An in vivo RNA interference screen identifies gene networks controlling Drosophila melanogaster blood cell homeostasis.

Abstract

In metazoans, the hematopoietic system plays a key role both in normal development and in defense of the organism. In Drosophila, the cellular immune response involves three types of blood cells: plasmatocytes, crystal cells and lamellocytes. This last cell type is barely present in healthy larvae, but its production is strongly induced upon wasp parasitization or in mutant contexts affecting larval blood cell homeostasis. Notably, several zygotic mutations leading to melanotic mass (or "tumor") formation in larvae have been associated to the deregulated differentiation of lamellocytes. To gain further insights into the gene regulatory network and the mechanisms controlling larval blood cell homeostasis, we conducted a tissue-specific loss of function screen using hemocyte-specific Gal4 drivers and UAS-dsRNA transgenic lines.
By targeting around 10% of the Drosophila genes, this in vivo RNA interference screen allowed us to recover 59 melanotic tumor suppressor genes. In line with previous studies, we show that melanotic tumor formation is associated with the precocious differentiation of stem-cell like blood progenitors in the larval hematopoietic organ (the lymph gland) and the spurious differentiation of lamellocytes. We also find that melanotic tumor formation can be elicited by defects either in the fat body, the embryo-derived hemocytes or the lymph gland. In addition, we provide a definitive confirmation that lymph gland is not the only source of lamellocytes as embryo-derived plasmatocytes can differentiate into lamellocytes either upon wasp infection or upon loss of function of the Friend of GATA cofactor U-shaped.
In this study, we identify 55 genes whose function had not been linked to blood cell development or function before in Drosophila. Moreover our analyses reveal an unanticipated plasticity of embryo-derived plasmatocytes, thereby shedding new light on blood cell lineage relationship, and pinpoint the Friend of GATA transcription cofactor U-shaped as a key regulator of the plasmatocyte to lamellocyte transformation.

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Open Access
RESEARCH ARTICLE
An in vivo RNA interference screen identifies gene
networks controlling Drosophila melanogaster
blood cell homeostasis
© 2010 Avet-Rochex et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Com-
mons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduc-
tion in any medium, provided the original work is properly cited.
Research article
Amélie Avet-Rochex†1,2,3, Karène Boyer†1,2, Cédric Polesello1,2, Vanessa Gobert1,2, Dani Osman1,2, Fernando Roch1,2,
Benoit Augé1,2, Jennifer Zanet1,2,3, Marc Haenlin*1,2 and Lucas Waltzer*1,2
Abstract
Background: In metazoans, the hematopoietic system plays a key role both in normal development and in defense of
the organism. In Drosophila, the cellular immune response involves three types of blood cells: plasmatocytes, crystal
cells and lamellocytes. This last cell type is barely present in healthy larvae, but its production is strongly induced upon
wasp parasitization or in mutant contexts affecting larval blood cell homeostasis. Notably, several zygotic mutations
leading to melanotic mass (or "tumor") formation in larvae have been associated to the deregulated differentiation of
lamellocytes. To gain further insights into the gene regulatory network and the mechanisms controlling larval blood
cell homeostasis, we conducted a tissue-specific loss of function screen using hemocyte-specific Gal4 drivers and UAS-
dsRNA transgenic lines.
Results: By targeting around 10% of the Drosophila genes, this in vivo RNA interference screen allowed us to recover 59
melanotic tumor suppressor genes. In line with previous studies, we show that melanotic tumor formation is
associated with the precocious differentiation of stem-cell like blood progenitors in the larval hematopoietic organ
(the lymph gland) and the spurious differentiation of lamellocytes. We also find that melanotic tumor formation can be
elicited by defects either in the fat body, the embryo-derived hemocytes or the lymph gland. In addition, we provide a
definitive confirmation that lymph gland is not the only source of lamellocytes as embryo-derived plasmatocytes can
differentiate into lamellocytes either upon wasp infection or upon loss of function of the Friend of GATA cofactor U-
shaped.
Conclusions: In this study, we identify 55 genes whose function had not been linked to blood cell development or
function before in Drosophila. Moreover our analyses reveal an unanticipated plasticity of embryo-derived
plasmatocytes, thereby shedding new light on blood cell lineage relationship, and pinpoint the Friend of GATA
transcription cofactor U-shaped as a key regulator of the plasmatocyte to lamellocyte transformation.
Background
In metazoan, blood cells play a critical role in establishing
the proper response against invading pathogens or in
removing both cancerous and apoptotic cells [1]. Con-
versely, deregulations of the hematopoietic differentia-
tion program are at the origin of numerous pathologies,
including leukemia and auto-immune diseases [2]. As
many key signaling pathways and transcription factors
controlling blood cell development and functions have
been conserved from humans to Drosophila [3], this
organism has emerged as an attractive model to investi-
gate the genetic basis controlling blood cell homeostasis.
Drosophila hematopoiesis occurs in two spatially and
temporally distinct phases. In the early embryo, blood
cell progenitors (prohemocytes) arise from the head
mesoderm [4]. These hemocytes subsist in the larva
either in circulation in the hemolymph or attached to the
inner surface of the integument, forming the so-called
sessile islands that can be mobilized upon infection [5,6].
* Correspondence: haenlin@cict.fr, waltzer@cict.fr
1 Université de Toulouse, UPS, CBD (Centre de Biologie du Développement),
Bât4R3, 118 route de Narbonne, 31062 Toulouse, France
2 Université de Toulouse, UPS, CBD (Centre de Biologie du Développement),
Bât4R3, 118 route de Narbonne, 31062 Toulouse, France
† Contributed equally
Full list of author information is available at the end of the article

