Background For insects the sense of smell and associated olfactory-driven behaviours are essential for survival. Insects detect odorants with families of olfactory receptor proteins that are very different to those of mammals, and there are likely to be other unique genes and genetic pathways involved in the function and development of the insect olfactory system. Methodology/Principal Findings We have performed a genetic screen of a set of 505 Drosophila melanogaster gene trap insertion lines to identify novel genes expressed in the adult olfactory organs. We identified 16 lines with expression in the olfactory organs, many of which exhibited expression of the trapped genes in olfactory receptor neurons. Phenotypic analysis showed that six of the lines have decreased olfactory responses in a behavioural assay, and for one of these we showed that precise excision of the P element reverts the phenotype to wild type, confirming a role for the trapped gene in olfaction. To confirm the identity of the genes trapped in the lines we performed molecular analysis of some of the insertion sites. While for many lines the reported insertion sites were correct, we also demonstrated that for a number of lines the reported location of the element was incorrect, and in three lines there were in fact two pGT element insertions. Conclusions/Significance We identified 16 new genes expressed in the Drosophila olfactory organs, the majority in neurons, and for several of the gene trap lines demonstrated a defect in olfactory-driven behaviour. Further characterisation of these genes and their roles in olfactory system function and development will increase our understanding of how the insect olfactory system has evolved to perform the same essential function to that of mammals, but using very different molecular genetic mechanisms.
References
[1]
de Bruyne M, Carlson JR (1999) Odor coding in a model olfactory organ: the Drosophila maxillary palp. J Neurosci 19: 4520–4532.
[2]
de Bruyne M, Foster K, Carlson JR (2001) Odor coding in the Drosophila antenna. Neuron 30: 537–552.
[3]
Clyne PJ, Warr CG, Freeman MR, Lessing D, Kim J, et al. (1999) A novel family of divergent seven-transmembrane proteins: candidate odorant receptors in Drosophila. Neuron 22: 327–338.
[4]
Vosshall LB, Amrein H, Morozov PS, Rzhetsky A, Axel R (1999) A spatial map of olfactory receptor expression in the Drosophila antenna. Cell 96: 725–736.
[5]
Benton R, Sachse S, Michnick SW, Vosshall LB (2006) Atypical membrane topology and heteromeric function of Drosophila odorant receptors in vivo. PLoS Biol 4: e20.
[6]
Smart R, Kiely A, Beale M, Vargas E, Carraher C, et al. (2008) Drosophila odorant receptors are novel seven transmembrane domain proteins that can signal independently of heterotrimeric G proteins. Insect Biochem Mol Biol 38: 770–780.
[7]
Sato K, Pellegrino M, Nakagawa T, Nakagawa T, Vosshall LB, et al. (2008) Insect olfactory receptors are heteromeric ligand-gated ion channels. Nature 452: 1002–1006.
[8]
Wicher D, Sch?fer R, Bauernfeind R, Stensmyr MC, Heller R, et al. (2008) Drosophila odorant receptors are both ligand-gated and cyclic-nucleotide-activated cation channels. Nature 452: 1007–1011.
Spradling AC, Stern D, Beaton A, Rhem EJ, Laverty T, et al. (1999) The Berkeley Drosophila Genome Project gene disruption project. Single P element insertions mutating 25% of vital Drosophila genes. Genetics 153: 135–177.
[11]
Lukacsovich T, Asztalos Z, Awano W, Baba K, Kondo S, et al. (2001) Dual-Tagging Gene Trap of Novel Genes in Drosophila melanogaster. Genetics 157: 727–742.
[12]
Flybase Database. Available: www.flybase.net. Accessed 2012 Jan 12.
[13]
Lee T, Luo L (1999) Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22: 451–461.
Woodard C, Huang T, Sun H, Helfand S, Carlson JR (1989) Genetic analysis of olfactory behavior in Drosophila: A new screen yields the ota mutants. Genetics 123: 315–326.
[16]
Bainton RJ, Tsai LT, Singh CM, Moore MS, Neckameyer WS, et al. (2000) Dopamine modulates acute responses to cocaine, nicotine and ethanol in Drosophila. Curr Biol 10: 187–194.
[17]
Leal SM, Neckameyer WS (2002) Pharmacological Evidence for GABAergic Regulation of Specific Behaviors in Drosophila melanogaster. J Neurobiol 50: 245–261.
