LETTERS

Reactive oxygen species prime Drosophilahaematopoietic progenitors for differentiationEdward Owusu-Ansah1{ & Utpal Banerjee1,2,3,4

Reactive oxygen species (ROS), produced during various electrontransfer reactions in vivo, are generally considered to be deleteriousto cells1. In the mammalian haematopoietic system, haematopoie-tic stem cells contain low levels of ROS. However, unexpectedly,the common myeloid progenitors (CMPs) produce significantlyincreased levels of ROS2. The functional significance of thisdifference in ROS level in the two progenitor types remains un-resolved2,3. Here we show that Drosophila multipotent haemato-poietic progenitors, which are largely akin to the mammalian mye-loid progenitors4, display increased levels of ROS under in vivophysiological conditions, which are downregulated on differenti-ation. Scavenging the ROS from these haematopoietic progenitorsby using in vivo genetic tools retards their differentiation intomature blood cells. Conversely, increasing the haematopoietic pro-genitor ROS beyond their basal level triggers precocious differenti-ation into all three mature blood cell types found in Drosophila,through a signalling pathway that involves JNK and FoxOactivation as well as Polycomb downregulation. We conclude thatthe developmentally regulated, moderately high ROS level in theprogenitor population sensitizes them to differentiation, and

establishes a signalling role for ROS in the regulation of haemato-poietic cell fate. Our results lead to a model that could be extendedto reveal a probable signalling role for ROS in the differentiation ofCMPs in mammalian haematopoietic development and oxidativestress response.

The Drosophila lymph gland is a specialized haematopoietic organwhich produces three blood cell types—plasmatocytes, crystal cells andlamellocytes—with functions reminiscent of the vertebrate myeloidlineage5,6. During the first and early second larval instars, the lymphgland comprises only the progenitor population (Fig. 1a, bottom dia-gram). However, by late third instar, multipotent stem-like progenitorcells become restricted to the medial region of the primary lymph glandlobe, in an area referred to as the medullary zone; whereas a peripheral

1Department of Molecular, Cell and Developmental Biology, 2Molecular Biology Institute, 3Department of Biological Chemistry, 4Eli and Edythe Broad Center of Regenerative Medicineand Stem Cell Research, University of California, Los Angeles, California 90095, USA. {Present address: Department of Genetics, Harvard Medical School, Boston, Massachusetts02115, USA.

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Haematopoietic progenitorsCells in transitionPlasmatocytesCrystal cellsLamellocytesCells in the nichePericardial cellsDorsal vessel

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dome>GFP dome>GFP>Gtpx-1

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Figure 1 | ROS profile of third-instar lymph glands. a, Schematics of latethird- (top) and early second-instar (bottom panel) lymph glands. Mostprogenitors are seen in the second instar and in the third instar, primary lobecentral domain, the medullary zone. Differentiated cells are restricted to thecortical zone. Primary lobes are anterior, followed posteriorly by secondaryand tertiary lobes. b, Medullary zone progenitors show increased ROS (red).Dotted outlines are based on high-power laser images. c, Expression of themedullary zone marker, dome-gal4, UAS-23EYFP (green; genotypeabbreviated ‘dome.GFP’ for clarity) overlaps with the ROS dye (red) inmedullary zone cells (therefore yellow). d, As b, the progenitors in themedullary zone show increased ROS levels (red). e, hmlD-gal4, UAS-23EGFP is restricted to cells in the cortical zone (green). Most of the cellsthat are marked by hmlD-gal4, UAS-23EGFP are low in ROS (thereforegreen) compared with cells in the medullary zone (red). A ring of hmlD-gal4,UAS-23EGFP-expressing cells can be seen along the edge of the medullaryzone that is both GFP and ROS positive (therefore yellow). These appear tobe cells in a state of transition between a stem-like and a differentiated cellfate. f, P1 expression (red) in plasmatocytes in the cortical zone. Expressionof dome-gal4, UAS-23EYFP (green) is restricted to the medullary zone ascortical zone cells (red) downregulate this marker. g, Overexpression of theantioxidant protein (GTPx-1) in progenitors (genotype: dome-gal4, UAS-23EYFP; UAS-Gtpx1) causes pronounced reduction in the number of cellsthat express P1 (red). Some cells occupying the cortical zone region continueto express dome-gal4, UAS-23EYFP whereas many others downregulate thismarker without yet expressing the differentiation marker P1. h, Inhypomorphic Sod2/Sod2 homozygotes, in which a major ROS scavengerexpression is reduced, P1 is expanded throughout the gland (red), notrestricted to the cortical zone. This image represents the central domain.Scale bars, 50 mm.