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A second hematopoietic wave occurs in the larva in a spe-
cialized organ called the lymph gland [5]. In third instar
larvae, the lymph gland is composed of a pair of primary
lobes and several more posterior secondary lobes. Each
primary lobe is subdivided into three zones: (1) the corti-
cal zone, containing differentiated hemocytes; (2) the
medullary zone, containing prohemocytes; and (3) the
posterior signaling center, a small group of cells whose
activity is required to maintain medullary zone cells into
a progenitor state [7-9]. The smaller posterior lobes, pre-
senting no organized structure, consist mainly of pro-
hemocytes [7]. In normal conditions, both the circulating
and sessile cells in the larva are only of embryonic origin
[6] whereas hemocytes from the lymph gland are released
into circulation only at pupariation [5]. Finally, in the
adult, no hematopoietic tissue has been described and
hemocytes of both embryonic and lymph gland origin are
observed [6].
Prohemocytes give rise to three terminally differenti-
ated cell types: plasmatocytes, crystal cells and lamello-
cytes [3]. Plasmatocytes, which comprise 90-95% of the
larval circulating blood cells, are phagocytic cells that
engulf apoptotic bodies and pathogens [10-13]. Crystal
cells secrete components of the melanization cascade, an
insect-specific immune reaction involved in wound heal-
ing and in the encapsulation of large foreign bodies [14-
16]. Lamellocytes are large flattened non-phagocytic cells
normally scarcely present in the larva but their develop-
ment is massively induced upon certain immune chal-
lenges such as infection of the larvae by eggs of the
parasitoid wasp Leptopilina boulardi [17]. Parasitization
elicits lymph gland overgrowth, massive production of
lamellocytes, and precocious hemocyte release from the
lymph gland into the circulation. Together with the other
blood cell types, lamellocytes form a melanotic capsule
around the parasitoid egg to prevent its development
[5,18]. While it was initially proposed that lamellocytes
might represent an ultimate state of plasmatocyte differ-
entiation [17,19-21] further evidence suggests that they
derive only from lymph gland progenitors [5,8,18,22,23].
Yet this view has been recently challenged as cells from
the sessile islands were shown to differentiate into lamel-
locytes after wasp infection [24].
Interfering with normal blood cell development and/or
function can trigger an aberrant immune response char-
acterized by lymph gland overgrowth and massive differ-
entiation of lamellocytes [25]. This response culminates
with the premature disintegration of the lymph gland and
the formation in the larvae of "melanotic tumors" (also
called melanotic masses, melanotic nodules or pseudotu-
mors) constituted by melanized aggregates of hemocytes,
mostly lamellocytes, sometimes surrounding cells from
other tissues. Melanotic masses are easily observable
through the larval cuticle and a large number of « melan-
otic tumor suppressor genes », were identified based on
such phenotype [25]. Unfortunately, the nature of the
mutated gene has not been ascertained in the majority of
the cases and the contribution of blood cells to the phe-
notype has seldom been evaluated [26]. Notwithstanding,
available evidences suggest that presence of melanotic
tumors reflects defects in the hematopoietic develop-
mental program and/or in the immune surveillance of
self-tissues. Accordingly, melanotic mutations have been
classically subdivided in two categories [25,27]: (1) class I
mutations modify a non-hematopoietic tissue and induce
a kind of "autoimmune response", as the mutations in
kurtz or spaghetti [26,28], (2) class II mutations affect
internal regulatory pathways within the hemocytes them-
selves, such as gain of function mutations in JAK/STAT
or Toll signaling pathway [29-34]. Hence, melanotic
tumor suppressor genes are potential candidates for regu-
lating both hematopoiesis and blood cell function.
In this work, we conducted a large-scale screen for mel-
anotic tumor suppressor genes aimed specifically at the
identification of genes involved in blood cell homeostasis,
taking advantage of recently developed UAS-dsRNA
transgenic line collections. Down-regulation of the tar-
geted genes was specifically induced in the blood cells or
both in the blood cells and the fat body using different
Gal4 drivers. By individually inactivating the function of
around 10% of the Drosophila genes, we recovered 59
melanotic tumor suppressor genes. This approach
allowed us to pinpoint several new pathways controlling
blood cell homeostasis. By analyzing some of these candi-
dates, we further demonstrate that melanotic masses can
be induced by defects in a specific subset of cells and
demonstrate that embryonic-derived plasmatocytes can
differentiate into lamellocytes.
Results
A loss of function screen for melanotic tumor suppressor
genes
To identify new genes regulating Drosophila blood cell
homeostasis, we performed a screen for melanotic tumor
suppressor genes (i.e. genes whose loss of function
induces melanotic mass formation in third instar larvae).
For this, we used of a collection of RNAi transgenes
(UAS-dsRNA) that consist of short gene fragments (300-
500 bp) cloned as inverted repeats and expressed via the
binary Gal4/UAS system http://www.shigen.nig.ac.fly/
nigfly, thus allowing tissue-specific gene knock-down.
We took advantage of this collection to induce RNAi in
blood cells using three different drivers: srp-Gal4, cg-
Gal4, and hmlΔ-Gal4 [35-37]. spr-Gal4 is expressed in all
the embryo-derived hemocytes from the early embryonic
stages, as well as in the larval lymph gland and fat body
[35]. cg-Gal4 is expressed in the plasmatocytes both in
the late embryo and in the larvae, including in the lymph

