Department of Molecular, Cell and Developmental Biology, Department of Biological Chemistry and Molecular Biology Institute, University of California, Los Angeles, CA 90095, USA
Author for correspondence (e-mail:
banerjee{at}mbi.ucla.edu)
Accepted 29 March 2005
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SUMMARY |
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Key words: Drosophila, Hematopoiesis, Blood, Hemocyte, Lymph gland
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Introduction |
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In Drosophila, mature hemocytes arise from two spatially and
temporally distinct phases of hematopoietic development, one early in the
embryonic head mesoderm and another at later larval stages in a specialized
organ called the lymph gland (Lebestky et
al., 2000; Rizki,
1978
; Tepass et al.,
1994
). The embryonic phase of hematopoiesis gives rise to mature,
circulating hemocytes of the larval stages, whereas lymph gland hemocytes,
under normal, non-immune conditions, do not enter circulation until the onset
of metamorphosis (Holz et al.,
2003
). Once in circulation, these lymph gland-derived hemocytes,
along with a subset of hemocytes derived from the embryonic head mesoderm, can
persist into the adult stage (Holz et al.,
2003
). The lymph gland originates in the cardiogenic mesoderm of
the embryo and subsequently grows by cellular proliferation during the larval
instars. Although the function of the lymph gland as a hematopoietic organ is
well established (reviewed by Evans et
al., 2003
; Lanot et al.,
2001
; Shrestha and Gateff,
1982
; Sorrentino et al.,
2002
), surprisingly little is known about the spatial and temporal
events regulating this process. This paucity of detail is primarily due to a
lack of lineage-specific genetic markers, which is in sharp contrast to the
level of detail afforded to other Drosophila tissues such as the
salivary glands, imaginal discs and ovaries, not to mention that seen for
vertebrate hematopoietic systems. In this report, we address the
spatiotemporal events of lymph gland hematopoiesis through an in-depth
characterization of known and novel genetic markers.
In Drosophila, there are at least three terminally differentiated
hemocyte types: plasmatocytes, crystal cells and lamellocytes
(Evans et al., 2003;
Rizki, 1956
). Each type is
thought to be derived from a common precursor that expresses and requires the
GATA factor Serpent (Srp) (Rehorn et al.,
1996
; Tepass et al.,
1994
). Plasmatocytes represent 90-95% of all mature
Drosophila hemocytes and function in the phagocytic removal of dead
cells and microbial pathogens (Rizki,
1978
; Tepass et al.,
1994
). The specification of plasmatocytes requires the
transcription factors Glial cells missing (Gcm) and Gcm2
(Alfonso and Jones, 2002
;
Bernardoni et al., 1997
).
Additionally, circulating plasmatocytes have been shown to express Peroxidasin
(Pxn) (Nelson et al., 1994
), a
component of the extracellular matrix, and an uncharacterized surface marker
called P1 antigen (Asha et al.,
2003
; Vilmos et al.,
2004
). Crystal cells, which constitute
5% of the hemocyte
population, are non-phagocytic cells that facilitate innate immune and
wound-healing responses by mediating the process of melanization
(Lanot et al., 2001
;
Rizki, 1978
;
Russo et al., 1996
). Crystal
cell differentiation requires the cell-autonomous expression of the
transcription factor Lozenge, a Runt-domain protein that shares homology with
mammalian Runx proteins, including Acute Myeloid Leukemia 1 (AML1 or Runx1)
(Daga et al., 1996
;
de Bruijn and Speck, 2004
;
Lebestky et al., 2000
).
Additionally, mature crystal cells express Prophenoloxidase A1 (ProPOA1), an
oxidoreductase related to hemocyanins and vertebrate tyrosinases that mediates
melanization reactions upon activation
(Rizki and Rizki, 1985
;
Soderhall and Cerenius, 1998
).
Lamellocytes are relatively large (15-40 µm across), flat, adherent cells
that primarily function in the encapsulation and neutralization of objects too
large to be engulfed by plasmatocytes
(Rizki and Rizki, 1992
).
Lamellocytes have not been found in embryos or adults and are rarely observed
during larval stages, although large numbers of these cells can be induced to
differentiate in larvae upon challenge with parasitic wasp eggs
(Lanot et al., 2001
;
Sorrentino et al., 2002
).
