926
RESEARCH ARTICLE
Development 140, 926-937 (2013) doi:10.1242/dev.086785
© 2013. Published by The Company of Biologists Ltd
Notch cooperates with Lozenge/Runx to lock haemocytes
into a differentiation programme
Ana Terriente-Felix*, Jinghua Li*, Stephanie Collins, Amy Mulligan, Ian Reekie, Fred Bernard‡, Alena Krejci§
and Sarah Bray¶
SUMMARY
The diverse functions of Notch signalling imply that it must elicit context-specific programmes of gene expression. With the aim of
investigating how Notch drives cells to differentiate, we have used a genome-wide approach to identify direct Notch targets in
Drosophila haemocytes (blood cells), where Notch promotes crystal cell differentiation. Many of the identified Notch-regulated
enhancers contain Runx and GATA motifs, and we demonstrate that binding of the Runx protein Lozenge (Lz) is required for enhancers
to 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 morphological
characteristics 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 target
genes that correctly locks cells into the differentiation programme.
INTRODUCTION
The Notch pathway operates during many different developmental
decisions, as exemplified by haematopoiesis, where Notch
regulates both the emergence of stem cells and the subsequent cell
fate 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 receptor
activation results in proteolytic release of the Notch intracellular
domain (NICD), one of the primary outcomes of Notch activation
is a change in transcription (reviewed by Bray, 2006; Kopan and
Ilagan, 2009; Kovall, 2008; van Tetering and Vooijs, 2011).
Recent studies have revealed large numbers of Notch-responsive
genes in progenitor cells or in cancers (Hamidi et al., 2011; Krejcí
et al., 2009; Li et al., 2012; Palomero et al., 2006; Wang et al.,
2011) that appear to be involved in preventing differentiation and
cross-regulation of other signalling pathways (Hamidi et al., 2011;
Krejcí et al., 2009; Li et al., 2012). It is not yet clear whether a
different spectrum of Notch targets is involved in promoting
differentiation and, if so, what such targets tell us about the
mechanisms involved.
To address these issues we have turned to a simple system,
Drosophila blood cells (haemocytes), where Notch activity
promotes crystal cell differentiation. The second wave of
haemocyte development occurs in the lymph gland (Fig. 1A)
(Crozatier and Meister, 2007; Evans and Banerjee, 2003; Jung et
al., 2005) and primarily gives rise to two cell types: crystal cells
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 URA
2578, 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
and plasmatocytes (Fig. 1B). Previous studies have shown that
Notch activity is required for expression of the Runx protein
Lozenge (Lz) in haemocytes (Lebestky et al., 2003). As Lz is
necessary for crystal cell development, this suggested a simple
mechanism to explain how Notch directs crystal cell
differentiation. However, there is as yet no evidence that lz is
directly regulated by Notch pathway (Lebestky et al., 2003;
Muratoglu et al., 2007). Furthermore, perturbations to Notch at late
stages prevent crystal cell differentiation and compromise cell
survival, suggesting that Notch activity may also be required in
parallel to or subsequent to Lz, although no other downstream
targets have been identified (Krzemien et al., 2010; Mukherjee et
al., 2011). Likewise, although Notch1 appears to function upstream
of Runx in several steps during mammalian haematopoiesis, no
evidence 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 to
integrate with Notch1 activity in specifying the T-cell lineage (Guo
et al., 2008) and Notch1 targets in leukaemic T-cell have signatures
suggestive of co-regulation by Runx and Notch (Wang et al., 2011).
Whether this reflects direct cooperation between Notch and Runx
has not been established.
Drosophila haemocytes therefore offer a simple model with
which to investigate how Notch coordinates differentiation and the
relationship it has with Lz/Runx in this process. To address these
issues, we first identified direct transcriptional targets of Notch in
haemocyte-related cells. Many of these direct Notch targets were
associated with Lz/Runx-binding motifs and we demonstrate that
Notch and Lz act in combination to regulate the enhancers.
Furthermore, our analysis of target gene functions reveals that
Notch simultaneously prevents cells adopting the alternate
plasmatocyte fate, by upregulating klumpfuss (a ERG/WT1 family
member) and promotes characteristics associated with
differentiation, by upregulating pebbled/hindsight (a RREB1
homologue). Therefore, through these combined targets, Notch and
Lz operate to tie cells into the differentiation programme,
converting them from an unstable to a committed state.
DEVELOPMENT
KEY WORDS: Lozenge/Runx, Notch, Chromatin immunoprecipitation, Haemocyte, Drosophila
RESEARCH ARTICLE
Fate commitment by Notch and Lz
Genome-wide expression and ChIP analysis
Kc 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% penicillinstreptomycin at 25°C. Expression and ChIP-chip array experiments were
performed as described previously (Krejcí et al., 2009). In brief, chromatin
was prepared after 30 minutes of Notch activation by 2 mM EDTA and
XChIP performed with α-Su(H) antibody (Krejcí et al., 2009). Precipitated
genomic fragments and a fraction of the input DNA were amplified by
ligation-mediated PCR, labelled with Cy3 or Cy5, mixed and hybridized to
Nimblegen Drosophila 2.1 M tiling arrays, which have 50-75 bp probes
distributed 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 significant
differences in expression levels (P≤0.05) or peaks of Su(H) occupancy. For
the latter, Tamalpais (Bieda et al., 2006) was used to identify significant
ChIP peaks (minimum cut-off of five adjacent probes, P<0.05) and after
excluding atypical peaks clustered in a small region of Chromosome 2R, the
analysis yielded a set of 185 peaks. A custom-written Perl script was used
to determine genes in the vicinity of peaks. Peaks were assigned to genes
only when they were within 10 kb of an upregulated gene on either strand.
Results have been deposited in Gene Expression Omnibus with series
Accession Numbers GSE43132 (ChIP) and GSE9964 (expression array).
Motif and GO analysis
CONSENSUS Matrix-based motif discovery in RSAT was used to look for
over-represented motifs (Thomas-Chollier et al., 2011). A position-weighted
matrix (PWM) for Su(H), Srp and Lz was generated from a compilation of
binding 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 calculate
the distance between Su(H) PWM and other PWMs within 600 bp window
of Su(H) ChIP peaks. Analysis of gene ontology enrichments was
performed using the functional enrichment chart at the DAVID
Bioinformatics Resource. Results were filtered for enrichment (over
threefold) and for significance using modified Fisher’s Exact Test (EASE)
with Benjamini correction (http://david.abcc.ncifcrf.gov). Representative
examples of Biological Functions (P≤0.005) are depicted to avoid
repetitions between similar categories. All enriched molecular function
categories with P≤0.1 are shown.
RNAi experiments and chromatin immunoprecipitation
For treating Kc cells with RNAi, double-stranded RNA duplexes
corresponding to 400-800bp exonic regions were produced using T7
promoter-containing primers and subsequently transcribed with a
MEGAscript T7 Kit (Ambion). Cells were treated with dsRNA for 72 hours
before harvesting. Three biological replicates were performed in all
experiments. RNA isolation, real-time PCR and ChIP experiments were
performed as described (Krejcí and Bray, 2007). Antibodies for ChIP were
goat α-Su(H) (Santa Cruz Biotechnology, sc15813) and mouse α-Lz
(DSHB).
Fly strains and immunofluorescence staining of lymph glands
Alleles and fly stocks, as described in FlyBase (http://flybase.org/), were
lz-Gal4 (Jackson Behan et al., 2005), He-Gal4 (Kurucz et al., 2003), pxnGal4 (Stramer et al., 2005), UAS-mamDN (Helms et al., 1999), UASSu(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), UASmycRNAi (TRiP.JF01761), UAS-whiteRNAi (TRiP.GL00094), UASCD8GFP, klu212lR51C and Klu-Gal4 (Klein and Campos-Ortega 1997),
ush(−7462/-25)-lacZ (Muratoglu et al., 2007), and NRE-GRins (Housden et
al., 2012).