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gland cortical zone, and also drives at high levels in the
larval fat body [7,36]. hmlΔ-Gal4 is expressed only at the
larval stages, in almost all the circulating blood cells and
in the cortical zone of the lymph gland [38]. All together
these three drivers, whose expression patterns are illus-
trated in Additional file 1, Figure S1, allow targeting most
of the tissues involved in hematopoietic development and
cellular immunity in Drosophila.
Based on pilot experiments, our screen was first per-
formed with srp-Gal4 and hmlΔ-Gal4 on UAS-dsRNA
transgenes predicted to target 1341 of the 13825 pre-
dicted protein-coding genes of Drosophila (Drosophila
genome release 5; http://flybase.org) (Additional file 4,
Table S1). All the hits were subsequently retested with the
three drivers on a higher number of larvae. Given that all
together 2.5% of the ± 145000 larvae that we screened
had melanotic nodules, we selected as positives only
those lines that scored two folds above this baseline, i.e.
with a tumor index ≥5% (Figure 1A). Thereby we identi-
fied 96 genes whose RNAi-induced down-regulation with
one of the three drivers induced melanotic masses in at
least 5% of the emerging larvae (Additional file 5, Table
S2). Interestingly four of these genes (cactus, DREF, ush
and ND75) were already known to be implicated in mel-
anotic mass formation and/or lamellocyte differentiation
[39-42], thereby validating our screening strategy. Figure
1B displays some representative larvae harboring melan-
otic tumors that we obtained in the screen. We did not
consider melanotic spots (on the cuticle, gut, trachea....)
as genuine melanotic tumors since they were shown to
arise independently of a modification in larval blood cell
homeostasis [26]. While smaller nodules were circulating
freely in the hemocel, larger ones were most often local-
ized to the posterior part of the larvae. In some rare
cases, we observed lymph gland melanization or disinte-
gration of the fat body (Figure 1B, right most panel).
Moreover, for a given gene, the penetrance of the pheno-
type is largely dependent on the Gal4 line (Additional file
5, Table S2). Indeed, the median tumor index for the 96
candidates was 1.5% with hmlΔ-Gal4, 9% with srp-Gal4
and 19% with cg-Gal4. Of note, cg-Gal4 also caused
severe growth delay or lethality before the third instar lar-
val stage with a few candidates, which impaired the analy-
sis of melanotic mass formation in such cases and
excluded its use in the screen first step (Additional file 5,
Table S2).
Validation of the candidates
Expression of long double stranded RNA can cause non-
specific phenotypes due to off-target effects (OTE) [43].
A common measure of dsRNA targeting specificity is the
specificity score, S19, which is the number of all on-target
19-mer matches divided by the total number of matches
of a given RNAi hairpin [43]. Among the candidates we
isolated, 43 of the 96 hairpins tested had no predicted off-
target (S19 = 1), 25 had a S19 above 0.99, 22 between 0.99
and 0.8 and 16 below 0.8, suggesting that the vast major-
ity of the dsRNA we used were specific (Additional file 5,
Table S2). Besides OTE, another potential source of false
positives is the dsRNA transgene insertion itself, which
might interfere with expression of nearby genes and pro-
duce melanotic nodules. To validate our hits, we obtained
independent secondary UAS-dsRNA lines for the entire
set of candidate genes, except two for which no second-
ary lines were available: cactus, a negative regulator of the
Toll pathway well known as a melanotic tumor suppres-
sor gene [32,34,40], and CG9663, which codes for an
ABCG transporter. Of note, 59 of these 94 secondary
UAS-dsRNA targeted a non-overlapping sequence in the
candidate gene mRNA as compared to the original set
(Additional file 5, Table S2). Using these secondary RNAi
lines, we could phenocopy formation of melanotic masses
for 58 genes, including 40 of them (70%) using non-over-
lapping dsRNA (Additional file 6, Table S3). Among the
18 genes where overlapping dsRNA were used, mutant
alleles for two of them induced melanotic tumor forma-
tion (Dref and Aos1) and 10 code for proteins that inter-
act with an other melanotic suppressors validated by
non-overlapping dsRNA or genetic means (see below and
Figure 2). As for the 6 remaining genes in that category
(CG14512, CG15784, CG31044, CG8444, Cp7Fa and fne),
we cannot exclude an off-target effect although dsRNA
lines targeting CG14512, CG8444 and fne have an S19
score of 1 and above 0.9 for CG15784 and Cp7Fa. Hence,
together with cactus, our screen allowed us to identify 59
genes potentially controlling larval blood cell homeosta-
sis.
We also sought to validate these candidates by checking
the phenotypes of genetic mutants affecting their activity.
However, in most cases, no mutants have been described
for these genes or the available mutations result in lethal-
ity before the third instar larval stage, thereby precluding
this kind of analysis. Nonetheless, beside the already
described mutations in cactus [32,34,40], we could con-
firm presence of melanotic nodules in larvae carrying
zygotic mutations for five genes: u-shaped (previously
associated only to lamellocyte differentiation) [41], pyra-
mus, RfC4, tiggrin and Aos1 (Additional file 6, Table S3).
These results support the idea that the use of UAS-
dsRNA allows efficient identification of a large panel of
genes participating in blood cell development.
Identification of gene networks controlling blood cell
homeostasis
To gain insights into the putative functions of the candi-
date genes and into the different pathways regulating
blood cell homeostasis, we built an interaction network
between the candidates. Accordingly, we searched for

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Figure 1 An RNAi screen for melanotic suppressor genes. (A) Distribution of tumor indices for the 342 UAS-dsRNA lines retested with the three
drivers (hmlΔ-Gal4, srp-Gal4, cg-Gal4). For each of the three driver lines, the candidates are classified by decreasing tumor index (% of larvae carrying
at least one melanotic nodule). The dotted line indicates the 5% threshold that we used to select 96 candidates for secondary validations. (B) Example
of melanotic mass mutant phenotypes recovered in the screen. Third instar larvae are shown with their corresponding genotypes. Small melanotic
masses are indicated by an arrow. Note the dissociation of the fat body in the cg-Gal4; UAS-ds-hyx larva.

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high confidence yeast two-hybrid, biochemical or genetic
interactions data using various databases (DroID,
BioGrid, Flybase....) as well as manual text mining for
each of the 59 genes and their mammalian or yeast
orthologs. We only considered first order (direct) and
second order (through one intermediate) interactions
between genes contained in our hit list or previously
identified as melanotic tumor suppressors. This approach
allowed us to uncover several nodes of interactions
between them (Figure 2) as 47 of the candidates are
linked to at least one other gene in the network. Interest-
ingly, 14 of the candidate genes code for proteins that are
part of complexes with previously described melanotic
tumor suppressors, thereby confirming that they are gen-
Figure 2 An interaction network of the melanotic suppressors. The 59 confirmed melanotic suppressors identified in the screen are depicted as
grey nodes together with their (inferred) name. Additional factors not identified in the screen but linking two or more melanotic suppressors are rep-
resented as white nodes. Factors previously associated with melanotic mass development and/or lamellocyte differentiation are underlined. Candi-
dates that were confirmed using non-overlapping dsRNA are indicated in bold. The different melanotic suppressors are grouped in 9 categories
(dashed lines) based on GO annotation and data mining. Factors belonging to a well-defined molecular complex are boxed together. Physical or func-
tional interactions between the different factors are represented by edges.

