Institut für Allgemeine Zoologie und Genetik der Westfälischen Wilhelms-Universität, Schloßplatz 5, 48149 Münster, Germany
Author for correspondence (e-mail:
klapper{at}uni-muenster.de)
Accepted 1 July 2003
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SUMMARY |
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Key words: Drosophila, Hemocytes, Clonal analysis, Cell lineage, Transplantation, Blood, GFP, Lymph gland, Macrophage, Hematopoiesis
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Introduction |
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Recent analyses have revealed both genetic and functional similarities
between several aspects of insect and mammalian hematopoiesis (reviewed by
Franc, 2002;
Hoffmann et al., 1999
;
Traver and Zon, 2002
). In both
systems, the family of GATA transcription factors (Serpent/GATA), their
co-factors (U-shaped/FOG), the AML1 domain family transcription factors
(Lozenge/Runx1) (reviewed by Fossett and
Schulz, 2001
) as well as Notch signaling
(Duvic et al., 2002
) are
essential for blood-cell determination and differentiation into specific
cytotypes. In Drosophila, hematopoiesis takes place at two different
stages of ontogenesis: a first population of hemocytes arises from the head
mesoderm during early embryogenesis, followed by a second population that
derives from the mesodermal lymph glands at a later stage of development
(Traver and Zon, 2002
).
During embryogenesis of Drosophila, a proportion of the mesodermal
cells originating from the head region migrate along specific pathways and
subsequently disperses throughout the body
(Hartenstein and Jan, 1992;
Tepass et al., 1994
). These
embryonic hemocytes (EH) either differentiate into small spherical cells with
phagocytic capacities, so-called plasmatocytes, or into crystal cells that are
involved in the melanization of pathogens
(Alfonso and Jones, 2002
;
Franc et al., 1996
;
Franc, 1999
;
Lanot et al., 2001
;
Lebestky et al., 2000
). The
Drosophila GATA homolog serpent (srp) is expressed
in all embryonic hemocyte precursors and is also required for the development
of plasmatocytes and crystal cells (Rehorn
et al., 1996
; Sam et al.,
1996
).
In larvae, five major types of hemocytes have been described
(Lanot et al., 2001;
Rizki, 1957
;
Rizki, 1978
;
Rizki and Rizki, 1980
;
Rizki and Rizki, 1984
;
Rizki and Rizki, 1992
;
Rizki et al., 1980
;
Shrestha and Gateff, 1982
):
(1) plasmatocytes, which make up to 95% of the circulating hemocytes; (2)
podocytes, which develop from plasmatocytes at the end of the third larval
instar and are characterized by their pseudopodia-like extensions; (3) crystal
cells; (4) lamellocytes, large flat cells that presumably differentiate from
plasmatocytes in response to parasitic infections; and (5) small sessile
cells, found in segmental clusters on the integument. It has been proposed
that the larval hemocytes are produced and released by the lymph glands
(Bairati, 1964
;
Rizki, 1978
;
Rizki and Rizki, 1980
;
Rizki and Rizki, 1984
;
Shrestha and Gateff, 1982
;
Stark and Marshall, 1930
).
However, as the release of blood cells by the lymph gland into the hemocoel or
dorsal vessel has never been directly observed prior to late larval or early
pupal stages (Lanot et al.,
2001
), the function of the lymph gland as a source of larval
hemocytes has been questioned by several authors
(el Shatoury, 1955
;
Srdic and Reinhardt,
1980
).
The lymph glands, which are of mesodermal origin, are formed along the
anterior part of the dorsal vessel during embryogenesis
(Campos-Ortega and Hartenstein,
1997; Poulson,
1945
; Poulson,
1950
; Rugendorff et al.,
1994
; Stark and Marshall,
1930
). In the larva, four to six pairs of lymph gland lobes are
located lateral to the tube of the dorsal vessel
(el Shatoury, 1955
;
Rizki, 1978
). Whereas the
anteriormost pair of the larval lymph gland lobes contains active secretory
cells, plasmatocytes, crystal cells and undifferentiated prohemocytes, the
posterior lobes predominantly contain prohemocytes
(Lanot et al., 2001
). As the
lymph glands are eliminated at metamorphosis
(Lanot et al., 2001
;
Robertson, 1936
) and there is
no evidence for an imaginal hematopoietic organ, it is commonly believed that
the lymph gland-derived hemocytes (LGH) persist through metamorphosis.