Lymph glands were dissected from third instar larvae (cultured at 29ºC
from 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% Triton
X100), lymph glands were removed from contaminating tissue and mounted
onto poly-lysine-coated slides. After blocking for 1 hour in PBTN (PBT +
4% horse serum) they were stained overnight with primary antibodies at
4°C. Primary antibodies were as follows: mouse anti-Hnt (1/20), mouse
anti-Lz (1/20), mouse anti-Lam (all from DSHB), mouse anti-P1 (1/30; a
gift 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). After
three 15-minutes washes in PBT, fluorescently conjugated secondary
antibodies (Jackson Labs) were applied for 1.5 hours at room temperature
followed by three 15-minute washes in PBT and one wash in PBS. Finally,
the sample was mounted in Vectashield containing DAPI for imaging with
Nikon D-Eclipse C1 or Leica SP2 confocal microscopes. For phenotypic
experiments, cell and nuclear dimensions were measured in over 10 lymph
glands using IMARIS. Typically, 300-1000 cells were scored for each
genotype.
Construct design and mutagenesis
ChIP-enriched regions from klu, CG32369, rgr, peb and other putative
targets were amplified from Drosophila genomic DNA, using primers
containing restriction enzyme sequences (see supplementary material Table
S2), and cloned into pGreenRabbit/pRedRabbit vectors (Housden et al.,
2012) for in vivo reporter assays or pGL3min for luciferase assays. Sitedirected mutagenesis was performed using a PCR-based approach with
primers overlapping the Su(H)/Srp/Lz-binding site to be mutated with the
sequences changed as follows: CGTGGGAA to CGTTGTTA for the Su(H)
motif; GGATAAC to GTTCTAC for the Srp motif and AACCACA to
AATGACC for the Lz motif. Luciferase assays were performed as
described previously (Krejcí and Bray, 2007).
RESULTS
Notch responsive genes in Drosophila Kc cells
As Drosophila Kc cells exhibit characteristics of haemocytes
(Echalier and Ohanessian, 1969; Lunstrum et al., 1988; Nelson et
al., 1994; Schneider, 1972), express several haemocyte markers
(including Hemese, Hemolectin and Serpent, Fig. 1D) (Cherbas et
al., 2011; Jung et al., 2005) and contain detectable levels of Lz
(Fig. 1E), they provide a suitable model for investigating the Notch
response 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 in
maintaining progenitor characteristics in collaboration with
transcription factor Twist (Bernard et al., 2010), enabling us to
discover how the Notch response differs between cell types.
To characterize Notch-responsive genes in Kc cells, we used a
similar strategy to that used previously, activating Notch using a
calcium chelator (Gupta-Rossi et al., 2001; Rand et al., 2000) and
monitoring mRNA expression changes 30 minutes later (Krejcí et
al., 2009; Krejcí and Bray, 2007). Probes prepared from control
and Notch-activated mRNA populations were hybridized in pairwise combinations to Drosophila transcriptome microarrays to
identify genes that were significantly upregulated (P≤0.05). In
parallel, we identified genomic regions that were occupied after
Notch activation by Suppressor of Hairless [Su(H)], the core
transcription factor in the Notch pathway (reviewed by Bray, 2006;
Kopan and Ilagan, 2009; Kovall, 2008; van Tetering and Vooijs,
2011), using chromatin immunoprecipitation (ChIP) and
hybridizing bound DNA to genomic tiling arrays. Integrating these
data, the 185 Su(H) occupied regions, ‘peaks’, were assigned to
genes if they were located within or close to genes that were
upregulated (Fig. 2A). This identified 69 assigned peak genes
(APGs) that fulfilled the criteria of Su(H) binding within 10 kb and
upregulation with the Notch activation regime [supplementary
material Table S1; ranked by AvgM, fold difference in expression
(log2)]. Several APG were validated using quantitative PCR to
DEVELOPMENT
MATERIALS AND METHODS
927
RESEARCH ARTICLE
Fig. 1. Haemocyte development: role of Notch and relationship to
Kc167 cells. (A) Drosophila lymph gland where larval haemocyte
development gives rise to crystal cells (red) and plasmatocytes (green).
Boxed area indicates the region shown in confocal images in this and
subsequent figures. (B) The haemocyte lineage, specific markers and
relevant Gal4 drivers are indicated. Notch and Lozenge (Lz) are required
for crystal cells (red nuclei) to develop from Serpent (Srp)-expressing prohaemocytes. (C) Expression of a general Notch-responsive reporter, NREGFP (green), indicates that Notch is active in Lz-expressing (red) crystal
cell precursors. Arrows indicate examples of cells co-expressing Lz and
NRE-GFP. (D) ModEncode measurements of relative mRNA expression
levels of the indicated genes (He, hemese; Hml, hemolectin; Nim, Nimrod;
srp, serpent; dome, domeless; twi, twist; N, Notch; vg, vestigial) in Kc167
cells compared with DmD8 (muscle precursor related) (Cherbas et al.,
2011); haemocyte-related genes Hemolectin (Hml) and Hemese (He) are
specifically expressed in Kc cells. (E) Lz is present in Kc167 cells; western
blot of total cell extracts from the indicated cells was probed with α-Lz
and 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.
confirm Su(H) binding in repeat ChIPs (e.g. Fig. 4C) and, using
this method, we also detected Su(H) binding near one additional
upregulated 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 their
upregulation in cells transfected with NICD (supplementary
material Fig. S1) (although this approach is hampered by the fact
that transfection efficiency is less than 30% in these cells).
A comparison with results from similar experiments in DmD8
cells demonstrated relatively little overlap of Su(H)-occupied sites
or of upregulated genes. Thus, although the number of occupied
sites in the two cell types was of similar magnitude, 260 in DmD8
and 185 in Kc cells, only 28 peaks were overlapping between the
Development 140 (4)
two. This suggests that the accessibility of sites to Su(H) is likely
to be a major factor in determining the outcome from Notch
activation. Most overlapping peaks were in proximity to
upregulated genes, corresponding to 17 APGs that were common
to both Kc and DmD8 cells (Fig. 2A). These included several of the
E(spl) genes, myc and Notch (supplementary material Table S1).
Such genes appear to be widespread targets of Notch in many
different organisms, including humans, and are likely to identify
mechanisms 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 also
substantial differences in the Notch-responsive genes in the two
cell types, indicative of disparate functional outcomes from Notch
activation. We note also that, as we were monitoring Su(H)
occupancy and not recruitment of NICD to these targets (owing to
NICD antibody not performing well in ChIP), we cannot formally
rule out the possibility that expression from some of the Su(H)bound upregulated genes may be independent of direct binding by
NICD. Furthermore, 58% Su(H) peaks had no upregulated genes
within 10 kb, and although some may be false positives, it suggests
that some Su(H) sites may be associated with genes that require
other co-factors or represent Notch-independent Su(H) targets.
To gain a global overview of the Su(H)-bound responsive genes
in Kc cells, we analysed their functional characteristics using gene
ontology (GO) annotations (http://david.abcc.ncifcrf.gov/)
(Fig. 2B). Although the cohort of targets differed, several enriched
Biological Process categories were similar to DmD8 cells,
including cell surface receptor-linked signalling and imaginal disc
morphogenesis. The cross-regulation of other signalling pathways,
especially Ras signal transduction, therefore emerges as a common
theme, with Gap1 and pointed being among the Kc-regulated
genes. Pattern specification and cell migration were, however,
more enriched in Kc than in DmD8 cells. Similarly, there was
enrichment for the Molecular Function categories ‘transcription
factors’ and ‘actin binding’. The former are candidates to
coordinate the programme of differentiation in haemocytes, and
many encode zinc-finger transcription factors (Interpro zinc-finger
C2H2 type; fivefold enriched) such as klumpfuss (klu), pebbled
(peb/hnt), regular (rgr) and Hnf4. We note, however, that lz was not
among the genes bound by Su(H) or upregulated within
30 minutes, despite its expression in the lymph gland being
dependent on Notch activity (Lebestky et al., 2003).
