notch cooperates with lozenge/runx to lock haemocytes into ... · directly regulated by notch...

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Development 140, 926-937 (2013) doi:10.1242/dev.086785© 2013. Published by The Company of Biologists Ltd

INTRODUCTIONThe Notch pathway operates during many different developmentaldecisions, as exemplified by haematopoiesis, where Notchregulates both the emergence of stem cells and the subsequent cellfate choices and differentiation (reviewed by Gering and Patient,2010; Maillard et al., 2005; Pajcini et al., 2011; Radtke et al.,2010). With a simple transduction pathway, where receptoractivation results in proteolytic release of the Notch intracellulardomain (NICD), one of the primary outcomes of Notch activationis a change in transcription (reviewed by Bray, 2006; Kopan andIlagan, 2009; Kovall, 2008; van Tetering and Vooijs, 2011).Recent studies have revealed large numbers of Notch-responsivegenes in progenitor cells or in cancers (Hamidi et al., 2011; Krejcíet al., 2009; Li et al., 2012; Palomero et al., 2006; Wang et al.,2011) that appear to be involved in preventing differentiation andcross-regulation of other signalling pathways (Hamidi et al., 2011;Krejcí et al., 2009; Li et al., 2012). It is not yet clear whether adifferent spectrum of Notch targets is involved in promotingdifferentiation and, if so, what such targets tell us about themechanisms involved.

To address these issues we have turned to a simple system,Drosophila blood cells (haemocytes), where Notch activitypromotes crystal cell differentiation. The second wave ofhaemocyte development occurs in the lymph gland (Fig. 1A)(Crozatier and Meister, 2007; Evans and Banerjee, 2003; Jung etal., 2005) and primarily gives rise to two cell types: crystal cells

and plasmatocytes (Fig. 1B). Previous studies have shown thatNotch activity is required for expression of the Runx proteinLozenge (Lz) in haemocytes (Lebestky et al., 2003). As Lz isnecessary for crystal cell development, this suggested a simplemechanism to explain how Notch directs crystal celldifferentiation. However, there is as yet no evidence that lz isdirectly regulated by Notch pathway (Lebestky et al., 2003;Muratoglu et al., 2007). Furthermore, perturbations to Notch at latestages prevent crystal cell differentiation and compromise cellsurvival, suggesting that Notch activity may also be required inparallel to or subsequent to Lz, although no other downstreamtargets have been identified (Krzemien et al., 2010; Mukherjee etal., 2011). Likewise, although Notch1 appears to function upstreamof Runx in several steps during mammalian haematopoiesis, noevidence that Runx genes are direct targets of Notch1 has emerged(e.g. Burns et al., 2009; Burns et al., 2005; Nottingham et al., 2007;Robert-Moreno et al., 2005). Indeed Runx factors are suggested tointegrate with Notch1 activity in specifying the T-cell lineage (Guoet al., 2008) and Notch1 targets in leukaemic T-cell have signaturessuggestive of co-regulation by Runx and Notch (Wang et al., 2011).Whether this reflects direct cooperation between Notch and Runxhas not been established.

Drosophila haemocytes therefore offer a simple model withwhich to investigate how Notch coordinates differentiation and therelationship it has with Lz/Runx in this process. To address theseissues, we first identified direct transcriptional targets of Notch inhaemocyte-related cells. Many of these direct Notch targets wereassociated with Lz/Runx-binding motifs and we demonstrate thatNotch and Lz act in combination to regulate the enhancers.Furthermore, our analysis of target gene functions reveals thatNotch simultaneously prevents cells adopting the alternateplasmatocyte fate, by upregulating klumpfuss (a ERG/WT1 familymember) and promotes characteristics associated withdifferentiation, by upregulating pebbled/hindsight (a RREB1homologue). Therefore, through these combined targets, Notch andLz operate to tie cells into the differentiation programme,converting them from an unstable to a committed state.

Department of Physiology Development and Neuroscience, University of Cambridge,Downing Street, Cambridge CB2 3DY, UK.

*These authors contributed equally to this work‡Present address: Institut Pasteur, Developmental Biology Department, CNRS URA2578, F-75015 Paris, France§Present address: University of South Bohemia, Faculty of Science, Ceske Budejovice,Czech Republic and Biology Centre, Czech Academy of Sciences, Ceske Budejovice,Czech Republic¶Author for correspondence ([email protected])

Accepted 7 December 2012

SUMMARYThe diverse functions of Notch signalling imply that it must elicit context-specific programmes of gene expression. With the aim ofinvestigating how Notch drives cells to differentiate, we have used a genome-wide approach to identify direct Notch targets inDrosophila haemocytes (blood cells), where Notch promotes crystal cell differentiation. Many of the identified Notch-regulatedenhancers contain Runx and GATA motifs, and we demonstrate that binding of the Runx protein Lozenge (Lz) is required for enhancersto be competent to respond to Notch. Functional studies of targets, such as klumpfuss (ERG/WT1 family) and pebbled/hindsight(RREB1 homologue), show that Notch acts both to prevent the cells adopting alternate cell fates and to promote morphologicalcharacteristics associated with crystal cell differentiation. Inappropriate activity of Klumpfuss perturbs the differentiation programme,resulting in melanotic tumours. Thus, by acting as a master regulator, Lz directs Notch to activate selectively a combination of targetgenes that correctly locks cells into the differentiation programme.

KEY WORDS: Lozenge/Runx, Notch, Chromatin immunoprecipitation, Haemocyte, Drosophila

Notch cooperates with Lozenge/Runx to lock haemocytesinto a differentiation programmeAna Terriente-Felix*, Jinghua Li*, Stephanie Collins, Amy Mulligan, Ian Reekie, Fred Bernard‡, Alena Krejci§

and Sarah Bray¶

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MATERIALS AND METHODSGenome-wide expression and ChIP analysisKc 167 cells, obtained from the Drosophila Genomics Resource Center(DGRC) were cultured in Schneider’s Drosophila Medium (Invitrogen)supplemented with 5% foetal calf serum (Kc 167) and 1% penicillin-streptomycin at 25°C. Expression and ChIP-chip array experiments wereperformed as described previously (Krejcí et al., 2009). In brief, chromatinwas prepared after 30 minutes of Notch activation by 2 mM EDTA andXChIP performed with α-Su(H) antibody (Krejcí et al., 2009). Precipitatedgenomic fragments and a fraction of the input DNA were amplified byligation-mediated PCR, labelled with Cy3 or Cy5, mixed and hybridized toNimblegen Drosophila 2.1 M tiling arrays, which have 50-75 bp probesdistributed at 55 bp intervals across the Drosophila genome. In all cases,three independent biological replicates were analysed and data normalized,as described previously, before identification of statistically significantdifferences in expression levels (P≤0.05) or peaks of Su(H) occupancy. Forthe latter, Tamalpais (Bieda et al., 2006) was used to identify significantChIP peaks (minimum cut-off of five adjacent probes, P<0.05) and afterexcluding atypical peaks clustered in a small region of Chromosome 2R, theanalysis yielded a set of 185 peaks. A custom-written Perl script was usedto determine genes in the vicinity of peaks. Peaks were assigned to genesonly when they were within 10 kb of an upregulated gene on either strand.Results have been deposited in Gene Expression Omnibus with seriesAccession Numbers GSE43132 (ChIP) and GSE9964 (expression array).