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uine melanotic tumor suppressor genes. Namely, these 14
genes code for the cytoplasmic ribosomal constituents
RpL26, RpL6 and RpS5b, which associate with RpS6 [44],
the mitochondrial ribosomal constituents mRpL13,
mRpL41, mRpL52 and mRpS30, which bind to mRpL55
[45], the COP9 signalosome component CSN1b, which
associates with CSN5 and CSN8 [46,47], Hsp70Bc and
EF1α (100)E, which are linked to Hsc70-4 [48], the repli-
cation factor RfC4, which associates with RfC1 [49], as
well as Wds, l(2)dtl and Cul4, which bind to DDB1 [50].
In addition, it is worth noting that DREF was shown to
regulate the transcription of two melanotic suppressors,
ddb1 and rfc1, in cell culture [49,50]. Finally, several can-
didates are indirectly connected to such melanotic sup-
pressors. For instance, the translation factors (EF1α(110),
EF1γ and eRF1) or the factors involved in ribosome
assembly (Dim1, Nol8, l(1)G0020, Rrp7 and PPAN) are
functionally linked to RpS6 function.
Given that our screen allowed identification of several
sets of interconnected genes, we asked whether con-
versely known partners of the genes we identified also
behave as melanotic tumor suppressor genes. As a root,
we chose Cct2, a component of the well defined and evo-
lutionarily conserved chaperonin complex TriC/CCT
(TCP1-ring Complex or Chaperonin Containing TCP1)
[51], which had not been linked to melanotic mass devel-
opment before. The CCT complex is composed of eight
subunits (Tcp1 and Cct2-8) and is a cytosolic chaperonin
complex regulating protein folding. We obtained UAS-
dsRNA lines against 5 other CCT subunits (Tcp1, Cct4,
Cct5, Cct7 and Cct8) and tested their capacity to induce
melanotic nodules upon expression under the control of
the three hematopoietic drivers used in the screen. As
summarized Table 1, downregulation of any of the six
CCT components tested induced melanotic masses.
These results show that the CCT complex plays a pivotal
role in regulating larval blood cell homeostasis and indi-
cate that the different candidates from the screen can be
used as entry points to explore the network of genes
implicated in melanotic mass formation.
For further analysis of melanotic tumor formation pro-
cess, we focused our attention on 5 genes that induced
melanotic mass to high frequency and might represent
different classes of melanotic tumor suppressor genes:
cct2, cul4, hyx, mRpS30 and ush.
Melanotic masses are associated to lamellocyte production
and premature lymph gland differentiation
To confirm that the melanotic masses we observed arise
from a modification in larval blood cell homeostasis, we
bled larvae expressing dsRNA for cct2, cul4, hyx, mRpS30
and ush and looked for lamellocyte differentiation. Figure
3 shows representative bleeds obtained from third instar
larvae expressing the indicated dsRNA under the control
of srp-Gal4 (similar results were obtained with hmlΔ-
Gal4 and cg-Gal4, data not shown). Lamellocytes are
normally absent in healthy larvae and can be easily distin-
guished from other blood cell types based on their mor-
phology (large flattened cells) and the expression of high
levels of actin as well as specific markers such as msn-
lacZ and α-ps4 [5,52,53]. Phalloidin staining and mor-
phological examination of larval blood smears showed
that all the larvae with melanotic nodules contained
numerous lamellocytes in circulation, whereas this blood
cell type was scarcely found in wild type controls. As
expected, msn-lacZ was strongly expressed in the
induced population of lamellocytes. In addition, we
observed a reproducible, albeit weaker, expression of
msn-lacZ in smaller blood cells unveiling a β-gal+ popula-
tion that is not present in wild type larvae. These results
suggest that melanotic tumor formation is also associated
to the activation of msn-lacZ in plasmatocytes or in cir-
culating lamellocyte progenitors (see below). Finally,
analysis of the melanotic masses themselves confirmed
that they contained numerous lamellocytes (Additional
file 2, Figure S2).
Table 1: Melanotic masses induction upon loss of CCT complex component
UAS-dsRNA driver
CGSYMBOLS19
srp-Gal4hmlΔ-Gal4 cg-Gal4
CG7033 Cct21 +++-++ a
CG5525Cct40.99+++- ++++ a
CG8439 Cct50.99 ++++++ a
CG8351Cct7 0.99 ++++-+++
CG8258 Cct8 0.99 ++- +++
CG5374TCP10.99++++++++ a
Melanotic masses indices: (-) < 5%; (+) 5-10%; (++) 10-20%; (+++) 20-50%; (++++) > 50%.
a Dead second and third instar larvae were observed and no or only few adults emerged.

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We also monitored lymph gland differentiation status.
Consistent with previous reports, we observed preco-
cious disintegration of the lymph gland in most larvae
harboring melanotic masses. By collecting larvae without
macroscopically apparent or with smaller nodules, we
were able to recover intact lymph gland and found that,
contrary to wild type, larvae expressing UAS-dsRNA con-
tained lamellocytes in their lymph glands, as revealed by
in situ hybridization against α-ps4 (Figure 4A-F). In addi-
tion, tepIV staining revealed that the medullary zone,
which contains stem-like blood progenitors, was mark-
edly reduced or absent (Figure 4G-L). Thus, similar to
wasp-egg parasitization [8,18,22], dsRNA-induced cap-
sule formation affect larval blood cell homeostasis by
activating lamellocyte development and premature differ-
entiation of lymph gland prohemocytes.
Melanotic capsule formation is induced in response to
defect in specific tissues
The three Gal4 drivers we used in our screen are predom-
inantly expressed in blood cells, but they are also
expressed in other territories (e.g. the fat body for srp-
Gal4 and cg-Gal4) and in overlapping patterns within the
hematopoietic lineages (embryonic and larval hemocytes
for srp-Gal4, differentiated embryonic and larval plasma-
tocytes for cg-Gal4, differentiated larval hemocytes for
hmlΔ-Gal4). We thus asked whether melanotic nodule
formation reflected a general response to a defect in any
tissue or was directly elicited by the modification of a
(particular) blood cell type. To investigate this issue, we
used a battery of Gal4 lines that are either not expressed
in hematopoietic tissues (fb-Gal4, cad-Gal4, repo-Gal4,
elav-Gal4, MS1096) or in restricted hematopoietic com-
partments (sn-Gal4, gcm-Gal4, tepIV-Gal4), or in over-
lapping patterns with the previous drivers (sn-Gal4, gcm-
Gal4, tepIV-Gal4, fb-Gal4, cad-Gal4) (Table 2). These
different drivers were used to assess through which cell
type the targeted loss of cct2, cul4, hyx, mRps30 or ush
can induce melanotic tumors.
We could induce both melanotic nodules and lamello-
cyte differentiation by targeting cul4 or cct2 dsRNA spe-
cifically in the lymph gland prohemocytes with the tepIV-
Gal4 line (see Additional file 1, Figure S1 for its expres-
sion pattern), indicating that these genes may participate
in blood cell progenitor fate maintenance and/or restrict
their differentiation potential. Interestingly, targeting
dsRNA expression only in plasmatocytes during embryo-
genesis either with sn-Gal4 [[54], Zanet et al., in prepara-
tion] or gcm-Gal4 [35] (which are not maintained in
larval hemocytes, see Figure 5 and Additional file 1, Fig-
ure S1) induced lamellocyte differentiation and melanotic
mass formation in the case of ush, hyx, cct2 and cul4.
Therefore, loss of function restricted to embryo-derived
plasmatocytes or to lymph gland prohemocytes is suffi-
cient to induce these phenotypes.
In addition we observed melanotic mass formation and
lamellocyte differentiation upon expression of the dsRNA
against mRpS30 or cul4 specifically in the fat body using
the FB-Gal4 line (see Additional file 1, Figure S1 for its
expression pattern). However, induction of melanotic
tumors or lamellocytes was never observed for any of the
five genes when dsRNA were expressed under the control
of MS1096, repo-Gal4, elav-Gal4 or cad-Gal4, which
respectively drive expression in the wing discs, the glial
cells, the central nervous system and the gut. Thus gene
knock down in the fat body, which plays a key role in
innate immune response, can elicit a non-autonomous
cellular immune response that culminates with the for-
mation of melanotic nodules by the hemocytes. These
results also show that the genes we tested act as melan-
otic tumor suppressors only in the immune system (blood
Figure 3 Blood cell phenotypes associated to melanotic mass for-
mation. Blood smears from third instar larvae carrying the msn-lacZ
transgene and expressing UAS-dsRNA targeting the indicated gene un-
der the control of srp-Gal4. Hemocyte actin cytoskeleton was visual-
ized using phalloidin (red) and expression of the lamellocyte marker
msn-lacZ was revealed by fluorescent immunolabeling against β-Gal
(green). Nuclei were stained with DAPI. Arrowheads indicate atypical
hemocytes that express β-Gal but do not exhibit the large flattened
morphology of lamellocytes.