Kc Notch targets are expressed in differentiating
crystal cells
None of the identified Kc Su(H) targets had been previously
associated with haemocyte fates and several were as yet
uncharacterized. To determine their relevance for blood cell
differentiation in vivo, we investigated their expression in third
instar lymph glands. First we used a Notch activity-sensing reporter
(NRE-GFP; Housden et al., 2012) to confirm that Notch is
specifically active in Lz-expressing crystal cell precursors (Fig. 1C),
although we note that NRE-GFP expression was detected only in a
subset of Lz+ cells, suggesting that there is a transient phase of
Notch activity. Selecting two highly upregulated transcription
factors, klu and peb/hnt, we compared their expression to the Lz
crystal cell lineage marker (Fig. 2C,D) and found that both were
expressed 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
DEVELOPMENT
928
Fate commitment by Notch and Lz
RESEARCH ARTICLE
929
Fig. 2. Identification of Notch targets in haemocytes. (A) Left: Venn
diagram illustrating overlap between genes in proximity to Su(H)-bound
regions [blue: anti-Su(H) ChIP, 185 bound regions] and upregulated after
30 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 in
DmD8 cells (green) is limited to 17 common genes. (B) Examples of overrepresented GO (Biological Process, upper graph; Molecular Function,
lower graph) categories in Kc direct Notch targets. (C-F) Expression of
indicated targets and/or target enhancers in crystal cell lineage (marked
by lz>GFP (C, E, F; green) and anti-Lz (D; green). See supplementary
material Table S1 for a list of direct targets. Scale bar: 10 μm.
cells with Peb/Hnt only. Similarly, the majority of Klu-expressing
cells (klu-Gal4 UAS-GFP) contained Lz. Thus, these Kc identified
APG are expressed in a relevant lineage in vivo, where their
expression appears to persist. For example, Peb/Hnt protein
expression was detected in mature crystal cells, based on its
colocalization with anti-ProPO (supplementary material Fig. S2)
Requirement for Lz/Runx
The differences between the Notch-responsive genes in Kc and
DmD8 cells suggests that intrinsic factors alter which target
enhancers can be regulated. One approach to identify such coregulators is to look for motifs that are enriched within the regions
bound by Su(H) in each cell type. Taking as our input the 300 bp
flanking the mid-point of each Su(H) ChIP peak (a 600 bp
window), we used CONSENSUS Matrix-based motif discovery
in RSAT to look for over-represented motifs (Thomas-Chollier et
al., 2011). This returned three assemblies. The first, CGTGGGAA,
corresponds to Su(H) motif. The second, GATAAAGT, resembles
a GATA-factor binding site, implicating Serpent (or one of the
three other Drosophila GATA factors). The third, ACCATAGT, a
variant on the established Runx motif suggesting that Lz could be
a co-factor, was detected under a subset of conditions. We
therefore used a position weight matrix to locate putative binding
motifs for Lz/Runx and GATA, and analysed their relationship to
Su(H) motifs in regions defined by Kc peaks, using for
comparison the motif for Twist (Twi), a muscle-specific
transcription factor. Approximately 40% of Su(H) motifs in Kc
peaks were within 100 bp of Lz and 36% were within 100 bp of a
GATA site, whereas fewer than 25% had a Twi site in similar
proximity (under conditions where the total numbers of each motif
in the genome were similar; Fig. 4A). Indeed, 49% of the assigned
genes are associated with Kc peaks that contain GATA and Lz
motifs, as well as Su(H). A further 17% are associated with peaks
containing only Su(H) and Lz motifs, and 13% are associated with
peaks containing only Su(H) and GATA motifs. By contrast, fewer
than 39% of APGs in DmD8 muscle-related cells have GATA and
Lz in proximity to Su(H), whereas 57% have a Twi motif. The
DEVELOPMENT
and with other markers in a recent study (Benmimoun et al., 2012).
In addition, expression of peb/hnt was upregulated in NICDexpressing clones and was suppressed when Notch was ablated by
RNAi (supplementary material Fig. S2).
To further investigate whether the APGs were subject to
Notch-mediated regulation in the lymph gland, reporter genes
were generated containing putative Notch-responsive enhancers
(NREs), as defined by Su(H) ChIP peaks, upstream of GFP or
mCherry (Fig. 3A) (Housden et al., 2012). All exhibited
expression in crystal cell precursors (Lz-expressing cells)
(Fig. 3C,E,G,I), although levels of expression varied. For
example, peb/hntNRE, kluNRE, mycNRE and CG32369NRE all
directed 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 low
levels in Lz-expressing cells (supplementary material Fig. S3)
but these levels could be augmented by expression of the
constitutively active Su(H)VP16 (supplementary material Fig.
S3). To confirm that the identified enhancers were regulated by
Notch, we tested consequences of mutating all the Su(H) motifs
in klu, CG32369, peb/hnt, myc and rgr NREs (Fig. 3D,F,H,J;
supplementary material Fig. S3A). With kluNRE, CG32369NRE
mycNRE and rgrNRE, the Su(H) motif mutations completely
abolished expression in the lymph gland (Fig. 3D,F,J;
supplementary material Fig. S3B). Effects on peb/hntNRE were
intermediate (Fig. 3H), with some residual expression detected
from the mutated NRE in a subset of glands (2/15). Moreover,
luciferase assays revealed that mutated NREs had lost their
response to NICD (Fig. 3B). These results confirmed the
importance of Su(H) sites for expression in haemocytes,
demonstrating that these targets are Notch responsive.
RESEARCH ARTICLE
Development 140 (4)
Fig. 3. Notch-responsive enhancers direct expression in crystal cell lineage and require Su(H) motifs for activity. (A) Examples of genomic
regions from representative Notch targets showing Su(H) ChIP-enriched regions in Kc cells (purple; fold enrichment relative to total input −0.1-2.5, log2
scale). Matches to the Su(H)-binding motif are indicated (bar height indicates affinity class of sites). Gene models are depicted in black. Blue bars
represent the regions (NRE) that were cloned to test responsiveness in vivo (for distribution of motifs see Fig. 5). Blue arrows in klu indicate fragments
analysed 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; single
channel, 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.
DEVELOPMENT
930
Fate commitment by Notch and Lz
RESEARCH ARTICLE
931
highly conserved Lz/Runx and GATA factors may therefore
provide the crucial specificity for Notch responses in Kc
cells/haemocytes.
One hypothesis is that Lz (and GATA) are necessary to make the
enhancers capable of responding to Su(H)/Notch. As we were
interested whether targets required cooperation between Notch and
Runx, we focused our analysis on Lz, for which there was a
monoclonal antibody that was suitable for ChIP. First, we
investigated whether Lz was present at candidate enhancers prior
to Notch activation. Fragments that encompass Lz motifs in peak
regions from klu, CG32369, rgr, peb/hnt were significantly
enriched in the Lz ChIP, compared with control regions (Fig. 4B).
We therefore asked whether reducing Lz, by treating cells with
RNAi (Fig. 1E), would interfere with recruitment of Su(H) to target
enhancers. Little residual Lz binding was detectable at target
enhancers in Lz RNAi-treated cells (Fig. 4B). Su(H) binding at klu,
CG32369, rgr, peb/hnt enhancers was similarly compromised in
Lz-depleted cells (Fig. 4C), whereas it remained unchanged at
E(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 argue
that 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, we
first checked for direct interactions by co-immunoprecipitation, but
were unable to detect any co-purification of Su(H) in Lz
immunoprecipitates or vice versa (supplementary material Fig. S4).
Second, we assessed whether Lz had an influence on chromatin
accessibility, as measured by sensitivity to DNase I treatment.
Focusing initially on klu, we tested the effects from two different
concentrations 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 tested
whether this sensitivity was altered in Lz RNAi-treated cells and
found that the NRE was less sensitive to DNase I under these
conditions, suggesting that Lz alters the DNA accessibility
(Fig. 4D). Finally, we tested whether other enhancers showed
similar Lz-dependant DNase I accessibility. Although the effects
were most robust with klu, both CG32369 and rgr also showed
some decrease in accessibility following Lz RNAi (Fig. 4E).
