TOOLS AND TECHNIQUES

A high-sensitivity bi-directional reporter to monitor NF-κB activityin cell culture and zebrafish in real timePaola Kuri1, Kornelia Ellwanger2, Thomas A. Kufer2, Maria Leptin1,3,4,* and Baubak Bajoghli1,*

ABSTRACTNuclear factor (NF)-κB transcription factors play major roles innumerous biological processes including development and immunity.Here, we engineered a novel bi-directional NF-κB-responsivereporter, pSGNluc, in which a high-affinity NF-κB promoterfragment simultaneously drives expression of luciferase and GFP.Treatment with TNFα (also known as TNF) induced a strong, dose-dependent luciferase signal in cell culture. The degree of inductionover background was comparable to that of other NF-κB-drivenluciferase reporters, but the absolute level of expression was at least20-fold higher. This extends the sensitivity range of otherwise difficultassays mediated exclusively by endogenously expressed receptors,as we show for Nod1 signaling in HEK293 cells. To measure NF-κBactivity in the living organism, we established a transgenic zebrafishline carrying the pSGNluc construct. Live in toto imaging of transgenicembryos revealed the activation patterns of NF-κB signaling duringembryonic development and as responses to inflammatory stimuli.Taken together, by integrating qualitative and quantitative NF-κBreporter activity, pSGNluc is a valuable tool for studying NF-κBsignaling at high spatiotemporal resolution in cultured cells and livinganimals that goes beyond the possibilities provided by currentlyavailable reporters.

KEY WORDS: Nod1, Tri-DAP, pSGNluc, Proctodeum, Zebrafish

INTRODUCTIONNuclear factor (NF)-κB transcription factors are essential for anumber of biological processes such as inflammatory and immuneresponses, cell growth, apoptosis and development. Theirinappropriate activation has been linked to autoimmunity, chronicinflammation and various types of cancer (Ben-Neriah and Karin,2011; Hayden and Ghosh, 2008; Napetschnig and Wu, 2013).NF-κB transcription factors are expressed in many cell types.

Five NF-κB polypeptides, p65 (RelA), c-Rel, RelB, p50 (encodedby NFKB1) and p52 (encoded by NFKB2), can combine to form 15different transcription factors through homo- and hetero-dimerization (Hoffmann and Baltimore, 2006). The transcriptionalactivation of target genes depends on the nuclear translocation ofNF-κB dimers. In the absence of stimulatory signals, inhibitory κBprotein family members (e.g. IκBα and IκBβ, also known asNFKBIA and NFKBIB, respectively) keep NF-κB transcription

factors sequestered in the cytoplasm, thereby preventing theirbinding to DNA. Activation of different signaling pathways byproinflammatory signals such as pathogen-associated molecularpatterns, danger-associated molecular patterns or proinflammatorycytokines (e.g. interleukin-1), results in IκB kinase (IKK)activation. This kinase complex phosphorylates the IκBs,inducing their proteasomal degradation and resulting in release ofNF-κB transcription factors and their translocation into the nucleus(Vallabhapurapu and Karin, 2009). All NF-κB transcription factorsshare the ability to bind to the κB site consensus sequence 5′-GGGRNWYYCC-3′ (where R is a purine, Y is a pyrimidine, W isan adenine or thymine and N is an unspecified base) (Hoffmann andBaltimore, 2006). However, the binding affinity of NF-κBtranscription factors to different κB site sequences varies,indicating a relationship between binding affinity, specificity andfunction (Siggers et al., 2012).

Various assays have been established to determine the activity ofthe canonical NF-κB pathway. For example, IκBα degradationassayed by western blotting, and analysis of NF-κB binding activityin the absence and presence of IκBα by electrophoretic mobilityshift assay (Vancurova et al., 2001). However, these time-consuming methods are restricted to the analysis of small samplenumbers. An IκBα-firefly luciferase fusion reporter (IκBα-FLuc)allows IKK activation to be monitored in real time (Gross andPiwnica-Worms, 2005). Non-canonical NF-κB activation cannot bemeasured using these methods since it occurs independently ofIκBα degradation (Hayden and Ghosh, 2004). To assess NF-κBtranscription factor dynamics, cell lines that stably express the NF-κB subunit p65 fused to GFP (Bartfeld et al., 2010) are used toquantify the nuclear translocation of p65 in response to stimulation.These assays, however, do not provide direct levels of downstreamtranscriptional activation by NF-κB. The more commonly usedmethod to assay NF-κB activation is based on reporter genes (e.g.luciferase or β-galactosidase) driven by promoters containingmultiple repeats of an NF-κB consensus sequence (Badr et al.,2009; Bowie and O’Neill, 2000; Matsuda et al., 2007; Munoz et al.,1994; Schindler and Baichwal, 1994). The sensitivity andspecificity for NF-κB activation varies between these reporters. Inthis work, we have used a palindromic κB site sequence with highbinding affinity for multiple NF-κB proteins and developed a newbi-directional NF-κB-responsive promoter to simultaneously drivethe GFP and luciferase reporter genes. We named this reporterpSGNluc. We found that pSGNluc is highly sensitive and induciblein response to various stimuli in cell culture. In addition, weestablished a transgenic zebrafish line carrying the pSGNlucreporter. We show that GFP and luciferase reporters can be usedfor different purposes, allowing us to visualize and quantify the NF-κB activity during embryonic development and in response toinflammation in live embryos. In toto imaging of zebrafish embryoscarrying pSGNluc revealed a dynamic NF-κB activity during earlydevelopment. Our data also suggest that Iκbαa (encoded byReceived 12 September 2016; Accepted 8 December 2016

1Directors’ Research Unit, European Molecular Biology Laboratory (EMBL),Meyerhofstrasse 1, 69117 Heidelberg, Germany. 2Department of Immunology,Institute of Nutritional Medicine, University of Hohenheim, 70593 Stuttgart, Germany.3Institute of Genetics, University of Cologne, Zulpicherstrasse 47a, 50674 Cologne,Germany. 4EMBO, Meyerhofstrasse 1, 69117 Heidelberg, Germany.

*Authors for correspondence (bajoghli@embl.de; maria.leptin@embo.org)

B.B., 0000-0002-7368-7523

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nfkbiaa), a member of the family of NF-κB inhibitory proteins,regulates the NF-κB activity in the proctodeum and skin.