Genetically, mature lamellocytes have commonly been identified by their
expression of a reporter in the misshapen locus
(Braun et al., 1997
;
Lanot et al., 2001
;
Sorrentino, 2002), which encodes a component of the JUN kinase signaling
cascade. Additionally, lamellocytes express an uncharacterized surface marker
called L1 antigen (Asha et al.,
2003
). All Drosophila hemocytes specifically express the
marker Hemese (He) (Kurucz et al.,
2003
), while a majority of plasmatocytes and crystal cells express
the Collagens Viking and Cg25C (Le Parco
et al., 1986
; Yasothornsrikul
et al., 1997
) and the Von Willebrand-like factor Hemolectin (Hml)
(Goto et al., 2003
;
Goto et al., 2001
;
Sinenko and Mathey-Prevot,
2004
).
Several signaling pathways have been associated with the
Drosophila hematopoietic process. Hyperactivation of the JAK homolog
Hopscotch (Hop) causes extensive hemocyte proliferation, lamellocyte
differentiation and melanized pseudotumor formation at high frequency
(Harrison et al., 1995;
Hou et al., 2002
;
Luo et al., 1995
). Despite
this apparent role in regulating hemocyte proliferation, hop
loss-of-function has no overt effect upon the numbers of circulating hemocytes
or the growth of the lymph gland
(Remillieux-Leschelle et al.,
2002
; Sorrentino et al.,
2004
). Loss of function does, however, impair the ability of the
lymph gland to mount an effective immune response to parasitization
(Sorrentino et al., 2004
). In
mammals, JAK/STAT signaling is required for various aspects of hematopoiesis
and immunity and dysregulation of this pathway has been associated with
numerous malignancies including lymphomas and leukemias
(Rane and Reddy, 2002
;
Ward et al., 2000
).
Misregulation of the Drosophila Toll pathway, which is related to
vertebrate NF
B/I
B signaling pathways, also causes significant
hematopoietic defects that include aberrant proliferation and differentiation
(Gerttula et al., 1988
;
Qiu et al., 1998
). Finally,
the receptor tyrosine kinase Pvr, which shares homology with vertebrate PDGF
and VEGF receptors, is known to function in embryonic hemocytes where it
controls migration and cell survival
(Bruckner et al., 2004
;
Cho et al., 2002
;
Heino et al., 2001
;
Sears et al., 2003
). The Pvr
pathway may also have a role in proliferation control because misexpression of
the ligand Pvf2 causes significant expansion of the lymph gland
(Munier et al., 2002
). Taken
together, it is clear that the JAK/STAT, Toll and Pvr pathways play
significant roles in Drosophila hematopoiesis.
A considerable amount is known about the role of the Notch signaling
pathway in controlling lymph gland hematopoiesis. In addition to a role in
proliferation, Notch signaling is required for the early specification of the
lymph gland (Mandal et al.,
2004) and the determination of the crystal cell hemocyte lineage,
both in the lymph gland and in the embryonic head mesoderm
(Duvic et al., 2002
;
Lebestky et al., 2003
).
Furthermore, the study of Notch function in hematopoiesis has led to the
identification of the first subdomain or compartment in the lymph gland, which
was termed the posterior signaling center (PSC)
(Lebestky et al., 2003
). The
PSC consists of a small cluster of cells at the posterior tip of each of the
primary (anterior-most) lymph gland lobes and is defined by the expression of
the Notch ligand Serrate (Ser). Serrate signaling through Notch mediates the
commitment of prohemocytes to the crystal cell lineage via the expression of
Lozenge. Recently, the transcription factor Collier, which shares homology
with mammalian Early B-cell Factor (EBF), has been shown to be expressed in
the PSC earlier than Ser and to control Ser expression in these cells
(Crozatier et al., 2004
).
collier mutant larvae fail to produce lamellocytes, even upon
parasitization, indicating a role of the PSC in both crystal cell and
lamellocyte specification.
In this report, we provide a description of the lymph gland as a developmental system based on the dynamic expression and function of a number of known as well as uncharacterized hematopoietic and pro-hemocytic markers. This molecular genetic and structural analysis reveals novel features and provides a comprehensive picture of, and a mechanistic basis for, the spatial and temporal events that give rise to blood cells from their precursors within the lymph gland.