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zone, referred to as the cortical zone, contains differentiated bloodcells. By late third instar, the progenitors within the medullary zoneare essentially quiescent, whereas the mature, differentiated populationin the cortical zone proliferates extensively5. The posterior signallingcentre is a group of about 30 cells (Fig. 1a, top diagram) that secretesseveral signalling molecules7–9 and serves as a stem-cell niche regulating

the balance between cells that maintain ‘stemness’ and those thatdifferentiate8,9.

Although several studies have identified factors that regulate thedifferentiation and maintenance of Drosophila blood cells and thestem-like progenitor population that generates them8–11, intrinsicfactors within the stem-like progenitors are less explored. Our inter-rogation of these intrinsic factors is the central theme of this investi-gation. We observed that by the third instar, the progenitorpopulation in the normal wild-type lymph gland medullary zonecontains significantly increased ROS levels compared with theirneighbouring differentiated progeny that express mature blood cellmarkers in the cortical zone (Fig. 1b–e). ROS are not increasedduring the earlier larval instars but increase as the progenitor cellsbecome quiescent and subside as they differentiate (Fig. 1b–e). Thisfirst suggested to us that the rise in ROS primes the relatively qui-escent stem-like progenitor cells for differentiation. We reduced ROSby expressing antioxidant scavenger proteins GTPx-1 (ref. 12)(Fig. 1f, g) or catalase (Supplementary Fig. 1), specifically in the

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dome>GFP; UAS-RNAiND75UAS-Gtpx-1

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Figure 2 | Increased ROS production triggers precocious differentiation ofthe multipotent progenitors. In all panels, the progenitor populationexpresses the medullary zone marker, dome-gal4, UAS-23EYFP (green). Ind–g, the green channel has been omitted for clarity. The two genotypes usedin these panels are control lymph glands (dome-gal4, UAS-23EYFP),referred to as wild-type in this legend, and experimental lymph glands whichexpress an RNAi construct to ND75 (dome-gal4, UAS-23EYFP; UAS-RNAiND75), abbreviated as ND75 RNAi. Scale bars, 50 mm. a, P1 is notexpressed (note absence of red) in early second-instar wild-type lymphglands. b, P1 expression (red) is robustly induced in early second-instarND75 RNAi lymph glands. c–e, Disruption of ND75 triggers precociousdifferentiation. c, In wild-type third-instar lymph glands, plasmatocytesmarked with P1 (red) and crystal cells with ProPO (grey) are restricted to thecortical zone. These differentiated cell types are rarely, if ever, found insecondary and tertiary lobes. d, In third-instar ND75 RNAi lymph glands,there is a marked increase in P1- (red) and ProPO- (grey) expressing cellsthroughout the primary as well as the secondary and tertiary lobes (tertiarylobes are shown in Supplementary Fig. 4). e, Lamellocytes, marked by L1(red) are prominently seen in third-instar ND75 RNAi lymph glands. Crystalcells are shown in grey. Lamellocytes are rarely found in secondary lobes.f, g, Scavenging ROS suppresses the differentiation associated with ND75disruption. Overexpression of Gtpx-1 in ND75 RNAi lymph glands (in f andg) potently suppresses differentiation into all three lineages as there is adecrease in P1 (red in f), ProPO (grey in g) and L1 (red in g) expression.Compare f, g with d. e, Controls for titration of GAL4 are shown inSupplementary Fig. 6.

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Figure 3 | Disrupting JNK signalling suppresses the ROS-dependentdifferentiation phenotype. The progenitor population also expresses themedullary zone marker, dome-gal4, UAS-23EYFP (green), in a–d, but thishas been omitted for clarity. Lymph glands that express an RNAi constructto ND75 (dome-gal4, UAS-23EYFP; UAS-RNAiND75), are abbreviated asND75 RNAi. Scale bars, 50 mm. a, b, JNK signalling is activated upon ROSincrease. Expression of puc-lacZ (red) in wild-type lymph glands (a) andND75 RNAi lymph glands (b). Levels of puc-lacZ, which is a transcriptionalreporter of JNK signalling, are markedly increased in ND75 RNAi cells.c, d, JNK signalling is required for triggering differentiation associated withND75 disruption. Expressing a dominant negative construct of JNK in theprecursor population ameliorates the effect of complex I disruption as thenumbers of plasmatocytes (red in c), crystal cells (grey in c and d) andlamellocytes (red in d) are reduced virtually to wild-type levels. Comparec, d with Fig. 2d, e. e, Suppression of the number of crystal cells formed inND75 RNAi UAS-DNbsk lymph glands relative to ND75 RNAi lymphglands. Error bars, s.e.m. (n 5 10).