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cells and fat body) and not in other tissues, perhaps
reflecting that only immune tissues can elicit melanotic
capsule formation (see discussion).
All together, it appears that there are at least three dif-
ferent means of inducing melanotic mass formation:
affecting the fat body, impinging on prohemocyte devel-
opment or modifying differentiated blood cells.
Embryo-derived plasmatocytes can cell autonomously
differentiate into lamellocytes
The fact that some of the genes we recovered in the
screen induced melanotic mass formation and lamello-
cyte differentiation when they were knocked-down spe-
cifically in embryonic plasmatocytes raised several
questions. In particular we wondered whether these
genes knock-downs provoked a cell-autonomous trans-
formation of embryo-derived plasmatocytes into lamello-
cytes. Alternatively, these knock-downs might non-
autonomously induce lamellocyte differentiation in the
lymph gland. To test these possibilities, we monitored the
fate of embryo-derived hemocytes that expressed the
UAS-dsRNA as compared to that of the lymph gland-
derived hemocytes. Accordingly, we used the flip-out
technique to permanently label embryonic plasmatocytes
and follow their fate in larval stages. Flies carrying the
embryonic-specific plasmatocyte drivers sn-Gal4 or gcm-
Figure 4 Lymph gland differentiation status. Expression of the lamellocyte differentiation marker α-ps4 (A-F) or of the prohemocyte marker tepIV
(G-L) was revealed by in situ hybridization on lymph glands from early third instar larvae expressing UAS-dsRNA targeting the indicated gene under the
control of srp-Gal4.
Table 2: Melanotic masses and lamellocytes are induced in response to defects in specific tissues
UAS-dsRNA
driverexpression patternwild type
cct2 cul4Hyx mRpS30 ush
srp-Gal4 EH, LH, LG, FB- / -+ / + + / ++ / + + / ++ / +
cg-Gal4EH, LH, LG (CZ), FB- / - + / + + / + + / ++ / + + / +
hmlΔ-Gal4 LH, LG (CZ), HG- / - + / + + / ++ / + + / ++ / +
sn-Gal4EH - / -- / - + / ++ / +- / - + / +
gcm-Gal4 EH, GC - / -+ / + + / ++ / + - / -- / +
tepIV-Gal4LG (MZ), CNS- / - + / + + / +Lethal - / - - / -
fb-Gal4FB, WD, CNS- / - - / -+ / + - / - + / +- / -
cad-Gal4 HG - / -- / - - / -- / -- / -- / -
repo-Gal4 GC- / -- / - - / -- / -- / -- / -
elav-Gal4CNS, PNS - / - lethal - / -Lethallethal- / -
MS1096WD - / - - / - - / -- / - - / -- / -
CNS: central nervous system, HG: hindgut, PNS: peripheral nervous sytem, CZ: cortical zone, EH: embryonic hemocytes; FB: fat body, GC: glial
cells, LH: larval hemocytes, LG: lymph gland, MZ: medullary zone, WD: wing disc. - / -: absence of melanotic masses or lamellocytes;+ /+:
presence of melanotic masses and lamellocytes; - / +: rare melanotic masses but presence of lamellocytes.

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Gal4 recombined with a UAS-FLP were crossed to a
strain bearing a flip-out cassette (Act5C > FRT > CD2 >
FRT > Gal4) and a UAS-GFP. This technique allowed us
to recover GFP-expressing circulating blood cells in third
instar larvae in wild type conditions, whereas GFP-
expressing cells were never observed in the absence of
UAS-FLP (Figure 5A, B and D). Labeling with the plasma-
tocyte-specific marker P1/NimC1 confirmed that these
cells were plasmatocytes (Additional file 3, Figure S3). Of
note, probably due to the limited efficiency of the FLIP-
FRT recombination, only a fraction of the blood cells was
GFP+. Also, sn-Gal4 reproducibly gave higher frequency
of GFP+ cells than gcm-Gal4. Importantly, we never
observed GFP+ cells in the lymph glands demonstrating
that these drivers are not sporadically expressed in this
compartment during larval development and that
embryo-derived plasmatocytes do not normally enter the
lymph gland (Figure 5E-H).
We then made use of this technique to label the cells
expressing a dsRNA targeting ush. As reported above
(Table 2), expression of ush dsRNA under the control of
gcm-Gal4 or sn-Gal4 was sufficient to induce the differ-
entiation of lamellocytes in circulation, as revealed by in
situ hybridization against α-ps4 and morphological analy-
sis (Figure 5I and 5K). In the absence of Flipase, no GFP+
cells were observed in circulation or in the lymph gland,
indicating that these two drivers are not re-activated
upon lamellocyte differentiation (Figure 5I, K, M and
5O). Strikingly, we observed GFP+ lamellocytes in circu-
lation in the presence of Flipase (Figure 5J and 5L) and
these GFP+ cells were also recovered in larvae with intact
lymph glands. Therefore ush loss of function is sufficient
to induce the cell autonomous transformation of embryo-
derived plasmatocytes into lamellocytes. In addition,
while gcm-Gal4-driven ush dsRNA induced lamellocyte
production in circulation but not in the lymph gland (Fig-
ure 5J and 5N), we observed lamellocyte differentiation in
both compartments using sn-Gal4 (Figure 5L and 5P).
Again, none of the lamellocytes in the lymph gland were
GFP+, indicating that they do not arise from sn-Gal4-
expressing blood cells. This indicates that embryo-
derived plasmatocytes participate in the production of
lamellocytes both through cell autonomous and non-
autonomous processes. Since sn-Gal4 gave rise to more
GFP+ cells than gcm-Gal4, it is possible that the non-
autonomous induction of lamellocytes in the lymph
gland is elicited in response to a threshold level of signal-
ization by circulating hemocytes. To assess whether
Figure 5 Fate of embryonic blood cells. (A-T) Blood smears (A-D, I-L and Q-T) and lymph glands (E-H and M-P) of third instar larvae. The Act > FRT >
CD2 > FRT > GAL4 cassette and the UAS-FLP and UAS-GFP transgenes were used to permanently label the cells that express sn-Gal4 or gcm-Gal4. Im-
munostaining against GFP (green, also displayed in white on right panel of each blood smear) was used to monitor gcm-Gal4, UAS-GFP or sn-Gal4,
UAS-GFP expression. In situ hybridization against α-ps4 (red) was used to reveal lamellocyte differentiation. Nuclei were counterstained with DAPI. GFP
labeling alone is shown to the right. (A-H) larvae raised in wild-type conditions, (I-P) larvae carrying a UAS-dsRNA transgene against ush, (Q-T) larvae
submitted to parasitization by L. boulardi. (A, E, I, M, Q) gcm-Gal4, UAS-GFP; Act > FRT > CD2 > FRT > GAL4; (B, F, J, N R) UAS-FLP; gcm-Gal4, UAS-GFP; Act
> FRT > CD2 > FRT > GAL4; (C, G, K, O, S) sn-Gal4, UAS-GFP; Act > FRT > CD2 > FRT > GAL4; (D, H, L, P, T) UAS-FLP; sn-Gal4, UAS-GFP; Act > FRT > CD2 > FRT >
GAL4;