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Materials and methods |
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Immunohistochemistry
Lymph glands were stained as previously described
(Lebestky et al., 2000). The
following antibodies were used in the described experiments: rabbit
-Srp (D. Hoshizaki), rabbit
-ßgal (Cappell), mouse
-ßgal (Promega), mouse
-Lz, mouse
-Pxn (J. Fessler
and L. Fessler), mouse
-Hemese, mouse
-P1 and mouse
-L1
(I. Ando), mouse
-Cut (Developmental Studies Hybridoma Bank), rat
-DE-cadherin (V. Hartenstein), rabbit
-U-shaped (R. Schulz), rat
-Pvr (B. Shilo and P. Garrity) and rat
-ProPO (H. Müller).
Alexa Fluor 488-, Alexa Fluor 546- (Molecular Probes) and Cy3- (Jackson
Laboratory) conjugated secondary antibodies were used. Lymph glands were
mounted in Vectashield (Vector Laboratories) alone or Vectashield with
To-Pro-3 (Molecular Probes) for nuclear staining. Unless otherwise stated,
late third instar larvae were used. For second instar lymph gland staining,
first instar larvae were collected upon hatching at 1 hour intervals,
maintained at 25°C, and dissected between 40 and 44 hours
post-hatching.
BrdU analysis
Dissected lymph glands were incubated in the 75 µg/ml BrdU in PBS for 1
hour, fixed immediately in 4% formaldehyde/PBS for 30 minutes, washed three
times for 10 minutes each in PBS, blocked in 10% normal goat serum/PBS, then
incubated in mouse -GFP antibody (Molecular Probes) or
-Pxn
overnight. Lymph glands were washed four times in PBS, fixed again in 2%
formaldehyde/PBS for 15 minutes, washed three times for 5 minutes, and then
incubated in 2 M HCl for 30 minutes to denature BrdU-labeled DNA. Lymph glands
were then washed four times for 10 minutes each and stained with rat
-BrdU (Abcam) antibody, followed by standard secondary antibody
staining and mounting.
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Results |
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Maturing hemocytes are restricted to the cortical zone of primary lymph gland lobes
Mature hemocytes have been shown to express several markers, including
collagens (Asha et al., 2003;
Fessler et al., 1994
;
Yasothornsrikul et al., 1997
),
Hemolectin (Goto et al., 2003
;
Goto et al., 2001
), Lozenge
(Lebestky et al., 2000
),
Peroxidasin (Nelson et al.,
1994
) and P1 antigen (Asha et
al., 2003
; Vilmos et al.,
2004
). We found that the expression of the reporter
Collagen-gal4 (Cg-gal4)
(Asha et al., 2003
), which is
expressed by both plasmatocytes and crystal cells, is restricted to the
periphery of the primary lymph gland lobe
(Fig. 2A). Comparison of
Cg-gal4 expression in G147 lymph glands, in which the
medullary zone and cortical zone can be distinguished, revealed that maturing
hemocytes are restricted to the cortical zone
(Fig. 2B,B'). In fact,
the expression of each of the maturation markers mentioned above is found to
be restricted to the cortical zone. The reporter hml-gal4
(Goto et al., 2003
) and Pxn,
which are expressed by the plasmatocyte and crystal cell lineages, are
extensively expressed in this region (Fig.
2C,D). Likewise, the expression of the crystal cell lineage marker
Lozenge is restricted in this manner (Fig.
2E). The spatial restriction of maturing crystal cells to the
cortical zone was verified by several means, including the distribution of
melanized lymph gland crystal cells in the Black cells background
(Fig. 2F) and analysis of the
terminal marker ProPOA1 (not shown). The cortical zone is also the site of P1
antigen expression (Fig. 2G), a
marker of the plasmatocyte lineage. The uncharacterized GFP fusion line
ZCL2826 also exhibits preferential expression in the cortical zone
(Fig. 2H). Last, we found that
the homeobox transcription factor Cut
(Blochlinger et al., 1993
) is
preferentially expressed in the cortical zone of the primary lobe
(Fig. 2I). Although the role of
Cut in Drosophila hematopoiesis is currently unknown, homologs of Cut
are known to be regulators of the myeloid hematopoietic lineage in both mice
and humans (Bjerregaard et al.,
2003
; Sinclair et al.,
2001
). Cells of the rare third cell type, lamellocytes, are also
restricted to the cortical zone (Fig.
