Cellular Microbiology (2007) 9(5), 11171126

doi:10.1111/j.1462-5822.2007.00930.x
First published online 29 March 2007

Microreview
Drosophila haematopoiesis
Michle Crozatier1 and Marie Meister2*
1
Centre de Biologie du Dveloppement, UMR 5547 and
IFR 109, CNRS/Universit Paul Sabatier, 118 route de
Narbonne, 31062 Toulouse, France.
2
Museum of Zoology, 29 boulevard de la Victoire, 67000
Strasbourg, France.
Summary
Like in vertebrates, Drosophila haematopoiesis
occurs in two waves. It gives rise to three types of
haemocytes: plasmatocytes (phagocytosis), crystal
cells (melanization) and lamellocytes (encapsulation
of parasites). A first population of haemocytes, specified during embryogenesis, gives rise to an invariant
number of plasmatocytes and crystal cells. A second
population of haemocytes is specified during larval
development in a specialized haematopoietic organ,
the lymph gland. All three types of haemocytes can be
specified in this organ, but lamellocytes only differentiate in response to parasitism. Thus, larval in contrast to embryonic haematopoiesis can be modulated
by physiological constraints. Molecular cascades
controlling embryonic haematopoiesis are relatively
well established and require transactivators such as
GATA, FOG and Runx factors, which are also
co-opted in mammalian haematopoiesis. Mechanisms
involved during larval haematopoiesis are less well
understood although a number of chromatin remodelling factors and signalling pathways (JAK/STAT,
Toll, Hedgehog, Notch) are required. In healthy larvae
a pool of progenitors is maintained within the lymph
gland, under the control of a signalling centre which
expresses Collier, Serrate, Antennapedia and Hedgehog, and controls haemocyte homeostasis. Its key
role in haemocyte homeostasis is reminiscent of
interactions described in vertebrates between haematopoietic stem cells and their microenvironment
(niche).

Received 22 December, 2006; revised 2 February, 2007; accepted 5

February, 2007. *For correspondence. E-mail Marie.Meister@
zool-ulp.u-strasbg.fr; Tel. (+33) 3 90 24 04 84; Fax (+33) 3 90 24
05 58.
Journal compilation 2007 Blackwell Publishing Ltd
No claim to original French government works

Introduction
In the past half century, Drosophila has successfully
served as an experimental model to unravel many developmental processes and has paved the way to inumerable studies on mammalian development. It is now used
as a model system to decipher molecular pathways that
regulate innate immunity, and again these studies have
highlighted key players in immune functions in both invertebrate and vertebrate systems (review in Lemaitre and
Hoffmann, 2007). However, only very recently has an
interest in Drosophila haematopoiesis arisen with the first
genetic and molecular reports dating back only some
10 years. It has become rapidly evident that many parallels can be drawn between vertebrate blood cell development and Drosophila haematopoietic processes (review
in Evans et al., 2003). To illustrate this, vertebrate haematopoiesis occurs in two successive waves named
primitive and definitive haematopoiesis, which are paralleled by Drosophila embryonic and larval haematopoiesis.
The signalling pathways and transactivators that regulate
both processes share many conserved components, and
eventually, the blood cells, or haemocytes, that are produced in Drosophila have features and exert functions
similar to those of the mammalian myeloid lineages
(review in Meister and Lagueux, 2003).
Description of the haematopoietic system
Different types of haemocytes and functions
The development of Drosophila comprises four distinct
stages, embryonic, three larval, pupal and adult stages.
Each of them exhibits a specific haemocyte composition.
Compared with the complexity of the blood cell lineages in
mammals, the Drosophila system is rather simple as only
three types of mature haemocytes can be distinguished
(Shrestha and Gateff, 1982; Rizki and Rizki, 1984; Lanot
et al., 2001; Hartenstein, 2006). Plasmatocytes, the major
function of which is phagocytosis, are the predominant
haemocyte population. In the embryo they engulf apoptotic corpses formed during developmental processes
(Tepass et al., 1994; Franc et al., 1996; 1999; Sears et al.,
2003). In larvae and adults they are responsible for
phagocytosis of invading bacteria or fungi (Ramet et al.,
2001; Kocks et al., 2005). At the pupal stage they play a

1118 M. Crozatier and M. Meister

fundamental role as they engulf and recycle doomed cells
during metamorphosis (Lanot et al., 2001). In addition to
their phagocytic function, plasmatocytes also produce
and secrete a number of peptides/proteins, such as extracellular matrix proteins and antimicrobial peptides following an infection (Fessler et al., 1994; Dimarcq et al.,
1997). Last but not least, they are believed to secrete
signals which inform distant tissues of an infection
(Agaisse et al., 2003; Irving et al., 2005). Secretory cells
(Lanot et al., 2001) have been described within the lymph
gland, as well as podocytes (Shrestha and Gateff, 1982)
in circulation in late larvae. They most likely correspond to
variations of the plasmatocyte lineage. The second type of
haemocytes, called crystal cells, contain conspicuous
crystalline structures and make up some 5% of the
haemocyte population in embryos and larvae (Shrestha
and Gateff, 1982; Lebestky et al., 2000) and disappear at
the onset of metamorphosis. The crystals are composed
of prophenoloxidase, the zymogen that is ultimately
cleaved by a proteolytic cascade called the melanization
cascade (Ashida and Brey, 1997). Melanization is an
immune reaction that results in the deposit of a black
pigment (melanin) during wound healing, or during
capsule formation (see below) and it is phenoloxidase
which catalyses the enzymatic reactions that produce
melanin. Finally, the third haemocyte type, the lamellocyte, can only be found at the larval stage. Lamellocytes
are rarely observed in healthy larvae, but differentiate
massively following infestation by parasitoid wasps which
lay their eggs in young larvae (Rizki and Rizki, 1992;
Lanot et al., 2001; Sorrentino et al., 2002; Crozatier et al.,
2004). Lamellocytes form a multilayered capsule around
the wasp egg which has been initially detected as a
foreign body probably by plasmatocytes (Russo et al.,
1996), the immune supervisors that constantly patrol in
circulation. The caspsule is then melanized through the
activity of the phenoloxidase. The parasite is killed within
the caspule, possibly via the production of cytotoxic intermediates during melanin synthesis (Nappi and Vass,
1993). Thus, lamellocytes are specifically devoted to the
fight against invaders too large to be phagocytosed by
plasmatocytes.
Origin of haemocytes
As mentioned above, two waves of haematopoiesis occur
in Drosophila. A first wave called embryonic haematopoiesis takes place in the head mesoderm of early embryos
(Fig. 1a) and gives rise to a fixed number of lineages and
haemocytes, including some 700 plasmatocytes that
migrate throughout the embryo (Tepass et al., 1994), and
about 30 crystal cells which remain located around the
proventriculus, an anterior structure of the midgut (Lebestky et al., 2000). The second wave of haematopoiesis,