RESULTSDesign of the pSGNluc reporterMost available NF-κB-responsive reporters use multimerized κBsite sequences upstream of a minimal promoter to drive a fireflyluciferase gene. The sequence and number of κB sites vary betweendifferent NF-κB reporters. According to a large dataset of potentialκB site sequences for multiple NF-κB dimers (Siggers et al., 2012),the predicted binding affinity of NF-κB dimers to the κB sitesequences used to generate these NF-κB reporters is moderate(Fig. 1A). We hypothesized that a binding site with high affinity fora range of different NF-κB dimers might allow us to make a moresensitive reporter. The study by Siggers et al. showed that thesequence 5′-GGGAATTCCC-3′, originally identified as a κB siteupstream of the type VII collagen gene (Kon et al., 1999), is able tointeract strongly with different combinations of hetero- and homo-dimers of NF-κB subunits. In designing a new reporter plasmid, wetook advantage of the palindromic nature of this sequence to create abi-directional reporter. In this plasmid, which we named pSGNluc,two minimal promoters flank a DNA fragment containing eighttandem copies of the 5′-GGGAATTCCC-3′ sequence (Fig. 1B).GFP was cloned downstream of one promoter to visualizeexpression in living cells. To enable quantitative analysis ofexpression levels, luciferase was cloned downstream of the minimalpromoter on the opposing side (Fig. 1C). The pSGNluc reportershould therefore simultaneously express both marker genes from thebi-directional promoter, allowing both the spatial and temporalmonitoring and quantification of NF-κB activity in living cells.

The pSGNluc reporter is strongly inducible and highlysensitive in cell cultureTo examine the efficiency of the new NF-κB-responsive reporter incell culture, we transfected the pSGNluc vector in HEK293T cells.Transfected, unstimulated cells showed weak or no GFP expression(Fig. 2A), indicating a low background activity of the pSGNlucreporter. Exposure to 10 ng/ml of TNFα (also known as TNF) led toa strong upregulation of GFP expression in transfected cells, asshown by live-cell microscopy and flow cytometry data (Fig. 2A,B).We also measured the level of luciferase activity of pSGNluc,

and compared it to two other NF-κB-driven luciferase reporters,pGL4.32 (Promega) and IgκB-Luci (Munoz et al., 1994) as well asan IL-8 reporter (Bowie et al., 2000). We treated cells transfectedwith each of these constructs with 10 ng/ml of TNFα and measuredthe luciferase activity in cell lysates 24 h after treatment. Wecalculated the inducibility of each of the reporters (measured as theratio of luciferase with and without treatment) and observed thatpSGNluc was induced to the same extent as pGL4.32 whencompared to non-treated controls. The IgκB-Luci and IL8-Lucreporters displayed the highest and lowest inducibility levels,respectively (Fig. 2C). All transfected cells showed low levels ofbackground signal without stimulation, although the pSGNlucreporter was expressed at up to 6-fold higher levels than the IL-8reporter (Table S1). To compare the sensitivity of the NF-κB-responsive reporters, the cells were stimulated with increasingTNFα concentrations. The luciferase level of all four reportersincreased in a TNFα-dose-dependent manner after a 24 h treatment.The absolute level of luciferase expressed with the pSGNlucreporter in response to TNFα at 1 ng/ml was 21-, 34- and 73-foldhigher than for the IgκB-Luci, pGL4.32 and IL-8 reporters(Fig. 2D,E).

We then determined the kinetics of NF-κB activation in responseto 10 ng/ml TNFα. The pSGNluc reporter reached luciferaseactivity levels that were significantly higher than other reporterstested from 6 h after treatment (Fig. 2F), illustrating an increasedsensitivity even at early time points after stimulation.

We also tested the response of the reporter to other inflammatorysignals. Nod1 is an intracellular pattern recognition receptor thatinduces NF-κB activation upon detection of bacterialpeptidoglycans (Chamaillard et al., 2003; Girardin et al., 2003).HEK293T cells have low levels of endogenous Nod1 (Viala et al.,2004). In many studies, HEK293T cells are therefore transfectedwith Nod1 expression plasmids (Masumoto et al., 2006; Zureket al., 2011) to increase the NF-κB response towards Nod1stimulation. However, the amount of Nod1 receptor in the cell iscrucial and its overexpression can result in autoactivation (Zureket al., 2011). We tested whether the high sensitivity of pSGNlucwould allow the detection of NF-κB activation mediated by theendogenously expressed Nod1. HEK293T cells were transfectedwith pSGNluc and then treated with L-Ala-γ-D-Glu-meso-diaminopimelic acid (Tri-DAP) (Chamaillard et al., 2003;Girardin et al., 2003). The levels of luciferase activity reached bypSGNluc after Tri-DAP stimulation were 13- to 55-fold higher thanthose of the pGL4.32, IgκB-Luci and IL-8 reporters (Fig. 3A;Table S2), representing a 24±8-fold (mean±s.d.) increase inluciferase activity in cells carrying the pSGNluc reporter(Fig. 3B). The fact that these inductions were small compared tothose achieved by TNFα might suggest that Tri-DAP is a weakeractivator of NF-κB signaling. However, when we looked at the GFPexpression pattern of transfected cells we observed that, instead of ahomogeneous low expression level that could be expected fromgeneral weak response, only a fraction of transfected cells respondedto Tri-DAP stimulation (Fig. 3C,D). Time-lapse imaging revealedthat these cells expressed GFP at similar levels to cells treated withTNFα (Fig. 3E; Movie 1), albeit with a delayed onset. This indicatesthat the average level of luciferase activity in all cells after treatmentwas low not because the Nod1 ligand Tri-DAP elicits a weakresponse, but because only a limited number of cells responded tothis treatment.

Overall, cell culture assays showed that pSGNluc is inducibleafter treatment with inflammatory stimulants to a degree that wascomparable with other reporters (measured as fold induction overbackground expression), but is much more sensitive than other NF-κB-driven luciferase reporters. In particular, we found pSGNlucsuitable to monitor NF-κB activation mediated by endogenousNod1 in HEK293T cells. Furthermore, the bi-directional promoterallows the concomitant monitoring of NF-κB activation byexpression of the two reporters, GFP and luciferase, and enablessingle-cell analysis of NF-κB activation and easier time-resolvedanalysis.