2J,K), based upon cell morphology and the expression of a
msn-lacZ reporter (msn06946). In summary, based
on the expression patterns of several genetic markers that identify the three
major blood cell lineages, we propose that the cortical zone is a specific
site for hemocyte maturation.
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We have also found that the PSC can be defined just as definitively by the
characteristic absence of several markers. For example, the RTK receptor Pvr,
which is expressed throughout the lymph gland, is notably absent from the PSC
(Fig. 3K). Likewise,
dome-gal4 is not expressed in the PSC
(Fig. 3L), further suggesting
that this population of cells is biased toward the production of ligands
rather than receptor proteins. Maturation markers such as Cg-gal4,
which are expressed throughout the cortical zone, were never found to be
expressed by PSC cells (Fig.
3M). Additionally, we found that the expression levels of the
hemocyte marker Hemese (Kurucz et al.,
2003) and the Friend-of-GATA protein U-shaped
(Fossett et al., 2001
) are
dramatically reduced in the PSC when compared with other hemocytes of the
lymph gland (Fig. 3N,O). Taken
together, both the expression and lack of expression of a number of genetic
markers defines the cells of the PSC as a unique hemocyte population.
Hematopoietic differentiation in secondary lymph gland lobes
In contrast to primary lobes of the third instar, maturing hemocytes are
generally not seen in the secondary lobes. Correspondingly, secondary lobes
often have a smooth and compact appearance
(Fig. 1H), much like the
medullary zone of the primary lobe. Consistent with this appearance, secondary
lymph gland lobes also express high levels of DE-cadherin
(Fig. 4A). The size of the
secondary lobe, however, varies from animal to animal and this correlates with
the presence or absence of maturation markers. Smaller secondary lobes contain
a few or no cells expressing maturation markers, whereas larger secondary
lobes usually exhibit groups of differentiating cells. Direct comparison of
DE-cadherin expression in secondary lobes with that of Cg-gal4,
hml-gal4 or Lz revealed that the expression of these maturation markers
occurs only in areas in which DE-cadherin is downregulated
(Fig. 4B-E'). Therefore,
although there is no apparent distinction between cortical and medullary zones
in differentiating secondary lobes, there is a significant correlation between
the expression of maturation markers and the downregulation of DE-cadherin, as
is observed in primary lobes.
Temporal analysis of lymph gland hematopoietic development
The relatively late `snapshot' of lymph gland development in the third
larval instar establishes the existence of spatial zones within the lymph
gland that are characterized by differences in structure as well as gene
expression (Fig. 4F). In order
to understand how these zones form over time, lymph glands of second instar
larvae, the earliest time at which we are able to dissect and stain, were
examined for the expression of hematopoietic markers. As expected, Srp and Odd
are expressed throughout the lymph gland during the second instar
(Fig. 1C,D) as they are in the
late embryo and third instar lymph gland
(Fig. 1A,B,E). Likewise, the
hemocyte-specific marker Hemese is expressed throughout the lymph gland at
this stage (Fig. 5A), although
it is not present in the embryonic lymph gland (not shown).
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By studying the temporal sequence of expression of hemocyte-specific
markers, one can describe stages in the maturation of a hemocyte. As an
example, the maturation steps of a typical plasmatocyte are indicated in
Fig. 5E. It should be noted,
however, that not all hemocytes of a particular lineage are identical. For
example, in the late third instar lymph gland, the large majority of mature
plasmatocytes (80%) expresses both Pxn and hml-gal4, but the
remainder expresses only Pxn (
15%) or hml-gal4 (
5%) alone.
Thus, while plasmatocytes as a group can be characterized by the expression of
representative markers (shown in Fig.
5E), populations expressing subsets of these markers indeed exist.
It remains unclear at this time whether this heterogeneity in the hemocyte
population is reflective of specific functional differences.