embryonic
haemocytes

lymph gland
precursors

b
+

gcm

stage 5

stage 7

stage 10

gcm

gcm

gcm

gcm gcm
lz+
10% 10%
+

lz+

gcm

gcm

gcm

gcm

gcm

80%

lz+
6%

gcm

4%

90%

6%

94%

crystal cells

plasmatocytes

stage 14

Fig. 1. Embryonic haematopoiesis.

a. Blastoderm fate map of the embryonic haemocytes (red) and
lymph gland-derived haemocyte territories (orange), located within
the mesoderm anlage (yellow) which is represented by the ventral
region of the blastoderm embryo (from Holz et al., 2003;
reproduced with permission of the Company of Biologists).
b. Schematic representation of blood cell fate resolution during
Drosophila embryogenesis. Initially all prohaemocytes express gcm.
Subsequently, gcm transcription is turned off and lz starts to be
activated in the first row of prohaemocytes but not in the others
which will differentiate into plasmatocytes. 60% of these lz +
progenitors maintain lz expression via an autoactivation loop and
will differentiate into crystal cells, while in the remaining 40% the
presence of residual Gcm interferes with lz expression and
promotes plasmatocyte differentiation (from Bataille et al., 2005;
reproduced with permission of the Company of Biologists).

or larval haematopoiesis, takes place in a dedicated

organ called the lymph gland (Shrestha and Gateff, 1982;
Lanot et al., 2001; Jung et al., 2005). In third instar larvae,
the lymph gland is composed of two to seven pairs of
haemocyte-containing lobes which are located alongside
the dorsal vessel, the aorta/heart tube of the open circulatory system of Drosophila, where they are associated
with pericardial cells that are proposed to have nephrocyte function (review in Evans et al., 2003). The lymph
gland has a mesodermal origin and forms during embryogenesis (Fig. 1a). From the end of embryogenesis until
the second larval instar the lymph gland is composed of a
single pair of lobes called the anterior or primary lobes

Journal compilation 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 11171126

No claim to original French government works

Drosophila haematopoiesis 1119

Late embryo

Late larva
cortical zone
medullary zone
PSC

cardiac tube
pericardial cells

primary lobes

secondary lobes

b
PSC
medullary zone
(prohaemocytes)
cortical zone
(crystal cells)

d
plasmatocytes
crystal cells
cortical
zone
medullary
zone

lamellocytes
plasmatocytes
crystal cells

JAK/STAT

PSC

PSC
N
prohaemocytes
normal conditions

prohaemocytes
parasitized conditions

S2?

S1
wasp
egg

Fig. 2. Model for larval haematopoiesis.

a. Schematic representation of the lymph gland at the end of embryogenesis and the end of third larval stage. Anterior is to the left.
b. Confocal imaging of a third instar larva lymph gland: the medullary zone is identified by the expression of a membrane-targeted GFP
(domeless-Gal4; UAS-mCD8 GFP, green), the PSC is marked by Col expression (blue), crystal cells in the cortical zone are marked by
antiprophenoloxidase expression (red). GFP and Col are also expressed in non-overlapping cells in the secondary lobes.
c. In third instar larvae during normal development, PSC cells (blue) act, in a non-cell-autonomous manner, to maintain JAK/STAT signalling in
medullary zone cells (green). JAK/STAT activity is required to prevent premature differentiation of multipotent prohaemocytes into
plasmatocytes and crystal cells that would migrate into the cortical zone. Notch (N) signalling mediated by Ser expression in the PSC is
required to maintain high levels of col transcription.
d. Reprogramming of prohaemocytes in response to parasitization: a signal likely emitted by circulating plasmatocytes (red circles) upon their
detection of wasp eggs is perceived by prohaemocytes either directly (S2) or via the PSC (S1> S2 relay), or both. In either case, Col activity
in the PSC is required for prohaemocytes to adopt the lamellocyte fate. c and d were adapted from (Krzemien et al., 2007).

(Fig. 2a). In early third instar larvae new pairs of posterior or secondary lobes develop. Haemocyte differentiation in the lymph gland is first observed in primary lobes
of early third instar larvae (Lebestky et al., 2000; Jung
et al., 2005). In healthy larvae very few haemocytes differentiate in the secondary lobes whereas an immune
challenge such as wasp infestation (see below) triggers

the premature differentiation of all haemocyte types in

these lobes. At the onset of metamorphosis most prohaemocytes differentiate into plasmatocytes, the lymph
gland desintegrates and delivers its content into the circulation (Lanot et al., 2001). There is, so far, no reported
evidence for the existence of an adult haematopoietic
organ.