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embryo-derived plasmatocytes also give rise to lamello-
cytes upon a natural immune challenge, we infected wild
type larvae with eggs from the parasitoid wasp L. bou-
lardi. As shown Figure 5Q-T, wasp infection induced
lamellocyte differentiation and flip-out analysis showed
that some of these lamellocytes are derived from cells
that had expressed gcm-Gal4 or sn-Gal4. Thus, we con-
clude that embryo-derived plasmatocytes can differenti-
ate into lamellocytes both upon intrinsic modification of
the blood cell developmental program or in response to
parasitoid infection.
To get further insights into the lamellocyte differentia-
tion process induced by melanotic tumor suppressor
genes or wasp infection, we analyzed circulating blood
cell differentiation status by monitoring the expression of
the plasmatocyte-specific marker P1/NimC1 and the
lamellocyte-specific marker msn-lacZ. In control larvae,
most blood cells expressed P1/NimC1 and only back-
ground β-gal staining was detected (Figure 6A). On the
contrary, in third instar larvae expressing dsRNA against
ush or cul4 under the control of srp-Gal4, msn-lacZ was
expressed in most hemocytes (Figure 6B and 5C). Inter-
estingly, a large fraction of these β-Gal+ hemocytes also
expressed P1, albeit often at lower levels. Similarly, 24 h
after infection by L. boulardi, we found that most P1+
cells also expressed msn-lacZ (Figure 6D). However, cells
expressing both markers were rare 48 h after infestation:
almost all the β-Gal+ cells corresponded to typical lamel-
locytes with large flattened morphology, big nuclei and
no P1 staining, whereas P1+ hemocytes had the charac-
teristic small and round morphology of plasmatocytes
(Figure 6E). Thus, together with the above results, these
data suggest that lamellocyte differentiate from plasmato-
cytes via a stepwise process implicating activation of
msn-lacZ, change in cell morphology and repression of
P1 expression.
Discussion
In this study we have conducted a loss of function screen
to identify factors that regulate Drosophila blood cell
development and/or function. Thus far, screens aiming at
uncovering genes controlling hemocyte development
relied primarily on the use of zygotic mutants potentially
acting in other tissues ([25] and references therein) or on
misexpression of factors potentially not expressed in
blood cells [55,56]. Likewise, the conclusion that a given
melanotic suppressor gene was specifically affecting
blood cells rather than an other tissue was based mostly
on the targeted expression of dominant negative (e.g. ush
or lwr with cg-Gal4) [41,57], rescue experiments (e.g.
vsp35 with hml-Gal4, or ADGF-A with cg-Gal4) [58,59],
or other indirect evidences (e.g. cactus, yantar, zfrp8)
[40,60,61]. To target more specifically genes expressed in
hemocytes and to be able to study genes required for
embryonic or early larval viability, we choose a tissue-
specific loss of function approach that relied on the use of
a collection of UAS-dsRNA transgenic lines and of three
different Gal4 drivers expressed in the hemocytes. To the
best of our knowledge this is the first time that such cell-
targeted loss of function approach is used to identify new
regulators of blood cell function and development.
By screening 1340 genes by RNA interference, we
recovered 96 candidate melanotic tumor suppressor
genes among which 59 were confirmed with secondary
RNAi lines and/or by genetic means. This corresponds to
a hit rate of 7.1% (4.4% if we only consider confirmed
hits). For comparison, a recent genome wide RNAi screen
for genes involved in intestinal pathogenic bacterial infec-
tion resulted in 8.6% of hits [62], while a gain of function
screen in larval hemocytes led to 3.2% of hits [55]. Yet, in
neither case a systematic validation of the candidates was
carried out. In a genome-wide RNAi screen for genes
affecting adult thorax development (19.6% of hits, exclud-
ing genes required for viability) [63], 63% of the 73 candi-
Figure 6 Plasmatocyte and lamellocyte relationships. (A-E) Dou-
ble fluorescent immunostainings on blood smears from early third in-
star larvae showing the expression of the plasmatocyte specific marker
P1 (green) and of the lamellocyte specific marker msn-lacZ (red). Nuclei
were counterstained with DAPI (blue). (A) wild type larvae, (B-C) larvae
expressing dsRNA against ush (B) or cul4 (C) under the control of srp-
Gal4, (D-E): larvae infected by L. boulardi, 24 h (D) or 48 h (E) after para-
sitization.