However, we were not able to detect similar sensitivity at the
peb/hnt NRE (Fig. 4E). Taken together, the data indicate therefore
that the effects of Lz on Su(H) recruitment are likely to be indirect
and most likely involve a change in the chromatin accessibility at
least at some loci, such as klu.
To address whether Notch is required in combination with Lz,
we compared the consequences on Peb/Hnt expression of
perturbing Notch function in Lz-expressing cells using a dominantnegative form of Mam (MamDN) with the effect of LzEnR, a
constitutive repressor form of Lz (Wildonger et al., 2005). The
MamDN peptide occupies the Mam-binding groove on the Su(H)NICD complex, blocking the functional Mam protein, an essential
co-activator of Notch, from binding. Expression of MamDN alone
or of LzEnR was sufficient to severely reduce levels of Peb/Hnt
expression (e.g. Fig. 4F,G). Only 56.5% of Lz>GFP-expressing
cells stained positive for Peb/Hnt in the presence of MamDN, and
only 66.6% in the presence of LzEnR, compared with 97.2% in
control glands (supplementary material Fig. S5). Importantly, coexpression of wild-type Lz with MamDN was unable to rescue the
Peb/Hnt expression (Fig. 4H). Thus, reduced Notch activity cannot
be compensated for by increased Lz. This implies that the
combination of Notch activity and Lz is required for Peb/Hnt
DEVELOPMENT
Fig. 4. Lz influences Su(H) recruitment at
Notch-responsive enhancers and is required
with Notch in vivo. (A) Lz and GATA motifs are
located in proximity to Su(H). Graph showing
percentage of detected motifs located within the
indicated distance (bp) of the Su(H) motif within
Kc ChIP peaks (blue, GATA; green, Lz; grey, Twist).
(B) Lz is present at NREs. Fold enrichment of the
indicated NRE in α-Lz ChIP relative to the adjacent
coding sequence (cds) in Kc cells (dark shading)
and after lz RNAi (light shading). (C) Depletion of
Lz compromises Su(H) binding. Fold enrichment
of the indicated NRE in anti-Su(H) ChIP relative to
the cds in Kc cells (dark shading) and after lz RNAi
(light shading). (D,E) Effects of Lz depletion on
DNase I sensitivity of the indicated regions in klu
(D) and of NRE from other genes (E). Chromatin
from control and Lz RNAi-treated cells was
subjected to digestion with 0, 4 or 12 U of DNase I
and 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) or
Lz and MamDN (H) were expressed (lz>GFP
indicates lz-Gal4 UAS-GFP). Scale bar: 10 μm. Data
are mean±s.e.m.
932
RESEARCH ARTICLE
Development 140 (4)
expression and establishes that the phenotype caused by MamDN
cannot be attributed to failure in Lz. To confirm the relevance of
Lz/Runx in vivo, we tested the consequences of mutating the
identified Lz motifs on expression from the klu, peb/hnt and
CG32369 NREs (Fig. 5). Expression from all three NREs was
abolished when Lz/Runx sites were eliminated (kluNRE[mLz],
CG32369NRE[mLz], pebNRE[mLz]; Fig. 5). Altogether, these data
indicate that Lz confers specificity on the Notch response in
differentiating haemocytes. We note that klu is also regulated by
Lz in another context, through a different enhancer (Wildonger et
al., 2005).
Finally we also tested the relevance of GATA motifs for
expression from klu, peb/hnt and CG32369 NREs. Expression was
reduced by mutations disrupting the GATA motifs (Fig. 5), but with
variable effects: CG32369NRE[mGATA] lost all detectable
expression, kluNRE[mGATA] exhibited low level expression in few
cells and peb/hntNRE[mGATA] had moderate levels of activity.
Nevertheless, the results make it likely that a GATA factor, possibly
Srp, also cooperates with Notch on these enhancers.
Inhibition of alternate plasmatocyte fates by the
Notch target klu
The identified Kc Notch targets are expressed in response to Notch
and Lz in the crystal cell lineage. So far, relatively little is known
about events downstream of Notch activation in these cells,
although recent studies have demonstrated that Notch is required
not only for crystal cell specification, but also for their expansion
and maintenance (Mukherjee et al., 2011). Target gene functions
should therefore reveal how Notch implements its role in promoting
the specific differentiation programme.
klu is one of the genes that is most highly upregulated in Kc cells
and encodes a zinc-finger protein related to the Wilms Tumor 1
(WT-1)/Early growth response (EGR) gene family, which regulate
differentiation 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 and
lymphoid leukaemias (see Huff, 2011; Tosello et al., 2009; Yang et
al., 2007). We investigated whether klu has a similar crucial role in
haemocytes by expressing a dominant-negative form of the Klu
protein (KluDN; Kaspar et al., 2008; Klein and Campos-Ortega,
1997) in the developing crystal cells with lz-Gal4. A characteristic
of lz-Gal4-expressing cells is that they are devoid of P1, a marker
for the alternate plasmatocyte lineage (Fig. 6A) (Krzemien et al.,
2010; Minakhina and Steward, 2010). Strikingly, in the presence of
KluDN, 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 lymph
glands from klu mutant larvae (kluG4/klu212lR51C; supplementary
material Fig. S7) and when klu was ablated using short hairpin
RNAi (Fig. 6C). These results suggest that Klu is important for
repressing the alternate plasmatocyte fate in crystal cell precursors.
Studies of Klu function in PNS development demonstrated that
a constitutive repressor form of Klu, KluEnR, behaved as a strong
gain of function for Klu when overexpressed (Kaspar et al., 2008).
We therefore tested the consequences of KluEnR expression using
He-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 expression
exhibited altered morphology (Fig. 6E). Furthermore, many larvae
showed striking accumulations of melanotic cells, resembling
melanotic tumours (Fig. 6F-H). However, the KluEnR expression
was not sufficient to fully convert the cells to crystal cells, as there
was little increase in the number of He>GFP cells that contained
Peb/Hnt or Lz (supplementary material Fig. S6). Thus, KluEnR
suppresses plasmatocyte characteristics but is not sufficient to fully
direct the cells into crystal cell lineage.
One possible mechanism through which Klu might prevent
plasmatocyte differentiation is by repressing ush, the Drosophila
homologue of FOG1, because a decrease in ush expression is
thought to be important for crystal cell maturation in the embryo
(Fossett et al., 2003; Fossett et al., 2001; Waltzer et al., 2003). We
therefore investigated whether, by restoring Ush in the presence of
KluEnR, we could suppress the melanotic cell masses. The number
of larvae with melanotic masses was significantly reduced in
combination with ush (Fig. 6H), supporting the hypothesis that one
mechanism through which Klu prevents plasmatocyte fates is by
DEVELOPMENT
Fig. 5. Notch-responsive enhancers require Lz and
Srp sites for full activity. (A-C) Consequences on
indicated NRE activity of mutating Lz (mLz) or GATA
(mGATA) motifs. Expression of the resulting GFP
reporters (green) in crystal cell lineage (α-Lz, purple).
Diagrams above depict the location of the Lz- (green
rectangles) and GATA (blue triangles)-binding motifs
within each enhancer relative to Su(H) motifs (pink
ovals). Scale bar: 10 μm.
Fate commitment by Notch and Lz
RESEARCH ARTICLE
933
antagonizing ush. Although expression of ush (ush-lacZ) was
widespread and heterogeneous in the lymph gland (supplementary
material Fig. S7), the majority of Lz-positive cells had little or no
ush-lacZ expression, consistent with the diminished expression in
crystal cells observed in the embryo (supplementary material Fig.
S7). Broad (pxn-Gal4) expression of KluDN alone (supplementary
material Fig. S7) or MamDN alone (supplementary material Fig.
S7) had little effect on ush expression in Lz-positive cells, although
there was more variability in levels in the former. However,
combined overexpression of kluDN and mamDN together resulted
in a significant increase in the number of Lz-expressing cells
exhibiting strong ush-lacZ (30% of Lz-expressing cells;
supplementary material Fig. S7). This suggests that Ush may be
one factor involved in the switch downstream of Klu, but suggests
that its regulation may require additional inputs from Notch.