Journal compilation 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 11171126

No claim to original French government works

1120 M. Crozatier and M. Meister

Recently, an elegant study based on transplantations of
cells from donor marked embryos into recipient embryos
clarified the origin of both larval and adult plasmatocytes
(Holz et al., 2003; Fig. 1a). This study demonstrated that
circulating plasmatocytes in larvae essentially derive from
the embryonic haematopoietic wave. As the number of
circulating haemocytes in larvae is about 10-fold higher
than that of embryos, it is assumed that cell division
occurs in circulation, which has already been described
(Rizki, 1957; Qiu et al., 1998), to account for this increase.
Labelled plasmatocytes from the lymph gland are not
released before the end of larval stages. Thus, in pupae
and adults the haemocyte population is composed of a
mixture of both embryonic and lymph gland-derived
haemocytes.
Embryonic haematopoiesis
The embryonic haemocyte anlage is determined very
early, before the blastoderm stage (Holz et al., 2003). A
good candidate for the determining factor of this cell fate
is Serpent (Srp), one of the five members of the GATA
transcription factor family in Drosophila, as srp expression
is observed in this domain from the blastoderm stage
onward (Bataille et al., 2005; de Velasco et al., 2006). It
has also been shown that srp is compulsory for haematopoietic development (Rehorn et al., 1996; Lebestky et al.,
2000). However, as srp is expressed in many other
tissues, there must be additional genes that induce
haemocyte determination at the blastoderm stage. Thus,
both srp expression and their relative position in the
embryo define cells of this anlage as haemocyte precursors which will further differentiate into plasmatocytes or
crystal cells. In vertebrates, GATA-1 to -3 play fundamental roles in haematopoietic development, namely proliferation and survival of stem cells, as well as differentiation of
several cell types (review in Orkin, 1998). Three transcription factors have been shown to participate in the secondary cell fate decision in Drosophila. (i) Lozenge (Lz) is a
member of the Runx family (Daga et al., 1996) and has
high homology to human AML1/Runx1, one of the most
frequent targets of chromosomal translocations leading to
acute myeloid leukemia (review in Blyth et al., 2005). lz
mutants do not develop crystal cells, neither in embryos
nor in the lymph gland (Lebestky et al., 2000). Thus, lz is
specifically required during haematopoiesis for the differentiation of crystal cells. (ii) U-shaped (Ush) is a member
of the family of Friend-of-GATA (FOG) multiple zinc-finger
proteins known to regulate GATA activity in mammals
(review in Cantor and Orkin, 2005). In mice, FOG-1 specifically regulates erythropoiesis and megakaryopoiesis in
conjonction with GATA-1 (Tsang et al., 1998). It was first
shown that Ush antagonizes crystal cell development
(Fossett et al., 2001), and more recent evidence suggests

that combinatorial regulation of crystal cell differentiation

is achieved in the embryo via subtle interplay between
Ush, Lz and the two isoforms of Srp, only one of which is
able to physically interact with Ush (Waltzer et al., 2002;
2003; Fossett et al., 2003). (iii) Glial cells missing (Gcm)
and Gcm2 are two closely related zinc-finger transactivators first identified for their role in glial cell specification in
the nervous system (Jones et al., 1995). It was subsequently established that Gcm plays an instructive role in
the development of the plasmatocyte lineage in embryos
(Bernardoni et al., 1997; Lebestky et al., 2000). This function is shared with Gcm2 which has a redundant function
(Alfonso and Jones, 2002).
The precise succession of early events that govern
lineage choice determination was recently analysed in the
embryonic haemocyte anlage (Bataille et al., 2005;
Fig. 1b). At the blastoderm stage, all Srp-positive cells
also express gcm, but this expression is rapidly downregulated in the anteriormost row of prohaemocytes which
then initiate lz expression. Lz-positive progenitors are a
mixed-lineage population whose fate depends on the relative level of Lz/Gcm-Gcm2, as Gcm-Gcm2 play a key role
in controlling the size of crystal cell population by inhibiting lz activation and maintenance. It must be emphasized
that srp remains expressed in haemocytes throughout
their lifetime (Lebestky et al., 2000) and also regulates
lineage-specific genes in combination with other transactivators (Waltzer et al., 2002; 2003).
Full maturation of plasmatocytes and their migration
throughout the embryo requires the activity of the PDGF/
VEGF Receptor (PVR (Duchek et al., 2001), a Receptor
Tyrosine Kinase which is specifically expressed in embryonic haemocytes (Heino et al., 2001; Cho et al., 2002;
Bruckner et al., 2004). In pvr loss-of-function embryos
plasmatocytes stop migrating and many of them undergo
apoptosis, and are subsequently phagocytosed by their
peers (Bruckner et al., 2004). Three ligands of PVR,
PVF1-3, are encoded in the Drosophila genome, which
are most closely related to mammalian VEGF (Duchek
et al., 2001). All three are expressed along haemocyte
migration routes, and their simultaneous mutation results
in the same phenotype as pvr loss-of-function (Cho et al.,
2002). Thus, PVR has at least two functions in embryonic
haemocytes: (i) it mediates anti-apoptotic survival of
blood cells throughout embryonic development; (ii) it is
required for their migration to the posterior end of the
embryo.
Although the processes which they control are not
strictly comparable, it appears that homologous transcription factors (GATA, FOG and Runx factors), or signalling
cascades (Receptor Tyrosine Kinase pathways) have
been co-opted both in Drosophila primitive haematopoiesis and in mammalian haematopoietic or endothelial
development.

Journal compilation 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 11171126

