Luciferase-based transcription factor reporters have provided significant insight into the function and regulation of the molecular clock [27–33]. Because luciferase activity is short-lived [34], and can be measured in behaving animals over extended periods of time, these reporters are powerful tools for measuring changes in molecular activities in vivo. Despite the advantages of this approach, it has rarely been applied outside of the core molecular clock to measure the general activity of transcription factors over the day:night cycle. We previously used a luciferase-based reporter gene under the control of dCREB2 binding sites (CRE-luc) to show that dCREB2 activity cycles [13]. An earlier report of NF-kB activity in flies utilized a reporter similar to the CRE-luc reporter, but it was not measured across the day/night cycle [26]. Until now, the major disadvantage of this approach has been the inability to refine these measurements to specific tissues in vivo. To gain spatial control of CRE-luc reporter activity, we take advantage of the UAS-GAL4 [35] and FLP-FRT [36] systems to restrict luciferase expression to specific populations of cells. We use the same strategy to generate a reporter fly based on a consensus sequence for NF-kB. In this study, we provide evidence in vivo that both dCREB2 and NF-kB transcriptional activities oscillate in multiple subsets of cells in the Drosophila nervous system. We show, furthermore, that these oscillations are under circadian control, and are modulated in per mutant flies. Results We generated modified versions of the CRE-luc reporter [13] to measure transcriptional activity in vivo in defined subsets of cells. A reporter transgene was placed under the control of the FLP-FRT and GAL-UAS systems to allow for spatial control over reporter expression (Fig. 1A). The new reporters contain three binding sites for dCREB2 (or NF-kB) upstream of the CaSpeR TATAA element. Downstream of where transcription initiates, a Cavener sequence [37] and translation initiation codon are positioned 59 to an FRT-flanked cassette containing the coding sequence for mCherry (but without its normal start codon). The coding region terminates with two tandem stop codons. Translation that initiates at the ATG codon reads through the first FRT site and the mCherry sequence before terminating at the tandem stop codons. The open reading frame for firefly luciferase (without its normal start codon) is placed downstream of the cassette. In the absence of FLP, translation should terminate at the tandem stop codons just 39 to the mCherry sequence. In the presence of FLP, site-specific recombination between the FRT sites should remove most of the cassette, leaving one FRT site. Translation initiated at the first ATG codon should then read through the remaining FRT sequence, a poly-glycine encoding linker, and into the luciferase sequence, producing a fusion protein with luminescent activity. As a result, specific reporter expression should be activated when FLP recombinase is expressed under the control of an anatomically defined GAL4 driver. To test if FLP recombinase can activate luciferase expression, we combined the CRE-F-luc transgene (Fig. 1A #1) with a heat- shock inducible transgene expressing FLP recombinase, and measured luciferase activity over time in live flies (Fig. 1B). While flies carrying the CRE-F-luc transgene alone show no detectible luminescence, flies carrying an additional hs-FLP transgene exhibit substantial leaky reporter expression when maintained at 22uC. This activity increases significantly when these flies are exposed to heat shock. Similar to the previous reporter [13], the CRE-F-luc reporter shows an oscillating pattern of day-night activity, characterized by daytime and nightime peaks. Light output dampens over time with luciferin degradation. Oscillating dCREB2 and NF-kB Activity While FLP-dependence for reporter activation is important, the real utility of this system requires that GAL4-UAS mediated spatial control can be layered onto the reporter. In order to test this, we combined the CRE-F-luc transgene with a UAS-driven FLP recombinase transgene [38]. Baseline expression in live UAS- FLP/+;CRE-F-luc/+ flies is detectable but low (,100 RLU/hr), and does not significantly increase in hs-GAl4/UAS-FLP/CRE-F- luc flies in the absence of heat shock (Fig. 1C). However, luciferase activity increases significantly in these triply transgenic flies following heat shock. Therefore, FLP recombinase expression under UAS control activates the reporter. To verify the specificity of the fully assembled reporter, UAS- FLP;CRE-F-luc flies were crossed to an eye specific driver line, gmrlong-GAL4. Luciferase activity was then measured from isolated fly heads. This activity shows a substantial gmrlong-driver-dependent increase. However, there is no significant difference between groups when isolated bodies are measured (Fig. 1D), indicating specific reporter expression. Measurements of dissected head and eye tissue confirm that the signal is produced only in the eye tissue (Fig. S1; see Methods S1 for details). In order to characterize the range of signal detection, and to assess the contribution of various cell populations to circadian oscillations in dCREB2 activity, we screened a number of GAL4 driver lines for reporter activity. Flies containing both the UAS- FLP and CRE-F-luc transgenes were crossed to different GAL4 driver lines, or to wild type flies as a baseline control. In most of the lines tested, significant luciferase activity is detected above baseline (Table S1). A significant increase in reporter activity above baseline is detectible when GAL4 is driven in small populations of neurons in the ellipsoid body (c42-GAL4 or c232- GAL4) or in the mushroom body (MB, ok107-GAL4) (Table S1, Fig. 3C), indicating that the reporter can be used to measure activity in small subsets of cells. However, no signal is detectable using the npf-GAL4 driver (Table S1), which expresses in only a handful of cells [39]. Notably, all drivers that produce detectible activity exhibit oscillating activity with the same general temporal pattern, peaking at around zeitgeber time (ZT) 4 (mid-day) and ZT16 (mid-evening). We used the same strategy to build a reporter for general NF-kB activity. Our reporter is similar to a previous NF-kB luciferase reporter, except that it is FLP-dependent, and based on a more generalized NF-kB binding site [26]. As with the CRE-F-luc reporter, luciferase activity is undetectable in flies that contain only the NFkB-F-luc reporter, but FLP expression stimulates activity (Fig. 2A). When ubiquitously expressed under the control of an actin-GAL4 driver, NF-kB reporter activity oscillates in a 24 hr rhythm under a light:dark regimen, peaking shortly after lights on (ZT1) (Fig. 2B). We observe a similar pattern when the reporter is activated ubiquitously by hs-FLP (Fig. S2). To verify that the NF-kB-F-luc reporter responds to NF-kB activity, we asked if established modulators of NF-kB would affect reporter activity. Because the innate immune response activates NF-kB [40,26], bacterial infection should stimulate activity measured from NFkB-F-luc reporter flies. As the fat body is the center for innate immunity in Drosophila, we used a fat body- specific driver (cg-GAL4) to express the reporter in this tissue. These flies were inoculated with either a mixture of gram-negative and gram-positive bacteria, or received a sham injury. Bacterial infection, when compared to mock injection, significantly increases NFkB-F-luc activity (Fig. 2C,D). This response peaks between 6 and 20 hours following infection, and lasts for about 30 hours. To validate the specificity of the reporter, we asked if inhibiting NF-kB activity would decrease NFkB-F-luc activity. Pyrrolidine dithiocarbamate (PDTC) is a potent NF-kB inhibitor, suppressing PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e45130