Other Notch targets implement cellular
programmes associated with crystal cell fates
One characteristic of crystal cells is that they undergo DNA
replication but do not appear to be mitotically active, suggesting
that they are in endocycle (Krzemien et al., 2010). In the follicular
epithelium, 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 might
function similarly in the lymph gland. Endocycling cells possess
an increased DNA content relative to neighbouring mitotic cells,
evident in the DAPI staining. We first investigated whether Notch
activity affected the DNA content in lymph-gland nuclei, by
expressing Su(H)VP16 in Lz-expressing cells (lz>GFP). This had
the striking consequence of increasing the dimensions of the
nucleus (based on DAPI staining and nuclear Lamin; Fig. 7A,B,E)
and also enlarged the patch of Fibrillarin (Fig. 7A″,B″), a
nucleolar marker. We noted that there was also considerable
heterogeneity among the lz>GFP population even in the absence
of additional Su(H)VP16 (Fig. 7C), suggesting that nuclear
enlargement is a feature of their differentiation. To investigate
whether peb/hnt has a role in this nuclear enlargement, we ablated
peb/hnt, by expressing peb/hnt-RNAi, in the presence of the
Su(H)VP16. This resulted in a significant reduction in the nuclear
dimensions compared with expression of Su(H)VP16 alone
(Fig. 7D; supplementary material Fig. S8). Thus, peb/hnt is in part
responsible for the effects of Su(H)VP16 on nuclear size. Then,
we examined the consequences of perturbing peb/hnt on nuclear
dimensions in the largest lz>GFP-expressing cells (Fig. 7E,F).
DEVELOPMENT
Fig. 6. Regulation of klu by Notch blocks plasmatocyte
fates. (A-A⬙) Expression of the plasmatocye marker P1 (red, A;
white, A⬘) does not overlap with Lz (lz>GFP, green in A; white in
A⬙) in wild-type glands (arrows). (B-C⬙) P1 is detected in Lz cells
that 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 HeGal4-expressing cells (He-Gal4 UAS-nlsGFP, He>GFP) in control
glands (arrows). (E-E⬙) Expression of KluEnR compromises P1
expression in many of the He>GFP cells (arrows). (F,G) Expression
of 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 melanotic
masses (large, larvae had at least one large mass; small, larvae
had only small dots of melanin). Co-expression of Ush reduces
the percentage of KluEnR larvae that exhibit melanotic masses.
Over 80 larvae were scored for each genotype. See
supplementary material Fig. S6 for Lz expression in KluEnR. Scale
bar: 10 μm.
934
RESEARCH ARTICLE
Development 140 (4)
Knockdown of peb/hnt led to a significant reduction in the nuclear
diameters in these cells, consistent with a role in promoting
nuclear 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 some
tissues (Maines et al., 2004; Pierce et al., 2004). Indeed when
overexpressed in Lz>GFP cells, Myc caused an increase in cell,
nuclear and nucleolus size (supplementary material Fig. S8). When
we assessed the consequences of myc-RNAi on the Su(H)VP16
phenotype, there was also a reduction in nuclear size
(supplementary material Fig. S8). Therefore, it appears that myc
may also be important in implementing the Notch-dependent
changes in nuclear size/ploidy that appear intrinsic to the crystal
cell differentiation programme, although quantitative
DEVELOPMENT
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)VP16
results in enlarged nuclei (compare arrows). (C) Size of nuclei based on DAPI staining in lz>GFP-expressing lymph glands, GFP-expressing nuclei (crystal
cell 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) size
obtained by expressing Su(H)VP16 (with lz-Gal4) is suppressed by co-expressing RNAi targeting peb/hnt or myc. RNAi targeting white was used as
control (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 for
LzG4>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, using
used 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 and
whisker 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 earlier
stages in haemocyte development, where the target genes are likely to differ, as it will operate in a different context. Scale bar: 10 μm.
measurements of DNA synthesis in individual lz>GFP cells will be
needed to confirm its role.
DISCUSSION
As signalling pathways, such as Notch, are used iteratively during
development, one of the challenges is to understand their contextspecific effects. Taking a genome-wide approach, we have
elucidated the transcriptional outputs of Notch in Drosophila Kc
cells, a model for haemocyte development, and shown that, in
vivo, the targets are involved in locking cells into a specific fate,
by shutting down the alternatives and by promoting specific
characteristics of the differentiated cells (Fig. 7H). The context
specificity for this programme is provided by the Runx factor Lz,
probably acting in combination with GATA factors, as mutation
of Lz or GATA sites eliminates expression from the Notchregulated enhancers. Furthermore, ablation of Lz compromises the
recruitment of Su(H) to these targets enhancers. Thus, Lz appears
to act as a lineage master regulator for Notch in the manner
proposed for cell-specific factors acting with BMP and Wnt
signals in regeneration of haematopoietic lineages (Trompouki et
al., 2011) and with TGFβ in differentiation (Mullen et al., 2011).
Our analysis of haemocyte Su(H) targets has thus uncovered a
cooperative activity between Lz and Notch. In most previous
studies, Notch and Runx have been shown to act in a hierarchical
manner, with Notch functioning upstream of Runx/Lz (Burns et
al., 2009; Burns et al., 2005; Lebestky et al., 2003; Nottingham
et al., 2007; Robert-Moreno et al., 2005). However, a similar
cooperative mechanism may operate at late stages in thymocyte
development, where Runx1 confers the capability to promote Tcell fate in response to Notch activity (Guo et al., 2008). Analysis
of Notch-regulated targets in T-ALL cells also uncovered a
signature that was suggestive of Notch and Runx co-regulation in
these malignant cells (Wang et al., 2011). Although Lz/Runx
expression is itself dependent on Notch activity, there is as yet
no evidence to suggest direct regulation in other systems and we
have 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 first
downstream of Notch and second in combination with Notch at
target enhancers suggests that a feed-forward ratchet mechanism
may contribute to cell fate specification both in Drosophila
crystal cells and mammalian lineages. This also highlights the
fact that Notch is needed at several different stages in such
lineages, and that the specific targets regulated are likely to differ
depending on the stage.
Among the newly identified Notch-regulated genes, many are
transcription factors (Klu, Peb/Hnt, Myc, Hnf4, Rgr, p53) whose
expression and functions illustrate their importance for the crystal
cell differentiation programme. Strikingly, our analysis and
manipulation of Klu, a zinc-finger protein related to the ERG/WT1
family (Klein and Campos-Ortega, 1997), revealed that it is
necessary to inhibit alternate cell fates in the Lz-expressing cells.
In some respects, this is surprising as lineage-tracing experiments
indicated that most, if not all, Lz-expressing cells were fated to
become crystal cells in healthy animals owing to signalling events
at much earlier stages (Krzemien et al., 2010; Minakhina and
Steward, 2010). Why is Klu required? One possibility is that Lz+
cells are in a transitional state prior to Notch activation/Klu
expression, retaining the potential to adopt alternate fates
depending on environmental challenges. For example, hypoxia
appears to be important for crystal cell differentiation to be
maintained (Mukherjee et al., 2011) and wasp infection can reroute
RESEARCH ARTICLE
935
differentiation to lamellocyte fates (e.g. Krzemień et al., 2007).