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dates retested with secondary RNAi lines were
confirmed, which is similar to our confirmation rate (94
candidates retested with secondary RNAi lines, 62% con-
firmed). Since in 31% of the cases, the primary and sec-
ondary RNAi lines targeted overlapping sequences, we
cannot rule out that some of the melanotic masses might
be caused by off-target effects. The analysis of the expres-
sion pattern of the candidates and of the RNAi efficiency
might help resolving this issue. Interestingly though, data
mining showed that most of the confirmed genes are
functionally connected to one another or to a previously
identified melanotic suppressor, further substantiating
our conclusion that they work in a common process.
Thus, altogether, this screen allowed us to find 59 genes
that likely contribute to larval blood cell homeostasis,
among which 55 had not been directly associated with
blood cell development or function before.
In contrast to the customary partition of the melanotic
tumor suppressor into class I/ class II genes [25,27], we
propose that the capacity to elicit melanotic mass devel-
opment is restricted to the blood cells and the fat body
(i.e. immune tissues). Indeed, none of the candidates we
tested induced lamellocytes or melanotic masses when
knocked-down in other territories. Similarly, knocking
down the melanotic tumor suppressors ddb1 or DREF by
RNAi in several non-immune tissues did not induce mel-
anotic masses [42,50], and HopTuml or a dominant nega-
tive form of GCM induced melanotic masses when
ectopically expressed respectively in blood cells or in the
fat body, but not in other tissues [31,64,65]. All together,
there is no strong evidence that melanotic capsule can
arise from genetic defects outside the hematopoietic sys-
tem or the fat body. Indeed, the rare "class I" mutations
that have been studied (kurtz, tuW and tu-Szts) have been
shown to affect fat body integrity [28,66]. While our
results demonstrate for the first time that fat body-spe-
cific loss of function can cause lamellocyte differentiation
and melanotic mass production, the mechanisms
involved remain unclear. The fat body may be a specific
source of signaling molecules that activate lamellocyte
differentiation or more generally control hemocyte differ-
entiation. Conversely, several lines of evidence show that
hemocytes can signal to the fat body to regulate the
humoral immune response [12,13,67,68]. This cross talk
between the fat body and the hematopoietic system is
likely to play a crucial role in coordinating the cellular
and humoral immune response to ensure efficient
defense of the organism.
Our results show that, within the hematopoietic sys-
tem, loss of function in larval lymph gland prohemocytes,
in differentiated larval blood cells or in embryonic plas-
matocytes is sufficient to induce melanotic masses in the
larvae. The only resilient blood cell type seemed to be the
crystal cells, as we never observed nodules or lamello-
cytes induction with the lz-Gal4 driver, even by overex-
pressing with this driver the two paradigmatic melanotic
tumor inducers Toll10b and HopTum (AAR, unpublished
observations). Thus melanotic tumor formation can serve
as a read out to identify genes potentially controlling sev-
eral steps of blood cell development. For instance, it may
help defining the gene regulatory network that control
the maintenance of a pool of stem-like blood cells in the
lymph gland cortical zone [8,9]. Actually, the respiratory
chain component ND75 that we recovered in the screen,
was recently shown to participate in the maintenance of
these progenitors by controlling the levels of reactive oxy-
gen species [39].
An important finding that stems out of the analysis of
some genes identified in the screen is that impinging on
the function of embryo-derived hemocytes is sufficient to
cause lamellocyte differentiation in the larvae. Our cell
lineage analysis demonstrates that embryo-derived plas-
matocytes cell-autonomously give rise to lamellocytes in
response to a genetic defect (ush loss) or to wasp infec-
tion. These results are consistent with and extend the
recent observation that embryo-derived hemocytes can
differentiate in lamellocytes after wasp infection [24].
Whereas Markus et al. proposed that lamellocytes differ-
entiate from hemocyte precursors [24], our analysis
strongly suggests that they derive from plasmatocytes
through a step-wise process. In addition, we found that
the presence of "mutant" embryo-derived blood cells
induced non-autonomously the differentiation of lamel-
locytes in the lymph gland, indicating that circulating
hemocytes signal to this hematopoietic organ. Mutations
in the EBF transcription factor Collier (which is
expressed in the posterior signaling centre) or in the JAK/
STAT signaling pathway (which is active in the medullary
zone) were shown to induce precocious differentiation of
the lymph gland progenitors and to suppress lamellocyte
fate, strongly suggesting that lamellocyte differentiate
solely in the lymph gland from a pool of progenitors
[8,22]. Alternatively, we propose that the posterior signal-
ing centre may orchestrate differentiation into lamello-
cytes of both circulating and lymph gland blood cells.
Thereby, full blown lamellocyte differentiation and mel-
anotic nodule formation would result from a cross talk
between the patrolling larval blood cells and the lymph
gland.
Finally our result shed new light on the function of the
Friend of GATA transcription cofactor Ush, which had
already been implicated in several steps of blood cell
development [41,69-72]. Consistent with our results, it
was shown that hypomorphic ush zygotic mutations or
the ectopic expression of a dominant negative form of
Ush under the control of cg-Gal4 induced lamellocyte
differentiation [41]. Moreover, it was proposed that Ush
was required in the lymph gland to prevent lamellocyte

