The GAL4/UAS gene expression system is a precise means of targeted gene expression employed to study biological phenomena in Drosophila. A modified GAL4/UAS system can be conditionally regulated using a temporal and regional gene expression targeting (TARGET) system that responds to heat shock induction. However heat shock-related temperature shifts sometimes cause unexpected physiological responses that confound behavioral analyses. We describe here the construction of a drug-inducible version of this system that takes advantage of tissue-specific GAL4 driver lines to yield either RU486-activated LexA-progesterone receptor chimeras (LexPR) or β-estradiol-activated LexA-estrogen receptor chimeras (XVE). Upon induction, these chimeras bind to a LexA operator (LexAop) and activate transgene expression. Using GFP expression as a marker for induction in fly brain cells, both approaches are capable of tightly and precisely modulating transgene expression in a temporal and dosage-dependent manner. Additionally, tissue-specific GAL4 drivers resulted in target gene expression that was restricted to those specific tissues. Constitutive expression of the active PKA catalytic subunit using these systems altered the sleep pattern of flies, demonstrating that both systems can regulate transgene expression that precisely mimics regulation that was previously engineered using the GeneSwitch/UAS system. Unlike the limited number of GeneSwitch drivers, this approach allows for the usage of the multitudinous, tissue-specific GAL4 lines for studying temporal gene regulation and tissue-specific gene expression. Together, these new inducible systems provide additional, highly valuable tools available to study gene function in Drosophila.
References
[1]
Bellen HJ, Levis RW, He Y, Carlson JW, Evans-Holm M, et al. (2011) The Drosophila gene disruption project: progress using transposons with distinctive site specificities. Genetics 188: 731–743.
[2]
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.
[3]
Brand AH, Perrimon N (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118: 401–415.
[4]
McGuire SE, Le PT, Osborn AJ, Matsumoto K, Davis RL (2003) Spatiotemporal rescue of memory dysfunction in Drosophila. Science 302: 1765–1768.
[5]
Bubliy OA, Loeschcke V (2005) Correlated responses to selection for stress resistance and longevity in a laboratory population of Drosophila melanogaster. J Evol Biol 18: 789–803.
[6]
Le Bourg E (2007) Hormetic effects of repeated exposures to cold at young age on longevity, aging and resistance to heat or cold shocks in Drosophila melanogaster. Biogerontology 8: 431–444.
[7]
Munch SB, Salinas S (2009) Latitudinal variation in lifespan within species is explained by the metabolic theory of ecology. Proc Natl Acad Sci U S A 106: 13860–13864.
[8]
Simon AF, Shih C, Mack A, Benzer S (2003) Steroid control of longevity in Drosophila melanogaster. Science 299: 1407–1410.
[9]
Wang HD, Kazemi-Esfarjani P, Benzer S (2004) Multiple-stress analysis for isolation of Drosophila longevity genes. Proc Natl Acad Sci U S A 101: 12610–12615.
[10]
Boothroyd CE, Wijnen H, Naef F, Saez L, Young MW (2007) Integration of light and temperature in the regulation of circadian gene expression in Drosophila. PLoS Genet 3: e54.
[11]
Busza A, Murad A, Emery P (2007) Interactions between circadian neurons control temperature synchronization of Drosophila behavior. J Neurosci 27: 10722–10733.
[12]
Yoshii T, Vanin S, Costa R, Helfrich-Forster C (2009) Synergic entrainment of Drosophila's circadian clock by light and temperature. J Biol Rhythms 24: 452–464.
[13]
Zhang Y, Liu Y, Bilodeau-Wentworth D, Hardin PE, Emery P (2010) Light and temperature control the contribution of specific DN1 neurons to Drosophila circadian behavior. Curr Biol 20: 600–605.
[14]
Ito C, Goto SG, Tomioka K, Numata H (2011) Temperature entrainment of the circadian cuticle deposition rhythm in Drosophila melanogaster. J Biol Rhythms 26: 14–23.
[15]
Shih C-T, Lin H-W, Chiang A-S (2011) Statistical analysis and modeling of the temperature-dependent sleep behavior of drosophila. Computer Physics Communications 182: 195–197.
[16]
Howlader G, Paranjpe DA, Sharma VK (2006) Non-ventral lateral neuron-based, non-PDF-mediated clocks control circadian egg-laying rhythm in Drosophila melanogaster. J Biol Rhythms 21: 13–20.
[17]
Marshall KE, Sinclair BJ (2010) Repeated stress exposure results in a survival-reproduction trade-off in Drosophila melanogaster. Proc Biol Sci 277: 963–969.
[18]
Peng IF, Berke BA, Zhu Y, Lee WH, Chen W, et al. (2007) Temperature-dependent developmental plasticity of Drosophila neurons: cell-autonomous roles of membrane excitability, Ca2+ influx, and cAMP signaling. J Neurosci 27: 12611–12622.
[19]
Wang X, Green DS, Roberts SP, de Belle JS (2007) Thermal disruption of mushroom body development and odor learning in Drosophila. PLoS One 2: e1125.
[20]
Folguera G, Mensch J, Munoz JL, Ceballos SG, Hasson E, et al. (2010) Ontogenetic stage-dependent effect of temperature on developmental and metabolic rates in a holometabolous insect. J Insect Physiol 56: 1679–1684.
