the only P450s that could not be P450-mediated drug metabolism in mammals (16), are also well amplified from either embryos or larvae. Cyp308a1 and characterized. Cyp313a2 could not be amplified from cDNA isolated from Compared to plants and mammals, much less is known about embryos, larvae, or adults, while Cyp6t3 could only be amplified functions of the different insect P450 enzymes. One exception is from cDNA isolated from adults. Using DIG-labeled RNA the involvement of P450s in the biosynthesis of the major insect probes for the 82 P450s amplified, in situ hybridization was hormone 20-hydroxyecdysone (20H) from plant sterols, where in performed on the different stages of embryogenesis, feeding Drosophila melanogaster at least six P450s are involved (17). third instar larval stage and wandering third instar larval stage. Cyp302a1, Cyp306a1, Cyp315a1, and Cyp307a2 are all expressed in the prothoracic glands of late embryos and larvae and catalyze steps in the ecdysone biosynthetic pathway (18–21), while Author contributions: H.C., T.S., P.B., and P.J.D. designed research; H.C., T.S., S.P., M.S., and Cyp314a1 (shade), responsible for catalyzing the conversion P.J.D. performed research; H.C., T.S., and P.J.D. analyzed data; and H.C., T.S., and P.J.D. from ecdysone to 20H, is expressed in the midgut, Malpighian wrote the paper. tubules, and the fat body (22). Cyp307a1 (spook) has also been The authors declare no conflict of interest. suggested to play a role in the synthesis of ecdysone, but it is not This article is a PNAS Direct Submission. expressed in larvae (21). P450s involved in other important 1To whom correspondence should be addressed. E-mail: [email protected]. endogenous processes in D. melanogaster have also been iden- This article contains supporting information online at www.pnas.org/cgi/content/full/ tified, including Cyp303a1, required for the development and 0812141106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812141106 PNAS ͉ April 7, 2009 ͉ vol. 106 ͉ no. 14 ͉ 5731–5736 Downloaded by guest on September 30, 2021 A B Fig. 1. Venn diagram representing unique and overlapping tissue expression of P450 genes expressed in the 3 key metabolic tissues of third instar larvae. FB, fat body; MG,midgut; MT, Malpighian tubules. Expression patterns were obtained for 50 P450s during em- bryogenesis and 58 P450s during the third instar larval stages (Table S1). Many P450s Are Expressed in the Larval Midgut, Malpighian Tubules, and Fat Body. Hybridization signals for 35 P450s were detected in combinations of the midgut, Malpighian tubules, and fat body during third instar larval stages (Fig. 1; see Table S1). This is Fig. 2. P450s expressed in the midgut and Malpighian tubules are detected consistent with a presumed function of P450s in detoxification in specific compartments by in situ hybridization. (A) Spatial patterns of P450s processes, as the metabolism of both exogenous and endogenous expressed in Malpighian tubules. (B) Spatial patterns of P450s in the midgut. compounds is likely to occur in these tissues (44, 45). Seventeen P450s were detected in the larval Malpighian tubules (see Fig. 1 and Table S1), which are suggested to be the primary organs of (51). The anterior portion of the midgut (m1–4) is suggested to excretion in insects (46). It has recently been demonstrated that play some role in immunity (52, 53), although the functions of the Malpighian tubules are important for the metabolism and many midgut regions are not well established (54, 55). Using in detoxification of xenobiotics, and might also be involved in situ hybridization, we detected expression of different P450s in immunity (44, 47). There are at least 5 defined domains in the a number of specific but different midgut regions in third instar Malpighian tubules (48). Of the 17 P450s detected in the larvae (Fig. 2B), with a predominance of P450s in the anterior Malpighian tubules, 15 are expressed in all whole tubules, except (m1–m4) (55% of all midgut expressed P450s) and middle Cyp28d1, which is expressed only in the lower tubule and ureter, (m5–m10) midgut regions (49% of all midgut expressed P450s and Cyp6w1 which is expressed only in the main segment and the expressed). transitional segment (Fig. 2A). Because of the initial segments of While expression patterns for the majority of P450s is consis- the tubules often being lost during dissection and in situ hybrid- tent between individual larvae, expression patterns for 2 P450s, ization, expression in the initial segment was not characterized. Cyp6d4
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