Establishment of pSGNluc transgenic zebrafish to monitorNF-κB activity in vivoMembers of the NF-κB transcription factor family are expressedalmost ubiquitously during embryonic development. In transgenicmice carrying an NF-κB-responsive reporter, the spatial activity ofNF-κB signaling has been observed either in fixed tissues (Schmidt-Ullrich et al., 1996) or in live animals with low spatiotemporalresolution (Carlsen et al., 2002). Given that zebrafish NF-κBproteins can bind to the mammalian consensus κB site (Correa et al.,2004), we created transgenic zebrafish carrying the pSGNlucreporter to observe NF-κB activity in the living organism. Thistransgenic fish line is hereafter called Tg(8×Hs.NFκB:GFP,

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Luciferase) in accordance with the Zebrafish nomenclatureguidelines. We used light-sheet microscopy to monitor theactivity of the pSGNluc reporter during early zebrafish embryonicdevelopment at high spatiotemporal resolution. Embryos carryingthe transgene expressed GFP in a dynamic pattern. Live in totoimaging of the transgenic embryos from mid-gastrula to thebeginning of the segmentation stage [approximately from 6 to12 hours post-fertilization (hpf )] revealed that there already wasubiquitous and weak GFP expression at the shield stage, with theentire animal pole labeled (Fig. 4A). Cells retained this level ofexpression throughout the gastrula stage and into the segmentation

stage (Fig. 4B–E). The first increase in the GFP signal was observedin the nasal vesicle (Fig. 4F) and in the proctodeum beginningshortly afterwards (Fig. 4G). Using confocal microscopy, weobserved strong GFP expression in microvillous sensory neuronsthat are connected to the forebrain (Fig. 4H), which diminished by2 days post-fertilization (dpf ) (Fig. 4I). At 2 dpf, the signal wasstrongly present in the lateral line (Fig. 4I, yellow arrows), theproctodeum and epithelial cells around the edges of the dorsal andventral fins. This expression pattern remained at 3 dpf (Fig. 4J). By5 dpf, cells in the intestinal lining began to express GFP, andexpression in the lateral line and fins remained (Fig. 4K, white

Fig. 1. The pSGNluc reporter plasmid.(A) Z-score for κB site sequences presentin the IL-8-Luc, IgκB-Luci, pGL4.32 andpSGNluc plasmids for their binding tomultiple NF-κB dimers; data are from theprotein-binding microarray dataset inSiggers et al. (2012). (B) Sequence ofDNA fragment containing eight tandemcopies of the κB site (black boxes).(C) Schematic representation of thepSGNluc vector showing the eight copiesof the κB site (black), two minimal CMVpromoters (gray), GFP (green) andluciferase (brown). The construct containstwo I-SceI meganuclease restriction sitesfor the generation of transgenic animals.

Fig. 2. High sensitivity and inducibility of the pSGNluc reporter in response to TNFα stimulation. (A,B) HEK293T cells were transfected with the pSGNlucand mCherry-expressing plasmids and treated with TNFα (10 ng/ml) where indicated. 24 h later, fluorescence signals were examined by fluorescencemicroscopy (A) and flow cytometry (B). Expression of mCherry indicates the transfection efficiency. (C–F) HEK293T cells were transfected with the indicatedreporter plasmids and a β-galactosidase plasmid for normalization. Directly after transfection cells were either left untreated or stimulated with TNFα (10 ng/ml). ThenRLU levels were measured 24 h later (see Materials and Methods). (C) The induction was calculated by dividing nRLU values from cells 24 h post stimulationwith TNFα (10 ng/ml) by nRLU values of unstimulated cells. Each dot represents the mean induction of independent experiments conducted in triplicates. (D)Sensitivity matrix of NF-κB-responsive reporters based on nRLU upregulation after 24 h stimulation with TNFα (1 ng/ml). (E) TNFα titration and (F) kinetics of10 ng/ml TNFα-induced reporter activation. Values represent mean±s.e.m. (n=3). Scale bars: 80 µm. n.s., not significant.

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arrowheads). The onset of expression in the gut coincides withthe stage in which the larva begins to feed. After the onset ofhematopoiesis, embryos showed a dim GFP signal. A similar levelof GFP was also observed in keratinocytes. Zebrafish embryoscarrying a previously published NF-κB-responsive reporter alsoexpressed GFP in the skin (Candel et al., 2014; Hatzold et al., 2016).However, the signal in the skin was stronger in embryos carrying thelatter reporter than in Tg(8×Hs.NFκB:GFP,Luciferase) embryos(Fig. S1A). In mice, NF-κB proteins are mainly present in basalkeratinocytes (Takao et al., 2003), which are mitotically active andprovide new cells that gradually undergo differentiation toward theskin surface. To better characterize the GFP signal in the skin, wetook advantage of the Tg(krt19:dTomato) fish, in which basalkeratinocytes are fluorescently labeled (Fischer et al., 2014). In vivoimaging of double-transgenic Tg(8×Hs.NFκB:GFP,Luciferase;krt19:dTomato) embryos revealed that GFP is expressed at a lowlevel in basal keratinocytes, albeit in a mosaic fashion (Fig. S2). AsNF-κB signaling has more than one role in keratinocytes, it remainsto be determined whether this expression pattern is associated withkeratinocyte homeostasis or inflammation.To test the specificity of pSGNluc-driven GFP expression as a

proxy for NF-κB activation in zebrafish embryos, we interfered withIκBα function. IκBα, an NF-κB inhibitory protein, binds to NF-κBdimers and prevents their translocation into the nucleus (Arenzana-Seisdedos et al., 1997). Knockdown of the zebrafish ortholog iκbαashould therefore enable NF-κB proteins to move to the nucleus and

activate transcription of target genes (He et al., 2015). In zebrafish,iκbαa is expressed in the proctodeum and nasal vesicle (Fig. S3).We injected a previously published morpholino (MO) (He et al.,2015) to interfere with Iκbαa function in Tg(8×Hs.NFκB:GFP,Luciferase) embryos. In iκbαa morphants, GFP expression wasenhanced in the skin and proctodeum (Fig. 5A–C). Although ourRNA in situ hybridization analysis did not detect iκbαa expressionin the skin, previous microarray analysis has shown that both theiκbαa and iκbαb (also known as nfkbiab) genes are expressed in thezebrafish skin (Lü et al., 2012). At high doses, injection of the iκbαamorpholino led to severe defects in notochord formation (Fig. 5D).This phenotype is consistent with those previously observed inzebrafish for NF-κB loss- or gain-of-function analysis (Correa et al.,2005, 2004). The tissue-specific increase in GFP levels resultingfrom knockdown of the NF-κB inhibitor iκbαa suggests thatreporter activity in the Tg(8×Hs.NFκB:GFP,Luciferase) line islinked to NF-κB activation, and that the dynamic activity of NF-κBsignaling in various tissues during embryonic development can bemonitored through live imaging of this transgenic line.