In the third instar, Pxn is a prototypical hemocyte maturation marker, while immature cells of the medullary zone express dome-gal4. Comparing the expression of these two markers in the second instar revealed an interesting developmental progression. As mentioned above, a group of cells along the peripheral edge of these early lymph glands already express Pxn. These developing hemocytes downregulate the expression of dome-gal4 (Fig. 5F,F'), as they do in the third instar. Next to these developing hemocytes is a group of cells that expresses dome-gal4 but not Pxn; these cells are most similar to medullary zone cells of the third instar and are therefore prohemocytes. Interestingly, there also exists a group of cells in the second instar that expresses neither Pxn nor dome-gal4. This population is most easily seen in the medial parts of the gland, close to the centrally placed dorsal vessel (Fig. 5F'). These cells resemble earlier precursors in the embryo, except they express the marker Hemese (Fig. 5G). We call these cells pre-prohemocytes. Our interpretation of the expression data is that pre-prohemocytes upregulate dome-gal4 to become prohemocytes. As prohemocytes begin to mature into hemocytes, dome-gal4 expression is downregulated, while the expression of maturation markers is initiated. The prohemocyte and hemocyte populations continue to be represented in the third instar as components of the medullary and cortical zones, respectively.
The cells of the PSC are already distinguishable in the late embryo by
their expression of collier
(Crozatier et al., 2004). We
found that the canonical PSC marker Ser-lacZ is not expressed in the
embryonic lymph gland (not shown) and is only expressed in a small number of
cells in the second instar (Fig.
5H). This relatively late onset of expression is consistent with
collier acting genetically upstream of Ser
(Crozatier et al., 2004
).
Another finding was that the earliest expression of upd3-gal4
parallels the expression of Ser-lacZ and is restricted to the PSC
region (Fig. 5I,J). Finally,
Pvr and dome-gal4 are excluded from the PSC in the second instar
(Fig. 5H,K,K'), similar
to that seen in the third instar.
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Role of Pvr in hemocyte maturation
Genetic manipulation of Pvr function provided valuable insight into its
involvement in the regulation of temporal events of lymph gland development.
To analyze Pvr function, FLP/FRT-based Pvr-mutant clones were
generated in the lymph gland early in the first instar and then examined
during the third instar for the expression of maturation markers. We found
that loss of Pvr function abolishes P1 antigen and Pxn expression
(Fig. 6A,A',B,B'),
but not Hemese expression (Fig.
6C,C'). The crystal cell markers Lz
(Fig. 6D,D') and ProPOA1
(not shown) are also expressed normally in Pvr-mutant clones,
consistent with the observation that mature crystal cells lack or downregulate
Pvr (not shown). The fact that Pvr-mutant cells express Hemese and
can differentiate into crystal cells suggests that Pvr specifically controls
plasmatocyte differentiation. Pvr-mutant cells do not become TUNEL
positive (Fig. 6E,E') but
do express the hemocyte marker Hemese and can differentiate into crystal
cells, all suggesting that the observed block in plasmatocyte differentiation
within the mutant clone is not due to cell death. Additionally, Pvr-mutant
clones were large (Fig. 6) and
not significantly different in size from their wild-type twin spots (not
shown). Thus, the primary role of Pvr is not in the control of cell
proliferation. Targeting Pvr by RNA interference (RNAi) revealed the same
phenotypic features (not shown), confirming that Pvr controls the transition
of Hemese-positive cells to plasmatocyte fate.
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Discussion |
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Our analysis of the lymph gland revealed three key features that arise during development. The first feature is the presence of three distinct zones in the primary lymph gland lobe of third instar larvae. Two of these zones, which we term the cortical and medullary zones, exhibit structural characteristics that make them morphologically distinct. These zones, as well as the third zone, the PSC, are also distinguishable by the expression of specific markers. The second key feature is the finding that cells expressing maturation markers such as Lz, ProPOA1, Pxn, hml-gal4 and Cg-gal4 are restricted to the cortical zone. The medullary zone is consistently devoid of maturation marker expression and is therefore defined as a region composed of immature hemocytes (prohemocytes). The finding of different developmental populations within the lymph gland (prohemoctyes and their derived hemocytes) is similar to the situation in vertebrates where it is known that hematopoietic stem cells and other blood precursors give rise to various mature cell types. Additionally, Drosophila hemocyte maturation is akin to the progressive maturation of myeloid and lymphoid lineages in vertebrate hematopoiesis. The third key feature of lymph gland hematopoiesis is the dynamic pattern of cellular proliferation observed in the third instar. At this stage, the vast majority of S-phase cells in the primary lobe are located in the cortical zone, suggesting a strong correlation between proliferation and hemocyte differentiation. Compared with earlier developmental stages, cell proliferation in the medullary zone actually decreases by the late third instar, suggesting that these cells have entered a quiescent state. Thus, proliferation in the lymph gland appears to be regulated such that growth, quiescence and expansion phases are evident throughout its development (Fig. 7F).