Notch-induced upregulation of Klu may be important to lock cells
into the crystal cell programme, by inhibiting activity of alternate
lineage regulators. A similar role has been proposed for the
mammalian homologues EGR1/2, which are part of an antagonistic
regulatory 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 the
crystal cell differentiation programme. In particular, the transcription
factor Peb/Hnt is important for the change in nuclear size/DNA
content that probably reflects endoreplication in crystal cells
(Krzemien et al., 2010), a process that may also require Myc. As both
Peb/Hnt and Myc are involved in regulating the switch from mitotic
to 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 at
multiple stages in development. Elsewhere, Myc has been shown to
regulate polyploidy and differentiation in several cell types, including
megakaryocytes and in T-ALL cells (e.g. Munoz-Alonso et al., 2012;
Palomero et al., 2006; Pierce et al., 2004; Zanet et al., 2005). Other
targets include Hnf4, which was previously implicated in causing
haemocyte expansion downstream of AML (Runx)-ETO (Sinenko et
al., 2010), and several genes involved in cell motility (e.g. CLIP-120),
as well as other conserved genes with unknown functions, such as
CG32369 (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 likely
to be the major factor in determining the outcome from Notch
activation. Nevertheless, despite these differences, a cohort of
genes was upregulated in both cell types. Besides the well
characterized E(spl) genes, other common targets included myc and
Notch itself. myc has emerged as a frequent and important target of
Notch 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 in
T-cell precursors (Yashiro-Ohtani et al., 2009), as well as in C.
elegans (Christensen et al., 1996), and is likely to be important in
maintaining Notch receptor levels in signalling cells as the process
of activation results in destruction of the receptor (Kopan and
Ilagan, 2009). Another common target in Kc and DmD8 cells
encodes Rgl, a member of the RalGEF family that is suggested to
couple Ras to Ral signalling (Ferro and Trabalzini, 2010). The
shared and related targets from the Kc and DmD8 cells may thus
identify core elements in the Notch response that are relevant in
many different contexts.
Despite these similarities, it is clear that there is a contextspecific response to Notch activation in Drosophila haemocytes
that ensures proper cell fate specification and stabilization in the
crystal cell lineage (Fig. 7H). Cooperating with the lineagedetermining factor Lz, Notch activation antagonizes alternate cell
fates, by eliciting expression of Klu, and promotes key aspects of
the differentiation programme, through other targets. Lz may
therefore be crucial in providing a transcriptional ‘priming’,
converting cells into a transitional state that can then be canalized
by the Notch activation.
Acknowledgements
We are very grateful to Bettina Fischer, Steve Russell and FlyChip for their help
with the genome-wide arrays, and to Boris Adryan and Robert Stojnic for
advice over motif analysis. We thank I. Ando, Utpal Banerjee, Nancy Fosset,
DEVELOPMENT
Fate commitment by Notch and Lz
RESEARCH ARTICLE
Pascal Heitzler, Paul Martin, Thomas Klein, Barry Yebovnik and other members
of the fly community for sharing fly stocks and antibodies, as well as the
Bloomington Stock Center and the Developmental Hybridoma Bank.
Funding
This work was supported by Medical Research Council programme grant to
S.J.B. [G0800034], S.C. was funded by a Biotechnology and Biological Sciences
Research Council studentship, J.L. by China Scholarship Council Cambridge
Scholarship and I.R. by Genetics Society summer studentship. Research in
A.K.’s lab is supported by Grantová agentura České republiky [P305/11/0126].
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.086785/-/DC1
References
Alberta, J. A., Springett, G. M., Rayburn, H., Natoli, T. A., Loring, J.,
Kreidberg, J. A. and Housman, D. (2003). Role of the WT1 tumor suppressor
in murine hematopoiesis. Blood 101, 2570-2574.
Benmimoun, B., Polesello, C., Waltzer, L. and Haenlin, M. (2012). Dual role for
Insulin/TOR signaling in the control of hematopoietic progenitor maintenance
in Drosophila. Development 139, 1713-1717.
Bernard, F., Krejci, A., Housden, B., Adryan, B. and Bray, S. J. (2010).
Specificity of Notch pathway activation: twist controls the transcriptional
output in adult muscle progenitors. Development 137, 2633-2642.
Bieda, M., Xu, X., Singer, M. A., Green, R. and Farnham, P. J. (2006). Unbiased
location analysis of E2F1-binding sites suggests a widespread role for E2F1 in
the human genome. Genome Res. 16, 595-605.
Bray, S. J. (1997). Expression and function of Enhancer of split bHLH proteins
during Drosophila neurogenesis. Perspect. Dev. Neurobiol. 4, 313-323.
Bray, S. J. (2006). Notch signalling: a simple pathway becomes complex. Nat. Rev.
Mol. Cell Biol. 7, 678-689.
Burns, C. E., Traver, D., Mayhall, E., Shepard, J. L. and Zon, L. I. (2005).
Hematopoietic stem cell fate is established by the Notch-Runx pathway. Genes
Dev. 19, 2331-2342.
Burns, C. E., Galloway, J. L., Smith, A. C., Keefe, M. D., Cashman, T. J., Paik, E.
J., Mayhall, E. A., Amsterdam, A. H. and Zon, L. I. (2009). A genetic screen in
zebrafish defines a hierarchical network of pathways required for
hematopoietic stem cell emergence. Blood 113, 5776-5782.
Cherbas, L., Willingham, A., Zhang, D., Yang, L., Zou, Y., Eads, B. D., Carlson,
J. W., Landolin, J. M., Kapranov, P., Dumais, J. et al. (2011). The
transcriptional diversity of 25 Drosophila cell lines. Genome Res. 21, 301-314.
Christensen, S., Kodoyianni, V., Bosenberg, M., Friedman, L. and Kimble, J.
(1996). lag-1, a gene required for lin-12 and glp-1 signaling in Caenorhabditis
elegans, is homologous to human CBF1 and Drosophila Su(H). Development
122, 1373-1383.
Crozatier, M. and Meister, M. (2007). Drosophila haematopoiesis. Cell. Microbiol.
9, 1117-1126.
Cubadda, Y., Heitzler, P., Ray, R. P., Bourouis, M., Ramain, P., Gelbart, W.,
Simpson, P. and Haenlin, M. (1997). u-shaped encodes a zinc finger protein
that regulates the proneural genes achaete and scute during the formation of
bristles in Drosophila. Genes Dev. 11, 3083-3095.
Davis, R. L. and Turner, D. L. (2001). Vertebrate hairy and Enhancer of split
related proteins: transcriptional repressors regulating cellular differentiation
and embryonic patterning. Oncogene 20, 8342-8357.
Echalier, G. and Ohanessian, A. (1969). [Isolation, in tissue culture, of
Drosophila melangaster cell lines]. C.R. Hebd. Seances Acad. Sci., Ser. D, Sci. Nat.
268, 1771-1773.
Evans, C. J. and Banerjee, U. (2003). Transcriptional regulation of hematopoiesis
in Drosophila. Blood Cells Mol. Dis. 30, 223-228.
Ferro, E. and Trabalzini, L. (2010). RalGDS family members couple Ras to Ral
signalling and that’s not all. Cell. Signal. 22, 1804-1810.
Fischer, A. and Gessler, M. (2007). Delta-Notch – and then? Protein interactions
and proposed modes of repression by Hes and Hey bHLH factors. Nucleic Acids
Res. 35, 4583-4596.
Fossett, N., Zhang, Q., Gajewski, K., Choi, C. Y., Kim, Y. and Schulz, R. A.
(2000). The multitype zinc-finger protein U-shaped functions in heart cell
specification in the Drosophila embryo. Proc. Natl. Acad. Sci. USA 97, 7348-53.
Fossett, N., Tevosian, S. G., Gajewski, K., Zhang, Q., Orkin, S. H. and Schulz,
R. A. (2001). The Friend of GATA proteins U-shaped, FOG-1, and FOG-2
function as negative regulators of blood, heart, and eye development in
Drosophila. Proc. Natl. Acad. Sci. USA 98, 7342-7347.
Fossett, N., Hyman, K., Gajewski, K., Orkin, S. H. and Schulz, R. A. (2003).
Combinatorial interactions of serpent, lozenge, and U-shaped regulate crystal
cell lineage commitment during Drosophila hematopoiesis. Proc. Natl. Acad.
Sci. USA 100, 11451-11456.
Development 140 (4)
Friedman, A. D. (2007). Transcriptional control of granulocyte and monocyte
development. Oncogene 26, 6816-6828.
Furriols, M. and Bray, S. J. (2000). Dissecting the mechanisms of suppressor of
hairless function. Dev. Biol. 227, 520-532.
Georgescu, C., Longabaugh, W. J., Scripture-Adams, D. D., David-Fung, E. S.,
Yui, M. A., Zarnegar, M. A., Bolouri, H. and Rothenberg, E. V. (2008). A gene
regulatory network armature for T lymphocyte specification. Proc. Natl. Acad.