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progenitor cell compartment using the GAL4/UAS system13, andfound that suppressing increased ROS levels in haematopoietic pro-genitors significantly retards their differentiation into plasmatocytes(Fig. 1f, g and Supplementary Fig. 1). As a corollary, mutating thegene encoding the antioxidant scavenger protein superoxide dismu-tase (Sod2)1 led to a significant increase in differentiated cells anddecrease in progenitors (Fig. 1h).

ROS levels in cells can be increased by the genetic disruption ofcomplex I proteins of the mitochondrial electron transport chain14,15,such as ND75 and ND42 (Supplementary Fig. 2). Unlike in wild type,where early second-instar lymph glands exclusively comprise un-differentiated cells (Fig. 2a), mitochondrial complex I depletion trig-gers premature differentiation of the progenitor population (Fig. 2b).This defect is even more evident in the third instar (compare Fig. 2c,d), where a complete depletion of the progenitors is seen as primarylobes are populated with differentiated plasmatocytes and crystalcells. The third differentiated cell type, the lamellocyte, defined bythe expression of the antigen L1, is rarely observed in the wild-typelymph gland (Supplementary Fig. 3) but is abundantly seen in themutant (Fig. 2e). Finally, the secondary and tertiary lobes, largelyundifferentiated in wild type, also embark on a robust program ofdifferentiation upon complex I depletion (Fig. 2d, e and Supplemen-tary Fig. 4). Importantly, the phenotype resulting from ND75 dis-ruption can be suppressed by the co-expression of the ROS scavengerprotein GTPx-1 (Fig. 2f, g; compare with Fig. 2d, e), which provides acausal link between increased ROS and the premature differentiationphenotype. Combining these results with those in Fig. 1, we concludethat the normally increased ROS levels in the stem-like progenitorsserve as an intrinsic factor that sensitizes the progenitors to differ-entiation into all three mature cell types. Any further increase ordecrease in the level of ROS away from the wild-type level enhancesor suppresses differentiation respectively.

In unrelated systems, increased ROS levels have been demon-strated to activate the JNK signal transduction pathway1,16,17.Consequently, we tested whether the mechanism by which the pro-genitors in the medullary zone differentiate when ROS levels increasecould involve this pathway. The gene puckered (puc) is a downstreamtarget of JNK signalling and its expression has been used extensivelyto monitor JNK activity18. Although puc transcripts are detectable byreverse transcriptase PCR (RT–PCR) (Supplementary Fig. 5), thepuc-lacZ reporter is very weakly expressed in wild type (Fig. 3a).After disruption of ND75, however, a robust transcriptional upregu-lation of puc-lacZ expression can be seen (Fig. 3b), indicating thatJNK signalling is induced in these cells in response to high ROS levels.

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Figure 4 | FoxO activation and Polycomb downregulation phenocopyaspects of the ROS-induced differentiation. Progenitor cells express themedullary zone marker, dome-gal4, UAS-23EYFP (green), omitted in somepanels for clarity. Lymph glands from dome-gal4, UAS-23EYFP larvae arereferred to as wild-type controls in this legend. Cells expressing RNAiconstruct to ND75 in the progenitor cells (dome-gal4, UAS-23EYFP; UAS-RNAiND75), are abbreviated as ND75 RNAi. Scale bars, 40 mm.a, b, Disruption of ND75 induces the FoxO reporter, Thor-lacZ. Wild type(a) does not express Thor-lacZ (absence of red). The asterisk points to Thor-lacZ expression in the ring gland, adjacent to the lymph gland. Expression ofThor-lacZ is significantly induced in ND75 RNAi lymph glands(b). c, d, Disruption of ND75 causes polycomb reporter expression. Thepolycomb reporter (red) is not expressed in wild type (c), but is induced inND75 RNAi (d). e–g, FoxO overexpression causes increase in plasmatocytesand crystal cells, but has no effect on lamellocytes. e, Overexpression ofFoxO in the progenitors (dome-gal4, UAS-23EYFP; UAS-foxo) causespremature differentiation into plasmatocytes, evident as earlier than normalP1 staining (red) in a second-instar lymph gland (compare with Fig. 2a).f, Progenitors expressing FoxO in the medullary zone also initiate extensivedifferentiation into plasmatocytes (red). Ectopic differentiation is also seenin secondary lobes (arrow, 2u). g, FoxO expression in the medullary zonecauses increase in crystal cell number (grey). However, only a few isolatedL1-positive cells (red) are evident. This image is at twice the magnification ofother panels to highlight the few lamellocytes (red). h, RNAi-mediateddownregulation of the expression of two polycomb proteins, Enhancer ofpolycomb, E(Pc), and polyhomeotic proximal, (Ph-p), leads to a robustincrease in lamellocytes that stain for L1 (red). i, j, When FoxO and the RNAiconstruct to E(Pc) are expressed together in the medullary zone, there is anincrease in all three mature cell markers. Co-expression of FoxO and anRNAi construct to E(Pc) trigger the full differentiation phenotype associatedwith complex I disruption as there is an increase in the number ofplasmatocytes (red in i), crystal cells (grey in j) and lamellocytes (red in j).