Drosophila blood cell precursors, prohemocytes and maturing hemocytes each exhibit extensive phases of proliferation. The competence of these cells to proliferate seems to be a distinct cellular characteristic that is superimposed upon the intrinsic maturation program. Based on the patterns of BrdU incorporation in developing primary and secondary lymph gland lobes, it is possible to envision at least two levels of proliferation control during hematopoiesis. We propose that the widespread cell proliferation observed in second instar lymph glands and in secondary lobes of third instar lymph glands occurs in response to a growth requirement that provides a sufficient number of prohemocytes for subsequent differentiation. The mechanisms promoting differentiation in the cortical zone also trigger cell proliferation, which accounts for the observed BrdU incorporation in this zone and serves to expand the effector hemocyte population. The quiescent cells of the medullary zone represent a pluripotent precursor population because they, similar to vertebrate hematopoietic precursors, rarely divide and give rise to multiple lineages and cell types.
Based on the analysis described above, we can propose a model by which
hemocytes mature in the lymph gland (Fig.
8). Hematopoietic precursors that populate the early lymph gland
are first distinguishable as Srp+, Odd+ (S+O+,
Fig. 8A) cells. These will
eventually give rise to a primary lymph gland lobe where the steps of hemocyte
maturation are most apparent. During the first or early second instar, these
S+O+ cells begin to express the hemocyte-specific marker
Hemese (He) and the tyrosine kinase receptor Pvr. Such cells can be called
pre-prohemocytes and, in the second instar, cells expressing only these
markers occupy a narrow region near the dorsal vessel. Subsequently, a subset
of these Srp+, Odd+, He+, Pvr+
(S+O+H+Pv+) pre-prohemocytes
initiate the expression of dome-gal4 (dg4),
thereby maturing into prohemocytes. The prohemocyte population
(S+O+H+Pv+dg4+)
can be subdivided into two developmental stages. Stage 1 prohemocytes, which
are abundantly seen in the second instar, are proliferative, whereas stage 2
prohemocytes, exemplified by the cells of the medullary zone, are quiescent.
As development continues, prohemocytes begin to downregulate
dome-gal4 and express maturation markers (M; becoming
S+O+H+Pv+dg4lowM+).
Eventually, dome-gal4 expression is lost entirely in these cells
(becoming
S+O+H+Pv+dg4-M+),
found generally in the cortical zone. Thus, the maturing hemocytes of the
cortical zone are derived from prohemocytes previously belonging to the
medullary zone. This is supported by lineage-tracing experiments that showed
cells expressing medullary zone markers can indeed give rise to cells of the
cortical zone. In turn, the medullary zone is derived from the earlier,
pre-prohemocytes. Early cortical zone cells continue to express successive
maturation markers (M) as they proceed towards terminal differentiation
(Fig. 8B). Depending on the
hemocyte type, examples of expressed maturation markers are Pxn, P1, Lz, L1,
msn-lacZ, etc. Our studies have shown that differentiation of the
plasmatocyte lineage requires Pvr, while previous work has shown that the
Notch pathway is crucial for the crystal cell fate
(Duvic et al., 2002;
Lebestky et al., 2003
). Both
the JAK/STAT and Notch pathways have been implicated in lamellocyte production
(Duvic et al., 2002
;
Sorrentino et al., 2004
).
Previous investigations have demonstrated that similar transcription factors and signal transduction pathways are used in the specification of blood lineages in both vertebrates and Drosophila. Given this relationship, Drosophila represents a powerful system for identifying genes crucial to the hematopoietic process that are conserved in the vertebrate system. The work presented here provides an analysis of hematopoietic development in the Drosophila lymph gland that not only identifies stage-specific markers, but also reveals developmental mechanisms underlying hemocyte specification and maturation. The prohemocyte population in Drosophila becomes mitotically quiescent, much as their multipotent precursor counterparts in mammalian systems. These conserved mechanisms further establish Drosophila as an excellent genetic model for the study of hematopoiesis.
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ACKNOWLEDGMENTS |
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Footnotes |
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