Motif and GO analysisCONSENSUS Matrix-based motif discovery in RSAT was used to look forover-represented motifs (Thomas-Chollier et al., 2011). A position-weightedmatrix (PWM) for Su(H), Srp and Lz was generated from a compilation ofbinding sites based on previously published data. Patser (Hertz and Stormo,1999) was used to search for matches to PWM in the Drosophila genome,with a threshold of 5.5. Custom-written Perl scripts were used to calculatethe distance between Su(H) PWM and other PWMs within 600 bp windowof Su(H) ChIP peaks. Analysis of gene ontology enrichments wasperformed using the functional enrichment chart at the DAVIDBioinformatics Resource. Results were filtered for enrichment (overthreefold) and for significance using modified Fisher’s Exact Test (EASE)with Benjamini correction (http://david.abcc.ncifcrf.gov). Representativeexamples of Biological Functions (P≤0.005) are depicted to avoidrepetitions between similar categories. All enriched molecular functioncategories with P≤0.1 are shown.

RNAi experiments and chromatin immunoprecipitationFor treating Kc cells with RNAi, double-stranded RNA duplexescorresponding to 400-800bp exonic regions were produced using T7promoter-containing primers and subsequently transcribed with aMEGAscript T7 Kit (Ambion). Cells were treated with dsRNA for 72 hoursbefore harvesting. Three biological replicates were performed in allexperiments. RNA isolation, real-time PCR and ChIP experiments wereperformed as described (Krejcí and Bray, 2007). Antibodies for ChIP weregoat α-Su(H) (Santa Cruz Biotechnology, sc15813) and mouse α-Lz(DSHB).

Fly strains and immunofluorescence staining of lymph glandsAlleles and fly stocks, as described in FlyBase (http://flybase.org/), werelz-Gal4 (Jackson Behan et al., 2005), He-Gal4 (Kurucz et al., 2003), pxn-Gal4 (Stramer et al., 2005), UAS-mamDN (Helms et al., 1999), UAS-Su(H)VP16 (Furriols and Bray, 2000), UAS-KluDN and UAS-kluEnR(Kaspar et al., 2008), UAS-lz (U. Banerjee), UAS-lzEnR (Wildonger et al.,2005), UAS-ush14A (Haenlin et al., 1997), UAS-ushN (Fossett et al., 2000),UAS-ush14A (Cubadda et al., 1997), UAS-hntRNAi (TRiP.JF03162), UAS-mycRNAi (TRiP.JF01761), UAS-whiteRNAi (TRiP.GL00094), UAS-CD8GFP, klu212lR51C and Klu-Gal4 (Klein and Campos-Ortega 1997),ush(−7462/-25)-lacZ (Muratoglu et al., 2007), and NRE-GRins (Housden etal., 2012).

Lymph glands were dissected from third instar larvae (cultured at 29ºCfrom 48-72 hours AEL), fixed for 10 minutes in 4% formaldehyde in PBS.After three washes in PBS and three washes in PBT (PBS + 1% TritonX100), lymph glands were removed from contaminating tissue and mounted

927RESEARCH ARTICLEFate commitment by Notch and Lz

onto poly-lysine-coated slides. After blocking for 1 hour in PBTN (PBT +4% horse serum) they were stained overnight with primary antibodies at4°C. Primary antibodies were as follows: mouse anti-Hnt (1/20), mouseanti-Lz (1/20), mouse anti-Lam (all from DSHB), mouse anti-P1 (1/30; agift from I. Ando, AFFILIATION), goat anti-GFP (1/600; Abcam, ab6673),mouse anti-Fib (1/500, Abcam, ab4566), rabbit anti-Ds-Red (1/25;Clontech, 632496) and rabbit anti-β-Galactosidase (1/5000; Cappel). Afterthree 15-minutes washes in PBT, fluorescently conjugated secondaryantibodies (Jackson Labs) were applied for 1.5 hours at room temperaturefollowed by three 15-minute washes in PBT and one wash in PBS. Finally,the sample was mounted in Vectashield containing DAPI for imaging withNikon D-Eclipse C1 or Leica SP2 confocal microscopes. For phenotypicexperiments, cell and nuclear dimensions were measured in over 10 lymphglands using IMARIS. Typically, 300-1000 cells were scored for eachgenotype.

Construct design and mutagenesisChIP-enriched regions from klu, CG32369, rgr, peb and other putativetargets were amplified from Drosophila genomic DNA, using primerscontaining restriction enzyme sequences (see supplementary material TableS2), and cloned into pGreenRabbit/pRedRabbit vectors (Housden et al.,2012) for in vivo reporter assays or pGL3min for luciferase assays. Site-directed mutagenesis was performed using a PCR-based approach withprimers overlapping the Su(H)/Srp/Lz-binding site to be mutated with thesequences changed as follows: CGTGGGAA to CGTTGTTA for the Su(H)motif; GGATAAC to GTTCTAC for the Srp motif and AACCACA toAATGACC for the Lz motif. Luciferase assays were performed asdescribed previously (Krejcí and Bray, 2007).

RESULTSNotch responsive genes in Drosophila Kc cellsAs Drosophila Kc cells exhibit characteristics of haemocytes(Echalier and Ohanessian, 1969; Lunstrum et al., 1988; Nelson etal., 1994; Schneider, 1972), express several haemocyte markers(including Hemese, Hemolectin and Serpent, Fig. 1D) (Cherbas etal., 2011; Jung et al., 2005) and contain detectable levels of Lz(Fig. 1E), they provide a suitable model for investigating the Notchresponse and its relationship with the Runx factor Lz. In addition,Kc cells differ substantially from the muscle-related DmD8 cells(Fig. 1D) (Cherbas et al., 2011), where Notch activity is involved inmaintaining progenitor characteristics in collaboration withtranscription factor Twist (Bernard et al., 2010), enabling us todiscover how the Notch response differs between cell types.

