Oscillating dCREB2 and NF-kB Activity Figure 2. Expression of the NF-kB-F-luc reporter is FLP dependent, oscillatory and NF-kB-dependent. (A) NF-kB-F-luc reporter activity is FLP-dependent. Reporter activity (Y-axis, relative light units) is plotted as a function of genotype (singly [NFkB-F-luc] or doubly transgenic flies [hs- FLP/+;NFkB-F-luc/+] and treatment (heat shock HS+ or not, HS2). The histogram bars show the mean hourly counts over a 3-day window (n = 24). (B) The ubiquitously expressed actin-GAL4 driver produces oscillatory NFkB-F-luc activity over a 24 h period. Luciferase activity is plotted as a function of time for singly (denoted no driver; NF-kB-F-luc) or doubly transgenic (denoted actin-GAL4; NFkB-F-luc/actin-GAL4) flies. n = 24 for each group (C) Bacterial infection affects reporter activity. Top panel: Reporter activity in the fat body is plotted for the first 48 hours after infection (filled circles) or sham treatment (open circles). The cg-GAL4 line (together with UAS-FLP and NFkB-F-luc) is used to activate the reporter in the fat body. n = 48 for each group. Bottom panel: The difference in reporter activity between the infected and sham-treated groups (infected RLU-sham RLU) over the same duration post-injection. (D) Quantification of reporter activity (mean hourly count) during the first and second days following infection. The mean luminescence counts in RLU are plotted as a function of time after infection. Values for each day are averaged and binned together. (E) There is a dose-dependent decrease in reporter activity in flies fed PDTC. The relative luminescence is plotted as a function of the PDTC dose fed to flies. Triply transgenic flies (UAS-FLP/+;NF-kB-F-luc/actin-GAL4) were fed different dosages of PDTC for 24 hours and then measured for luminescence 1 h after the end of feeding. n = 24 for each group, Error bars = S.E.M, **p,.001. doi:10.1371/journal.pone.0045130.g002 mutations in the period gene (per0, perS and perL) affect reporter activity. Flies were raised under a 12:12 light:dark cycle, and switched into constant darkness. As previously observed, neuronal NF-kB reporter activity is maintained under DD conditions in wild type flies (Fig. 4C). In contrast, NF-kB reporter activity in per0 flies becomes arrhythmic (Fig. 3D), activity in perS mutant flies shows a shorter period (,19 h, Fig. 3E), while perL flies have an extended free-running period (,28, Fig. 3F). In the original CRE-luc reporter, these mutations in per affected dCREB2 reporter activity in an almost identical manner [13]. dCREB2 reporter activity in neurons is similarly modulated in per0, perS, and perL mutants (Fig. S4). Collectively, this data suggest that dCREB2 and NF-kB binding activity are likely to be under circadian control in most, if not all, nervous system tissues. Because feeding behavior is also under circadian regulation [50], circadian changes in luminescence in dCREB2 and NF-kB reporter flies might be explained by diurnal variations in substrate availability due to feeding. To test this, we asked if oscillations in reporter activity persist after flies are removed from luciferin. UAS-FLP/+;CRE-F-luc/actin-GAL4 or UAS-FLP/+;NFkB-F- luc/actin-GAL4 reporter flies were kept on luciferin-containing food for 24 hours, and then transferred to 96-well plates containing non-luciferin food. Luminescence decayed rapidly, with a half-life of around 3.5–4 hours for the dCREB2 reporter flies, and 2.5 hours for the NF-kB reporter flies (Fig. 5). However, oscillations in residual activity from both reporters are detectable for multiple days after substrate removal (Fig. 5 insets). A similar pattern of activity occurs for UAS-FLP/+;CRE-F-luc/actin-GAL4 flies whether maintained on, or removed from luciferin-containing food, with largely two peaks during the daytime and the nighttime (Fig. 1B, 3, 5A). Daytime and nighttime peaks of luciferase activity also persist beyond 24 h after substrate removal in CRE-luc reporter flies (Fig. S5). Likewise, UAS-FLP/+;NFkB-F-luc/actin- GAL4 reporter flies show the same general activity pattern both on and off of luciferin, with one dominant peak occurring shortly after lights-on (Fig. 2B, 5). Upon substrate removal, reporter activity patterns become more variable, perhaps attributable to increased variability of signal when luciferin is low, greater variability in residual substrate between flies, or because substrate ingestion has some contribution to reporter activity patterns. Regardless, circadian variation in substrate feeding does not itself appear to explain circadian variation in reporter activity. Discussion We have used the FLP/FRT system to make spatially restricted transcription factor reporters. These transgenes contain reiterated consensus DNA binding sequences for single factors, thus providing a simpler, but cleaner picture of the activation state of specific transcription factors. The use of luciferase as the reporter moiety, which shortens the half-life of reporter activity, allows a more dynamic picture of transcription. This new reporter format produces detectable signal from behaving flies that is FLP- dependent (Fig. 1B–C, 2A, S1). This allows the activity of specific factors to be measured in defined cell populations. In general, the new CRE-F-luc reporter seems to be indistin- guishable from the original CRE-luc reporter [13]. Both reporters show identical temporal patterns of activity, and per mutations modulate them indistinguishably (Fig. 1B, 3A–D, S4). When the PLOS ONE | www.plosone.org 4 October 2012 | Volume 7 | Issue 10 | e45130