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The precocious progenitor cell differentiation caused bymitochondrial disruption is suppressed upon expressing a dominantnegative version of basket (bsk), the sole Drosophila homologue ofJNK (Fig. 3c, d; compare with Fig. 2d, e; see also SupplementaryFig. 5). This suppression was associated with a decrease in the levelof expression (Supplementary Fig. 5) of the stress response geneencoding phosphoenol pyruvate carboxykinase19; quantitatively a68% suppression of the ND75 crystal cell phenotype was observedwhen JNK function was removed as well (Fig. 3e). Although disrupt-ing JNK signalling suppressed differentiation, ROS levels remainincreased in the mutant cells (Supplementary Fig. 2f), as would beexpected from JNK functioning downstream of ROS.

In several systems and organisms, JNK function can be mediatedby activation of FoxO as well as through repression of Polycombactivity17,20,21. FoxO activation can be monitored by the expressionof its downstream target Thor, using Thor-lacZ as a transcriptionalread-out22–24. This reporter is undetectable in wild-type lymph glands(Fig. 4a) although Thor transcripts are detectable by RT–PCR(Supplementary Fig. 5); however, the reporter is robustly inducedwhen complex I is disrupted (Fig. 4b), which suggests that theincrease in ROS that is mediated by loss of complex I activatesFoxO. To monitor Polycomb de-repression, we used a Polycombreporter, which expresses lacZ when Polycomb proteins are down-regulated. Although undetectable in wild-type lymph glands (Fig. 4c),disrupting ND75 leads to lacZ expression (Fig. 4d), which suggeststhat Polycomb activity is downregulated by the altered ROS andresulting JNK activation. Direct FoxO overexpression causes aremarkable advancement in differentiation to a time as early as thesecond instar (Fig. 4e), never seen in wild type (Fig. 2a). By early thirdinstar, the entire primary and secondary lobes stained for plasmato-cyte (Fig. 4f) and crystal cell (Fig. 4g) markers when FoxO isexpressed in the progenitor population. Unlike with ROS increase,we did not find a significant increase in lamellocytes upon FoxOoverexpression. However, downregulating the expression of twopolycomb proteins, Polyhomeotic Proximal (Php-x) and Enhancerof Polycomb (E(Pc)), that function downstream of JNK21 markedlyincreased lamellocyte number (Fig. 4h) without affecting plasmato-cytes and crystal cells (data not shown). When FoxO and a transgenicRNA interference (RNAi) construct against E(Pc) are expressedtogether in the progenitor cell population, differentiation to all threecell types is evident (Fig. 4i, j). We conclude that FoxO activation andPolycomb downregulation act combinatorially downstream of JNKto trigger the full differentiation phenotype: an increase in plasma-tocytes and crystal cells due to FoxO activation, and an increase inlamellocytes primarily due to Polycomb downregulation.