In vivo monitoring of NF-κB activation in response toinflammationAs regulators of the immune and inflammatory processes, NF-κBtranscription factors are activated in response to a variety of signalsincluding pathogens, stresses and injuries. We therefore monitoredthe dynamics of NF-κB activation in response to injuries in

Fig. 3. Activation of the pSGNluc reporter in response to Nod1-mediated peptidoglycan recognition. (A,B) HEK293T cells were transfected with theindicated reporter plasmids and a β-galactosidase plasmid for normalization. Subsequently cells were either left untreated or stimulated with 10 µg/ml Tri-DAP.nRLU values were determined 24 h later (see Materials and Methods). (A) Tri-DAP (10 µg/ml)-induced reporter activation. Values represent mean±s.e.m. (n=3).(B) The induction was calculated by dividing nRLU values of stimulated cells by control cells values. Each dot represents the mean induction of independentexperiments conducted in triplicates and the overall mean±s.d. is shown. (C) Representative images of HEK293T cells transfected with the pSGNluc andmCherry-expressing plasmids. Expression of mCherry indicates the transfection efficiency. (D) GFP expression in cells after Tri-DAP stimulation (mean±s.e.m.;n=3). The data from Fig. 2B and this figure come from the same experiment and the control group in the top panel of Fig. 2B is therefore also the control for thispanel. (E) Time course of GFP expression in pSGNluc-transfected cells without stimulation (top panel), and cells treated with Tri-DAP at 10 µg/ml (middle panel)or TNFα at 5 ng/ml (bottom panel). Images are taken from Movie 1. Red arrows indicate the onset of GFP expression. Scale bars: 80 µm (C); 100 µm (E).

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Tg(8×Hs.NFκB:GFP,Luciferase) embryos. In a tailfin-woundingassay, GFP expression was upregulated in the area around thewound edge of injured embryos (Fig. 6A; Movie 2). Most epithelialcells at the wound edge exhibited strong NF-κB activation whenexamined 24 h later. To test the inducibility of the reporter, we firstcompared the kinetics of NF-κB activation in this assay with thosefor a previously developed reporter in zebrafish (Banerjee andLeptin, 2014). The Tg(NFκB-EGFP) line, previously used tomonitor NF-κB activation upon UV radiation in zebrafish embryos,contains three copies of κB site sequences derived from the IgκB-Luci vector (Banerjee and Leptin, 2014). No GFP is detectablein Tg(NFκB-EGFP) embryos during embryonic development. In

response to sterile injury, Tg(NFκB-EGFP) embryos showed weakGFP expression 40 h after wounding. In contrast, Tg(8×Hs.NFκB:GFP,Luciferase) embryos showed a strong increase in GFPexpression 40 h after injury (Fig. S1B). Thus, Tg(8×Hs.NFκB:GFP,Luciferase) embryos show a more sensitive response to localinjuries than the Tg(NFκB-EGFP).

We also tested the efficiency of the pSGNluc reporter in responseto infection. Non-pathogenic Gram-negative Escherichia coliexpressing red fluorescent protein were injected into thenotochord or the inner ear. No GFP expression driven bypSGNluc was detectable at 3 dpf either in the notochord(Fig. 6B, upper panel) or the inner ear (Fig. 6C, left panel) ofuntreated animals. Injection of PBS alone resulted in GFPexpression in the notochord (Fig. 6B, middle panel) indicatingthat a sterile injury alone activates NF-κB signaling. However, evenhigher GFP levels were observed in notochords infected withE. coli when examined 18 h post infection (Fig. 6B, bottom panel).This is in agreement with previous studies showing thatmicroinjection of bacteria in these tissues results in aninflammatory response that includes the recruitment of leukocytes(Levraud et al., 2008; Nguyen-Chi et al., 2014). We also observedan accumulation of GFP-expressing immune cells (Fig. 6C, right

Fig. 4. Dynamic NF-κB activity during early zebrafish embryonicdevelopment. (A–E) Light-sheet images of Tg(8×Hs.NFκB:GFP,Luciferase)embryos show ubiquitous, weak NF-κB activity from 6 hpf until 12 hpf. (F) Thefirst strong tissue-specific signal was observed in the nasal vesicle (nv) ataround 16 hpf. (G–K) Confocal images of Tg(8×Hs.NFκB:GFP,Luciferase)embryos from 1 dpf until 5 dpf. GFP was detected in the proctodeum (p) at1 dpf (G). The GFP signal in the nasal vesicle (G) originates from microvilloussensory neurons (H). E–G and I–K show embryos in lateral view. Anterior is tothe left. H shows the head of embryos from the frontal side. Yellow arrowsindicate GFP in the lateral line. White arrowheads indicate GFP in the intestine.Scale bars: 100 µm (A–F); 300 µm (G); 50 µm (H); 500 µm (I–K).

Fig. 5. Specificity of Tg(8xHs.NFκB:GFP,Luciferase) for NF-κB activityin zebrafish embryos. (A,B) 0.3 mM iκbαa morpholino was injected intotransgenic embryos. Morphants at 3 dpf had increased expression of GFPin the skin (A) and proctodeum (B, arrows). GFP fluorescence is shown as afalse-color heat map. The intensity of the laser was equal for both images toallow direct comparison of the GFP signal in morphants and controls.(C) Quantitative comparison of the GFP signal in the proctodeum of embryosinjected with 0.3 mM standard control or iκbαa morpholino. Each dotrepresents an individual embryo at 2 dpf (mean±s.d.). (D) Injection of 1 mMiκbαa morpholino results in higher GFP expression and embryonicmalformation. GFP fluorescence is shown as a heat map as indicated in A.Scale bars: 500 µm (A); 50 µm (B); 300 µm (D).