Sci. USA 105, 20100-20105.
Gering, M. and Patient, R. (2010). Notch signalling and haematopoietic stem
cell formation during embryogenesis. J. Cell. Physiol. 222, 11-16.
Guo, Y., Maillard, I., Chakraborti, S., Rothenberg, E. V. and Speck, N. A.
(2008). Core binding factors are necessary for natural killer cell development
and cooperate with Notch signaling during T-cell specification. Blood 112,
480-492.
Gupta-Rossi, N., Le Bail, O., Gonen, H., Brou, C., Logeat, F., Six, E.,
Ciechanover, A. and Israël, A. (2001). Functional interaction between SEL-10,
an F-box protein, and the nuclear form of activated Notch1 receptor. J. Biol.
Chem. 276, 34371-34378.
Haenlin, M., Cubadda, Y., Blondeau, F., Heitzler, P., Lutz, Y., Simpson, P. and
Ramain, P. (1997). Transcriptional activity of pannier is regulated negatively by
heterodimerization of the GATA DNA-binding domain with a cofactor
encoded by the u-shaped gene of Drosophila. Genes Dev. 11, 3096-3108.
Hamidi, H., Gustafason, D., Pellegrini, M. and Gasson, J. (2011). Identification
of novel targets of CSL-dependent Notch signaling in hematopoiesis. PLoS
ONE 6, e20022.
Helms, W., Lee, H., Ammerman, M., Parks, A. L., Muskavitch, M. A. and
Yedvobnick, B. (1999). Engineered truncations in the Drosophila mastermind
protein disrupt Notch pathway function. Dev. Biol. 215, 358-374.
Hertz, G. Z. and Stormo, G. D. (1999). Identifying DNA and protein patterns
with statistically significant alignments of multiple sequences. Bioinformatics
15, 563-577.
Housden, B. E., Millen, K. and Bray, S. J. (2012). Drosophila reporter vectors
compatible with PhiC31 integrase transgenesis techniques and their use to
generate new notch reporter fly lines. G3 (Bethesda), 2, 79-82.
Huff, V. (2011). Wilms’ tumours: about tumour suppressor genes, an oncogene
and a chameleon gene. Nat. Rev. Cancer 11, 111-121.
Jackson Behan, K., Fair, J., Singh, S., Bogwitz, M., Perry, T., Grubor, V.,
Cunningham, F., Nichols, C. D., Cheung, T. L., Batterham, P. et al. (2005).
Alternative splicing removes an Ets interaction domain from Lozenge during
Drosophila eye development. Dev. Genes Evol. 215, 423-435.
Jung, S. H., Evans, C. J., Uemura, C. and Banerjee, U. (2005). The Drosophila
lymph gland as a developmental model of hematopoiesis. Development 132,
2521-2533.
Kageyama, R., Ohtsuka, T. and Kobayashi, T. (2007). The Hes gene family:
repressors and oscillators that orchestrate embryogenesis. Development 134,
1243-1251.
Kaspar, M., Schneider, M., Chia, W. and Klein, T. (2008). Klumpfuss is involved
in the determination of sensory organ precursors in Drosophila. Dev. Biol. 324,
177-191.
Klein, T. and Campos-Ortega, J. A. (1997). klumpfuss, a Drosophila gene
encoding a member of the EGR family of transcription factors, is involved in
bristle and leg development. Development 124, 3123-3134.
Klinakis, A., Szabolcs, M., Politi, K., Kiaris, H., Artavanis-Tsakonas, S. and
Efstratiadis, A. (2006). Myc is a Notch1 transcriptional target and a requisite
for Notch1-induced mammary tumorigenesis in mice. Proc. Natl. Acad. Sci. USA
103, 9262-9267.
Kopan, R. and Ilagan, M. X. (2009). The canonical Notch signaling pathway:
unfolding the activation mechanism. Cell 137, 216-233.
Kovall, R. A. (2008). More complicated than it looks: assembly of Notch pathway
transcription complexes. Oncogene 27, 5099-5109.
Krejcí, A. and Bray, S. (2007). Notch activation stimulates transient and selective
binding of Su(H)/CSL to target enhancers. Genes Dev. 21, 1322-1327.
Krejcí, A., Bernard, F., Housden, B. E., Collins, S. and Bray, S. J. (2009). Direct
response to Notch activation: signaling crosstalk and incoherent logic. Sci.
Signal. 2, ra1.
Krzemień, J., Dubois, L., Makki, R., Meister, M., Vincent, A. and Crozatier, M.
(2007). Control of blood cell homeostasis in Drosophila larvae by the posterior
signalling centre. Nature 446, 325-328.
Krzemien, J., Oyallon, J., Crozatier, M. and Vincent, A. (2010). Hematopoietic
progenitors and hemocyte lineages in the Drosophila lymph gland. Dev. Biol.
346, 310-319.
Kurucz, E., Zettervall, C. J., Sinka, R., Vilmos, P., Pivarcsi, A., Ekengren, S.,
Hegedüs, Z., Ando, I. and Hultmark, D. (2003). Hemese, a hemocyte-specific
transmembrane protein, affects the cellular immune response in Drosophila.
Proc. Natl. Acad. Sci. USA 100, 2622-2627.
Laslo, P., Spooner, C. J., Warmflash, A., Lancki, D. W., Lee, H. J., Sciammas, R.,
Gantner, B. N., Dinner, A. R. and Singh, H. (2006). Multilineage
transcriptional priming and determination of alternate hematopoietic cell
fates. Cell 126, 755-766.
DEVELOPMENT
936
Lebestky, T., Jung, S. H. and Banerjee, U. (2003). A Serrate-expressing
signaling center controls Drosophila hematopoiesis. Genes Dev. 17, 348-353.
Li, Y., Hibbs, M. A., Gard, A. L., Shylo, N. A. and Yun, K. (2012). Genome-wide
analysis of N1ICD/RBPJ targets in vivo reveals direct transcriptional regulation
of Wnt, SHH, and hippo pathway effectors by Notch1. Stem Cells 30, 741-752.
Lunstrum, G. P., Bächinger, H. P., Fessler, L. I., Duncan, K. G., Nelson, R. E.
and Fessler, J. H. (1988). Drosophila basement membrane procollagen IV. I.
Protein characterization and distribution. J. Biol. Chem. 263, 18318-18327.
Maillard, I., Fang, T. and Pear, W. S. (2005). Regulation of lymphoid
development, differentiation, and function by the Notch pathway. Annu. Rev.
Immunol. 23, 945-974.
Maines, J. Z., Stevens, L. M., Tong, X. and Stein, D. (2004). Drosophila dMyc is
required for ovary cell growth and endoreplication. Development 131, 775-786.
Minakhina, S. and Steward, R. (2010). Hematopoietic stem cells in Drosophila.
Development 137, 27-31.
Mukherjee, T., Kim, W. S., Mandal, L. and Banerjee, U. (2011). Interaction
between Notch and Hif-alpha in development and survival of Drosophila
blood cells. Science 332, 1210-1213.
Mullen, A. C., Orlando, D. A., Newman, J. J., Lovén, J., Kumar, R. M.,
Bilodeau, S., Reddy, J., Guenther, M. G., DeKoter, R. P. and Young, R. A.
(2011). Master transcription factors determine cell-type-specific responses to
TGF-β signaling. Cell 147, 565-576.
Muñoz-Alonso, M. J., Ceballos, L., Bretones, G., Frade, P., León, J. and
Gandarillas, A. (2012). MYC accelerates p21CIP-induced megakaryocytic
differentiation involving early mitosis arrest in leukemia cells. J. Cell. Physiol.
227, 2069-2078.
Muratoglu, S., Hough, B., Mon, S. T. and Fossett, N. (2007). The GATA factor
Serpent cross-regulates lozenge and u-shaped expression during Drosophila
blood cell development. Dev. Biol. 311, 636-649.