To characterize Notch-responsive genes in Kc cells, we used asimilar strategy to that used previously, activating Notch using acalcium chelator (Gupta-Rossi et al., 2001; Rand et al., 2000) andmonitoring mRNA expression changes 30 minutes later (Krejcí etal., 2009; Krejcí and Bray, 2007). Probes prepared from controland Notch-activated mRNA populations were hybridized in pair-wise combinations to Drosophila transcriptome microarrays toidentify genes that were significantly upregulated (P≤0.05). Inparallel, we identified genomic regions that were occupied afterNotch activation by Suppressor of Hairless [Su(H)], the coretranscription factor in the Notch pathway (reviewed by Bray, 2006;Kopan and Ilagan, 2009; Kovall, 2008; van Tetering and Vooijs,2011), using chromatin immunoprecipitation (ChIP) andhybridizing bound DNA to genomic tiling arrays. Integrating thesedata, the 185 Su(H) occupied regions, ‘peaks’, were assigned togenes if they were located within or close to genes that wereupregulated (Fig. 2A). This identified 69 assigned peak genes(APGs) that fulfilled the criteria of Su(H) binding within 10 kb andupregulation with the Notch activation regime [supplementarymaterial Table S1; ranked by AvgM, fold difference in expression(log2)]. Several APG were validated using quantitative PCR to D

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confirm Su(H) binding in repeat ChIPs (e.g. Fig. 4C) and, usingthis method, we also detected Su(H) binding near one additionalupregulated gene [pebbled (peb), also known as hindsight (hnt)].As calcium chelation is a non-specific method of activating Notch,a subset, including peb/hnt, were further validated by testing theirupregulation in cells transfected with NICD (supplementarymaterial Fig. S1) (although this approach is hampered by the factthat transfection efficiency is less than 30% in these cells).

A comparison with results from similar experiments in DmD8cells demonstrated relatively little overlap of Su(H)-occupied sitesor of upregulated genes. Thus, although the number of occupiedsites in the two cell types was of similar magnitude, 260 in DmD8and 185 in Kc cells, only 28 peaks were overlapping between the

RESEARCH ARTICLE Development 140 (4)

two. This suggests that the accessibility of sites to Su(H) is likelyto be a major factor in determining the outcome from Notchactivation. Most overlapping peaks were in proximity toupregulated genes, corresponding to 17 APGs that were commonto both Kc and DmD8 cells (Fig. 2A). These included several of theE(spl) genes, myc and Notch (supplementary material Table S1).Such genes appear to be widespread targets of Notch in manydifferent organisms, including humans, and are likely to identifymechanisms of fundamental importance to Notch signalling (Bray,1997; Davis and Turner, 2001; Fischer and Gessler, 2007;Kageyama et al., 2007; Klinakis et al., 2006; Krejcí et al., 2009;Palomero et al., 2006; Satoh et al., 2004; Weng et al., 2006;Yashiro-Ohtani et al., 2009). Nevertheless, there were alsosubstantial differences in the Notch-responsive genes in the twocell types, indicative of disparate functional outcomes from Notchactivation. We note also that, as we were monitoring Su(H)occupancy and not recruitment of NICD to these targets (owing toNICD antibody not performing well in ChIP), we cannot formallyrule out the possibility that expression from some of the Su(H)-bound upregulated genes may be independent of direct binding byNICD. Furthermore, 58% Su(H) peaks had no upregulated geneswithin 10 kb, and although some may be false positives, it suggeststhat some Su(H) sites may be associated with genes that requireother co-factors or represent Notch-independent Su(H) targets.

To gain a global overview of the Su(H)-bound responsive genesin Kc cells, we analysed their functional characteristics using geneontology (GO) annotations (http://david.abcc.ncifcrf.gov/)(Fig. 2B). Although the cohort of targets differed, several enrichedBiological Process categories were similar to DmD8 cells,including cell surface receptor-linked signalling and imaginal discmorphogenesis. The cross-regulation of other signalling pathways,especially Ras signal transduction, therefore emerges as a commontheme, with Gap1 and pointed being among the Kc-regulatedgenes. Pattern specification and cell migration were, however,more enriched in Kc than in DmD8 cells. Similarly, there wasenrichment for the Molecular Function categories ‘transcriptionfactors’ and ‘actin binding’. The former are candidates tocoordinate the programme of differentiation in haemocytes, andmany encode zinc-finger transcription factors (Interpro zinc-fingerC2H2 type; fivefold enriched) such as klumpfuss (klu), pebbled(peb/hnt), regular (rgr) and Hnf4. We note, however, that lz was notamong the genes bound by Su(H) or upregulated within30 minutes, despite its expression in the lymph gland beingdependent on Notch activity (Lebestky et al., 2003).

Kc Notch targets are expressed in differentiatingcrystal cellsNone of the identified Kc Su(H) targets had been previouslyassociated with haemocyte fates and several were as yetuncharacterized. To determine their relevance for blood celldifferentiation in vivo, we investigated their expression in thirdinstar lymph glands. First we used a Notch activity-sensing reporter(NRE-GFP; Housden et al., 2012) to confirm that Notch isspecifically active in Lz-expressing crystal cell precursors (Fig. 1C),although we note that NRE-GFP expression was detected only in asubset of Lz+ cells, suggesting that there is a transient phase ofNotch activity. Selecting two highly upregulated transcriptionfactors, klu and peb/hnt, we compared their expression to the Lzcrystal cell lineage marker (Fig. 2C,D) and found that both wereexpressed in the lymph gland in a pattern that overlapped with Lz.Indeed, peb/hnt protein was present in all Lz-expressing cells(detected with lz-Ga4 UAS-GFP), although there were also a few