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panels) in the inner ear suggesting that NF-κB signaling is stronglyactivated in response to local infection.

Quantification of NF-κB activity in live animalsOne advantage of the Tg(8×Hs.NFκB:GFP,Luciferase) fishcompared to previously available zebrafish NF-κB-responsivereporters is the option for quantification of NF-κB activity bybioluminescence. We measured bioluminescence in vivo with a

previously published method that is non-invasive and is easilyscalable (Lahiri et al., 2014). Briefly, living zebrafish larvae wereassayed in 96-multiwell plates by adding beetle luciferin potassiumsalt solution directly to thewater. In the first 4 dpf, concomitant withthe broadening of the GFP expression pattern (Fig. 4), luciferaseexpression increased ∼10-fold (Fig. 7A). Treatment with JSH-23, adrug that has been previously used to inhibit NF-κB activity inzebrafish embryos (He et al., 2015), significantly reduced luciferaselevels at 2 dpf (Fig. 7B). Next, we measured the luciferase activity iniκbαa morphants. Morpholino injection resulted in a significantincrease in luciferase activity in 2 dpf embryos compared to bothuninjected and control-injected embryos (Fig. 7C). Theseexperiments confirmed that the palindromic sequence drivesexpression of both GFP and luciferase genes, and that thebioluminescence can be used to quantify NF-κB activity in theliving fish.

The pSGNluc reporter does not interfere with cytokineexpressionBoth in vitro and in vivo data confirmed that pSGNluc is a sensitivereporter for monitoring NF-κB activation, likely due to the highbinding affinity of NF-κB proteins for the κB site sequence used inthis reporter. One could assume that the strong binding affinity ofpSGNluc reporter sites might interfere with the expression ofendogenous genes by outcompeting the transcription factorcomplex, especially in NF-κB target genes that exhibit weaktranscriptional activity. To test this possibility in vitro, weperformed a competition assay by transfecting HEK293T cellswith different amounts of NF-κB-responsive plasmids, andmeasuring interleukin-8 (IL-8) production and luciferase activity24 h after stimulation with 10 ng/ml TNFα. We observed a positivecorrelation between the amount of transfected NF-κB-responsiveplasmids and luciferase activity (Fig. S4A). However, increasingamounts of transfected NF-κB-responsive plasmids did notnegatively affect IL-8 secretion (Fig. S4B). Surprisingly, even atrend towards higher IL-8 secretion was visible. We next testedwhether cytokine expression was negatively affected in theTg(8xHs.NFκB:GFP,Luciferase) fish. We compared mRNAlevels of cytokine genes in transgenic and wild-type siblingsbefore and after inflammatory stimuli. We used UV irradiation toinduce inflammation in zebrafish embryos (Banerjee and Leptin,2014), since NF-κB signaling plays a major role in the induction ofseveral cytokines during the inflammatory responses to thisstimulus. As expected, Tg(8×Hs.NFκB:GFP,Luciferase) embryosexposed to UV light showed an increase of NF-κB activity(Fig. 8A). We used quantitative real-time RT-PCR (qPCR) tocompare the mRNA level of selected cytokines in transgenic andwild-type siblings (Fig. 8B). The cytokine genes interleukin-10(il10), il12b and tnfa are direct target genes of NF-κB (Banerjee andLeptin, 2014; Kennedy et al., 1997; Rivas and Ullrich, 1992).Consistent with a previous study (Banerjee and Leptin, 2014), il10and tnfa, but not il12b, were significantly upregulated in UV-treatedembryos (Fig. 8B). The results from qPCR showed that thecytokines were expressed at comparable levels in the transgenic andwild-type siblings. Taken together, these data suggest that thepSGNluc reporter does not significantly interfere with the NF-κBresponse in cell culture and zebrafish embryos.

DISCUSSIONWe have established a new NF-κB-responsive reporter system.Compared to other NF-κB-driven luciferase reporters, pSGNluc hasthree major advantages. First, pSGNluc is inducible in response to

Fig. 6. In vivo visualization of NF-κB activity upon local wounding andinfection. (A) Time-lapse images derived fromMovie 2 show NF-κB activationafter tailfin cut. GFP fluorescence in the top panel is shown as a heat map.GFP is upregulated locally near the wound. (B,C) Injection of HcRed-expressing E. coli into the notochord (B) or inner ear (C) of Tg(8×Hs.NFκB:GFP,Luciferase) elicits NF-κB activation 20 h after infection. The bacterialinfection results in the accumulation of immune cells in the inner ear. GFP wasnot expressed in the notochord or inner ear of uninfected control zebrafish.The white dashed line in (C) demarcates the inner ear. Anterior is to the left.Scale bars: 150 µm (A,C); 200 µm (B).

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inflammatory stimuli, such as TNFα, at levels comparable to otheravailable reporters. Second, the reporter is significantly moresensitive to both the proinflammatory cytokine TNFα and theNod1-specific inflammatory activator Tri-DAP than othercommonly used NF-κB reporters. This is likely because thesequence and copy number of the κB site enhance the sensitivity ofthe NF-κB-responsive reporter (Matsuda et al., 2007). Third, the bi-directional property of the NF-κB-responsive promoter enablessimultaneous expression of two reporter genes: GFP expressionshows the kinetics and spatial aspects of a response in individualcells in real time, while the luciferase assay allows a quantitativeevaluation of the global NF-κB activation throughout all stimulatedcells or tissues. This dual reporter system allows simultaneousqualitative and quantitative monitoring of NF-κB activity inresponse to inflammatory stimuli, the integration of which canprovide information beyond on–off activation. For example, thebroad GFP expression and high luciferase levels observed in cells inculture after TNFα treatment shows that the majority of transfectedcells responded to stimulation. In the case of Tri-DAP exposure, theluciferase measurement suggested that NF-κB signaling was notstrongly induced after exposure; however, the mosaic GFPexpression after treatment showed that some cells do respondstrongly. It is not clear why only a subset of cells respond to Tri-DAP, but we suspect it may be related to differences in drug uptake.Tri-DAP enters epithelial cells via clathrin-mediated endocytosis,then, it requires endosomal processing and an optimal pH for itstranslocation into the cytosol (Sorbara and Philpott, 2011). Thismay be an inefficient process, explaining why penetration of Tri-DAP into the cytoplasm of HEK293T cells might not proceeduniformly in all cells.Two transgenic zebrafish lines carrying NF-κB-responsive

