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Fat cells reactivate quiescent neuroblasts via TOR andglial insulin relays in DrosophilaRita Sousa-Nunes1, Lih Ling Yee1 & Alex P. Gould1

Many stem, progenitor and cancer cells undergo periods of mitoticquiescence from which they can be reactivated1–5. The signals trig-gering entry into and exit from this reversible dormant state are notwell understood. In the developing Drosophila central nervous sys-tem, multipotent self-renewing progenitors called neuroblasts6–9

undergo quiescence in a stereotypical spatiotemporal pattern10.Entry into quiescence is regulated by Hox proteins and an internalneuroblast timer11–13. Exit from quiescence (reactivation) is subjectto a nutritional checkpoint requiring dietary amino acids14. Organco-cultures also implicate an unidentified signal from an adipose/hepatic-like tissue called the fat body14. Here we provide in vivoevidence that Slimfast amino-acid sensing and Target of rapamycin(TOR) signalling15 activate a fat-body-derived signal (FDS) requiredfor neuroblast reactivation. Downstream of this signal, Insulin-likereceptor signalling and the Phosphatidylinositol 3-kinase (PI3K)/TOR network are required in neuroblasts for exit from quiescence.We demonstrate that nutritionally regulated glial cells provide thesource of Insulin-like peptides (ILPs) relevant for timely neuroblastreactivation but not for overall larval growth. Conversely, ILPssecreted into the haemolymph by median neurosecretory cells sys-temically control organismal size16–18 but do not reactivate neuro-blasts. Drosophila thus contains two segregated ILP pools, oneregulating proliferation within the central nervous system and theother controlling tissue growth systemically. Our findings support amodel in which amino acids trigger the cell cycle re-entry of neuralprogenitors via a fat-body–glia–neuroblasts relay. This mechanismindicates that dietary nutrients and remote organs, as well as localniches, are key regulators of transitions in stem-cell behaviour.

In fed larvae, Drosophila neuroblasts (Fig. 1a) exit quiescence fromthe late first instar (L1) stage onwards. This reactivation involves cellenlargement and entry into S phase, monitored in this study using thethymidine analogue 5-ethynyl-29-deoxyuridine (EdU). Consistentwith a previous study10, we observed that reactivated neuroblastlineages (neuroblasts and their progeny; Fig. 1b) reproducibly incor-porated EdU in a characteristic spatiotemporal sequence: central brainR thoracic R abdominal neuromeres (Fig. 1c and Supplementary Fig.1). Mushroom-body neuroblasts and one ventrolateral neuroblast,however, are known not to undergo quiescence and to continue divid-ing for several days in the absence of dietary amino acids14 (Fig. 1a, c, f).This indicates that dietary amino acids are more than mere ‘fuel’,providing a specific signal that reactivates neuroblasts. However,explanted central nervous systems (CNSs) incubated with amino acidsdo not undergo neuroblast reactivation unless co-cultured with fatbodies from larvae raised on a diet containing amino acids14. We there-fore tested the in vivo requirement for a fat-body-derived signal (FDS)in neuroblast reactivation by blocking vesicular trafficking and thussignalling from this organ using a dominant-negative Shibire dynamin(SHIDN). This strongly reduced neuroblast EdU incorporation, indi-cating that exit from quiescence in vivo requires an FDS (Fig. 1d, e). Onecandidate we tested was Ilp6, known to be expressed by the fat body19,20,but neither fat-body-specific overexpression nor RNA interference ofthis gene significantly affected neuroblast reactivation (Supplementary

Table 1 and data not shown). Fat-body cells are known to sense aminoacids via the cationic amino-acid transporter Slimfast (SLIF), whichactivates the TOR signalling pathway, in turn leading to the productionof a systemic growth signal15,21. We found that fat-body-specific over-expression of the TOR activator Ras homologue enriched in brain(RHEB), or of an activated form of the p110 PI3K catalytic subunit,or of the p60 adaptor subunit, had no significant effect on neuroblastreactivation in fed animals or in larvae raised on a nutrient-restricteddiet lacking amino acids (Fig. 1e, f and data not shown). In contrast,global inactivation of Tor, fat-body-specific Slif knockdown or fat-body-specific expression of the TOR inhibitors Tuberous sclerosiscomplex 1 and 2 (Tsc1/2) all strongly reduced neuroblasts from exitingquiescence (Fig. 1d, e). Together, these results show that a SLIF/TOR-dependent FDS is required for neuroblasts to exit quiescence and thatthis may be equivalent to the FDS known to regulate larval growth.