No claim to original French government works

Drosophila haematopoiesis 1121

Larval haematopoiesis
Ontogeny of the lymph gland
In early embryos the lateral thoracic mesoderm gives rise
to the lymph gland precursors (Holz et al., 2003; Fig. 1a).
collier (col), which encodes the Drosophila orthologue of
the vertebrate transcription factor Early B Cell Factor
(Hagman et al., 1993; Crozatier et al., 1996), is the earliest marker of these precursors (Crozatier et al., 2004). Col
protein is first observed in two discrete clusters of cells in
the dorsal mesoderm of thoracic segments T2 and T3, at
the germ-band extension stage. These two clusters of
Col-expressing cells are growing closer until ultimately
coalescing to form the primary lobes of the lymph gland.
The expression of the Odd-skipped transactivator,
another lymph gland marker, is detected slightly later than
col (Mandal et al., 2007). When the primary lobes are
formed, the lymph gland precursors start to express the
transcription factor Srp whose expression will be maintained during larval development (Lebestky et al., 2000;
Jung et al., 2005). In late embryos, the lymph gland consists of a single pair of lobes (primary lobes) containing
some 20 cells and col expression becomes restricted to
two to three posteriorly located cells per lobe, which prefigure the Posterior Signaling Center (PSC; Fig. 2a). The
PSC was previously identified by Serrate (Ser, a ligand of
Notch) expression in third instar lymph gland (Lebestky
et al., 2003; see below).
Evidence for a fruit fly haemangioblast
In vertebrate embryo it has repeatedly been proposed that
blood and vascular cells likely derive from a common
progenitor cell called the haemangioblast, which arises in
the yolk sac and in the aorta-gonadal-mesonephros mesenchyme (Medvinsky and Dzierzak, 1996; Kennedy et al.,
1997; Huber et al., 2004). Although a close relationship
between blood and vascular progenitors is well established, the existence of the haemangioblast is still controversial (Jaffredo et al., 2005; Bollerot et al., 2005; Ueno
and Weissman, 2006). However, very recently, the in vivo
evidence that a single cell can divide and give rise to both
a blood cell and an endothelial cell was described in
zebrafish (Vogeli et al., 2006). Recently, a clonal analysis
of the early steps of larval haematopoiesis in Drosophila
indicated that cardioblasts (prospective vascular cells)
and lymph gland cells can arise from the division of a
single progenitor cell, which the authors proposed to call
haemangioblast (Mandal et al., 2004). A previous study
(Alvarez et al., 2003) based on lineage tracing experiments using a cardiac marker showed that thoracic myocardial cells can give rise to mixed two-cell clones
composed of one cardioblast and one non-labelled cell
which could correspond to a lymph gland cell based on

the study performed by Mandal et al. (2004). Thus, the

ontogeny of larval haemocytes in Drosophila seems to
parallel that of vertebrate blood cells.
The PSC controls blood cell homeostasis
A recent description of the third instar lymph gland, based
on the expression pattern of various markers revealed the
existence of three distinct zones in the primary lobes
(Jung et al., 2005; Fig. 2a and b). The first zone identified
was called the PSC (see previous description) and was
defined by the expression of Ser (Lebestky et al., 2003)
and Col (Crozatier et al., 2004) in a small cluster of cells
located posteriorly. Two other zones called the medullary
zone and the cortical zone are composed of prohaemocytes and differentiated haemocytes, respectively
(Fig. 2a and b). In second instar larvae the lymph gland
consists only of the medullary zone and the PSC. Lineage
tracing experiments (Jung et al., 2005) established that in
third instar larvae the cortical zone cells derive from medullary zone prohaemocytes whereas the PSC cells remain
a distinct population of signalling haemocytes that do not
contribute to the cortical zone. PSC cells are distinguishable from other lymph gland cells from the end of embryogenesis, when they are the only cells that maintain col
transcription (Crozatier et al., 2004) and express the
homeotic gene Antennapedia (Antp) (Perrin et al., 2004;
Mandal et al., 2007). The identity of PSC cells, which is
established very early during embryonic development, is
under the control of Antp, the expression of which is
maintained throughout larval development (Mandal et al.,
2007). Analysis of col mutant lymph glands further established that col is required for the maintenance of the PSC
during larval development, and that the PSC plays a
crucial role in the specification of lamellocytes after wasp
parasitism (Crozatier et al., 2004). Two recent studies
indicate that the PSC is essential for the control of
haemocyte homeostasis in healthy larvae (Krzemien
et al., 2007; Mandal et al., 2007). For this, communication
between the PSC and haematopoietic progenitors is
crucial and requires: (i) a restricted expression of col to
the PSC through the localized expression of Ser; (ii) the
localized expression of the signalling molecule Hedgehog
(Hh) in the PSC and the non-automomous activation of
the Hh pathway in the prohaemocytes (Mandal et al.,
2007); and (iii) PSC-triggered activation of the JAK/STAT
signalling pathway in prohaemocytes in order to prevent
their premature differentiation (Krzemien et al., 2007;
Fig. 2c). How these signalling pathways are interconnected and control larval haemocyte homeostasis is not
yet deciphered. The observation of thin cytoplasmic
extensions called fillopodia, that extend from the PSC
cells, suggest that direct cellular contacts might be
involved (Krzemien et al., 2007; Mandal et al., 2007).

Journal compilation 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 11171126

No claim to original French government works

1122 M. Crozatier and M. Meister

Egg-laying by parasitoid wasps in Drosophila larvae
triggers the production of lamellocytes. Strikingly, this
occurs simultaneously with premature loss of the medullary zone and downregulation of the JAK/STAT pathway
(Krzemien et al., 2007; Fig. 2d) suggesting that the production of lamellocytes takes place at the expense of the
pool of prohaemocytes which, during normal development
are still present in the lymph gland when it bursts at
metamorphosis. This reprogramming, which bypasses the
normal haemocyte homeostasis, reveals an unexpected
level of plasticity of Drosophila prohaemocytes.
In mammals, the existence of a microenvironment for
blood cells within the bone marrow, in which stromal cells
influence the proliferation and differentiation of the
haematopoietic stem cells (HSCs) has been proposed
(Nagasawa, 2006; Wilson and Trumpp, 2006). The
fact that prohaemocytes and differentiated haemocytes
coexist within the lymph gland, together with the key role
of the PSC in controlling blood cell homeostasis are reminiscent of interactions between haematopoietic progenitors and their microenvironment in vertebrates. This
further highlights the interest of Drosophila as a model
system for studying haematopoietic processes.
Specification of larval haemocytes
Whereas srp and ush, the roles of which are well documented in embryos, are expressed in all lymph gland cells
during both embryonic and larval stages, their functions
within the lymph gland are not established yet. So far, Lz
is the only transcription factor known to be involved in
crystal cell differentiation both in embryos and in larvae. In
the lymph gland, Notch activity is also required in this
process (Duvic et al., 2002; Lebestky et al., 2003) and it
was shown that cell-autonomous activation of Notch triggers lz transcription, which in turn induces crystal cell
differentiation. While Notch requirement in the formation
of crystal cells in the lymph gland is clearly established, its
involvement in embryonic haematopoiesis is still controversial (Lebestky et al., 2003; Bataille et al., 2005). The
control of plasmatocyte specification is not understood in
larvae. Indeed, although gcm-gcm2 are key players in this
process during embryonic haematopoiesis, they are not
expressed in the lymph gland (Bataille et al., 2005) which
precludes any function in larval haematopoiesis. In addition, whereas pvr function in embryos is required for plasmatocyte survival and/or migration, clonal analysis in the
lymph gland of pvr mutant cells suggests that PVR is
specifically required for plasmatocyte differentiation (Jung
et al., 2005).
Finally, few genes have been described for their
involvement in lamellocyte specification. It was recently
shown that Hemese and Yantar, which, respectively,
encode a protein with a transmembrane domain and a