reporters have been generated previously (Banerjee and Leptin,2014; Kanther et al., 2011). Both lines contained constructs withmultimerized κB sites to trigger GFP expression. The κB sitesequences used in these reporters are predicted to have lower

binding affinities for NF-κB proteins than those used in thepSGNluc. One of these reporters (Banerjee and Leptin, 2014) doesnot show detectable levels of GFP expression during embryonicdevelopment and is less responsive to injury than the pSGNlucreporter. The second reporter was used to study the effect ofmicrobiota on NF-κB activity in the intestine (Kanther et al., 2011).This reporter drives GFP expression in the lateral line and intestineduring embryonic development, an activation pattern consistentwith our own observations for the pSGNluc reporter. However,there are also some differences between the two lines, including thedynamic expression in the olfactory neurons and stronger sporadicexpression in the epithelia, as well as the reduced expression in thepharyngeal arches seen for the pSGNluc line. The GFP expressionseen with the pSGNluc reporter in the first 30 hpf is consistentwith previously described early expression of NF-κB transcriptionfactors (Correa et al., 2004) and reflected published spatiotemporalexpression patterns of genes involved in NF-κB signaling at thesestages, such as c-rel (Correa et al., 2004) and ikk1 (Correa et al.,2005). This early NF-κB activation matches what is knownabout the functional requirements for NF-κB signaling duringembryogenesis: during gastrulation NF-κB activation is involved incoordinating the cell cycle and mesoendodermal cell movements(Liu et al., 2009) and lack of NF-κB signaling in the notochord leadsto embryonic dorsalization and deformities (Correa et al., 2004).The requirement of finely tuned NF-κB signaling during notochorddifferentiation is also supported by the defects caused by high dosesof iκbαa morpholino.

The weak NF-κB activity in keratinocytes and its upregulation iniκbαa morphants and in response to UV irradiation are also inagreement with what is known about the role of NF-κB signaling inkeratinocyte inflammation and homeostasis (Wullaert et al., 2011).The skin is exposed to multiple environmental stimuli, and NF-κBsignaling is required for the inflammatory response in this organ.Additionally, NF-κB is a regulator of keratinocyte proliferation andgrowth (Wullaert et al., 2011). The weak GFP signal in the skin is

Fig. 7. Quantification of NF-κB activity in live Tg(8×Hs.NFκB:GFP,Luciferase) embryos. (A) Luciferase activityduring embryonic development. (B) Embryos at 1 dpf weretreated with 100 µM JSH-23, and luciferase activity wasmeasured at 2 dpf. Data represent the mean±s.d. luciferaseactivity of three independent experiments conducted with>12 embryos. (C) Transgenic embryos were injected with0.3 mM standard control morpholino or iκbαamorpholino atthe one-cell stage and luciferase activity was measured at2 dpf. Luciferase activity was increased in iκbαamorphants.Each dot represents an individual embryo and the overallmean±s.d. is shown. cps, counts per second.

Fig. 8. UV irradiation results in activation of the NF-κB pathway. (A) The induction was calculated by dividing luciferase values from embryos at 24 h postUV irradiation by values of untreated transgenic (Tg) siblings at 2 dpf. The data represent the mean±s.d. induction of three independent experiments conductedwith >6 embryos. (B) Relative expression of il10, tnfa and il12b in transgenic (Tg) and wild-type (WT) siblings without and 24 h post UV irradiation. The β-actingene was used as a reference for normalization. Each dot represents an individual embryo and the overall mean±s.d. is shown.

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also in agreement with findings in transgenic mice carrying anNF-κB-responsive reporter (Carlsen et al., 2002). In mice, IκBα isexpressed in the skin and prevents the nuclear localization of NF-κBproteins. Newborn mice lacking IκBα exhibit increased NF-κBactivation in the skin, which triggers keratinocyte proliferation andepidermal hyperplasia (Klement et al., 1996). A similar phenotypewas observed in RelA-knockout mice or after overexpression of adominant-negative mutant version of Iκbα in the epidermalkeratinocytes (Seitz et al., 2000; Zhang et al., 2004). The tissue-specific response to iκbαa knockdown as well as to localinflammation suggest that the GFP signal in the Tg(8×Hs.NFκB:GFP,Luciferase) embryos recapitulates the activity of NF-κBsignaling in zebrafish embryos.A novel aspect of our reporter line compared to the existing

NF-κB-responsive reporters (Banerjee and Leptin, 2014; Kantheret al., 2011) is that it allows quantification of NF-κB activity bybioluminescence measurement. We showed that induction ofluciferase in vivo correlated with the increase in NF-κB activity asmeasured by GFP expression, and that it responded to theknockdown of the NF-κB inhibitor iκbαa in the same way asGFP did. Our assays measure luciferase activity in each larvaindividually in such a way that the larva is unharmed and cansurvive for several days, allowing continuous tracking of reporteractivity (Lahiri et al., 2014). This represents a significantimprovement over a previously published method to measure NF-κB activity in zebrafish with a luciferase reporter in which lysis ofthe embryo was required for activity measurement (Alcaraz-Perezet al., 2008). Bioluminescence imaging of NF-κB expression in aluciferase transgenic mouse model has been used as a tool to screenanti-NF-κB drug candidates (Robbins and Zhao, 2011). Thezebrafish is a model ideally suited for high-throughput, whole-organism drug screening (Zon and Peterson, 2005) through the useof fluorescence-based analysis both of reporter activity (Wang et al.,2015) and bacterial load (Ordas et al., 2015; Veneman et al., 2013).We therefore anticipate that the zebrafish transgenic pSGNluc willbecome a valuable tool to study many aspects of NF-κB signalingduring development and inflammation in real time. The transgenicreporter could also provide a cost-effective improvement for high-throughput assays in drug discovery.