However, up to now it was not possible to trace the different hemocyte populations throughout development. In this study, we performed transplantations of genetically labeled cells to follow the hemocytes from their formation up to the adult fly. Using this approach, we could show that the EH are already determined at the blastoderm stage. Both EH and LGH persist through metamorphosis and together represent the cellular components of the adult blood.
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Materials and methods |
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Transplantation experiments
For cell lineage analyses, single cells were transplanted at the cellular
blastoderm stage following the transplantation technique of Meise and Janning
(Meise and Janning, 1993). In
addition to single-cell transplantations, transplantations of five to ten
cells were carried out. The resulting clones of
ß-galactosidase-expressing cells were examined at the third larval instar
or in the adult fly. The preparation and staining procedure was carried out
according to Holz et al. (Holz et al.,
1997
). Living embryos and third-instar larvae were examined for
GFP expression and raised to adulthood. For detailed examination of GFP
expression, larvae and adult flies were dissected in transplantation solution
(Klapper, 2000
;
Meise and Janning, 1993
). An
Olympus inverse microscope CK40 equipped with an EGFP filter set (AHF
Analysentechnik) and a video enhancement system were used for fluorescence
analysis.
Immunohistochemical staining
To identify hemocytes, which are specified by their characteristic
expression of peroxidasin, we carried out double labeling. After the X-gal
staining that was employed to highlight the descendants of the transplanted
cell, we additionally performed an immunohistochemical staining using a mouse
anti-peroxidasin antibody (Nelson et al.,
1994) at a 1:10 dilution (anti-peroxidasin was kindly provided by
Rolf Reuter). Secondary antibodies conjugated to Biotin (Jackson
ImmunoResearch) were used at 1:200 dilutions. Biotinylated secondary
antibodies were detected by using the Vectastain Elite ABC kit and
HRP/diaminobenzidine (DAB) reaction. The immunohistochemical staining was
carried out following standard protocols.
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Results |
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Because it was shown that the EH originate from the head mesoderm region, we performed homotopic transplantations between 51 and 93% EL (EL, egg length; 0% EL, posterior pole) and 0 to 30% VD (VD, ventrodorsal; 0% VD, ventral midline). Sixty-five percent EL corresponds to the prospective position of the cephalic furrow, dividing the head from the trunk mesoderm, whereas 85% EL represents the anterior border of the mesoderm at the blastoderm stage. The mesodermal anlage extends dorsoventrally from the ventral midline to about 30% VD. To minimize possible heterotopic effects, we examined only transplantations in which the sites of cell removal and integration differ by no more than 5% EL. This corresponds to about five cell diameters at the blastoderm stage.
Of 2458 homotopic single-cell transplantations carried out between 51 and 93% EL, we detected 452 mesodermal and 33 endodermal clones in 3rd instar larvae (Table 1). In addition to these clones, we found some ectodermal clones at each position within the transplantation region. These ectodermal clones derived from transplantations of donor cells originating from the lateral border of the mesoderm anlage between 10 and 30% VD and were not further considered in this analysis. Besides 374 clones in mesodermal tissues, such as the fat body, somatic musculature and visceral musculature, we also detected 78 clones in hemocytes.
|
|
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The embryonic hemocytes are already determined at the blastoderm
stage
Previous transplantation experiments within the thoracic and abdominal
mesoderm revealed that the descendants of a single transplanted cell can give
rise to as many as four different mesodermal tissues
(Beer et al., 1987;
Holz et al., 1997
;
Klapper et al., 1998
). So far,
there is no evidence for a tissue-specific determination within the mesoderm
prior to the second postblastodermal mitosis. However, none of the 72 hemocyte
clones overlapped with other mesodermal derivatives, suggesting that the
embryonic hemocyte (EH) anlage might already be determined at the blastoderm
stage.
In order to test this possibility, we carried out heterotopic transplantations at the blastoderm stage. Single cells were transplanted either from outside the EH anlage into the EH anlage (Table 2B) or vice versa (Table 2C,D). Of 334 heterotopic single-cell transplantations into the EH primordium, 59 resulted in mesodermal clones (Table 2B). None of these clones contributed to hemocytes.