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differentiation in a putative plamatocyte/lamellocyte
common progenitor [41,69]. Yet the fate of the ush
mutant cells had not been tracked. Remarkably, we dem-
onstrate here that ush function is not restricted to the
lymph gland. Indeed ush loss in circulating plasmatocytes
during embryogenesis is sufficient to cause the cell-
autonomous transformation of these cells into lamello-
cytes and to promote lamellocyte development in the
lymph gland. Thus down-regulation of ush function in
the circulating larval blood cells could be an initiating
event in the immune response that leads to melanotic
mass formation. Our results also identify ush as the first
gene controlling the fate of the circulating larval blood
cells and lay the basis for the analysis of the gene net-
works controlling this hematopoietic compartment.
Conclusions
In this study, we show that lamellocyte differentiation and
melanotic tumor formation can be elicited specifically by
defects in different immune compartments (embryo-
derived blood cells, larval hematopoietic organ or fat
body). These results shed new lights on the coordination
of the cellular immune response and on blood cell lineage
relationships in Drosophila. Notably, we demonstrate
that embryo-derived plasmatocytes are plastic cells that
can differentiate into lamellocytes and that ush is a key
regulator of this process. Finally, this work pinpoints sev-
eral new genes and pathways controlling Drosophila
blood cell homeostasis. Their identification paves the way
for future experiments aiming at dissecting their mecha-
nism of action and their interplay with other known key
regulators of Drosophila hematopoiesis. It is anticipated
that deciphering the function of these genes in the differ-
ent blood cell types will shed new light on the mecha-
nisms controlling blood cell homeostasis and cellular
immune response in Drosophila and, by homology, in
mammals.
Methods
Fly strains and genetic crosses
Flies were raised at 25° on standard cornmeal and agar
media. The following strains were used: srp-Gal4 [73];
hmlΔ-Gal4 [37]; cg-Gal4 [36]; gcm-Gal4 [35]; sn-Gal4
(Zanet et al., in preparation); fb-Gal4 (from M. Meister);
cad-Gal4, tepIV-gal4 (from Kyoto DGRC); MS1096, en-
Gal4, repo-Gal4, elav-Gal4, Act5C > FRT > CD2 > FRT >
Gal4, UAS-FLP, msn-lacZ, UAS-EGFP, UAS-mCD8GFP
(from Bloomington). UAS-dsRNA transgenic lines were
obtained from the Japanese National Institute of Genetic
(NIG), the Vienna Drosophila Resource Center (VDRC)
and Bloomington.
A collection of 1941 UAS-dsRNA transgenic lines tar-
geting 1341 genes (Additional file 4, Table S1) obtained
from NIG was analyzed in our primary screen. For this,
5-7 virgin females carrying either the hmlΔ-Gal4 or the
srp-Gal4 driver were crossed to 3-4 males carrying the
different UAS-dsRNA transgenes. Vials were changed
every two days and presence of melanotic masses was
evaluated in the progeny by examinating under the dis-
section microscope an average of 20 wandering larvae.
Candidate UAS-dsRNA lines inducing melanization with
at least one driver were systematically retested with both
drivers as well as with cg-Gal4 and the corresponding
"tumor" indices (percentage of larvae harboring at least
one melanotic masse) were determined on a minimum of
50 larvae. For validation of the candidate genes, indepen-
dent secondary UAS-dsRNA lines or mutant alleles were
obtained from VDRC and Bloomington.
Hemocyte labeling
To observe the circulating larval hemocytes, third instar
larvae were thoroughly washed in PBS and ethanol 75%
and bled on polylysine-coated glass slides (Nunc). For
immunostaining and/or phalloidin labeling, hemocytes
were briefly air-dried and then fixed for 15 min in 4%
paraformaldehyde in PBS. After two washes in PBS,
hemocytes were permeabilized for 15 min in PBS, 0.3%
Triton (PBST), rinsed twice in PBST-1% BSA, and
blocked 15 min in PBST-1% BSA. Hemocytes were incu-
bated 2 h at room temperature or overnight at 4°C with
primary antibody. After several 15 min washes, cells were
incubated 2 h with secondary antibody and / or with
phalloidin SR101 (1:200) (SIGMA) and washed 4 times in
PBST. Slides were mounted in Vectashield-DAPI
medium. Double fluorescent immuno-staining and in
situ hybridization on circulating blood cells were per-
formed as described in [74].
For lymph gland analysis, third instar larvae were dis-
sected in ice cold PBS, fixed for15 min in 4% paraformal-
dehyde, washed three times 15 min in PBST and pre-
incubated for 1 h at 60°C in Hybridization Buffer (HB:
50% formamide, 2 × SSC, 1 mg/ml Torula RNA, 0.05 mg/
ml Heparin, 2% Roche blocking reagent, 0.1% CHAPS, 5
mM EDTA, 0.1% Tween 20). Larvae were then incubated
overnight at 60°C with DIG-labeled RNA probe, washed
twice for 1 h in 50% HB-50% PBST at 60°C and three
times 20 min in PBST-1% BSA at room temperature,
before 3 h incubation with sheep anti-DIG antibody con-
jugated to alkaline phosphatase (1:2000; Roche). Finally,
larvae were extensively washed in PBST and the in situ
hybridization signal was revealed with FastRed or NBT/
BCIP substrates. Lymph glands were mounted in 50%
glycerol-PBS or in Vectashield-DAPI.
The following primary antibodies were used: mouse
anti-P1/NimC1 (1:30) (kind gifts from I. Ando, [75]), rab-
bit anti-ϐ galactosidase (1:1000, Cappel), rabbit anti-GFP
(1:1000; Torrey). The corresponding secondary antibod-
ies coupled to Alexa fluor 488 or 555 (1:400) (Molecular

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Probes). For in in situ hybridization, we used DIG-UTP
labeled α-ps4 and tepIV anti-sense RNA probes [53,76].
For in situ hybridization followed by immunostaining,
primary antibodies were used 5 times more concentrated.
Wasp egg parasitization
To obtain synchronous larval populations, females were
left to lay eggs for 4-6 hours. Second instar larvae were
submitted to infection by L. boulardi during 2-4 h, and
then allowed to develop 24 or 48 h before being analyzed
as described above.
Additional material
Authors' contributions
AAR and KB performed most experiments, contributed to acquisition of all
data, analyzed and interpreted the data. CP participated in the design of the
experiments and interpretation of the results. CP, VG, DO, BA, FR participated in
the realization of the screen and provided important genetic tools. JZ provided
important genetic tools. MH and LW conceived and coordinated the project.
AAR, KB, CP, FR and MH have been involved in drafting the manuscript. LW
wrote the manuscript. All authors read and approved the final manuscript.
Acknowledgements
We deeply thank the National Institute of Genetics Fly Stock Center (Japan), the
Bloomington Drosophila Stock Center, the Vienna Drosophila Resource Centre
as well as S. Sinenko and I. Ando for fly stocks and reagents. We thank Toulouse
RIO imaging platform for assistance with confocal microscopy. This work was
supported by the CNRS and by grants from the Agence Nationale pour la
Recherche, Association pour la Recherche sur le Cancer and Association for
International Cancer Research.
Author Details
1Université de Toulouse, UPS, CBD (Centre de Biologie du Développement),
Bât4R3, 118 route de Narbonne, 31062 Toulouse, France, 2CNRS, CBD
UMR5547, 31062 Toulouse, France and 3King's College London, Guy's Campus,
London SE1 1UL, UK
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9.
Additional file 1 Figure S1. Expression pattern of different Gal4 lines in
the embryo and in the circulating hemocytes, lymph gland and fat body of
third instar larvae. The activity of the indicated Gal4 lines was revealed
using either a UAS-lacZ or a UAS-GFP reporter transgene whose expression
was detected respectively by in situ hybridization against lacZ in embryos or
by fluorescent immunostaining against GFP in third instar larvae. Circulat-
ing hemocyte actin cytoskeleton was labeled with phalloidin (red). Lymph
gland and fat body nuclei were counterstained with DAPI (blue).
Additional file 4 Table S1. Results from the primary and secondary
screens.
Additional file 5 Table S2. Validation of candidates with secondary UAS-
dsRNA lines.
Additional file 6 Table S3. List of confirmed melanotic suppressor genes.
Additional file 2 Figure S2. High magnification view of a melanotic mass
(induced using srp-Gal4, UAS-GFP; UAS-ds-Ush). GFP and phalloidin staining
show that the mass is surrounded by lamellocytes.
Additional file 3 Figure S3. Flip-out analysis of the expression pattern of
sn-Gal and gcm-Gal4 in circulating larval blood cells. Blood cells smears
from third instar larvae of the indicated genotypes were processed to reveal
nuclear-GFP (in green) and P1/NimC1 (in red) expression by double fluores-
cent immuno-labeling. Nuclei were counterstained with DAPI (blue).
Received: 19 February 2010 Accepted: 11 June 2010
Published: 11 June 2010
This article is available from: http://www.biomedcentral.com/1471-213X/10/65 © 2010 Avet-Rochex et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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doi: 10.1186/1471-213X-10-65
Cite this article as: Avet-Rochex et al., An in vivo RNA interference screen
identifies gene networks controlling Drosophila melanogaster blood cell
homeostasis BMC Developmental Biology 2010, 10:65