MATERIALS AND METHODSDNA constructsMultimerized NF-κB-binding sites with the idealized sequence 5′-GGGA-ATTCCC-3′ separated by 6 bp spacers were generated by oligonucleotideligation. A fragment containing eight NF-κB-binding sites was inserted in thepSGHluc plasmid (Bajoghli et al., 2004). The resulting plasmid (pSGNluc)contains eight NF-κB sites flanked by two 260 bp long minimal CMVpromoters. The bi-directional promoter drives the firefly luciferase genewith a3′ UTR of the globin gene and an SV40 polyA at one side, and GFP with abovine growth hormone (bGH) polyA at the other side. The sequence of thepSGNluc plasmid has been deposited at NCBI under the accession numberKY129798. The β-galactosidase expression plasmid is described in Philpottet al. (2000). pGL4.32 was purchased from Promega and IL-8 luciferasereporter is described in Bowie et al. (2000). The original name of IgκB-Luci is(IgK)3-conaluc and is described in Munoz et al. (1994).

Cell culture assaysIn vitro analysis was performed as described previously (Zurek et al., 2011).Briefly, cells of the human embryonic kidney cell line HEK293Twere platedat a density of 30,000 cells per well in a 96-multiwell plate. Transfectionmixes were prepared for triplicate assays. A total amount of 51 ng DNAwith13 ng of the respective NF-κB-responsive vector, 8.6 ng β-galactosidase- ormCherry-expressing plasmid, and 29.4 ng pcDNA3.1 was used per well.HEK293T cells were transfected with DNA mixes using X-tremeGENETM

9 DNA transfection reagent (Roche). Unless otherwise indicated,transfected cells were directly stimulated with either TNFα (Invivogen) orTri-DAP (Invivogen) at the indicated concentrations, and measurementswere performed after 24 h. For kinetic measurements, cells were transfected,incubated overnight and stimulated with TNFα at 10 ng/ml for 8, 6, 3, 2 or1 h before cell lysis. For the measurement of luciferase and β-galactosidaseactivities, cells were lysed at 24 h post transfection. Cells were lysed in100 µl lysis buffer (25 mM Tris-HCl pH 8.0, 8 mM MgCl2, 1% Triton X-100, 15% glycerol) per well. To measure the luciferase activity, 50 µl of thecell lysates were transferred to a white non-transparent 96-multiwell plateand luciferase activity was quantified as relative luminescence units (RLU)in a multiplate reader (Enspire, Perkin Elmer) upon automatic adding of100 µl luciferase substrate solution [lysis buffer containing 1.3 µM ATP and770 ng/ml D-luciferin (Sigma)]. To measure β-galactosidase activity, theremaining 50 µl cell lysate was supplemented with 100 µl of 1 mg/ml o-nitrophenyl-β-D-galactopyranosid (ONPG) in 60 mM Na2HPO4, 40 mMNaH2PO4, 10 mM KCl, 1 mM MgSO4 at pH 7.0 per well and incubated at37°C for 30 min. Then, absorption was measured in a photometer at 405 nm(620 nm reference). β-Galactosidase activity was used to normalizeluciferase activity (nRLU) in each well. Three independent experiments,each performed in triplicate, were conducted for each assay.

ELISATo determine IL-8 concentrations, supernatants of HEK293T cells withoutand with TNFα (10 ng/ml) stimulation were analyzed with the humanCXCL8/IL8 DuoSet ELISA kit (R&D Systems) according to themanufacturer’s protocol. The IL-8 concentration in unstimulated cells wasbelow the level of detection.

ZebrafishAll animal experiments described in the present study were conducted onembryos younger than 5 dpf under the rules of the European MolecularBiology Laboratory and the guidelines of the European Commission,Directive 2010/63/EU. The zebrafish strain used in this study was Daniorerio wild-type TL. The stable transgenic line Tg(krt19:dTomato) wasdescribed previously (Fischer et al., 2014). To develop the stable transgeniczebrafish line, pSGNluc plasmid was co-injected with I-SceI meganucleaseenzyme in 1× I-SceI buffer (New England, BioLabs) into the blastomere atthe one-cell stage as described previously (Aghaallaei et al., 2007). Threezebrafish founders were identified with similar GFP expression patterns.One founder was crossed with the wild-type TL strain and their progenywas used for this work. The transgenic zebrafish carrying the pSGNlucis named Tg(8xHs.NFκB:GFP,Luciferase)hdb5 in accordance with theZebrafish Nomenclature Guidelines (https://wiki.zfin.org/display/general/ZFIN+Zebrafish+Nomenclature+Guidelines) and was approved by theZebrafish Nomenclature Committee.

Morpholino and bacterial microinjectionsAntisense morpholino (MO) for zebrafish ikbαa (5′-TGCGGCTCTGTG-TAAATCCATGTTC-3′; He et al., 2015) and standard control morpholinowere obtained from Gene Tools. They were prepared as 1 mM and 3 mMstock solutions in double-distilled H2O, respectively. Morpholino at aconcentration of 0.3 mM or 1 mM together with 100 mM KCl was injectedinto the blastomere of zebrafish embryos at the one-cell stage. Non-pathogenic Gram-negative Escherichia coli BL21 expressing HcRed(Aghaallaei et al., 2010) were microinjected into the transgenic pSGNlucembryos at 3 dpf as described previously (Nguyen-Chi et al., 2014).

UV treatmentsUV treatment in zebrafish embryos was performed as described previously(Banerjee and Leptin, 2014). Briefly, embryos at 24 hpf (n=50) were sortedinto a petri dish and exposed to 24 mJ/cm2 of UV. Embryos were maintainedin E3 medium at 28°C for 24 h prior to total RNA extraction or luciferasemeasurement.

Drug treatmentJSH-23 (Selleckchem Inc) was diluted to a stock concentration of 10 mM inDMSO, aliquoted and stored at −80°C. For treatment, the drug was added

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directly to the E3 medium of manually-dechorionated 1 dpf embryos to afinal concentration of 100 µM. Luciferase activity was measured 24 h afterdrug exposure.