[21]
Huckesfeld S, Niederegger S, Schlegel P, Heinzel HG, Spiess R (2010) Feel the heat: The effect of temperature on development, behavior and central pattern generation in 3rd instar Calliphora vicina larvae. J Insect Physiol 57: 136–146.
[22]
Zars M, Zars T (2006) High and low temperatures have unequal reinforcing properties in Drosophila spatial learning. J Comp Physiol A Neuroethol Sens Neural Behav Physiol 192: 727–735.
[23]
Riveron J, Boto T, Alcorta E (2009) The effect of environmental temperature on olfactory perception in Drosophila melanogaster. J Insect Physiol 55: 943–951.
[24]
Garcia AM, Calder RB, Dolle ME, Lundell M, Kapahi P, et al. (2010) Age- and temperature-dependent somatic mutation accumulation in Drosophila melanogaster. PLoS genetics 6: e1000950.
[25]
Han DD, Stein D, Stevens LM (2000) Investigating the function of follicular subpopulations during Drosophila oogenesis through hormone-dependent enhancer-targeted cell ablation. Development 127: 573–583.
[26]
Osterwalder T, Yoon KS, White BH, Keshishian H (2001) A conditional tissue-specific transgene expression system using inducible GAL4. Proc Natl Acad Sci U S A 98: 12596–12601.
[27]
Roman G, Endo K, Zong L, Davis RL (2001) P[Switch], a system for spatial and temporal control of gene expression in Drosophila melanogaster. Proc Natl Acad Sci U S A 98: 12602–12607.
[28]
Nicholson L, Singh GK, Osterwalder T, Roman GW, Davis RL, et al. (2008) Spatial and temporal control of gene expression in Drosophila using the inducible GeneSwitch GAL4 system. I. Screen for larval nervous system drivers. Genetics 178: 215–234.
[29]
Emelyanov A, Parinov S (2008) Mifepristone-inducible LexPR system to drive and control gene expression in transgenic zebrafish. Dev Biol 320: 113–121.
Joiner WJ, Crocker A, White BH, Sehgal A (2006) Sleep in Drosophila is regulated by adult mushroom bodies. Nature 441: 757–760.
[32]
Ohlmeyer JT, Kalderon D (1997) Dual pathways for induction of wingless expression by protein kinase A and Hedgehog in Drosophila embryos. Genes & development 11: 2250–2258.
[33]
McGuire SE, Mao Z, Davis RL (2004) Spatiotemporal gene expression targeting with the TARGET and gene-switch systems in Drosophila. Sci STKE 2004: pl6.
[34]
Bello B, Resendez-Perez D, Gehring WJ (1998) Spatial and temporal targeting of gene expression in Drosophila by means of a tetracycline-dependent transactivator system. Development 125: 2193–2202.
[35]
Bieschke ET, Wheeler JC, Tower J (1998) Doxycycline-induced transgene expression during Drosophila development and aging. Mol Gen Genet 258: 571–579.
[36]
Urlinger S, Baron U, Thellmann M, Hasan MT, Bujard H, et al. (2000) Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. Proc Natl Acad Sci U S A 97: 7963–7968.
[37]
Burcin MM, O'Malley BW, Tsai SY (1998) A regulatory system for target gene expression. Front Biosci 3: c1–7.
[38]
Stebbins MJ, Urlinger S, Byrne G, Bello B, Hillen W, et al. (2001) Tetracycline-inducible systems for Drosophila. Proceedings of the National Academy of Sciences of the United States of America 98: 10775–10780.
[39]
Stebbins MJ, Yin JC (2001) Adaptable doxycycline-regulated gene expression systems for Drosophila. Gene 270: 103–111.
[40]
Madalan A, Yang X, Ferris J, Zhang S, Roman G (2012) G(o) activation is required for both appetitive and aversive memory acquisition in Drosophila. Learning & memory 19: 26–34.
[41]
Ford D, Hoe N, Landis GN, Tozer K, Luu A, et al. (2007) Alteration of Drosophila life span using conditional, tissue-specific expression of transgenes triggered by doxycyline or RU486/Mifepristone. Experimental gerontology 42: 483–497.
[42]
Mizuguchi H, Hayakawa T (2002) The tet-off system is more effective than the tet-on system for regulating transgene expression in a single adenovirus vector. The journal of gene medicine 4: 240–247.
[43]
Viktorinova I, Wimmer EA (2007) Comparative analysis of binary expression systems for directed gene expression in transgenic insects. Insect Biochem Mol Biol 37: 246–254.
[44]
Mao Z, Roman G, Zong L, Davis RL (2004) Pharmacogenetic rescue in time and space of the rutabaga memory impairment by using Gene-Switch. Proc Natl Acad Sci U S A 101: 198–203.
[45]
Markstein M, Pitsouli C, Villalta C, Celniker SE, Perrimon N (2008) Exploiting position effects and the gypsy retrovirus insulator to engineer precisely expressed transgenes. Nat Genet 40: 476–483.
[46]
Hendricks JC, Finn SM, Panckeri KA, Chavkin J, Williams JA, et al. (2000) Rest in Drosophila is a sleep-like state. Neuron 25: 129–138.