The analysis of ROS in the wild-type lymph gland highlights apreviously unappreciated role for ROS as an intrinsic factor thatregulates the differentiation of multipotent haematopoietic progeni-tors in Drosophila. Any further increase in ROS beyond thedevelopmentally regulated levels, owing to oxidative stress, will causethe progenitors to differentiate into one of three myeloid cell types. Itwas reported previously2 that the ROS levels in mammalian haema-topoietic stem cells is low but that in the CMPs is relatively high. TheDrosophila haematopoietic progenitors give rise entirely to a myeloidlineage and therefore are functionally more similar to CMPs thanthey are to haematopoietic stem cells. It is therefore a remarkableexample of conservation to find that they too have high ROS levels.The genetic analysis makes it clear that the high ROS in Drosophilahaematopoietic progenitors primes them towards differentiation. Itwill be interesting to determine whether such a mechanism operatesin mammalian CMPs. In mice, as in flies, a function of FoxO is toactivate antioxidant scavenger proteins. Consequently, deletion ofFoxO increases ROS levels in the mouse haematopoietic stem celland drives myeloid differentiation2. However, even in the mousehaematopoietic system, FoxO function is dose and context depend-ent, as ROS levels in CMPs are independent of FoxO2. Thus, althoughthe basic logic of increased ROS in myeloid progenitors is conserved

between flies and mice, the exact function of FoxO in this contextmay have diverged.

Our past work14 and that of others1,25,26 has hinted that ROS canfunction as signalling molecules at physiologically moderate levels. Thiswork supports and further extends this notion. Although excessiveROS is damaging to cells, developmentally regulated ROS productioncan be beneficial. The finding that ROS levels are moderately high innormal Drosophila haematopoietic progenitors and mammalian CMPsraises the possibility that wanton overdose of antioxidant productsmay in fact inhibit the formation of cells participating in the innateimmune response.

METHODS SUMMARY

Lymph glands were stained as previously described5,8 using the following

antibodies: mouse anti-P1 and L1, rat anti-ProPO, rabbit anti-b-gal (Cappell)

and mouse anti-b-gal (Promega). Cy3-, Cy5- and FITC-conjugated secondary

antibodies were from Jackson Laboratory. ROS staining was conducted as previ-

ously described14,15. Images were captured using a BioRad Radiance 2000 con-

focal microscope with LaserSharp 2000 acquisition software.

Full Methods and any associated references are available in the online version ofthe paper at www.nature.com/nature.

Received 3 May; accepted 23 July 2009.Published online 2 September 2009.

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3. Coffer, P. J. & Burgering, B. M. Stressed marrow: FoxOs stem tumour growth.Nature Cell Biol. 9, 251–253 (2007).

4. Evans, C. J., Hartenstein, V. & Banerjee, U. Thicker than blood: conservedmechanisms in Drosophila and vertebrate hematopoiesis. Dev. Cell 5, 673–690(2003).

5. Jung, S. H., Evans, C. J., Uemura, C. & Banerjee, U. The Drosophila lymph gland as adevelopmental model of hematopoiesis. Development 132, 2521–2533 (2005).

6. Wood, W. & Jacinto, A. Drosophila melanogaster embryonic haemocytes: mastersof multitasking. Nature Rev. Mol. Cell Biol. 8, 542–551 (2007).

7. Lebestky, T., Jung, S. H. & Banerjee, U. A. Serrate-expressing signaling centercontrols Drosophila hematopoiesis. Genes Dev. 17, 348–353 (2003).

8. Mandal, L., Martinez-Agosto, J. A., Evans, C. J., Hartenstein, V. & Banerjee, U. A.Hedgehog- and Antennapedia-dependent niche maintains Drosophilahaematopoietic precursors. Nature 446, 320–324 (2007).

9. Krzemien, J. et al. Control of blood cell homeostasis in Drosophila larvae by theposterior signalling centre. Nature 446, 325–328 (2007).

10. Lebestky, T., Chang, T., Hartenstein, V. & Banerjee, U. Specification of Drosophilahematopoietic lineage by conserved transcription factors. Science 288, 146–149(2000).

11. Bruckner, K. et al. The PDGF/VEGF receptor controls blood cell survival inDrosophila. Dev. Cell 7, 73–84 (2004).

12. Missirlis, F. et al. A putative glutathione peroxidase of Drosophila encodes athioredoxin peroxidase that provides resistance against oxidative stress but failsto complement a lack of catalase activity. Biol. Chem. 384, 463–472 (2003).

13. Brand, A. H. & Perrimon, N. Targeted gene expression as a means of altering cellfates and generating dominant phenotypes. Development 118, 401–415 (1993).

14. Owusu-Ansah, E., Yavari, A., Mandal, S. & Banerjee, U. Distinct mitochondrialretrograde signals control the G1-S cell cycle checkpoint. Nature Genet. 40,356–361 (2008).

15. Owusu-Ansah, E., Yavari, A. & Banerjee, U. A protocol for in vivo detection ofreactive oxygen species. Nature Protocols. doi:10.1038/nprot.2008.23 (2008).