Next we investigated the signalling pathways essential within neu-roblasts for their reactivation. Nutrient-dependent growth is regulatedin many species by the interconnected TOR and PI3K pathways22–24

(Supplementary Fig. 2). In fed larvae, we found that neuroblast inac-tivation of TOR signalling (by overexpression of TSC1/2), or PI3Ksignalling (by overexpression of p60, the Phosphatase and tensinhomologue PTEN, the Forkhead box subgroup O transcription factorFOXO or dominant-negative p110), all inhibited reactivation (Fig. 1e).Conversely, stimulation of neuroblast TOR signalling (by overexpres-sion of RHEB) or PI3K signalling (by overexpression of activatedp110 or Phosphoinositide-dependent kinase 1 (PDK1)) triggered pre-cocious exit from quiescence (Fig. 1e). RHEB overexpression had aparticularly early effect, preventing some neuroblasts from undergoingquiescence even in newly hatched larvae (Supplementary Fig. 3).Hence, TOR/PI3K signalling in neuroblasts is required to trigger theirtimely exit from quiescence. Importantly, neuroblast overexpression ofRHEB or activated p110 in nutrient-restricted larvae, which lack FDSactivity14, was sufficient to bypass the block to neuroblast reactivation(Fig. 1f). Notably, both genetic manipulations were even sufficient toreactivate neuroblasts in explanted CNSs, cultured without fat body orany other tissue (Fig. 1g). Together with the previous results this indi-cates that neuroblast TOR/PI3K signalling lies downstream of theamino-acid-dependent FDS during exit from quiescence.

To identify the mechanism bridging the FDS with neuroblast TOR/PI3K signalling, we tested the role of the Insulin-like receptor (InR) inneuroblasts (Supplementary Fig. 2). Importantly, a dominant-negativeInR inhibited neuroblast reactivation, whereas an activated form stimu-lated premature exit from quiescence (Fig. 1e). Furthermore, InR activa-tion was sufficient to bypass the nutrient restriction block to neuroblastreactivation (Fig. 1f). This indicates that at least one of the potential InRligands, the seven ILPs, may be the neuroblast reactivating signal(s). Bytesting various combinations of targeted Ilp null alleles25 and genomic Ilpdeficiencies25,26, we found that neuroblast reactivation was moderatelydelayed in larvae deficient for both Ilp2 and Ilp3 (Df(3L)Ilp2–3) or lack-ing Ilp6 activity (Fig. 2a). Stronger delays, as severe as those observed inInR31 mutants, were observed in larvae simultaneously lacking the activ-ities of Ilp2, 3 and 5 (Df(3L)Ilp2–3, Ilp5) or Ilp1–5 (Df(3L)Ilp1–5)

1Division of Developmental Neurobiology, Medical Research Council National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.

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(Fig. 2a). Despite the developmental delay in Df(3L)Ilp1–5 homozy-gotes25,26, neuroblast reactivation eventually begins in the normal spatialpattern—albeit heterochronically—in larvae with L3 morphology(Fig. 2b, compare timeline with Fig. 1c). Together, the genetic analysisshows that Ilp2, 3, 5 and 6 regulate the timing but not the spatial patternof neuroblast exit from quiescence. However, as removal of some ILPscan induce compensatory regulation of others25, the relative importanceof each cannot be assessed from loss-of-function studies alone.