protein with a putative RNA-processing function, are

required to repress the differentiation of lamellocytes
(Kurucz et al., 2003; Sinenko et al., 2004). Lamellocyte
production in the absence of wasp infestation was
observed in several mutant contexts affecting either chromatin remodelling or proliferation (review in Evans et al.,
2003). Mutations in the Drosophila CREB binding protein
(dCBP) gene enhance melanotic tumour formation when
associated with modulo mutations (Bantignies et al.,
2002). modulo encodes a chromatin-associated factor.
Melanotic tumours, more appropriately named melanotic
nodules (Minakhina and Steward, 2006), are generated
by lamellocytes and may represent inappropriate encapsulation responses against self tissue (Rizki and Rizki,
1979). Similarly, mutations in the multi-sex combs PcG
gene, or in the nurf301 gene that encodes a subunit of the
NURF chromatin remodelling complex, cause increases
in haemocyte proliferation and lamellocyte production,
often associated with melanotic nodules (Badenhorst
et al., 2002; Remillieux-Leschelle et al., 2002). Chromatin
modification is a global and relatively generalized mechanism of gene regulation; however, many of its effectors
play specific roles in haematopoietic development. How
chromatin modification is involved in the control of haematopoiesis needs to be established, but numerous
reports in mammals clearly established that it plays a
critical role in HSC self-renewal, and later during lineage
specification (reviews in Fisher, 2002 and Lessard et al.,
2004). Two transduction signalling pathways, the JAK/
STAT and Toll pathways, were proposed to regulate
haemocyte proliferation and lamellocyte differentiation
(Gerttula et al., 1988; Qiu et al., 1998; review in Hou et al.,
2002). Mutations that cause constitutive activation of the
JAK kinase Hopscotch lead to hyperproliferation of circulating and lymph gland haemocytes and to formation of
melanotic nodules (Harrison et al., 1995; Luo et al., 1995).
The same phenotype was also observed in gain-offunction mutations in the transmembrane receptor Toll
and loss-of-function mutations in cactus which encodes
the IkB homologue downstream of the Toll pathway (Qiu
et al., 1998). When, how and in which cells these two
signalling pathways are required during larval haematopoiesis still needs to be investigated.
Conclusions and perspectives
Whereas during Drosophila embryonic haematopoiesis
lineages, genes and control mechanisms are relatively
well understood, the processes which are involved during
larval haematopoiesis are still largely unknown. Several
features distinguish embryonic and larval haematopoiesis: (i) embryonic haematopoiesis leads to a fixed number
of both lineages and haemocytes; (ii) in larvae, the lamellocyte constitutes a cryptic fate that is only revealed in

Journal compilation 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 11171126

No claim to original French government works

Drosophila haematopoiesis 1123

the lymph gland following parasitism, which demonstrates
that larval haematopoiesis is flexible and is controlled by
physiological conditions. These discrepancies suggest
that both the mechanisms and the genetic controls that
govern embryonic and larval haematopoiesis may significantly diverge.
The key role of the PSC in maintaining, throughout
Drosophila larval development, a pool of uncommitted
haematopoietic progenitors that can generate the necessary supply of lamellocytes in response to parasitization is
reminiscent of the microenvironmental niches that supply
factors in order to maintain stem cell potential in vertebrates (Nagasawa, 2006; Wilson and Trumpp, 2006).
Requirement of the JAK/STAT pathway is also reminiscent of its role in the stem cell niches of Drosophila male
and female gonads (Lin, 2002; Decotto and Spradling,
2005). Expression of Ser in the PSC and the role of Notch
signalling in controlling germline stem cell proliferation in
nematodes and Drosophila (Ward et al., 2006), and HSCs
in vertebrates suggest even further parallels (Calvi et al.,
2003; Zhang et al., 2003). Finally, Hh signalling is required
in zebrafish for adult blood stem cell formation (Gering
and Patient, 2005). Altogether these parallels highlight the
fundamental interest of Drosophila as a model system for
studying haematopoietic processes.
The indisputable success of Drosophila as a model
organism largely relies on the possibility to apply both
genetic and genomic approaches. Indeed, production of
transgenic flies that express dsRNA in a targeted manner,
and generation of KO flies constitute new powerful tools
that add to the more classical transgenic and genetic
approaches which allow functional analyses in vivo. In
addition, Drosophila haematopoiesis is relatively simple
compared with mammalian haematopoiesis, as it involves
both a limited number of blood cell types and fewer
members of gene families than their vertebrate
counterparts.
Among the fundamental questions which must be
answered in the future, we would like to stress the following: (i) How are the different types of haemocytes specified in the lymph gland and how are the differentiation and
proliferation programs co-ordinated? (ii) How do circulating blood cells communicate with lymph gland cells to
influence their program, as observed following a wasp
infection for instance? (iii) How does the PSC control the
balance between prohaemocytes and differentiated
haemocytes? Can we use Drosophila as a model to better
understand the mode of action of a microenvironmental
niche in vivo? (iv) Do genuine haematopoietic stem cells
exist in the lymph gland?
The remarkable similarities between Drosophila and
vertebrate haematopoiesis clearly point to a common
ancestry in evolution. A better understanding of the evolution of haematopoiesis in metazoans will require the

study of this process in other invertebrate species. From

this point of view, the recent genomic sequencing of
several Drosophila species raises the question of the
evolution of cellular immunity among Drosophilidae, and
highlights Drosophila as an outstanding model for studying microevolution of haematopoietic processes.