Fig. 1. Haemocyte development: role of Notch and relationship toKc167 cells. (A) Drosophila lymph gland where larval haemocytedevelopment gives rise to crystal cells (red) and plasmatocytes (green).Boxed area indicates the region shown in confocal images in this andsubsequent figures. (B) The haemocyte lineage, specific markers andrelevant Gal4 drivers are indicated. Notch and Lozenge (Lz) are requiredfor crystal cells (red nuclei) to develop from Serpent (Srp)-expressing pro-haemocytes. (C) Expression of a general Notch-responsive reporter, NRE-GFP (green), indicates that Notch is active in Lz-expressing (red) crystalcell precursors. Arrows indicate examples of cells co-expressing Lz andNRE-GFP. (D) ModEncode measurements of relative mRNA expressionlevels of the indicated genes (He, hemese; Hml, hemolectin; Nim, Nimrod;srp, serpent; dome, domeless; twi, twist; N, Notch; vg, vestigial) in Kc167cells compared with DmD8 (muscle precursor related) (Cherbas et al.,2011); haemocyte-related genes Hemolectin (Hml) and Hemese (He) arespecifically expressed in Kc cells. (E) Lz is present in Kc167 cells; westernblot of total cell extracts from the indicated cells was probed with α-Lzand α-Tub. The quantified ratio of Lz/Tub is indicated for each lane(arbitrary units). Cells pre-treated with dsRNA to ablate Lz (Kc LzRNAi)have reduced protein. Scale bar: 10 μm.

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cells with Peb/Hnt only. Similarly, the majority of Klu-expressingcells (klu-Gal4 UAS-GFP) contained Lz. Thus, these Kc identifiedAPG are expressed in a relevant lineage in vivo, where theirexpression appears to persist. For example, Peb/Hnt proteinexpression was detected in mature crystal cells, based on itscolocalization with anti-ProPO (supplementary material Fig. S2)


and with other markers in a recent study (Benmimoun et al., 2012).In addition, expression of peb/hnt was upregulated in NICD-expressing clones and was suppressed when Notch was ablated byRNAi (supplementary material Fig. S2).

To further investigate whether the APGs were subject toNotch-mediated regulation in the lymph gland, reporter geneswere generated containing putative Notch-responsive enhancers(NREs), as defined by Su(H) ChIP peaks, upstream of GFP ormCherry (Fig. 3A) (Housden et al., 2012). All exhibitedexpression in crystal cell precursors (Lz-expressing cells)(Fig. 3C,E,G,I), although levels of expression varied. Forexample, peb/hntNRE, kluNRE, mycNRE and CG32369NRE alldirected expression at high levels in Lz+ cells (Fig. 2E,F;Fig. 3C,G,E,I). Other reporters, rgrNRE, Gap1NRE,CG6860NRE, CG11873NRE and hnf4NRE, were detected at lowlevels in Lz-expressing cells (supplementary material Fig. S3)but these levels could be augmented by expression of theconstitutively active Su(H)VP16 (supplementary material Fig.S3). To confirm that the identified enhancers were regulated byNotch, we tested consequences of mutating all the Su(H) motifsin klu, CG32369, peb/hnt, myc and rgr NREs (Fig. 3D,F,H,J;supplementary material Fig. S3A). With kluNRE, CG32369NREmycNRE and rgrNRE, the Su(H) motif mutations completelyabolished expression in the lymph gland (Fig. 3D,F,J;supplementary material Fig. S3B). Effects on peb/hntNRE wereintermediate (Fig. 3H), with some residual expression detectedfrom the mutated NRE in a subset of glands (2/15). Moreover,luciferase assays revealed that mutated NREs had lost theirresponse to NICD (Fig. 3B). These results confirmed theimportance of Su(H) sites for expression in haemocytes,demonstrating that these targets are Notch responsive.

Requirement for Lz/RunxThe differences between the Notch-responsive genes in Kc andDmD8 cells suggests that intrinsic factors alter which targetenhancers can be regulated. One approach to identify such co-regulators is to look for motifs that are enriched within the regionsbound by Su(H) in each cell type. Taking as our input the 300 bpflanking the mid-point of each Su(H) ChIP peak (a 600 bpwindow), we used CONSENSUS Matrix-based motif discoveryin RSAT to look for over-represented motifs (Thomas-Chollier etal., 2011). This returned three assemblies. The first, CGTGGGAA,corresponds to Su(H) motif. The second, GATAAAGT, resemblesa GATA-factor binding site, implicating Serpent (or one of thethree other Drosophila GATA factors). The third, ACCATAGT, avariant on the established Runx motif suggesting that Lz could bea co-factor, was detected under a subset of conditions. Wetherefore used a position weight matrix to locate putative bindingmotifs for Lz/Runx and GATA, and analysed their relationship toSu(H) motifs in regions defined by Kc peaks, using forcomparison the motif for Twist (Twi), a muscle-specifictranscription factor. Approximately 40% of Su(H) motifs in Kcpeaks were within 100 bp of Lz and 36% were within 100 bp of aGATA site, whereas fewer than 25% had a Twi site in similarproximity (under conditions where the total numbers of each motifin the genome were similar; Fig. 4A). Indeed, 49% of the assignedgenes are associated with Kc peaks that contain GATA and Lzmotifs, as well as Su(H). A further 17% are associated with peakscontaining only Su(H) and Lz motifs, and 13% are associated withpeaks containing only Su(H) and GATA motifs. By contrast, fewerthan 39% of APGs in DmD8 muscle-related cells have GATA andLz in proximity to Su(H), whereas 57% have a Twi motif. The

Fig. 2. Identification of Notch targets in haemocytes. (A) Left: Venndiagram illustrating overlap between genes in proximity to Su(H)-boundregions [blue: anti-Su(H) ChIP, 185 bound regions] and upregulated after30 minutes of Notch activation (mauve, 1302 upregulated transcripts)identifies 69 APGs that are putative direct Notch targets in Kc cells. Right:overlap between direct Notch targets identified in Kc cells (purple) and inDmD8 cells (green) is limited to 17 common genes. (B) Examples of over-represented GO (Biological Process, upper graph; Molecular Function,lower graph) categories in Kc direct Notch targets. (C-F) Expression ofindicated targets and/or target enhancers in crystal cell lineage (markedby lz>GFP (C, E, F; green) and anti-Lz (D; green). See supplementarymaterial Table S1 for a list of direct targets. Scale bar: 10 μm.