Brain median neurosecretory cells (mNSCs) (Fig. 1a) are an import-ant source of ILPs, secreted into the haemolymph in an FDS-dependentmanner to regulate larval growth16–18,21. They express Ilp1, 2, 3 and 5,although not all during the same development stages16–18. However, wefound that none of the seven ILPs could reactivate neuroblasts duringnutrient restriction when overexpressed in mNSCs (SupplementaryTable 2). Similarly, increasing mNSC secretion using the NaChBacsodium channel21 or altering mNSC size using PI3K inhibitors/activa-tors, which in turn alters body growth, did not significantly affectneuroblast reactivation under fed conditions (Fig. 2a, c, Supplemen-tary Fig. 1b and L. Y. Cheng and colleagues, manuscript submitted).Surprisingly, therefore, mNSCs are not the relevant ILP source forneuroblast reactivation. Nonetheless, Ilp3 and Ilp6 messenger RNAswere detected in the CNS cortex, at the early L2 stage, in a domain

distinct from the Ilp21 mNSCs (Supplementary Fig. 4). Two differentIlp3-lacZ transgenes17 indicate that Ilp3 is expressed in some glia(Repo1 cells) and neurons (Elav1 cells). An Ilp6-GAL4 insertion (seeMethods) indicates that Ilp6 is also expressed in glia, including thecortex glia surrounding neuroblasts and the glia of the blood-brainbarrier (BBB) (Fig. 3a).

We next assessed the ability of each of the seven ILPs to reactivateneuroblasts when overexpressed in glia or in neurons (SupplementaryTable 2). Pan-glial or pan-neuronal overexpression of ILP4, 5 or 6 ledto precocious reactivation under fed conditions (Fig. 3b, c). Each ofthese manipulations also bypassed the nutrient restriction block toneuroblast reactivation, as did overexpression of ILP2 in glia or inneurons, or ILP3 in neurons (Fig. 3d and Supplementary Table 2).In all of these ILP overexpressions, and even when ILP6 was expressedin the posterior Ultrabithorax domain (Fig. 3e), the temporal ratherthan the spatial pattern of reactivation was affected. Importantly,experiments blocking cell signalling with SHIDN indicate that gliarather than neurons are critical for neuroblast reactivation (Fig. 4a,b). Interestingly, glial-specific overexpression of ILP3–6 did not signifi-cantly alter larval mass (Fig. 2c). Thus, in contrast to mNSC-derivedILPs, glial-derived ILPs promote CNS growth without affecting bodygrowth.

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Figure 1 | TOR/PI3K signalling in fat body and neuroblasts regulatesreactivation. a, Diagram depicting larval fat body (FB) and CNS with centralbrain (CB), thoracic (Th) and abdominal (Ab) neuromeres, mNSCs,mushroom-body neuroblasts (MB NBs) and other neuroblasts (circles)indicated. b, Brain lobe (inset in Fig. 1a), showing EdU incorporation inpostembryonic neuroblasts (large cells; for example, dotted circle) and theirprogeny (smaller cells), labelled with nab-GAL4 driving membrane GFP

(Neuroblasts . mGFP). c, EdU incorporation time course from first-instar (L1)to third-instar (L3) larval stages in the wild-type (WT) CNS. OL, optic lobe.d, f, g, EdU-labelled CNSs from larvae expressing TOR/PI3K componentsdriven by Cg-GAL4 (Fat body .) or nab-GAL4 (Neuroblasts .). e, Histogramsof EdU1 voxels from thoracic CNSs of fed larvae, normalized to controls. Inthis and all subsequent figures, error bars are s.e.m.; *P , 0.05. See text,Methods and Supplementary Fig. 2 for details of molecules expressed.