Acknowledgements
We thank Joanna Krzemien for the confocal image and Patrick
Blader, Lucas Waltzer and Julien Royet for critical reading of the
manuscript and discussion. Work carried out in the authors laboratories was supported by CNRS and Ministre de la Recherche
(ACI Biologie Cellulaire, Molculaire et Structurale).

References
Agaisse, H., Petersen, U.M., Boutros, M., Mathey-Prevot, B.,
and Perrimon, N. (2003) Signaling role of hemocytes in
Drosophila JAK/STAT-dependent response to septic injury.
Dev Cell 5: 441450.
Alfonso, T.B., and Jones, B.W. (2002) gcm2 promotes glial
cell differentiation and is required with glial cells missing for
macrophage development in Drosophila. Dev Biol 248:
369383.
Alvarez, A.D., Shi, W., Wilson, B.A., and Skeath, J.B. (2003)
Pannier and pointedP2 act sequentially to regulate Drosophila heart development. Development 130: 30153026.
Ashida, M., and Brey, P.T. (1997) Recent advances in
research on the insect propheno-loxidase cascade. In
Molecular Mechanisms of Immune Response in Insects.
Brey, P.T., and Hultmark, D. (eds). London: Chapman &
Hall, pp. 135172.
Badenhorst, P., Voas, M., Rebay, I., and Wu, C. (2002)
Biological functions of the ISWI chromatin remodeling
complex NURF. Genes Dev 16: 31863198.
Bantignies, F., Goodman, R.H., and Smolik, S.M. (2002) The
interaction between the coactivator dCBP and Modulo, a
chromatin-associated factor, affects segmentation and
melanotic tumor formation in Drosophila. Proc Natl Acad
Sci USA 99: 28952900.
Bataille, L., Auge, B., Ferjoux, G., Haenlin, M., and Waltzer,
L. (2005) Resolving embryonic blood cell fate choice in
Drosophila: interplay of GCM and RUNX factors. Development 132: 46354644.
Bernardoni, R., Vivancos, B., and Giangrande, A. (1997)
Glide/gcm is expressed and required in the scavenger cell
lineage. Dev Biol 191: 118130.
Blyth, K., Cameron, E.R., and Neil, J.C. (2005) The RUNX
genes: gain or loss of function in cancer. Nat Rev Cancer
5: 376387.
Bollerot, K., Pouget, C., and Jaffredo, T. (2005) The
embryonic origins of hematopoietic stem cells: a tale of
hemangioblast and hemogenic endothelium. APMIS 113:
790803.
Bruckner, K., Kockel, L., Duchek, P., Luque, C.M., Rorth, P.,
and Perrimon, N. (2004) The PDGF/VEGF receptor controls blood cell survival in Drosophila. Dev Cell 7: 7384.
Calvi, L.M., Adams, G.B., Weibrecht, K.W., Weber, J.M.,

Journal compilation 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 11171126

No claim to original French government works

1124 M. Crozatier and M. Meister

Olson, D.P., Knight, M.C., et al. (2003) Osteoblastic cells
regulate the haematopoietic stem cell niche. Nature 425:
841846.
Cantor, A.B., and Orkin, S.H. (2005) Coregulation of GATA
factors by the friend of GATA (FOG) family of multitype zinc
finger proteins. Semin Cell Dev Biol 16: 117128.
Cho, N.K., Keyes, L., Johnson, E., Heller, J., Ryner, L.,
Karim, F., and Krasnow, M.A. (2002) Developmental
control of blood cell migration by the Drosophila VEGF
pathway. Cell 108: 865876.
Crozatier, M., Valle, D., Dubois, L., Ibnsouda, S., and Vincent,
A. (1996) Collier, a novel regulator of Drosophila head
development, is expressed in a single mitotic domain. Curr
Biol 6: 707718.
Crozatier, M., Ubeda, J.M., Vincent, A., and Meister, M.
(2004) Cellular immune response to parasitization in
Drosophila requires the EBF orthologue collier. Plos Biol 2:
E196.
Daga, A., Karlovich, C.A., Dumstrei, K., and Banerjee, U.
(1996) Patterning of cells in the Drosophila eye by
Lozenge, which shares homologous domains with AML1.
Genes Dev 10: 11941205.
Decotto, E., and Spradling, A.C. (2005) The Drosophila
ovarian and testis stem cell niches: similar somatic stem
cells and signals. Dev Cell 9: 501510.
Dimarcq, J.L., Imler, J.L., Lanot, R., Ezekowitz, R.A.,
Hoffmann, J.A., Janeway, C.A., and Lagueux, M. (1997)
Treatment of l (2) mbn Drosophila tumorous blood cells
with the steroid hormone ecdysone amplifies the inducibility of antimicrobial peptide gene expression. Insect
Biochem Mol Biol 27: 877886.
Duchek, P., Somogyi, K., Jekely, G., Beccari, S., and Rorth,
P. (2001) Guidance of cell migration by the Drosophila
PDGF/VEGF receptor. Cell 107: 1726.
Duvic, B., Hoffmann, J.A., Meister, M., and Royet, J. (2002)
Notch signaling controls lineage specification during
Drosophila larval hematopoiesis. Curr Biol 12: 19231927.
Evans, C.J., Hartenstein, V., and Banerjee, U. (2003) Thicker
than blood: conserved mechanisms in Drosophila and vertebrate hematopoiesis. Dev Cell 5: 673690.
Fessler, L.I., Nelson, R.E., and Fessler, J.H. (1994) Drosophila extracellular matrix. Methods Enzymol 245: 271294.
Fisher, A.G. (2002) Cellular identity and lineage choice. Nat
Rev Immunol 2: 977982.
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: 73427347.
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: 1145111456.
Franc, N.C., Dimarcq, J.L., Lagueux, M., Hoffmann, J., and
Ezekowitz, R.A. (1996) Croquemort, a novel Drosophila
hemocyte/macrophage receptor that recognizes apoptotic
cells. Immunity 4: 431443.
Franc, N.C., Heitzler, P., Ezekowitz, R.A., and White, K.
(1999) Requirement for croquemort in phagocytosis of
apoptotic cells in Drosophila. Science 284: 19911994.