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Fig. 3. Notch-responsive enhancers direct expression in crystal cell lineage and require Su(H) motifs for activity. (A) Examples of genomicregions from representative Notch targets showing Su(H) ChIP-enriched regions in Kc cells (purple; fold enrichment relative to total input −0.1-2.5, log2scale). Matches to the Su(H)-binding motif are indicated (bar height indicates affinity class of sites). Gene models are depicted in black. Blue barsrepresent the regions (NRE) that were cloned to test responsiveness in vivo (for distribution of motifs see Fig. 5). Blue arrows in klu indicate fragmentsanalysed in DNase I sensitivity assays (see Fig. 4D). (B) Fold change in luciferase activity in the presence of the NICD from each unmutated NRE (wt)indicated and from NRE with mutated (m) Su(H) motifs. (C-J′) Expression from the indicated NRE, either unmutated (C,C�,E,E�,G,G�,I,I�) or where Su(H)motifs have been mutated [mSu(H)] (D,D′,F,F′,H,H′,J,J′). Levels of enhancer activity are detected by fluorescence (GFP or mCherry, green, C-J; singlechannel, white, C�-J�) in crystal cell precursors marked by expression of Lz (red, C-J). See supplementary material Fig. S1 for additional NRE expression.Scale bar: 10 μm. D

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highly conserved Lz/Runx and GATA factors may thereforeprovide the crucial specificity for Notch responses in Kccells/haemocytes.

One hypothesis is that Lz (and GATA) are necessary to make theenhancers capable of responding to Su(H)/Notch. As we wereinterested whether targets required cooperation between Notch andRunx, we focused our analysis on Lz, for which there was amonoclonal antibody that was suitable for ChIP. First, weinvestigated whether Lz was present at candidate enhancers priorto Notch activation. Fragments that encompass Lz motifs in peakregions from klu, CG32369, rgr, peb/hnt were significantlyenriched in the Lz ChIP, compared with control regions (Fig. 4B).We therefore asked whether reducing Lz, by treating cells withRNAi (Fig. 1E), would interfere with recruitment of Su(H) to targetenhancers. Little residual Lz binding was detectable at targetenhancers in Lz RNAi-treated cells (Fig. 4B). Su(H) binding at klu,CG32369, rgr, peb/hnt enhancers was similarly compromised inLz-depleted cells (Fig. 4C), whereas it remained unchanged atE(spl)mβ, a region not linked to Lz sites (Fig. 4C). Conversely,there was no reduction in Lz binding in Su(H) RNAi-treated cells(supplementary material Fig. S4). Altogether, these results arguethat Lz binding precedes recruitment of the Su(H)/Notch complex,and that it enhances Su(H) recruitment to target enhancers.

To investigate the mechanism of Su(H) recruitment by Lz, wefirst checked for direct interactions by co-immunoprecipitation, butwere unable to detect any co-purification of Su(H) in Lzimmunoprecipitates or vice versa (supplementary material Fig. S4).Second, we assessed whether Lz had an influence on chromatinaccessibility, as measured by sensitivity to DNase I treatment.Focusing initially on klu, we tested the effects from two differentconcentrations of DNase I on digestion at different sites across the


locus. This revealed that two sites were more sensitive than others,the promoter and the NRE (Fig. 4D). We subsequently testedwhether this sensitivity was altered in Lz RNAi-treated cells andfound that the NRE was less sensitive to DNase I under theseconditions, suggesting that Lz alters the DNA accessibility(Fig. 4D). Finally, we tested whether other enhancers showedsimilar Lz-dependant DNase I accessibility. Although the effectswere most robust with klu, both CG32369 and rgr also showedsome decrease in accessibility following Lz RNAi (Fig. 4E).However, we were not able to detect similar sensitivity at thepeb/hnt NRE (Fig. 4E). Taken together, the data indicate thereforethat the effects of Lz on Su(H) recruitment are likely to be indirectand most likely involve a change in the chromatin accessibility atleast at some loci, such as klu.

To address whether Notch is required in combination with Lz,we compared the consequences on Peb/Hnt expression ofperturbing Notch function in Lz-expressing cells using a dominant-negative form of Mam (MamDN) with the effect of LzEnR, aconstitutive repressor form of Lz (Wildonger et al., 2005). TheMamDN peptide occupies the Mam-binding groove on the Su(H)-NICD complex, blocking the functional Mam protein, an essentialco-activator of Notch, from binding. Expression of MamDN aloneor of LzEnR was sufficient to severely reduce levels of Peb/Hntexpression (e.g. Fig. 4F,G). Only 56.5% of Lz>GFP-expressingcells stained positive for Peb/Hnt in the presence of MamDN, andonly 66.6% in the presence of LzEnR, compared with 97.2% incontrol glands (supplementary material Fig. S5). Importantly, co-expression of wild-type Lz with MamDN was unable to rescue thePeb/Hnt expression (Fig. 4H). Thus, reduced Notch activity cannotbe compensated for by increased Lz. This implies that thecombination of Notch activity and Lz is required for Peb/Hnt

Fig. 4. Lz influences Su(H) recruitment atNotch-responsive enhancers and is requiredwith Notch in vivo. (A) Lz and GATA motifs arelocated in proximity to Su(H). Graph showingpercentage of detected motifs located within theindicated distance (bp) of the Su(H) motif withinKc ChIP peaks (blue, GATA; green, Lz; grey, Twist).(B) Lz is present at NREs. Fold enrichment of theindicated NRE in α-Lz ChIP relative to the adjacentcoding sequence (cds) in Kc cells (dark shading)and after lz RNAi (light shading). (C) Depletion ofLz compromises Su(H) binding. Fold enrichmentof the indicated NRE in anti-Su(H) ChIP relative tothe cds in Kc cells (dark shading) and after lz RNAi(light shading). (D,E) Effects of Lz depletion onDNase I sensitivity of the indicated regions in klu(D) and of NRE from other genes (E). Chromatinfrom control and Lz RNAi-treated cells wassubjected to digestion with 0, 4 or 12 U of DNase Iand the levels of intact DNA quantified by qPCR.Location of klu fragments are indicated in Fig. 3A.(F-H) Peb/Hnt expression in control glands (F, lz >GFP) and in glands where MamDN only (G) orLz and MamDN (H) were expressed (lz>GFPindicates lz-Gal4 UAS-GFP). Scale bar: 10 μm. Dataare mean±s.e.m.