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Figure 2 | Insulin-like peptides but not mNSCs control neuroblastreactivation. a, EdU-labelled CNSs from various Ilp or InR mutants showdecreased reactivation whereas larvae with Ilp2-GAL4 driving UAS-p60(mNSCs . p60) do not. b, EdU incorporation time course in the CNS of

Df(3L)Ilp1–5 larvae. c, The mass of fed L3 larvae at the wandering (W) stage issignificantly altered by Ilp2-GAL4 (mNSCs .) driving PI3K signallingcomponents but not by repo-GAL4 (Glia .) driving Ilp genes.

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Figure 3 | CNS-specific Insulin-like peptides are sufficient for neuroblastreactivation. a, Panels show expression of Ilp3-nLacZ in subsets of neurons(XD311-11) and glia (XD311-1) and Ilp6-GAL4 (Ilp6 . nGFP andIlp6 . mGFP) in glia, including BBB and cortex glia. b, d, EdU-labelled CNSsfrom larvae overexpressing Ilp genes in various cell types (see Methods forGAL4 drivers used). c, Histograms of normalized EdU1 voxels in the thoracicCNS for the genotypes in b. e, Ilp6 overexpression in the Ultrabithorax domain

(Ubx . Ilp6) reactivates neuroblasts in the normal spatial pattern duringnutrient restriction (NR; left panel). Quiescent/enlarging neuroblasts in thecentral brain, far from the posterior Ubx domain (middle panels), extendcytoplasmic processes (arrowheads) towards the neuropil, close to long Ubx1

cell processes (right panel). The range of Ilp6 activity is difficult to determinefrom this experiment. Neurons, glia and neuroblasts are marked by Elav, Repoand Miranda respectively.

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Focusing on ILP6, we used CNS explant cultures to demonstratedirectly that glial overexpression was sufficient to substitute for theFDS during neuroblast exit from quiescence (Fig. 3d). In vivo, ILP6 wassufficient to induce reactivation during nutrient restriction when over-expressed via its own promoter or specifically in cortex glia but not inthe subperineurial BBB glia, nor in many other CNS cells that we tested(Fig. 3d and Supplementary Table 1). Hence, cortex glia possess theappropriate processing machinery and/or location to deliver reactivat-ing ILP6 to neuroblasts. Ilp6 mRNA is known to be up- rather thandownregulated in the larval fat body during starvation19 and, accord-ingly, Ilp6-GAL4 activity is increased in this tissue after nutrientrestriction (Fig. 4c). Conversely, we found that Ilp6-GAL4 is strongly

downregulated in CNS glia during nutrient restriction (Fig. 4c). Thus,dietary nutrients stimulate glia to express Ilp6 at the transcriptionallevel. Consistent with this, an important transducer of nutrient signals,the TOR/PI3K network, is necessary and sufficient in glia (but not inneurons) for neuroblast reactivation (Fig. 4a, b). Together, the geneticand expression analyses indicate that nutritionally regulated glia relaythe FDS to quiescent neuroblasts via ILPs.

This study used an integrative physiology approach to identify therelay mechanism regulating a nutritional checkpoint in neural pro-genitors. A central feature of the fat-body–glia–neuroblasts relaymodel is that glial insulin signalling bridges the amino-acid/TOR-dependent FDS with InR/PI3K/TOR signalling in neuroblasts(Fig. 4d). The importance of glial ILP signalling during neuroblastreactivation is also underscored by an independent study, publishedwhile this work was under revision27. As TOR signalling is alsorequired in neuroblasts and glia, direct amino-acid sensing by thesecell types may also impinge upon the linear tissue relay. This wouldthen constitute a feed-forward persistence detector28, ensuring thatneuroblasts exit quiescence only if high amino-acid levels are sustainedrather than transient. We also showed that the CNS ‘compartment’ inwhich glial ILPs promote growth is functionally isolated, perhaps bythe BBB, from the systemic compartment where mNSC ILPs regulatethe growth of other tissues. The existence of two functionally separateILP pools may explain why bovine insulin cannot reactivate neuro-blasts in CNS organ culture14, despite being able to activate DrosophilaInR in vitro29. Given that insulin/PI3K/TOR signalling componentsare highly conserved between insects and vertebrates, it will be import-ant to address whether mammalian adipose or hepatic tissues signal toglia and whether or not this involves an insulin/IGF relay to CNSprogenitors. In this regard, it is intriguing that brain-specific over-expression of IGF1 can stimulate cell-cycle re-entry of mammaliancortical neural progenitors30, indicating utilization of at least part ofthe mechanism identified here in Drosophila.