[18]
Edwards AC, Zwarts L, Yamamoto A, Callaerts P, Mackay TFC (2009) Mutations in many genes affect aggressive behaviour in Drosophila melanogaster. BMC Biology 7: 29.
[19]
Harbison ST, Seghal S (2008) Quantitative genetic analysis of sleep in Drosophila melanogaster. Genetics 178: 2341–2360.
[20]
Norga KK, Gurganus MC, Dilda CL, Yamamoto A, Lyman RF, et al. (2003) Quantitative analysis of bristle number in Drosophila mutants identifies genes involved in neural development. Current Biology 13: 1388–1397.
Yamamoto A, Zwarts L, Callaerts P, Norga K, Mackay TFC (2008) Neurogenetic networks for startle-induced locomotion in Drosophila melanogaster. Proc Natl Acad Sci USA 105: 12393–12398.
[23]
Robertson HM, Warr CG, Carlson JR (2003) Molecular evolution of the insect chemoreceptor gene superfamily in Drosophila melanogaster. Proc Natl Acad Sci USA 100: 14537–14542.
[24]
Yang P, Shaver SA, Hilliker AJ, Sokolowski MB (2000) Abnormal Turning Behavior in Drosophila Larvae: Identification and Molecular Analysis of scribbler (sbb). Genetics 155: 1161–1174.
[25]
Bellen HJ, Kooyer S, D'Evelyn D, Pearlman J (1992) The Drosophila Couch potato protein is expressed in nuclei of peripheral neuronal precursors and shows homology to RNA-binding proteins. Genes and Dev 6: 2125–2136.
[26]
Bellen HJ, Vaessin H, Bier E, Kolodkin A, D'Evelyn D, et al. (1992) The Drosophila couch potato gene: An essential gene required for normal adult behavior. Genetics 131: 365–375.
[27]
Skoulakis EM, Kalderon D, Davis RL (1993) Preferential expression in mushroom bodies of the catalytic subunit of protein kinase A and its role in learning and memory. Neuron 11: 197–208.
[28]
Li W, Tully T, Kalderon D (1996) Effects of a conditional Drosophila PKA mutant on olfactory learning and memory. Learn Mem 2: 320–333.
[29]
Sargsyan V, Getahun MN, Llanos SL, Olsson SB, Hansson BS, et al. (2011) Phosphorylation via PKC Regulates the Function of the Drosophila Odorant Co-Receptor. Front Cell Neurosci 5: 5.
[30]
Cho KS, Lee JH, Kim S, Kim D, Koh H, et al. (2001) Drosophila phosphoinositide-dependent kinase-1 regulates apoptosis and growth via the phosphoinositide 3-kinase-dependent signaling pathway. Proc Natl Acad Sci USA 98: 6144–6149.
[31]
Diao F, Waro G, Tsunoda S (2009) Fast inactivation of Shal (K(v)4) K+ channels is regulated by the novel interactor SKIP3 in Drosophila neurons. Mol Cell Neurosci 42: 33–44.
[32]
Salkoff L, Baker K, Butler A, Covarrubias M, Pak MD, et al. (1992) An essential “set” of K+ channels conserved in flies, mice and humans. Trends Neurosci 15: 161–166.
[33]
Nagel KI, Wilson RI (2011) Biophysical mechanisms underlying olfactory receptor neuron dynamics. Nat Neurosci 14: 208–216.
[34]
Greil F, van der Kraan I, Delrow J, Smothers JF, de Wit E, et al. (2003) Distinct HP1 and Su(var)3–9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location. Genes Dev 17: 2825–2838.
[35]
Kitajiri S, Sakamoto T, Belyantseva IA, Goodyear RJ, Stepanyan R, et al. (2010) Actin-bundling protein TRIOBP forms resilient rootlets of hair cell stereocilia essential for hearing. Cell 141: 786–798.
[36]
Shin OH, Han W, Wang Y, Südhof TC (2005) Evolutionarily conserved multiple C2 domain proteins with two transmembrane regions (MCTPs) and unusual Ca2+ binding properties. J Biol Chem 280: 1641–1651.
[37]
Bellen HJ, Levis RW, Liao G, He Y, Carlson JW, et al. (2004) The BDGP Gene Disruption Project: Single Transposon Insertions Associated With 40% of Drosophila Genes. Genetics 167: 761–781.
[38]
Phillips AM, Martin J, Bedo DG (1994) In Situ Hybidization to Polytene Chromosomes of Drosophila melanogaster and Other Dipteran Species. Methods in Molecular Biology 33: 193–209.