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expression and establishes that the phenotype caused by MamDNcannot be attributed to failure in Lz. To confirm the relevance ofLz/Runx in vivo, we tested the consequences of mutating theidentified Lz motifs on expression from the klu, peb/hnt andCG32369 NREs (Fig. 5). Expression from all three NREs wasabolished when Lz/Runx sites were eliminated (kluNRE[mLz],CG32369NRE[mLz], pebNRE[mLz]; Fig. 5). Altogether, these dataindicate that Lz confers specificity on the Notch response indifferentiating haemocytes. We note that klu is also regulated byLz in another context, through a different enhancer (Wildonger etal., 2005).

Finally we also tested the relevance of GATA motifs forexpression from klu, peb/hnt and CG32369 NREs. Expression wasreduced by mutations disrupting the GATA motifs (Fig. 5), but withvariable effects: CG32369NRE[mGATA] lost all detectableexpression, kluNRE[mGATA] exhibited low level expression in fewcells and peb/hntNRE[mGATA] had moderate levels of activity.Nevertheless, the results make it likely that a GATA factor, possiblySrp, also cooperates with Notch on these enhancers.

Inhibition of alternate plasmatocyte fates by theNotch target kluThe identified Kc Notch targets are expressed in response to Notchand Lz in the crystal cell lineage. So far, relatively little is knownabout events downstream of Notch activation in these cells,although recent studies have demonstrated that Notch is requirednot only for crystal cell specification, but also for their expansionand maintenance (Mukherjee et al., 2011). Target gene functionsshould therefore reveal how Notch implements its role in promotingthe specific differentiation programme.

klu is one of the genes that is most highly upregulated in Kc cellsand encodes a zinc-finger protein related to the Wilms Tumor 1(WT-1)/Early growth response (EGR) gene family, which regulatedifferentiation in haematopoetic lineages (Alberta et al., 2003;Friedman, 2007; Georgescu et al., 2008; Klein and Campos-Ortega,1997) and which exhibit altered activity in acute myeloid andlymphoid leukaemias (see Huff, 2011; Tosello et al., 2009; Yang etal., 2007). We investigated whether klu has a similar crucial role in


haemocytes by expressing a dominant-negative form of the Kluprotein (KluDN; Kaspar et al., 2008; Klein and Campos-Ortega,1997) in the developing crystal cells with lz-Gal4. A characteristicof lz-Gal4-expressing cells is that they are devoid of P1, a markerfor the alternate plasmatocyte lineage (Fig. 6A) (Krzemien et al.,2010; Minakhina and Steward, 2010). Strikingly, in the presence ofKluDN, many lz-Gal4-expressing cells were found to express P1(Fig. 6B), although they retained Lz (supplementary material Fig.S6). Similarly, we observed P1 in Lz-expressing cells in lymphglands from klu mutant larvae (kluG4/klu212lR51C; supplementarymaterial Fig. S7) and when klu was ablated using short hairpinRNAi (Fig. 6C). These results suggest that Klu is important forrepressing the alternate plasmatocyte fate in crystal cell precursors.

Studies of Klu function in PNS development demonstrated thata constitutive repressor form of Klu, KluEnR, behaved as a stronggain of function for Klu when overexpressed (Kaspar et al., 2008).We therefore tested the consequences of KluEnR expression usingHe-Gal4, which drives expression in P1-expressing cells (Fig. 6D).Many of the KluEnR-expressing cells had reduced P1 expression(Fig. 6E, arrows) and those with significant residual P1 expressionexhibited altered morphology (Fig. 6E). Furthermore, many larvaeshowed striking accumulations of melanotic cells, resemblingmelanotic tumours (Fig. 6F-H). However, the KluEnR expressionwas not sufficient to fully convert the cells to crystal cells, as therewas little increase in the number of He>GFP cells that containedPeb/Hnt or Lz (supplementary material Fig. S6). Thus, KluEnRsuppresses plasmatocyte characteristics but is not sufficient to fullydirect the cells into crystal cell lineage.

One possible mechanism through which Klu might preventplasmatocyte differentiation is by repressing ush, the Drosophilahomologue of FOG1, because a decrease in ush expression isthought to be important for crystal cell maturation in the embryo(Fossett et al., 2003; Fossett et al., 2001; Waltzer et al., 2003). Wetherefore investigated whether, by restoring Ush in the presence ofKluEnR, we could suppress the melanotic cell masses. The numberof larvae with melanotic masses was significantly reduced incombination with ush (Fig. 6H), supporting the hypothesis that onemechanism through which Klu prevents plasmatocyte fates is by

Fig. 5. Notch-responsive enhancers require Lz andSrp sites for full activity. (A-C) Consequences onindicated NRE activity of mutating Lz (mLz) or GATA(mGATA) motifs. Expression of the resulting GFPreporters (green) in crystal cell lineage (α-Lz, purple).Diagrams above depict the location of the Lz- (greenrectangles) and GATA (blue triangles)-binding motifswithin each enhancer relative to Su(H) motifs (pinkovals). Scale bar: 10 μm.

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antagonizing ush. Although expression of ush (ush-lacZ) waswidespread and heterogeneous in the lymph gland (supplementarymaterial Fig. S7), the majority of Lz-positive cells had little or noush-lacZ expression, consistent with the diminished expression incrystal cells observed in the embryo (supplementary material Fig.S7). Broad (pxn-Gal4) expression of KluDN alone (supplementarymaterial Fig. S7) or MamDN alone (supplementary material Fig.S7) had little effect on ush expression in Lz-positive cells, althoughthere was more variability in levels in the former. However,combined overexpression of kluDN and mamDN together resultedin a significant increase in the number of Lz-expressing cellsexhibiting strong ush-lacZ (30% of Lz-expressing cells;supplementary material Fig. S7). This suggests that Ush may beone factor involved in the switch downstream of Klu, but suggeststhat its regulation may require additional inputs from Notch.