16. Gotoh, Y. & Cooper, J. A. Reactive oxygen species- and dimerization-inducedactivation of apoptosis signal-regulating kinase 1 in tumor necrosis factor-a signaltransduction. J. Biol. Chem. 273, 17477–17482 (1998).

17. Essers, M. A. et al. FOXO transcription factor activation by oxidative stressmediated by the small GTPase Ral and JNK. EMBO J. 23, 4802–4812 (2004).

18. Martin-Blanco, E. et al. puckered encodes a phosphatase that mediates afeedback loop regulating JNK activity during dorsal closure in Drosophila. GenesDev. 12, 557–570 (1998).

19. Harvey, K. F. et al. FOXO-regulated transcription restricts overgrowth of Tscmutant organs. J. Cell Biol. 180, 691–696 (2008).

20. Wang, M. C., Bohmann, D. & Jasper, H. JNK extends life span and limits growth byantagonizing cellular and organism-wide responses to insulin signaling. Cell 121,115–125 (2005).

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22. Puig, O., Marr, M. T., Ruhf, M. L. & Tjian, R. Control of cell number by DrosophilaFOXO: downstream and feedback regulation of the insulin receptor pathway.Genes Dev. 17, 2006–2020 (2003).

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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We thank I. Ando and H. Muller for antibodies, and E. Hafen,A. Martinez-Arias, F. Missirlis, S. Noselli, R. Paro, S Sinenko, the National Instituteof Genetics Fly Stock Center (Japan) and the Bloomington Stock Center for flystocks. We acknowledge M. Kulkarni and C. Pitsouli of the Perrimon laboratory fortechnical assistance. Owing to space limitations, we apologize to our colleagueswhose work is not referenced. This study was supported by US National Institutesof Health grant R01HL067395 to U.B. and a T32 institutional postdoctoralfellowship T32-HL069766 to E.O.-A.

Author Contributions U.B. supervised the project. E.O.-A. conceived, designed andperformed all experiments. E.O.-A. and U.B. discussed results and wrote themanuscript.

Author Information Reprints and permissions information is available atwww.nature.com/reprints. Correspondence and requests for materials should beaddressed to U.B. (banerjee@mbi.ucla.edu).

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METHODSDrosophila stocks and genetics. The following fly stocks were used: domeless-gal4,

hmlD-gal4 UAS-2xeGFP, UAS-GTPx1, UAS-cat, puce69/TM3,Sb, the FLW-1 and

LW-1 lines (for assessing polycomb downregulation). Oregon R, w; P{tubP-

GAL80ts}10; TM2/TM6B, Tb, y1 w67c23; P{SUPor-P}Sod2KG06854, UAS-2xeYFP on

the X chromosomes, y1 w; P{lacW}Thork13517, w; P{UAS-bsk.K53R}20.1a and y1

w*; P{UAS-foxo.P}2 stocks are from the Bloomington stock centre. The RNAi

stocks (UAS-ND75RNAi, UAS-ND42RNAi, UAS-E(Pc)RNAi and UAS-ph-

pRNAi) were obtained from National Institute of Genetics Fly Stock Center

(Japan). In all instances, crosses were set up at 25 uC for a day. For the third-instarRNAi experiments and all suppression experiments, the larvae were shifted to

18 uC for 2 days, and then transferred to 29 uC until dissection. For the second-

instar experiments, larvae were transferred to 29 uC directly (without an interven-

ing shift to 18 uC). For the overexpression experiments (involving UAS-foxo and

UAS-Gtpx-1) the larvae were maintained at 25 uC until dissection. In all instances,

at least ten lymph glands were examined.

Gene expression analyses. Total RNA was extracted from third-instar lymph

glands using TRIzol (Invitrogen), followed by RNA cleanup with the RNAeasy

Kit (Qiagen). The RNA QuantiTect Reverse Transcription Kit (Qiagen) was used

for first-strand complementary DNA synthesis, and quantitative real-time PCR

was performed with the QuantiTect SYBR Green PCR Kit (Qiagen) and analysed

on an ABI Prism 7000 (Applied Biosystems) sequence detection system. The

levels of actin transcripts were used to normalize total complementary DNA

input. Relative quantification of transcript levels was calculated using the com-

parative Ct method. Results shown represent the mean of three replicates. For

semi-quantitative RT–PCR, transcripts were analysed after the twentieth cycle,

and actin transcripts served as loading controls. Primers used are shown in

Supplementary Table 1.

doi:10.1038/nature08313

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