Nelson, R. E., Fessler, L. I., Takagi, Y., Blumberg, B., Keene, D. R., Olson, P. F.,
Parker, C. G. and Fessler, J. H. (1994). Peroxidasin: a novel enzyme-matrix
protein of Drosophila development. EMBO J. 13, 3438-3447.
Nottingham, W. T., Jarratt, A., Burgess, M., Speck, C. L., Cheng, J. F.,
Prabhakar, S., Rubin, E. M., Li, P. S., Sloane-Stanley, J., Kong-A-San, J. et
al. (2007). Runx1-mediated hematopoietic stem-cell emergence is controlled
by a Gata/Ets/SCL-regulated enhancer. Blood 110, 4188-4197.
Pajcini, K. V., Speck, N. A. and Pear, W. S. (2011). Notch signaling in
mammalian hematopoietic stem cells. Leukemia 25, 1525-1532.
Palomero, T., Lim, W. K., Odom, D. T., Sulis, M. L., Real, P. J., Margolin, A.,
Barnes, K. C., O’Neil, J., Neuberg, D., Weng, A. P. et al. (2006). NOTCH1
directly regulates c-MYC and activates a feed-forward-loop transcriptional
network promoting leukemic cell growth. Proc. Natl. Acad. Sci. USA 103, 1826118266.
Pierce, S. B., Yost, C., Britton, J. S., Loo, L. W., Flynn, E. M., Edgar, B. A. and
Eisenman, R. N. (2004). dMyc is required for larval growth and
endoreplication in Drosophila. Development 131, 2317-2327.
Radtke, F., Fasnacht, N. and Macdonald, H. R. (2010). Notch signaling in the
immune system. Immunity 32, 14-27.
Rand, M. D., Grimm, L. M., Artavanis-Tsakonas, S., Patriub, V., Blacklow, S.
C., Sklar, J. and Aster, J. C. (2000). Calcium depletion dissociates and
activates heterodimeric notch receptors. Mol. Cell. Biol. 20, 1825-1835.
Robert-Moreno, A., Espinosa, L., de la Pompa, J. L. and Bigas, A. (2005).
RBPjkappa-dependent Notch function regulates Gata2 and is essential for the
formation of intra-embryonic hematopoietic cells. Development 132, 11171126.
Satoh, Y., Matsumura, I., Tanaka, H., Ezoe, S., Sugahara, H., Mizuki, M.,
Shibayama, H., Ishiko, E., Ishiko, J., Nakajima, K. et al. (2004). Roles for cMyc in self-renewal of hematopoietic stem cells. J. Biol. Chem. 279, 2498624993.
Schneider, I. (1972). Cell lines derived from late embryonic stages of Drosophila
melanogaster. J. Embryol. Exp. Morphol. 27, 353-365.
Shcherbata, H. R., Althauser, C., Findley, S. D. and Ruohola-Baker, H. (2004).
The mitotic-to-endocycle switch in Drosophila follicle cells is executed by
RESEARCH ARTICLE
937
Notch-dependent regulation of G1/S, G2/M and M/G1 cell-cycle transitions.
Development 131, 3169-3181.
Sinenko, S. A., Hung, T., Moroz, T., Tran, Q. M., Sidhu, S., Cheney, M. D.,
Speck, N. A. and Banerjee, U. (2010). Genetic manipulation of AML1-ETOinduced expansion of hematopoietic precursors in a Drosophila model. Blood
116, 4612-4620.
Song, Y. and Lu, B. (2011). Regulation of cell growth by Notch signaling and its
differential requirement in normal vs. tumor-forming stem cells in Drosophila.
Genes Dev. 25, 2644-2658.
Sorrentino, R. P., Tokusumi, T. and Schulz, R. A. (2007). The Friend of GATA
protein U-shaped functions as a hematopoietic tumor suppressor in
Drosophila. Dev. Biol. 311, 311-323.
Stramer, B., Wood, W., Galko, M. J., Redd, M. J., Jacinto, A., Parkhurst, S. M.
and Martin, P. (2005). Live imaging of wound inflammation in Drosophila
embryos reveals key roles for small GTPases during in vivo cell migration. J. Cell
Biol. 168, 567-573.
Sun, J. and Deng, W. M. (2007). Hindsight mediates the role of notch in
suppressing hedgehog signaling and cell proliferation. Dev. Cell 12, 431-442.
Sun, J., Smith, L., Armento, A. and Deng, W. M. (2008). Regulation of the
endocycle/gene amplification switch by Notch and ecdysone signaling. J. Cell
Biol. 182, 885-896.
Thomas-Chollier, M., Defrance, M., Medina-Rivera, A., Sand, O., Herrmann,
C., Thieffry, D. and van Helden, J. (2011). RSAT 2011: regulatory sequence
analysis tools. Nucleic Acids Res. 39, W86-W91.
Tosello, V., Mansour, M. R., Barnes, K., Paganin, M., Sulis, M. L., Jenkinson,
S., Allen, C. G., Gale, R. E., Linch, D. C., Palomero, T. et al. (2009). WT1
mutations in T-ALL. Blood 114, 1038-1045.
Trompouki, E., Bowman, T. V., Lawton, L. N., Fan, Z. P., Wu, D. C., DiBiase, A.,
Martin, C. S., Cech, J. N., Sessa, A. K., Leblanc, J. L. et al. (2011). Lineage
regulators direct BMP and Wnt pathways to cell-specific programs during
differentiation and regeneration. Cell 147, 577-589.
van Tetering, G. and Vooijs, M. (2011). Proteolytic cleavage of Notch: “HIT and
RUN”. Curr. Mol. Med. 11, 255-269.
Waltzer, L., Ferjoux, G., Bataillé, L. and Haenlin, M. (2003). Cooperation
between the GATA and RUNX factors Serpent and Lozenge during Drosophila
hematopoiesis. EMBO J. 22, 6516-6525.
Wang, H., Zou, J., Zhao, B., Johannsen, E., Ashworth, T., Wong, H., Pear, W.
S., Schug, J., Blacklow, S. C., Arnett, K. L. et al. (2011). Genome-wide
analysis reveals conserved and divergent features of Notch1/RBPJ binding in
human and murine T-lymphoblastic leukemia cells. Proc. Natl. Acad. Sci. USA
108, 14908-14913.
Weng, A. P., Millholland, J. M., Yashiro-Ohtani, Y., Arcangeli, M. L., Lau, A.,
Wai, C., Del Bianco, C., Rodriguez, C. G., Sai, H., Tobias, J. et al. (2006). cMyc is an important direct target of Notch1 in T-cell acute lymphoblastic
leukemia/lymphoma. Genes Dev. 20, 2096-2109.
Wildonger, J., Sosinsky, A., Honig, B. and Mann, R. S. (2005). Lozenge directly
activates argos and klumpfuss to regulate programmed cell death. Genes Dev.
19, 1034-1039.
Yang, L., Han, Y., Suarez Saiz, F. and Minden, M. D. (2007). A tumor suppressor
and oncogene: the WT1 story. Leukemia 21, 868-876.
Yashiro-Ohtani, Y., He, Y., Ohtani, T., Jones, M. E., Shestova, O., Xu, L., Fang,
T. C., Chiang, M. Y., Intlekofer, A. M., Blacklow, S. C. et al. (2009). Pre-TCR
signaling inactivates Notch1 transcription by antagonizing E2A. Genes Dev. 23,
1665-1676.
Yu, M., Riva, L., Xie, H., Schindler, Y., Moran, T. B., Cheng, Y., Yu, D., Hardison,
R., Weiss, M. J., Orkin, S. H. et al. (2009). Insights into GATA-1-mediated gene
activation versus repression via genome-wide chromatin occupancy analysis.
Mol. Cell 36, 682-695.
Zanet, J., Pibre, S., Jacquet, C., Ramirez, A., de Alborán, I. M. and
Gandarillas, A. (2005). Endogenous Myc controls mammalian epidermal cell
size, hyperproliferation, endoreplication and stem cell amplification. J. Cell Sci.
118, 1693-1704.
DEVELOPMENT
Fate commitment by Notch and Lz