Other Notch targets implement cellularprogrammes associated with crystal cell fatesOne characteristic of crystal cells is that they undergo DNAreplication but do not appear to be mitotically active, suggestingthat they are in endocycle (Krzemien et al., 2010). In the follicularepithelium, peb/hnt is required downstream of Notch for


implementing the switch to endoreplication (Sun and Deng, 2007;Sun et al., 2008). We therefore considered whether it mightfunction similarly in the lymph gland. Endocycling cells possessan increased DNA content relative to neighbouring mitotic cells,evident in the DAPI staining. We first investigated whether Notchactivity affected the DNA content in lymph-gland nuclei, byexpressing Su(H)VP16 in Lz-expressing cells (lz>GFP). This hadthe striking consequence of increasing the dimensions of thenucleus (based on DAPI staining and nuclear Lamin; Fig. 7A,B,E)and also enlarged the patch of Fibrillarin (Fig. 7A″,B″), anucleolar marker. We noted that there was also considerableheterogeneity among the lz>GFP population even in the absenceof additional Su(H)VP16 (Fig. 7C), suggesting that nuclearenlargement is a feature of their differentiation. To investigatewhether peb/hnt has a role in this nuclear enlargement, we ablatedpeb/hnt, by expressing peb/hnt-RNAi, in the presence of theSu(H)VP16. This resulted in a significant reduction in the nucleardimensions compared with expression of Su(H)VP16 alone(Fig. 7D; supplementary material Fig. S8). Thus, peb/hnt is in partresponsible for the effects of Su(H)VP16 on nuclear size. Then,we examined the consequences of perturbing peb/hnt on nucleardimensions in the largest lz>GFP-expressing cells (Fig. 7E,F).

Fig. 6. Regulation of klu by Notch blocks plasmatocytefates. (A-A�) Expression of the plasmatocye marker P1 (red, A;white, A�) does not overlap with Lz (lz>GFP, green in A; white inA�) in wild-type glands (arrows). (B-C�) P1 is detected in Lz cellsthat express KluDN (B-B�, e.g. arrows) or kluRNAi (C-C�, arrows),labelled as in A. (D-D�) Expression of P1 is detected in all He-Gal4-expressing cells (He-Gal4 UAS-nlsGFP, He>GFP) in controlglands (arrows). (E-E�) Expression of KluEnR compromises P1expression in many of the He>GFP cells (arrows). (F,G) Expressionof KluEnR leads to melanotic masses, (F) wild-type larvae, (G)KluEnR-expressing larvae with both large (arrows) and small(arrowhead) small masses. (H) Larvae were scored for melanoticmasses (large, larvae had at least one large mass; small, larvaehad only small dots of melanin). Co-expression of Ush reducesthe percentage of KluEnR larvae that exhibit melanotic masses.Over 80 larvae were scored for each genotype. Seesupplementary material Fig. S6 for Lz expression in KluEnR. Scalebar: 10 μm.

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Knockdown of peb/hnt led to a significant reduction in the nucleardiameters in these cells, consistent with a role in promotingnuclear enlargement associated with endoreplication (Fig. 7G).

Myc (also known as dimutive) is another of the Kc Notch targets,which is implicated in cell growth and regulates polyploidy in sometissues (Maines et al., 2004; Pierce et al., 2004). Indeed whenoverexpressed in Lz>GFP cells, Myc caused an increase in cell,


nuclear and nucleolus size (supplementary material Fig. S8). Whenwe assessed the consequences of myc-RNAi on the Su(H)VP16phenotype, there was also a reduction in nuclear size(supplementary material Fig. S8). Therefore, it appears that mycmay also be important in implementing the Notch-dependentchanges in nuclear size/ploidy that appear intrinsic to the crystalcell differentiation programme, although quantitative

Fig. 7. Notch regulates nuclear size via Hnt. (A-B�) Crystal cell lineage marked by lz>GFP (green, e.g. arrows) in control (A) and Su(H)VP16-expressing(B) lymph gland stained to detect DNA (DAPI, blue, single channel A�,B�) and nucleolus (Fibrillarin, red, single channel A�,B�). Expression of Su(H)VP16results in enlarged nuclei (compare arrows). (C) Size of nuclei based on DAPI staining in lz>GFP-expressing lymph glands, GFP-expressing nuclei (crystalcell lineage) are larger than average (P=3.331e-14, 15 LG, 75 Lz+ cells and 1833 non Lz+ cells). Box and whisker plot. Horizontal line indicates median,box indicates interquartile range (IQR), and whiskers indicate maximum and minimun within 1.5 IQR. (D) The measurable increase in nuclei (DAPI) sizeobtained by expressing Su(H)VP16 (with lz-Gal4) is suppressed by co-expressing RNAi targeting peb/hnt or myc. RNAi targeting white was used ascontrol (nuclear size in C,D was calculated by measuring the diameter/volume of DAPI staining; 15 LG, 732 cells for Lz>+; 11 LG, 1325 cells forLzG4>Su(H)VP16+whiteRNAi; 17 LG, 527 cells for LzG4>Su(H)VP16+hntRNAi). Asterisk indicates that results were significantly different, P<2.2e-16, usingused two-sample Kolmogorov-Smirnov test. Numbers are arbitrary units that differ between experiments owing to the method used to obtain images.Box and whisker plot as in C. (E-F�) Nuclear diameters (nuclear Lamin, red E,F; single channel E�,F�) in large lz>GFP crystal cells from control (E, whiteRNAi;F, peb/hntRNAi). (G) Knockdown of peb/hnt leads to a reduction in nuclear diameter. Asterisk indicates that results were significantly different,P=0.004799, using used two-sample Kolmogorov-Smirnov test (11 LG per genotype, 60 Lz+ cells for controls and 47 Lz+ cells for hntNRAi). Box andwhisker plot as in C. (H) The proposed role of Notch acting in combination with Lz in the crystal cell lineage. Notch activity is also required at earlierstages in haemocyte development, where the target genes are likely to differ, as it will operate in a different context. Scale bar: 10 μm.

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measurements of DNA synthesis in individual lz>GFP cells will beneeded to confirm its role.

DISCUSSIONAs signalling pathways, such as Notch, are used iteratively duringdevelopment, one of the challenges is to understand their context-specific effects. Taking a genome-wide approach, we haveelucidated the transcriptional outputs of Notch in Drosophila Kccells, a model for haemocyte development, and shown that, invivo, the targets are involved in locking cells into a specific fate,by shutting down the alternatives and by promoting specificcharacteristics of the differentiated cells (Fig. 7H). The contextspecificity for this programme is provided by the Runx factor Lz,probably acting in combination with GATA factors, as mutationof Lz or GATA sites eliminates expression from the Notch-regulated enhancers. Furthermore, ablation of Lz compromises therecruitment of Su(H) to these targets enhancers. Thus, Lz appearsto act as a lineage master regulator for Notch in the mannerproposed for cell-specific factors acting with BMP and Wntsignals in regeneration of haematopoietic lineages (Trompouki etal., 2011) and with TGFβ in differentiation (Mullen et al., 2011).