Gering, M., and Patient, R. (2005) Hedgehog signaling is

required for adult blood stem cell formation in zebrafish
embryos. Dev Cell 8: 389400.
Gerttula, S., Jin, Y.S., and Anderson, K.V. (1988) Zygotic
expression and activity of the Drosophila Toll gene, a gene
required maternally for embryonic dorsal-ventral pattern
formation. Genetics 119: 123133.
Hagman, J., Belanger, C., Travis, A., Turck, C.W., and Grosschedl, R. (1993) Cloning and functional characterization of
early B-cell factor, a regulator of lymphocyte-specific gene
expression. Genes Dev 7: 760773.
Harrison, D.A., Binari, R., Stines Nahreini, T., Gilman, M.,
and Perrimon, N. (1995) Activation of a Drosophila Janus
kinase (JAK) causes hematopoietic neoplasia and developmental defects. EMBO J 14: 28572865.
Hartenstein, V. (2006) Blood cells and blood cell development in the animal kingdom. Annu Rev Cell Dev Biol 22:
677712.
Heino, T.I., Karpanen, T., Wahlstrom, G., Pulkkinen, M.,
Eriksson, U., Alitalo, K., and Roos, C. (2001) The Drosophila VEGF receptor homolog is expressed in hemocytes.
Mech Dev 109: 6977.
Holz, A., Bossinger, B., Strasser, T., Janning, W., and
Klapper, R. (2003) The two origins of hemocytes in
Drosophila. Development 130: 49554962.
Hou, S.X., Zheng, Z., Chen, X., and Perrimon, N. (2002) The
Jak/STAT pathway in model organisms: emerging roles in
cell movement. Dev Cell 3: 765778.
Huber, T.L., Kouskoff, V., Fehling, H.J., Palis, J., and Keller,
G. (2004) Haemangioblast commitment is initiated in the
primitive streak of the mouse embryo. Nature 432: 625
630.
Irving, P., Ubeda, J.M., Doucet, D., Troxler, L., Lagueux, M.,
Zachary, D., et al. (2005) New insights into Drosophila
larval haemocyte functions through genome-wide analysis.
Cell Microbiol 7: 335350.
Jaffredo, T., Bollerot, K., Sugiyama, D., Gautier, R., and
Drevon, C. (2005) Tracing the hemangioblast during
embryogenesis: developmental relationships between
endothelial and hematopoietic cells. Int J Dev Biol 49:
269277.
Jones, B.W., Fetter, R.D., Tear, G., and Goodman, C.S.
(1995) Glial cells missing: a genetic switch that controls
glial versus neuronal fate. Cell 82: 10131023.
Jung, S.H., Evans, C.J., Uemura, C., and Banerjee, U. (2005)
The Drosophila lymph gland as a developmental model of
hematopoiesis. Development 132: 25212533.
Kennedy, M., Firpo, M., Choi, K., Wall, C., Robertson, S.,
Kabrun, N., and Keller, G. (1997) A common precursor for
primitive erythropoiesis and definitive haematopoiesis.
Nature 386: 488493.
Kocks, C., Cho, J.H., Nehme, N., Ulvila, J., Pearson, A.M.,
Meister, M., et al. (2005) Eater, a transmembrane protein
mediating phagocytosis of bacterial pathogens in
Drosophila. Cell 123: 335346.
Krzemien, J., Dubois, L., Makki, R., Meister, M., Vincent, A.,
and Crozatier, M. (2007) Control of blood cell homeostasis
in Drosophila larvae by the Posterior Signaling Center.
Nature 446: 325328.
Kurucz, E., Zettervall, C.J., Sinka, R., Vilmos, P., Pivarcsi, A.,
Ekengren, S., et al. (2003) Hemese, a hemocyte-specific

Journal compilation 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 11171126

No claim to original French government works

Drosophila haematopoiesis 1125

transmembrane protein, affects the cellular immune
response in Drosophila. Proc Natl Acad Sci USA 100:
26222627.
Lanot, R., Zachary, D., Holder, F., and Meister, M. (2001)
Postembryonic hematopoiesis in Drosophila. Dev Biol 230:
243257.
Lebestky, T., Chang, T., Hartenstein, V., and Banerjee, U.
(2000) Specification of Drosophila hematopoietic lineage
by conserved transcription factors. Science 288: 146149.
Lebestky, T., Jung, S.H., and Banerjee, U. (2003) A serrateexpressing signaling center controls Drosophila hematopoiesis. Genes Dev 17: 348353.
Lemaitre, B., and Hoffmann, J. (2007) The host defense of
Drosophila melanogaster. Annu Rev Immunol 25: in
press.
Lessard, J., Faubert, A., and Sauvageau, G. (2004) Genetic
programs regulating HSC specification, maintenance and
expansion. Oncogene 23: 71997209.
Lin, H. (2002) The stem-cell niche theory: lessons from flies.
Nat Rev Genet 3: 931940.
Luo, H., Hanratty, W.P., and Dearolf, C.R. (1995) An amino
acid substitution in the Drosophila hopTum-1 Jak kinase
causes leukemia-like hematopoietic defects. EMBO J 14:
14121420.
Mandal, L., Banerjee, U., and Hartenstein, V. (2004) Evidence for a fruit fly hemangioblast and similarities between
lymph-gland hematopoiesis in fruit fly and mammal aortagonadal-mesonephros mesoderm. Nat Genet 36: 1019
1023.
Mandal, L., Martinez-Agosto, J.A., Evans, C.J., Hartenstein,
V., and Banerjee, U. (2007) A hematopoietic niche defined
by Antennapedia expression uses Hedgehog for the maintenance of blood cell precursors in Drosophila. Nature 446:
320324.
Medvinsky, A., and Dzierzak, E. (1996) Definitive hematopoiesis is autonomously initiated by the AGM region. Cell
86: 897906.
Meister, M., and Lagueux, M. (2003) Drosophila blood cells.
Cell Microbiol 5: 573580.
Minakhina, S., and Steward, R. (2006) Melanotic mutants in
Drosophila: pathways and phenotypes. Genetics 174:
253263.
Nagasawa, T. (2006) Microenvironmental niches in the bone
marrow required for B-cell development. Nat Rev Immunol
6: 107116.
Nappi, A.J., and Vass, E. (1993) Melanogenesis and the
generation of cytotoxic molecules during insect cellular
immune reactions. Pigment Cell Res 6: 117126.
Orkin, S.H. (1998) Embryonic stem cells and transgenic mice
in the study of hematopoiesis. Int J Dev Biol 42: 927934.
Perrin, L., Monier, B., Ponzielli, R., Astier, M., and Semeriva,
M. (2004) Drosophila cardiac tube organogenesis requires
multiple phases of Hox activity. Dev Biol 272: 419431.
Qiu, P., Pan, P.C., and Govind, S. (1998) A role for the
Drosophila Toll/Cactus pathway in larval hematopoiesis.
Development 125: 19091920.
Ramet, M., Pearson, A., Manfruelli, P., Li, X., Koziel, H.,
Gobel, V., et al. (2001) Drosophila scavenger receptor CI is
a pattern recognition receptor for bacteria. Immunity 15:
10271038.
Rehorn, K.P., Thelen, H., Michelson, A.M., and Reuter, R.