Flow cytometry and fluorescence microscopyFor FACS analysis, HEK293T cells were plated in 24-well plates, directlytransfected with pSGNluc and pmCherry plasmids in a 1:1 ratio, andstimulated with TNFα or Tri-DAP at the indicated concentrations. 24 h later,cells were trypsinized, resuspended in 5% fetal calf serum (FCS) in PBS andanalyzed with the FACSCanto system (BDBiosciences). To image mCherryand GFP expression in vitro, HEK293T cells were plated on glasscoverslips, transfected and stimulated as above. After 24 h, stimulatedcells were fixed with 4% paraformaldehyde in PBS for 20 min. After a shortwash in 1×PBS, fixed cells were mounted with Mowiol4.88 (Sigma)supplemented with bis-benzimide to stain the nuclei. For live-cell imaging,HEK293T cells were plated in compartmentalised glass-bottom petri dishes(Greiner bio-one) before transfection and stimulation. Images were taken ona Zeiss Axiovert 200 M equipped with a Plan-Apochromat 20×0.8 NAM27objective and AxioCamMR3 camera (Carl Zeiss, Jena) or a Leica DMi8equipped with a FluotarL 20×0.4 NA objective (Leica) and Orca Flash4.0LT camera (Hamamatsu).

To image GFP expression in vivo, transgenic pSGNluc zebrafish embryoswere anesthetized with tricaine methanesulfonate and mounted in 1.5% low-melting-point agarose. In vivo imaging of the embryos was performed with aZeiss Lightsheet Z.1 and Zeiss LSM 780 NLO 2-Photon confocalmicroscopes. To visualize NF-κB activation upon infection or injury,time-lapse experiments were carried out with an Ultraview ERS spinningdisk (PerkinElmer) confocal microscope using a 40× water-immersionobjective. Images were analyzed with Imaris software as describedpreviously (Bajoghli et al., 2015).

Luciferase measurement in live embryosSingle embryos were transferred into individual wells of a 96-multiwellplate (Nunc) in 100 µl E3 medium (without methylene blue), supplementedwith 0.5 mM beetle luciferin potassium salt solution (Promega), and theplate was sealed using an adhesive Top Seal sheet (Packard).Bioluminescence from each embryo was then assayed at roomtemperature using a Top-count NXT scintillation counter (2-detectormodel; Packard).

Whole-mount in situ hybridizationRNA in situ hybridization of wild-type zebrafish embryos was performed asdescribed previously (Bajoghli et al., 2009) using digoxigenin-labeled RNAantisense probe for iκbαa (accession number, BC068382; nucleotides 230–903) and iκbαb (accession number, BC050175; nucleotides 242–689).

Quantitative RT-PCR analysisTotal RNA was extracted from pools of 25 embryos using TRIzol (LifeTechnologies) following the manufacturer’s protocol. 1 µg RNA sampleswere treated with 1 µl RQ1 RNase-Free DNase (Promega) before first-strandcDNA synthesis with random hexamer primers and Superscript III ReverseTranscriptase (Thermo Fisher). The first-strand cDNAwas directly used as atemplate in PCR reactions. qPCR was carried out using the SYBR Green kit(Applied Biosystems) on the ABI 7500 Real-Time PCR System. The datawere analyzed in Microsoft Excel using the ΔCt method with β-actin as areference gene for normalization. All primer sequences used in this studywere described previously (Banerjee and Leptin, 2014).

Statistical analysisPrism software (version 6, GraphPad Software Inc.) was used for graphingand statistical analysis. Unpaired, two-tailed Student t-tests were used tocompare the means of different data sets.

AcknowledgementsWe thank the Advanced Light Microscopy Facility (AMLF) at EMBL for continuoussupport and PerkinElmer and Zeiss for support of the AMLF. We are grateful toNicholas S. Foulkes (Institute of Toxicology and Genetics, Karlsruhe, Germany) forproviding the experimental materials for luciferase measurement in live zebrafish

embryos, Stephen A Renshaw (University of Sheffield, UK) for providing the Tg(NFκB:EGFP) fish and to Matthias Hammerschmidt (University of Cologne,Germany) for the Tg(krt19:dTomato) fish. We thank Francesca Peri for hosting ourzebrafish, Sinja Kraus and Omnia El Said Ibrahim for experimental help; YvonnePostma for technical support; Marvin Albert for help with the processing of SPIMdata; Joanna Natalia Buffoni and Cornelia Henkel for the care of zebrafish. We thankSanjita Banerjee for help in the generation of the zebrafish Tg(8×Hs.NFκB:GFP,Luciferase) line.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsP.K. established the zebrafish pSGNluc transgenic line, performed in vivo analyses,contributed to the design of the work and co-wrote the manuscript. K.E. performedthe reporter assays in human cells. T.A.K. conducted initial studies, providedscientific discussion and supervised the in vitro characterization work in humancells. M.L. provided scientific discussion, contributed to critical revision for importantintellectual content and final approval. B.B. designed and developed the pSGNlucreporter, conducted initial studies, planned and oversaw the work, interpreted thedata, designed figures, co-wrote the paper and all authors read and edited themanuscript.

FundingThe laboratory of M.L. is supported by the EuropeanMolecular Biology Organization(EMBO) and European Molecular Biology Laboratory (EMBL), and grants andfellowships to B.B. (European Commission, EMBL EU Marie Curie Action Cofund),P.K. (European Commission Marie-Curie Initial Training Network FishForPharma;FP7-PEOPLE-2011-ITN, grant PITN-GA-2011-289209). Preparatory work wasfunded by the Deutsche Forschungsgemeinschaft (DFG) SFB 670 ‘ZellautonomeImmunitat’. The laboratory of T.A.K. is supported by the DeutscheForschungsgemeinschaft (KU1945/4-1).

Data availabilityThe sequence of the pSGNluc plasmid has been deposited at NCBI under theaccession number KY129798 (https://www.ncbi.nlm.nih.gov/nuccore/KY129798)and the the Tg(8×Hsa.NFκB:GFP,Luciferase) line described here has beendeposited in the European Zebrafish Resource Center (EZRC) for public distribution(in the ZFIN network under https://zfin.org/ZDB-TGCONSTRCT-161209-5).

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.196485.supplemental

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