<em>In Vivo</em> Circadian Oscillation of dCREB2 and NF-κB Activity in the <em>Drosophila</em> Nervous System

<div><p>cAMP response element-binding protein (CREB) and nuclear factor kappa-B (NF-κB) are two ubiquitous transcription factors involved in a wide number of cellular processes, including the circadian system. Many previous studies on these factors use cellular assays that provide limited information on circadian activity or anatomical specificity. The ability to study transcription factors in defined tissue within intact animals will help to bridge the gap between cellular and <em>in vivo</em> data. We have used the GAL4-UAS and FLP-FRT systems to gain spatial control over reporter gene expression. Using a luciferase-based reporter, we show <em>in vivo</em> that <em>Drosophila</em> dCREB2- and NF-κB-mediated transcription oscillates in neuronal cells, glia, and in the mushroom body, a higher-order brain center in flies. This oscillation is under circadian control, cycling with a 24-hour rhythm, under both light-dark and dark-dark conditions. In light-light conditions, dCREB2 and NF-κB reporter flies exhibit a suppression of rhythmic activity. Furthermore, neuronal cycling of dCREB2 and NF-κB activity are modulated in <em>period</em> mutant flies, indicating these oscillations are controlled through the central clock. This study shows for the first time region-specific circadian oscillation of dCREB2/NF-κB activity in the <em>Drosophila</em> nervous system.</p> </div

Expression of the NF-κB-F-luc reporter is FLP dependent, oscillatory and NF-κB-dependent.

<p>(A) NF-κB-F-luc reporter activity is FLP-dependent. Reporter activity (Y-axis, relative light units) is plotted as a function of genotype (singly [NFκB-F-luc] or doubly transgenic flies [hs-FLP/+;NFκB-F-luc/+] and treatment (heat shock HS+ or not, HS−). The histogram bars show the mean hourly counts over a 3-day window (n = 24). (B) The ubiquitously expressed <i>actin</i>-GAL4 driver produces oscillatory NFκB-F-luc activity over a 24 h period. Luciferase activity is plotted as a function of time for singly (denoted no driver; NF-κB-F-luc) or doubly transgenic (denoted <i>actin</i>-GAL4; NFκB-F-luc/<i>actin</i>-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 <i>cg</i>-GAL4 line (together with UAS-FLP and NFκB-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-κB-F-luc/<i>actin</i>-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.</p

The NF-κB reporter activity oscillates under circadian control.

<p>(A) NF-κB reporter activity in multiple tissues oscillates in constant darkness. Reporter activity is plotted as a function of time for different genotypes. Triply transgenic flies, where the reporter is activated in all neurons (using the <i>elav</i>^c155-GAL4 driver, black squares), in a pan-glial pattern (using the <i>repo</i>-GAL4 driver, open circles), or in the mushroom body (using the <i>ok107</i>-GAL4 driver, open triangles), exhibit oscillatory activity when flies are maintained in light∶dark (first 24 h period) or dark∶dark (subsequent time) conditions. These plots are compared to those from doubly transgenic flies (solid line; UAS-FLP/+; NFκB-F-luc/+) that do not contain the tissue-specific driver. (B) The activity of the NF-κB reporter in different tissues is dampened under constant light. Reporter activity is shown as a function of time, with the shift to constant light occurring during the second 24 h period. The reporter is activated in triply transgenic flies in neurons (denoted <i>elav^c155</i>-GAL4, <i>elav^c155</i>-GAL4; UAS-FLP/+; NFκB-F-luc/+), glia (denoted <i>repo</i>-GAL4, UAS-FLP; NFκB-F-luc/<i>repo</i>-GAL4) or the mushroom body (denoted <i>ok107-GAL4</i>; UAS-FLP; NFκB-F-luc/+; <i>ok107</i>-GAL4/+) when compared to doubly transgenic flies without a GAL4 driver (solid line, UAS-FLP/+; NFκB-F-luc/+). (C–F) Neuronal reporter activity is plotted over time as flies are shifted from light∶dark to constant darkness. For all of these panels, the same transgenes (UAS-FLP/+; NFκB-F-luc/<i>elav</i>-GAL4) exist in all flies, but the flies are examined in a wild type (C), <i>per⁰</i> (D), <i>per^S</i> (E) or <i>per^L</i> (F) genetic background.</p

Oscillations in dCREB2 and NF-κB activity persist after luciferin removal.