METHODS SUMMARYFor GAL4/UAS experiments, Drosophila were raised at 29 uC unless otherwisestated. Larvae hatching within a 2 h window were transferred to cornmeal food(5.9% glucose, 6.6% cornmeal, 1.2% baker’s yeast, 0.7% agar in water) or nutrient-restricted medium (5% sucrose, 1% agar in PBS) and further synchronized byselecting L2 larvae morphologically from an L1/L2 moulting population. For EdUexperiments, dissected CNSs were incubated for 1 h in 10mM EdU/PBS, fixed for15 min in 4% formaldehyde/PBS and Alexa Fluor azide was detected according toinstructions (Click-iT EdU Imaging Kit, Invitrogen). CNS explants were culturedon 8-mm pore-size inserts in Schneider’s medium, 10% fetal calf serum, 2 mML-glutamine (Gibco) and 13 Pen Strep (Gibco) in 24-well Transwell plates(Costar) in a humidified chamber at 25 uC. For EdU quantifications, the ‘thoracic’region used corresponds to the ventral nerve cord from the level of the brain lobesdown to A1/A2. EdU1 voxels were quantified using Volocity (Improvision) from anaverage of ten CNSs per experimental genotype, normalized to controls processed inparallel (siblings or half-siblings), using Leica SP5 scans (LAS AF software) with a1.5-mm-step z-series. For larval mass measurements, triplicates of ,50 wanderingL3 male larvae per genotype were transferred to pre-weighed microfuge tubes andwet weights determined using a Precisa XB 120A balance. For all histograms, errorbars represent the s.e.m. and P values are from two-tailed Student’s t-tests with equalsample variance. Further details can be found in Methods.

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

Received 16 June 2010; accepted 24 January 2011.

Published online 23 February 2011.

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Figure 4 | Ilp6-expressing glia are nutritionally regulated. a, EdU-labelledCNSs from larvae expressing the components indicated. b, Histograms ofnormalized EdU1 voxels in the thoracic CNS for genotypes in a. c, Ilp6 . nGFPexpression in the CNS and fat body of fed versus nutrient-restricted (NR)larvae. d, Relay model for amino-acid-dependent fat body regulation of CNSand body growth. CNS-restricted (green) and systemic (purple) pools ofInsulin-like peptides (ILPs) are functionally segregated. Direct amino-acidsensing by glia and neuroblasts may contribute to neuroblast reactivation(dashed arrows). See text for details.

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

Acknowledgements We are grateful to A. Brand, S. Cohen, B. Edgar, U. Gaul, E. Hafen,C. Klambt, T. Lee, S. Leevers, P. Leopold, F. Matsuzaki, I. Miguel-Aliaga, T. Neufeld,R. Palmer, L. Partridge, L. Pick, E. Sanchez-Herrero, H. Stocker, N. Tapon and T. Xu, andalso to the Bloomington stock centre and Kyoto National Institute of Genetics (NIG) forDrosophila stocks, antibodies and plasmids. We also acknowledge I. Salecker, J.-P.Vincent, A. Bailey, E. Cinnamon, L. Cheng, R. Makki, A. Matheu, P. Pachnis, P. Serpenteand I. Stefana for providing advice, reagents and critical reading of the manuscript. Theauthors were supported by the Medical Research Council (U117584237).

Author Contributions R.S.-N. and A.P.G. designed the experiments, R.S.-N. and L.L.Y.performed the experiments and R.S.-N. and A.P.G. wrote the manuscript. All authorshave read and subscribe to the contents of the manuscript.

Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Readers are welcome to comment on the online version of this article atwww.nature.com/nature. Correspondence and requests for materials should beaddressed to A.P.G. (agould@nimr.mrc.ac.uk).

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METHODSRearing and staging of Drosophila larvae. To assist larval genotyping, lethalchromosomes were re-established over Dfd-YFP balancers. For EdU experiments,crosses were performed in cages with grape-juice plates (25% (v/v) grape-juice,1.25% (w/v) sucrose, 2.5% (w/v) agar) supplemented with live yeast paste. ForGAL4/UAS experiments, larvae hatched within a 2-h time window were trans-ferred to our standard cornmeal food (5.9% w/v glucose, 6.6% cornmeal, 1.2%baker’s yeast, 0.7% agar in water) or to nutrient-restricted (NR) medium (5%sucrose, 1% agar in PBS). Drosophila were raised at 29 uC throughout, with thefollowing exceptions owing to lethality at high temperature: tub-GAL80ts,repo . shiDN and tub-GAL80ts, repo . Ilp2 Drosophila were raised at 25 uC duringembryogenesis and 29 uC during larval development, other Ilp2 overexpressionswere performed at 25 uC throughout. At the time of dissection, development wasfurther synchronized by selecting L2 animals morphologically from a mixed L1/L2moulting population. Df(3L)Ilp2–3, Ilp53 and Df(3L)Ilp1–5 homozygotes developconsiderably slower than controls, so EdU-incorporation experiments used mor-phological staging after the L1/L2 and L2/L3 moults. Co-expression of Dcr-2 wasused to enhance knockdown efficiency for the slifANTI allele, in which antisense slifsequences are under UAS control from an EP element insertion. As absolutenumbers of reactivated neuroblasts can vary with small differences in temperatureand humidity, parallel control experiments were carried out for each geneticbackground (using the siblings or half-siblings of experimental animals), ratherthan using a single control.Drosophila strains. Stocks used in this study were: Tor2L19 (ref. 31), InR31 (ref. 32),Df(3L)Ilp1–5, Df(X)Ilp6 and Df(X)Ilp7 (ref. 33), Ilp11, Ilp21, Ilp31, Ilp41, Ilp51, Ilp53,Df(X)Ilp641, Df(X)Ilp668, Ilp71, Df(3L)Ilp2–3, Df(3L)Ilp1–4 (ref. 34), slifANTI (ref.35), UAS-Tsc1/2 (ref. 36), UAS-Rheb, UAS-InRDN 5 UAS-InRK1409A, UAS-InRACT 5 UAS-InRA1325D, FB driver 5 Cg-GAL4 (ref. 37), pan-glial driver 5

repo-GAL4 (ref. 38), OK107-GAL4 (ref. 39), eg-GAL4 (ref. 40), DopR-GAL4(ref. 41), btl-GAL4 (ref. 42), UAS-CD8::GFP, FRT82B, tub-GAL80ts, Sco/CyO,Dfd-YFP and Dr/TM6B, Sb, Dfd-YFP (Bloomington Drosophila Stock Center),UAS-p110DN 5 UAS-p110A2860C and UAS-p110ACT 5 UAS p110CAAX (ref. 43),UAS-Pten (ref. 44), UAS-p60 (ref. 45), UAS-PdkACT 5 UAS-Pdk1A467V (ref. 46),UAS-foxo (ref. 47), UAS-Dcr2 (VDRC), UAS-Ilp7 (ref. 48), UAS-Ilp1, UAS-Ilp2,UAS-Ilp3, UAS-Ilp4, UAS-Ilp5, UAS-Ilp6, Ilp3-nLacZpXD311-1 and Ilp3-nLacZpXD311-11 (both recapitulate endogenous Ilp3 expression in L3 mNSCs, ref.49), UAS-shiDN (ref. 50), NB driver 5 nab-GAL4NP4604 (ref. 51), cortex gliadriver 5 NP577-GAL4 and ensheathing glia driver 5 NP6520-GAL4 (ref. 52),Ilp6-GAL4 5 NP1079-GAL4 (NIG stock centre), subperineurial BBB glia driver 5