By contrast, transplantations from the EH anlage into the adjacent regions of the mesoderm frequently gave rise to hemocyte clones (Table 2C,D). These clones are indistinguishable from homotopic transplantation clones: the marked cells intermingle with other (unlabelled) hemocytes, exhibit the same morphology and colonize identical positions in third instar larvae (Fig. 2A,B). Taken together, these results demonstrate that the hemocytes are already determined at the blastoderm stage. A determination of other mesodermal cells towards an EH fate is not possible from the blastoderm stage onwards.
|
Two-hundred and twenty-nine homotopic single-cell transplantations were
carried out within the hemocyte anlage between 70 and 80% EL. In 101 of these
a hemocyte clone was identified in stage 17 embryos
(Fig. 3A). Up to 16 hemocytes
per embryo were labeled, indicating that the transplanted cell performed a
maximum of four postblastodermal mitoses. Of the 101 embryos showing a
hemocyte clone, 72 survived until the third larval instar. In 56 of these
larvae, the previously observed hemocyte clone was re-detected
(Fig. 3B,C). At this stage of
development, the clone sizes varied from 50 to 300 labeled hemocytes. This
reveals that the embryonic hemocytes (EH) performed up to five additional
mitoses during larval development. On the basis of their morphological
characteristics (spherical shape, presence of filamentous extensions) and
their motility, the observed hemocytes were classified as plasmatocytes and
podocytes (data not shown). We also observed clusters of sessile hemocytes,
possibly resembling the class of sessile hemocytes described by Lanot et al.
(Lanot et al., 2001).
|
The lymph glands release hemocytes at the onset of metamorphosis
Several analyses, mainly based on morphological observations, indicated
that the lymph glands produce and release hemocytes found in larvae
(Bairati, 1964;
Rizki, 1978
;
Shrestha and Gateff, 1982
;
Stark and Marshall, 1930
).
However, none of the hemocyte clones originating from the head mesoderm
labeled parts of the lymph glands at any time of development. Furthermore,
after transplantation of several thousand single mesodermal cells to sites
outside the embryonic hemocyte (EH) anlage described above, we never detected
clones contributing to hemocytes in third instar larvae
(Holz et al., 1997
;
Klapper, 2000
;
Klapper et al., 2001
;
Klapper et al., 1998
;
Klapper et al., 2002
). Besides
other mesodermal clones (data not shown), we obtained only three clones
contributing to the larval lymph glands. In all three cases, the
transplantations were carried out between 50 and 53% EL and resulted in clones
labelling one of the lymph gland lobes almost entirely. This indicates that
only a few progenitor cells give rise to a lymph gland lobe. Two of the clones
additionally labeled somatic muscles (Fig.
4A), revealing that the lymph gland in contrast to the
hemocytes originating from the head mesoderm is not determined at the
blastoderm stage. In none of these three clones were hemocytes outside the
lymph gland labeled.
|
Nine out of the 22 larvae with a lymph gland clone were examined repeatedly
until the onset of puparium formation (Fig.
4D-F). In seven of these larvae, labeled hemocytes became visible
at the late third larval instar, directly prior to pupation. This shows that
the lymph glands give rise to hemocytes, but that the release does not take
place prior to the onset of metamorphosis. Of the seven specimens, three
survived to adulthood and were examined once again. In all three individuals,
large hemocyte clones consisting of sessile as well as circulating
GFP-expressing cells were detected (Fig.
4G). On the basis of morphological criteria, the lymph gland
derived hemocytes (LGH) in adult flies were not distinguishable from the
persisting EH. Owing to the degradation of the lymph glands during
metamorphosis (Robertson,
1936), the labeling within this tissue were not redetected.
Taken together, our observations indicate that the embryonic hemocytes as well as the lymph gland hemocytes persist through metamorphosis. Thus, the blood of the adult fly is composed of two subpopulations of hemocytes that have two different spatial and temporal origins.