Our analysis of haemocyte Su(H) targets has thus uncovered acooperative activity between Lz and Notch. In most previousstudies, Notch and Runx have been shown to act in a hierarchicalmanner, with Notch functioning upstream of Runx/Lz (Burns etal., 2009; Burns et al., 2005; Lebestky et al., 2003; Nottinghamet al., 2007; Robert-Moreno et al., 2005). However, a similarcooperative mechanism may operate at late stages in thymocytedevelopment, where Runx1 confers the capability to promote T-cell fate in response to Notch activity (Guo et al., 2008). Analysisof Notch-regulated targets in T-ALL cells also uncovered asignature that was suggestive of Notch and Runx co-regulation inthese malignant cells (Wang et al., 2011). Although Lz/Runxexpression is itself dependent on Notch activity, there is as yetno evidence to suggest direct regulation in other systems and wehave not detected Lz as a Su(H)-bound target in our experiments,making it plausible that the regulation by Notch is indirect.Nevertheless, the observation that Lz/Runx is required firstdownstream of Notch and second in combination with Notch attarget enhancers suggests that a feed-forward ratchet mechanismmay contribute to cell fate specification both in Drosophilacrystal cells and mammalian lineages. This also highlights thefact that Notch is needed at several different stages in suchlineages, and that the specific targets regulated are likely to differdepending on the stage.

Among the newly identified Notch-regulated genes, many aretranscription factors (Klu, Peb/Hnt, Myc, Hnf4, Rgr, p53) whoseexpression and functions illustrate their importance for the crystalcell differentiation programme. Strikingly, our analysis andmanipulation of Klu, a zinc-finger protein related to the ERG/WT1family (Klein and Campos-Ortega, 1997), revealed that it isnecessary to inhibit alternate cell fates in the Lz-expressing cells.In some respects, this is surprising as lineage-tracing experimentsindicated that most, if not all, Lz-expressing cells were fated tobecome crystal cells in healthy animals owing to signalling eventsat much earlier stages (Krzemien et al., 2010; Minakhina andSteward, 2010). Why is Klu required? One possibility is that Lz+

cells are in a transitional state prior to Notch activation/Kluexpression, retaining the potential to adopt alternate fatesdepending on environmental challenges. For example, hypoxiaappears to be important for crystal cell differentiation to bemaintained (Mukherjee et al., 2011) and wasp infection can reroute


differentiation to lamellocyte fates (e.g. Krzemień et al., 2007).Notch-induced upregulation of Klu may be important to lock cellsinto the crystal cell programme, by inhibiting activity of alternatelineage regulators. A similar role has been proposed for themammalian homologues EGR1/2, which are part of an antagonisticregulatory circuit that maintains lineage fidelity, downstream of‘pioneer’ transcription factors, by repressing alternate fate choices(Laslo et al., 2006).

Other targets appear to be actively involved in promoting thecrystal cell differentiation programme. In particular, the transcriptionfactor Peb/Hnt is important for the change in nuclear size/DNAcontent that probably reflects endoreplication in crystal cells(Krzemien et al., 2010), a process that may also require Myc. As bothPeb/Hnt and Myc are involved in regulating the switch from mitoticto endocycle downstream of Notch in the Drosophila follicle cells(Maines et al., 2004; Shcherbata et al., 2004; Sun and Deng, 2007;Sun et al., 2008), this may be a conserved cassette that is deployed atmultiple stages in development. Elsewhere, Myc has been shown toregulate polyploidy and differentiation in several cell types, includingmegakaryocytes and in T-ALL cells (e.g. Munoz-Alonso et al., 2012;Palomero et al., 2006; Pierce et al., 2004; Zanet et al., 2005). Othertargets include Hnf4, which was previously implicated in causinghaemocyte expansion downstream of AML (Runx)-ETO (Sinenko etal., 2010), and several genes involved in cell motility (e.g. CLIP-120),as well as other conserved genes with unknown functions, such asCG32369 (LONRF1-3).

A comparison with Notch-regulated genes in a different cell type(DmD8 cells) demonstrates that differences in Su(H) binding,elicited by cooperating transcription factors such as Lz, are likelyto be the major factor in determining the outcome from Notchactivation. Nevertheless, despite these differences, a cohort ofgenes was upregulated in both cell types. Besides the wellcharacterized E(spl) genes, other common targets included myc andNotch itself. myc has emerged as a frequent and important target ofNotch in several tissues, as well as in cancers (Klinakis et al., 2006;Palomero et al., 2006; Song and Lu, 2011; Weng et al., 2006).Positive feedback on Notch expression has also been observed inT-cell precursors (Yashiro-Ohtani et al., 2009), as well as in C.elegans (Christensen et al., 1996), and is likely to be important inmaintaining Notch receptor levels in signalling cells as the processof activation results in destruction of the receptor (Kopan andIlagan, 2009). Another common target in Kc and DmD8 cellsencodes Rgl, a member of the RalGEF family that is suggested tocouple Ras to Ral signalling (Ferro and Trabalzini, 2010). Theshared and related targets from the Kc and DmD8 cells may thusidentify core elements in the Notch response that are relevant inmany different contexts.

Despite these similarities, it is clear that there is a context-specific response to Notch activation in Drosophila haemocytesthat ensures proper cell fate specification and stabilization in thecrystal cell lineage (Fig. 7H). Cooperating with the lineage-determining factor Lz, Notch activation antagonizes alternate cellfates, by eliciting expression of Klu, and promotes key aspects ofthe differentiation programme, through other targets. Lz maytherefore be crucial in providing a transcriptional ‘priming’,converting cells into a transitional state that can then be canalizedby the Notch activation.

AcknowledgementsWe are very grateful to Bettina Fischer, Steve Russell and FlyChip for their helpwith the genome-wide arrays, and to Boris Adryan and Robert Stojnic foradvice over motif analysis. We thank I. Ando, Utpal Banerjee, Nancy Fosset, D

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936 RESEARCH ARTICLE Development 140 (4)

Pascal Heitzler, Paul Martin, Thomas Klein, Barry Yebovnik and other membersof the fly community for sharing fly stocks and antibodies, as well as theBloomington Stock Center and the Developmental Hybridoma Bank.

FundingThis work was supported by Medical Research Council programme grant toS.J.B. [G0800034], S.C. was funded by a Biotechnology and Biological SciencesResearch Council studentship, J.L. by China Scholarship Council CambridgeScholarship and I.R. by Genetics Society summer studentship. Research inA.K.’s lab is supported by Grantová agentura České republiky [P305/11/0126].

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.086785/-/DC1

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