moody-GAL4 (ref. 53), midline glia driver 5 slit-GAL4 (ref. 54), midline glia/neuronal driver 5 sim-GAL4 (ref. 55), mNSC driver 5 Ilp2-GAL4 (ref. 56), pan-neuronal driver 5 n-syb-GAL4 (ref. 57), Ubx-GAL4 (ref. 58), wg-GAL4 (ref. 59),en-GAL4 was a gift from A. Brand via J.-P. Vincent, repo-FLP (ref. 60).EdU detection, immunostaining, in situ hybridization and imaging. L1 and L2tissues were immobilized on poly-L-Lysine-coated slides for all stainings, except forCNS explants. For EdU experiments, dissected CNSs were incubated for 1 h in 10mMEdU/PBS, fixed for 15 min in 4% formaldehyde/PBS, followed by detection of AlexaFluor azide according to the manufacturer’s instructions (Click-iT EdU Imaging Kit,Invitrogen) and washing in 0.1% Triton/PBS. Antibody staining and in situ hybrid-ization were performed according to standard protocols. Primary antibodies used inthis study were: rabbit anti-b-Galactosidase (Molecular Probes) 1/2,000; rabbit anti-GFP (Invitrogen) 1/1,000; mouse anti-Repo 1/20; mouse anti-Miranda 1/20 and ratanti-Elav 1/100 (Developmental Studies Hybridoma Bank); pre-adsorbed alkaline-phosphatase-conjugated sheep anti-digoxigenin (Roche) 1/2,000. Secondaryantibodies used were: F(ab9)2 fragments conjugated to either Alexa-Fluor-488,Alexa-Fluor-633 (Molecular Probes) or Cy3 (Jackson), used at 1/250–1/2,000. Livetissues were photographed in PBS. Fixed tissues labelled for fluorescence microscopywere mounted in Vectashield (Vector Laboratories) whereas those processed for insitu hybridization were mounted in 80% glycerol. Fluorescent images were acquiredwith a Leica SP5 confocal microscope (LAS AF software) and bright-field imageswere acquired with a Zeiss Axiophot2 microsope (AxioVision software). Images ofthe whole CNS are projections of a 1.5-mm-step z-series. Images of fat body and ofhigh-magnification double-labels of parts of the CNS are single sections except for theright panel of Fig. 3e, which is a projection of 13 sections from a z-series.CNS explant cultures. Explanted CNSs from larvae hatched within a 2-h windowwere cultured for 3–4 days on 8-mm pore-size inserts in 10mM EdU in Schneider’smedium, 10% fetal calf serum, 2 mM L-glutamine (Gibco) and 13 Pen Strep (Gibco),in 24-well Transwell plates (Costar) placed in a humidified chamber at 25 uC.Quantification of EdU incorporation. The ‘thoracic’ region used for EdU quan-tifications corresponds to the ventral nerve cord from brain-lobe level down to A1/A2, distinguishable from more posterior neuromeres by a sharp transition inneuroblast density (Fig. 1a). The numbers of EdU1 voxels per CNS were deter-mined using Volocity (Improvision) from Leica SP5 confocal microscope scans

(LAS AF software) using a 1.5-mm-step z-series. An average of ten CNSs werequantified per experimental genotype and controls (siblings or half-siblings) wereprocessed in parallel. Control and experimental values were normalized using theaverage number of control EdU1 voxels. For all histograms, error bars representstandard error of the mean (s.e.m.) of normalized values and asterisks indicateP , 0.05 using two-tailed Student’s t-tests with equal sample variance.Larval mass measurements. Wet weights were determined for wandering L3 malelarvae, sexed and genotyped in PBS, dabbed dry with tissue and transferred to pre-weighed microfuge tubes. For each data point, triplicate samples, each containingan average of 50 animals per genotype were weighed (Precisa XB 120A balance).

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