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Discussion |
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The embryonic hemocytes are already determined at the blastoderm
stage
It has previously been shown that the origin of the embryonic hemocytes
(EH) can be traced back to the head mesoderm of late stage 11 embryos by
morphological criteria (Tepass et al.,
1994). Owing to the fact that srp is expressed in a
narrow stripe within the cephalic mesoderm at the blastoderm stage and that a
loss of srp function leads to a complete loss of embryonic hemocytes,
the primordium of the EH was referred to the respective expression domain
(Rehorn et al., 1996
). By
homotopic single-cell transplantations we were able to restrict the anlage to
a sharply delimitated region located at 70 to 80% EL within the mesoderm
(Fig. 5), exactly corresponding
to the cephalic expression domain of srp. The fact that none of the
EH clones overlapped with other tissues indicated that the hemocytes are
already determined at the blastoderm stage. This was confirmed by heterotopic
transplantations from the EH anlage into the abdominal mesoderm, which also
gave rise to hemocytes. As mesodermal cells transplanted into the EH anlage
are not determined into EH, the determining factor is not able to induce a
hemocyte fate within these cells and seems to function cell-autonomously. A
good candidate for such a factor is Srp, a member of the GATA-binding
transcription factor family. However, as srp is also expressed in
many other tissues that do not give rise to hemocytes
(Abel et al., 1993
;
Rehorn et al., 1996
;
Sam et al., 1996
), there must
be additional genes that lead to a determination of the EH at the blastoderm
stage. The early determination of the EH is quite unusual, as all other
mesodermal tissues analysed so far including the anlage of the LGH
were not restricted to a tissue-specific fate prior to the second
postblastodermal mitoses (Beer et al.,
1987
; Holz et al.,
1997
; Klapper et al.,
1998
). This might be a developmental adaptation of the EH, which
at stage 12 are already differentiated into functional macrophages and are
responsible for the removal of apoptotic cells within developing tissues
(Abrams et al., 1993
;
Franc et al., 1996
;
Franc, 1999
;
Hartenstein and Jan, 1992
;
Tepass et al., 1994
).
|
The two origins of hemocytes
Previous studies, as well as our cell lineage analyses, reveal that the two
populations of hemocytes share many functional, morphological and genetic
similarities. In both cases, the determination of hemocytes depends on
srp (Lebestky et al.,
2000; Rehorn et al.,
1996
), while the specification towards the distinct blood cell
types is induced by the expression of lozenge (lz)
(Lebestky et al., 2000
),
glia cells missing (gcm) (Bernardoni et al., 1997) and the
gcm homolog gcm2 (Alfonso
and Jones, 2002
). Both EH and LGH differentiate into podocytes,
crystal cells and plasmatocytes (Lanot et
al., 2001
). Hemocytes of both populations have the capability to
adopt macrophage characteristics. However, despite all similarities, the
history of the two populations is quite different, as they originate from two
different mesodermal regions and are determined at different developmental
stages. In view of the fact that the lymph glands do not release hemocytes
before the onset of metamorphosis under nonimmune conditions, all hemocytes
found in the larval hemocoel represent EH. This was not taken into account in
several genetic analyses of embryonic and larval hemocytes. Thus, it may be
possible that some of these data have to be reconsidered, taking into account
that not lymph gland derived larval hemocytes were studied, but EH during
larval development.
The many similarities between EG and LGH raise the question why there are
two populations at all. As also observed in many other studies, we noted a
massive release of hemocytes by the lymph glands just at the onset of
pupation. Lanot et al. (Lanot et al.,
2001) could show that the lymph glands additionally have the
capacity to differentiate and release a special type of hemocytes, the
lamellocytes, under immune conditions even before the onset of metamorphosis.
Thus, because under nonimmune conditions the lymph glands do not release any
cells before the onset of pupation, it might be their primary role to provide
a reservoir of immune defensive hemocytes. The massive apoptosis and
accumulation of cell debris might be a secondary trigger to stimulate
proliferation and release of the lymph gland hemocytes.
To study Drosophila hematopoiesis, the knowledge of which hemocyte population is present at different stages of development is pivotal. To avoid misunderstandings associated with the confusing term `larval hemocytes', which has been used to describe all kinds of hemocytes in postembryonic development, we propose the use of the terms embryonic hemocytes (EH) and lymph gland hemocytes (LGH).
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ACKNOWLEDGMENTS |
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Footnotes |
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