<p>(A) Reporter activity in UAS-FLP/+;CRE-F-luc/<i>actin</i>-GAL4 flies starting ∼1 h following luciferin removal, plotted as a function of time. The inset shows a scaled view of the same data, with the first 24 h dropped. (B) Reporter activity in UAS-FLP/+;NFκB-F-luc/<i>actin</i>-GAL4 flies starting ∼1 h following luciferin removal. Inset shows a scaled view of the same data, with the first 24 h dropped. n = 24 flies for each group.</p

Developing a spatially restricted reporter system.

<p>(A) A cartoon of the reporter system. Three DNA binding sites are placed upstream of the CaSpeR TATAA sequence, followed by transcription (arrow) and translation (arrowhead) initiation sites, an FRT-flanked open reading frame for mCherry (ORF) including two tandem stop codons, and the luciferase ORF (#1). Targeted GAL4 expression (#3) drives the expression of UAS-FLP (#2), which catalyzes site-specific recombination at the FRT sites, activating the reporter. (B) CRE-F-luc reporter activity is FLP-dependent. Singly (CRE-F-luc) or doubly transgeneic flies (<i>hs</i>-FLP/+;CRE-F-luc/+) are maintained under 12∶12 LD conditions. Flies are exposed to heat-shock (+HS) or not (−HS), and measured for <i>in vivo</i> luminescence. The relative luminescence is plotted as a function of time, with daytime (white bars) and nighttime (black bars) durations indicated below the graph. Each data point represents the average hourly luminescence counts in relative light units (RLU) of 24 flies. (C) FLP protein expressed using the GAL4-UAS system can activate the reporter. Reporter activity (Y-axis, the mean hourly relative light units [RLUs] over a 4-day window) is plotted as a function of genotype (indicated with different colored bars) and treatment (+/− HS, heat shock). Error bars = S.E.M. *** p<0.001, Student' T-test, n = 24; n.s. signifies not significant. Similar statistical comparisons are made for the remaining figures. (D) Anatomical specificity of <i>gmr^long</i>-GAL4 driven reporters. <i>In vitro</i> luciferase activity measured in extracts made from isolated heads and bodies. The relative light units (Y-axis) are plotted as a function of the genotype (shown in gray [UAS-FLP/+; CRE-F-luc/+ or black [UAS-FLP/<i>gmr^long</i>-GAL4; CRE-F-luc/+]) or tissue source (head versus body).</p

cAMP production is decreased in FX mice.

<p>Platelets (Control n = 3, FX n = 5) and cortical membranes (n = 3 for each group) from wildtype and fmr1 knockout mice were stimulated with forskolin and levels of cAMP were measured (each n averaged in triplicate). Raw fluorescence (RF) is an inverse index of cAMP levels. The change in cAMP production was assessed using the fractional decrease in RF (FDRF) = (No Forskolin−Forskolin)/(No Forskolin). (a) Mouse platelet RF is comparable in the absence of forskolin [t(6) = 0.99; p = 0.36] but significantly different between groups (GroupxStimulus:[F(1,12) = 7.56;p = 0.018]) in the presence of forskolin [t(6) = −2.62; p = 0.040]. (b) Mouse platelet FDRF shows reduced cAMP induction in FX platelets [t(6) =  3.46; p = 0.014]. (c) Mouse cortex RF is comparable between groups in the absence [t(4) = 1.22;p = 0.29] or presence [t(4) = −1.92;p = 0.13] of forskolin. (d) Mouse cortex FDRF shows reduced FX cAMP induction [t(4) = 3.01; p = 0.04]. Error bars are SEM.</p

cAMP production is decreased in FX fly.

<p>Head membranes from wildtype, FX, FXR+, FXR++ flies were stimulated with forskolin and levels of cAMP were measured. Raw fluorescence (RF) is an inverse index of cAMP levels. The change in cAMP production was assessed using the fractional decrease in RF (FDRF) = (No Forskolin−Forskolin)/(No Forskolin). (a) RF-based cAMP measures in the fly head (Control n = 10, FX n = 3, FXR+ n = 6; FXR++ n = 6 sets of 20 heads) are comparable between groups in the absence of forskolin [F(3,21) = 1.96; p = 0.15], but not its presence [F(3,21) = 6.23;p = 0.003], since RF intensity in FX flies is larger than the others [t(21) = −4.10;p = 0.000]. (b) Fly head FDRF shows reduced FX cAMP induction relative to control membranes [FDRF:t(21) = 3.85;p = 0.001] and to the FX flies rescued with a single or multiple transgenes [FXR+FDRF:t(21) = 3.23;p = 0.004; FXR++FDRF:t(21) = 4.13;p = 0.000]. Error bars are SEM.</p