(1996) A molecular aspect of hematopoiesis and endoderm

development common to vertebrates and Drosophila.
Development 122: 40234031.
Remillieux-Leschelle, N., Santamaria, P., and Randsholt,
N.B. (2002) Regulation of larval hematopoiesis in Drosophila melanogaster: a role for the multi sex combs gene.
Genetics 162: 12591274.
Rizki, M.T.M. (1957) Alterations in the haemocyte population
of Drosophila melanogaster. J Morphol 100: 437458.
Rizki, R.M., and Rizki, T.M. (1979) Cell interactions in the
differentiation of a melanotic tumor in Drosophila. Differentiation 12: 167178.
Rizki, T.M., and Rizki, R.M. (1984) The cellular defense
system of Drosophila melanogaster. In Insect Ultrastructure. King, R.C., and Akai, H. (eds). New York: Plenum
Publishing Corporation, pp. 579604.
Rizki, T.M., and Rizki, R.M. (1992) Lamellocyte differentiation
in Drosophila larvae parasitized by Leptopilina. Dev Comp
Immunol 16: 103110.
Russo, J., Dupas, S., Frey, F., Carton, Y., and Brehelin, M.
(1996) Insect immunity: early events in the encapsulation
process of parasitoid (Leptopilina boulardi) eggs in resistant and susceptible strains of Drosophila. Parasitology
112 (Pt 1): 135142.
Sears, H.C., Kennedy, C.J., and Garrity, P.A. (2003)
Macrophage-mediated corpse engulfment is required for
normal Drosophila CNS morphogenesis. Development
130: 35573565.
Shrestha, R., and Gateff, E. (1982) Ultrastructure and
cytochemistry of the cell types in the larval hematopoietic
organs and hemolymph of Drosophila melanogaster. Dev
Growth Differ 24: 6582.
Sinenko, S.A., Kim, E.K., Wynn, R., Manfruelli, P., Ando, I.,
Wharton, K.A., et al. (2004) Yantar, a conserved argininerich protein is involved in Drosophila hemocyte
development. Dev Biol 273: 4862.
Sorrentino, R.P., Carton, Y., and Govind, S. (2002) Cellular
immune response to parasite infection in the Drosophila
lymph gland is developmentally regulated. Dev Biol 243:
6580.
Tepass, U., Fessler, L.I., Aziz, A., and Hartenstein, V. (1994)
Embryonic origin of hemocytes and their relationship to cell
death in Drosophila. Development 120: 18291837.
Tsang, A.P., Fujiwara, Y., Hom, D.B., and Orkin, S.H. (1998)
Failure of megakaryopoiesis and arrested erythropoiesis in
mice lacking the GATA-1 transcriptional cofactor FOG.
Genes Dev 12: 11761188.
Ueno, H., and Weissman, I.L. (2006) Clonal analysis of
mouse development reveals a polyclonal origin for yolk sac
blood islands. Dev Cell 11: 519533.
de Velasco, B., Erclik, T., Shy, D., Sclafani, J., Lipshitz, H.,
McInnes, R., and Hartenstein, V. (2006) Specification and
development of the pars intercerebralis and pars lateralis,
neuroendocrine command centers in the Drosophila brain.
Dev Biol 302: 309323.
Vogeli, K.M., Jin, S.W., Martin, G.R., and Stainier, D.Y.
(2006) A common progenitor for haematopoietic and
endothelial lineages in the zebrafish gastrula. Nature 443:
337339.
Waltzer, L., Bataille, L., Peyrefitte, S., and Haenlin, M. (2002)
Two isoforms of Serpent containing either one or two

Journal compilation 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 11171126

No claim to original French government works

1126 M. Crozatier and M. Meister

GATA zinc fingers have different roles in Drosophila
haematopoiesis. EMBO J 21: 54775486.
Waltzer, L., Ferjoux, G., Bataille, L., and Haenlin, M. (2003)
Cooperation between the GATA and RUNX factors Serpent
and Lozenge during Drosophila hematopoiesis. EMBO J
22: 65166525.
Ward, E.J., Shcherbata, H.R., Reynolds, S.H., Fischer, K.A.,
Hatfield, S.D., and Ruohola-Baker, H. (2006) Stem cells

signal to the niche through the notch pathway in the

Drosophila ovary. Curr Biol 16: 17.
Wilson, A., and Trumpp, A. (2006) Bone-marrow
haematopoietic-stem-cell niches. Nat Rev Immunol 6:
93106.
Zhang, J., Niu, C., Ye, L., Huang, H., He, X., Tong, W.G., et al.
(2003) Identification of the haematopoietic stem cell niche
and control of the niche size. Nature 425: 836841.

Journal compilation 2007 Blackwell Publishing Ltd, Cellular Microbiology, 9, 11171126

No claim to original French government works